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specific reaction of S-methylation on sulphhydryl groups, the microsomal enzymes involved also require participation of SAM. Thiol methylation is important in the metabolism of captopril, D-penicillamine, azathioprine and 6-mercap topurine (6MP), which, following this process will be excreted as sulphoxides or sulphones. In the context, we should also mention the so-called thiomethyl shunt, acting on compounds in which sulphur has been added from glutathione. It begins with the addition of glutathione, followed by conversion to the cysteine conjugate. Sequential steps include the cleavage of the conjugate by a cysteine conjugate ȕ-lyase to pyruvate, ammonia and thiol, and subsequent methylation of the thiol formed. There are three enzymes with different characteristics involved in this process: • the microsomal thiol-methyl transferase (TMT), a membrane- bound enzyme that catalyses demethylation of aliphatic sulphhydryl compounds (such as captopril and D-penicillamine); • the cytosolic thioether-S-methyltransferase (TEMT), and • the soluble thiopurine-methyl transferase (TPMT). This is a cytoplasmic enzyme catalysing the S-methylation of aromatic and heterocyclic sulphhydryl compounds, such as 6MP and azathioprine preferentially. Further details are provided in Chapter 4. S-methylation of sulphhydryl compounds also requires the presence and participation of SAM. It is important to mention that none of the 152 Chapter 3 endogenous sulphhydryl compounds (e.g. cysteine, GSH) can function as substrates, although a wide variety of exogenous sulphhydryl compounds may be S-methylated by the microsomal S-methyl transferases. Amino acid conjugation reactions generally involve one or two amino acids, viz. glycine or glutamic acid, the former being the more common. These reactions can occur with substrates containing either an alcohol, or a carboxyl moiety, especially substrates that contain aromatic groups. The general reaction is given in Figure 3.18: R COOH + ATP R CO AMP + PPi R CO AMP + CoASH R CO S CoA + AMP R CO S CoA + R' NH2 R CO NH R' + CoASH Fig.3.18 The amino acid conjugation of carboxylic acids It is evident from the above figure that amino acid conjugation is a special form of N-acylation, where the drug, and not the endogenous co-factor is activated. Exogenous carboxylic acids can form CoA derivatives in the body and can then react with endogenous amines, forming an amide (or peptide) bond. The general reaction can be written more succinctly as follows: R(CO)OH + NH2CH2(CO)OH  R(CO)NHCH2(CO)OH + H2O 3.3 Such conjugation with amino acids represents an important metabolic pathway in the eventual elimination of drug (and other xenobiotic) carboxylic acids. The substrates may be aromatic, arylaliphatic, and heterocyclic carboxylic acids and the resulting metabolites are water-soluble ionic conjugates. Usually, these amino acid conjugates are less toxic then their precursor acids and are readily excreted into the urine or bile. Conjugation reactions with amino acids may be limited by the amount of endogenous glycine, or by the amount of enzyme available to catalyse the reaction. The metabolic fate of the aforementioned carboxylic acids depends strongly on the size and type of substituents adjacent to the carboxylic group, according to the following guidelines: • most unbranched aliphatic acids are completely oxidized and do not usually form conjugates; Pathways of biotransformation – phase II reactions 153 • branched aliphatic and aryl aliphatic acids resist ȕ-oxidation, forming glycine or glucuronide conjugates, with glycine conjugation preferred for xenobiotic carboxylic acids at low doses. It is of interest to note that substituents on the Į-carbon atom favour glucuronidation over glycine conjugation. Glycine conjugation is not confined to xenobiotics but also occurs with endogenous compounds; conjugates of bile acids are formed by enzymatic action in the microsomal fraction. Some examples will be given at the end of the chapter. Besides glycine conjugation, other amino acids yielding conjugated metabolites are glutamine and cysteine. Glutamine conjugation reactions are, however, limited in the body to specific arylacetic acids, a representative case being phenylacetic acid, which can be converted to indolacetyl - glutamine in various species (including monkeys and humans). In the case of cysteine, the substrates are aromatic drugs, and the subsequent metabolites are the corresponding mercapturic acid. In contrast to the enhanced reactivity and toxicity of the various glucuronide, acetyl and glutathione conjugates, amino acid conjugates have not proven to be toxic. Moreover, it has been proposed that amino acid conjugation is an important detoxication pathway for reactive acyl CoA thioesters [6]. Sulphation This is a major conjugation pathway for phenols, but also contributes to the biotransformation of alcohols, amines, and to a lesser extent, thiols. It is also relevant in the metabolism of endogenous compounds such as catecholamine neurotransmitters, steroid hormones, thyroxine and bile acids. Moreover, the tyrosinyl group of peptides and proteins may represent sites of sulphation, resulting in possible changes in their properties. The resulting compounds are generally less active, and more polar, thus more readily excreted in the urine. Sulphate conjugation is a multistep process, comprising activation of inorganic sulphate, first, by converting it via ATP to adenosine-5’- phosphosulphate (APS), and further to the activated form, known as PAPS, 3’-phosphoadenosine-5’-phosphosulphate, as shown in equations 3.4 and 3.5. Each step involves a specific enzyme, present in cytosol. ATP-sulphurase ATP + SO 2- 4 APS +PPi 3.4 APS-phosphokinase APS + ATP PAPS + ADP 3.5 154 Chapter 3 The reaction by which sulphotransferases catalyse the transfer of a sulphuryl group from PAPS to an acceptor molecule is shown in the following reaction: sulphotransferases R-OH + PAPS R-OSO3H + P A P 3.6 The availability of PAPS and its precursor inorganic sulphate strongly determine the rate of reaction. In humans, sulphotransferases are found in the liver, small intestine, brain, kidneys, and platelets. Two forms of sulphotransferases are known to exist, namely a “thermolabile” (TL) form, responsible for the sulphation of dopamine (and other monoamines), and a “thermostable” (TS) form, which catalyses the sulphation of a variety of phenolic compounds. Further details concerning sulphotransferases appear in Chapter 4. It is important to note that the total pool of sulphate in the body is normally limited and can be easily exhausted. Thus, with increasing doses of a drug, sulphate conjugation will become a less significant pathway. For a competing substrate, at high doses glucuronidation usually predominates over sulphation, which instead prevails at low substrate doses. Sulphate conjugation is most common for phenols, and to a lesser extent for alcohols, yielding highly ionic and polar sulphates, metabolites that are readily excreted in the urine. In contrast, N-sulphates, analogous to the N-glucuronides, are able to promote cytotoxicity by facilitating the formation of reactive electrophilic intermediates. Sulphation of N-oxygenated aromatic amines is an activation process for some arylamines that can eliminate the sulphate to an electrophilic species capable of reacting with proteins or DNA. Different drug sulphate conjugates are excreted mostly in the urine, but in the case of steroids, biliary elimination is more prevalent. However, in the small intestine, through mediation of certain sulphatases, the parent drug or its metabolites may be reabsorbed into the portal circulation. The rate of sulphation varies inversely with an individual’s age. It should be noted that, especially after oral administration of a drug, the intestine represents an important site of sulphation. For drugs whose primary metabolic pathway is sulphation, the result is a pre-systemic first pass effect, which decreases the bioavailability. Some drugs in this category are acetaminophen, steroid hormones, Į-methyldopa, isoproterenol and albuterol. This feature is also important when one considers co- administration of certain drugs, where competition for intestinal sulphation might influence their bioavailability, either enhancing or reducing their therapeutic effects. (Examples and details are provided in Chapter 8). Pathways of biotransformation – phase II reactions 155 Fatty acid conjugation Fatty acid conjugation with stearic and palmitic acids has been shown to occur for 11-hydroxy-∆9-tetrahydrocannabinol (THC) (Figure 3.19): CH2 OH CH2 OR OH O C5H11 O O R = C (CH2)14 CH3 (palmitate,) C (CH2)16 CH3 (stearate) Fig.3.19 The conjugation of 11-hydroxy-∆9-tetrahydrocannabinol to the corresponding stearic and palmitic acids (Reproduced with the permission of Nelson Thornes from ‘Introduction to Drug Metabolism’, 2001, 3rd Ed., isbn 0 7487 6011 3 - Gibson & Skett - first published in 1986) These reactions are catalysed by the microsomal fraction from liver. However, the mechanistic aspects are still unknown and the range of compounds that could be involved in such conjugations is uncertain. Amino acid conjugation Results from the reaction of the carboxylic group of a xenobiotic with an amino acid (most frequently, glycine, glutamine, alanine and histidine). The reaction is given only by a relatively small group of substrate structures such as aromatic, heteroaromatic and arylacetic acids, and results in enhanced elimination and decreased toxicity of the parent drug, although not as effective as the main conjugation reactions (glucuronidation, GSH- conjugation). Condensation reactions Condensation reactions may not proceed under enzymatic control and have been found for amine and aldehyde substrates. A representative case is the condensation of dopamine and its own metabolite, 3,4-dihydroxy phenylethanal, to form an alkaloid that is a potent dopamine antagonist (Figure 3.20): 156 Chapter 3 HO H HO NH2 O NH HO HO HO CHO HO HO HO N HO OH OH Fig.3.20 Example of condensation reaction (Reproduced with the permission of Nelson Thornes from ‘Introduction to Drug Metabolism’, 2001, 3rd Ed., isbn 0 7487 6011 3 - Gibson & Skett -first published in 1986) Representative examples of combined phase I and phase II reactions Phenylbutazone The first biotransformation reactions are CYTP450-mediated hydroxylations: the aromatic hydroxylation yields an active metabolite, namely oxyphenbutazone, which is even more active than the parent drug, possessing potent anti-inflammatory effects (Fig. 3.21). In contrast, under conditions of aliphatic hydroxylation, the resulting metabolite, Ȗ-hydroxyphenylbutazone, is also active, but presents a different type of activity, namely uricosuric effects. Both hydroxylated metabolites subsequently undergo 4-glucuronoconjugation, the resulting metabolites being excreted in urine. Alternative, minor pathways, include either direct glucuronidation (bypassing phase I reactions) or a second hydroxylation of Pathways of biotransformation – phase II reactions 157 the metabolite that is hydroxylated on the aliphatic chain, yielding the inactive, dihydroxylated species [41]. OH O N N H3C (CH2)2 CH2 O hydroxylation oxyphenbutazone (active metabolite) O N 4-glucurono O N N conjugation N glucuronic acid H3C (CH2)2 CH2 O H3C (CH2)2 CH2 O phenylbutazone hydroxylation O N OH N H3C CH CH2 CH2 O γ-hydroxyphenbutazone (active metabolite, uricosuric) 4-glucurono conjugation hydroxylation OH O N OH N O N N H H glucuronic acid 3C CH CH2 C 2 O H3C CH CH2 CH dihydroxylated metabolite 2 O inactive OH Fig.3.21 Metabolization pathways of phenylbutazone 158 Chapter 3 Salicylate COOH OH oxidation salicylic acid ester or acyl COOH salicyluric acid glucuronide (the glycine conjugate) OH 75% HO ether or phenolic glucuronide gentisic acid (2,5-dihydroxybenzoic acid) unchanged COOH 10% OH OH glycine 2,3-dihydroxybenzoic acid conjugation COOH OH HO OH 2,3,5-trihydroxybenzoic acid gentisuric acid Fig.3.22 Biotransformation of salicylate Pathways of biotransformation – phase II reactions 159 Salicylate biotransformation (Fig. 3.22 above) takes place in many tissues, but particularly in the hepatic endoplasmic reticulum and mitochondria [42]. The three major metabolites are salicyluric acid (representing in fact the glycine conjugate), the ether or phenolic glucuronide and the ester or acyl glucuronide. A small fraction is oxidized to gentisic acid (2,5-dihydroxybenzoic acid), 2,3-dihydroxybenzoic acid and the 2,3,5-trihydroxylated acid. The gentisic acid may subsequently undergo glycine conjugation, as indicated above. It is remarkable that in this system, conjugation (Phase II) reactions take place without prior Phase I reactions (see the left-hand part of Fig. 3.22). Salicylates are excreted in the urine mostly as salicyluric acid (about 75%) and as free salicylic acid (about 10%). However, excretion of free salicylates is extremely variable, depending on both the dose and the urinary pH. An alkaline pH is favourable, leading to about 30% of the ingested drug being eliminated, whereas in acidic urine, elimination may be less than or equal to 2% [42]. Indomethacin Indomethacin is converted primarily to inactive metabolites, including those formed by O-demethylation (about 50%), conjugation with glucuronic acid (about 10%) and N-deacylation (Fig. 3.23). Some of these metabolites are detectable in plasma, and the free and conjugated metabolites are eliminated in the urine, bile and faeces. A noteworthy feature is that enterohepatic cycling of the conjugates, and probably of indomethacin itself, occurs. Between 10 and 20% of the drug is excreted unchanged in the urine (in part by tubular secretion) [43]. 160 Chapter 3 Cl O C N CH3 H3CO CH2 COOH N-deacylation indomethacin unchanged 30-40% 10-20% O-demethylation about 50% Cl

O C N CH3 HO CH2 COOH conjugation with glucuronic acid 10% Cl O C N CH3 GLU O CH2 COOH Fig.3.23 Pathways in the metabolism of indomethacin Pathways of biotransformation – phase II reactions 161 Sulindac O CH3 S H CH3 different types F CH2 COOH of conjugation sulindac O oxidation CH3 S sulphone sulphide H reduction CH3 F CH2 COO conjugates Fig.3.24 Biotransformation of sulindac The metabolism of sulindac (Fig. 3.24) is complex and highly species dependent variable. It undergoes two major biotransformations in addition to conjugation reactions. It is oxidised to the sulphone and then reversibly reduced to the sulphide. It is this latter metabolite that is the active moiety, although all three compounds are found in comparable concentrations in human plasma [44]. 162 Chapter 3 Ketorolac O C N COOH ketorolac unchanged O 10% C N COO GLU glucuronidated conjugate 90% Fig.3.25 Metabolism of ketorolac This drug is rapidly absorbed with an oral bioavailability of about 80%. Urinary excretion accounts for ~90% of eliminated drug, with the rest excreted unchanged and/or as a glucuronidated conjugate [45] (Fig. 3.25). The rate of elimination is reduced in the elderly and in patients with renal failure. Diclofenac Diclofenac is rapidly and completely absorbed after oral administration. There is a substantial first-pass effect, such that only about 50% of diclofenac is systemically available. Diclofenac is metabolised in the liver by a CYTP450 isozyme of the CYP2C subfamily, to the 4-hydroxy- metabolite, and other hydroxylated forms (Fig. 3.26). After glucuronidation and sulphation, the respective metabolites are excreted in the urine (65%) and bile (35%) [46]. Pathways of biotransformation – phase II reactions 163 CH2 COOH NH Cl Cl diclofenac in liver, by a cyt P450 isozyme of the CYP2C subfamily CH2 COOH HO NH Cl Cl glucuronidated sulphated conjugates conjugates excreted in the urine - 65% bile - 35% Fig.3.26 Main pathways in the biotransformation of diclofenac Piroxicam Piroxicam is also completely absorbed after oral administration and then extensively bound to plasma proteins (about 95%). The major metabolic transformation in humans is CYTP450-mediated hydroxylation of the pyridyl ring (predominantly by an isozyme of the CYP2C subfamily). The 164 Chapter 3 inactive metabolite and its glucuronide conjugate account for about 60% of the drug excreted in the urine or faeces [47] (Fig. 3.27). O O S CH N 3 N C NH OH O piroxicam major biotransformation by cyt P450 isozyme of the CYP2C subfamily mediated hydroxylation of the pyridyl ring (inactive metabolite) 60% of the excreted metabolites glucuronide conjugate Fig.3.27 Metabolism of piroxicam Tolmetin O CH3 C N CH2 COOH H3C tolmetin oxidation unchanged conjugated eliminated (major metabolic transformation) metabolites O CH3 C N CH2 COOH HOOC Fig.3.28 Pathways in the biotransformation of tolmetin Pathways of biotransformation – phase II reactions 165 After absorption, tolmetin is extensively (~99%) bound to plasma proteins. Virtually all of the drug can be recovered in the urine after 24 hours; some is unchanged, but most is conjugated or otherwise metabolised (Fig. 3.28). The major metabolic transformation involves oxidation of the p-methyl group to a carboxylic acid [48]. 3.6 CONCLUDING REMARKS In summary, drug metabolism is generally an extremely complicated process. Often, a drug is metabolised into many products, some major, others minor; furthermore, as indicated in some of the examples above, the drug may be excreted unchanged. Drug biotransformation may not necessarily produce a metabolite that is devoid of pharmacological activity. In the case of e.g. the antiarrhythmic encainide, hepatic oxidation produces two active metabolites, so both the parent compound and its products of metabolism contribute to the therapeutic effects produced [49]. Metabolism may convert an inactive agent (a prodrug) into the active agent responsible for producing the therapeutic effect. A representative example is given by enalapril [50]. As such it is inactive, but after serum hydrolysis, it is converted into the active, antihypertensive agent, enalaprilate, an inhibitor of angiotensin converting enzyme (See also Chapter 9). Most drugs, however, require structural modification to facilitate excretion, and the sum of these modification processes is called drug metabolism. The latter can be considered a detoxification function that the human body possesses to defend itself from environmental hostility. Drugs are usually lipophilic substances, so they can pass plasma membranes and reach the site of action. Drug metabolism is basically a process that introduces hydrophilic functionalities onto the drug molecule to facilitate excretion. These ‘functionalized’ intermediates are substrates for the phase II enzymes, generating conjugates that are more hydrophilic and thus excreted more rapidly. Drugs often undergo both Phase I and II reactions before excretion. Nevertheless, there are certain instances where the drug is directly conjugated, or even eliminated in an unchanged form. Although the liver is the primary site of metabolism, virtually all tissue cells have some metabolic activities. Other organs having significant metabolic activities include the gastrointestinal tract, kidneys and lung. When a drug is administered orally, it usually undergoes first-pass metabolism (it is metabolised in the GI tract or liver, before reaching the 166 Chapter 3 systemic circulation). First-pass metabolism limits the oral bioavailability of drugs, sometimes significantly. The number of enzymes and enzyme systems is vast and their manifold functions lead to a wide range of products when they act on both xenobiotics as well as endogenous compounds. It follows that a drug and an endogenous substance might compete for the same enzyme. Likewise, different enzymes might compete for the same substrate. The complexity of these interactions must be considered in accounting for both toxic and therapeutic actions of drugs [51]. Ultimately, drugs are excreted from the body through various routes, the major organ for drug excretion being the kidney, which excretes hydrophilic drugs and drug metabolites through glomerular filtration. Lipophilic drug molecules can be excreted through the kidneys into urine only after they are metabolised into more hydrophilic molecules. Drugs and their metabolites may also be excreted into the bile, this process usually being mediated by protein transporters. Some drugs may be reabsorbed into the body from the intestine. Also, some drug metabolites such as glucuronide conjugates, may be converted back to the “parent” drug in the intestine (through glucuronidase enzyme), and then reabsorbed into the systemic circulation. This drug recycling process is called enterohepatic recycling. This process, if extensive, may prolong the half-life of the drug. Also, a variety of orally administered drugs are excreted through faeces because they are not absorbed through the intestine. As a final conclusion, we underscore the highly complex nature of drug metabolism; in many cases, a complete profile of the metabolism of a drug is not attainable. The study of drug metabolism serves primarily two purposes, namely to elucidate the function and fate of the drug, and, in connection with drug design, to manipulate the metabolic process of a potential drug. The latter theme is explored in Chapter 9. The Phase I and Phase II reactions whose overall chemistry was described in this and the previous chapter take place under enzymatic control. In the next chapter, the focus turns to details of the interaction between a substrate and its metabolising enzyme, both from structural and kinetic viewpoints. Pathways of biotransformation – phase II reactions 167 References 1. Taylor JB, Kennewell PD. 1993. Drug metabolism. In: Modern Medicinal Chemistry. London: Ellis Harwood Ltd, p 110. 2. Ritter JM, Lewis LD, Mant T.GK. 1999. Drug metabolism. In: Radojicic R, Goodgame F, editors. 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Watanabe T, Sekizawa K, Sasaki H. 1995. The role of HMT (histamine N-methyltransferase) in airways: a review. Method Find Exp Clin 17:16-20. 38. Ziegler MG, Bao X, Kennedy BP, Joiner A, Enns R. 2002. Location, development, control, and function of extraadrenal phenylethanolamine N-methyltransferase. Ann NY Acad Sci 971:76-82. 39. Hodel A. 2001. Effects of glucocorticoids on adrenal chromaffin cells. J Neuroendocrinol 13:217-221. 40. Thompson MA, Weinshilboum RM, El Yazal J, Wood TC, Pang Y-P. 2001. Rabbit indolethylamine N-methyltransferase three-dimensional structure prediction: a model approach to bridge sequence to function in pharmacogenomic studies. J Mol Model [online computer file] 7:324-333. 41. Hanson GR. Analgesic, Antipyretic, and Anti-Inflammatory Drugs. 2000. In: Gennaro AR editor. Remington: The Science and Practice of Pharmacy, 20th ed. Philadelphia: Lippincott Williams & Wilkins, pp 1459-1460. 42. Knutson K. Topical Drugs. 2000. In: Gennaro AR editor. Remington: The Science and Practice of Pharmacy, 20th ed. Philadelphia: Lippincott Williams & Wilkins, p 1212. 43. Hanson GR. Analgesic, Antipyretic, and Anti-Inflammatory Drugs. 2000. In: Gennaro AR editor. Remington: The Science and Practice of Pharmacy, 20th ed. Philadelphia: Lippincott Williams & Wilkins, p 1457. 44. Hanson GR. Analgesic, Antipyretic, and Anti-Inflammatory Drugs. 2000. In: Gennaro AR editor. Remington: The Science and Practice of Pharmacy, 20th ed. Philadelphia: Lippincott Williams & Wilkins, p 1460. 45. Hanson GR. Analgesic, Antipyretic, and Anti-Inflammatory Drugs. 2000. In: Gennaro AR editor. Remington: The Science and Practice of Pharmacy, 20th ed. Philadelphia: Lippincott Williams & Wilkins, p 1445. 46. Hanson GR. Analgesic, Antipyretic, and Anti-Inflammatory Drugs. 2000. In: Gennaro AR editor. Remington: The Science and Practice of Pharmacy, 20th ed. Philadelphia: Lippincott Williams & Wilkins, p 1456. 47. Hanson GR. 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Chapter 4 ENZYMATIC SYSTEMS INVOLVED IN DRUG BIOTRANSFORMATION 4.1 INTRODUCTION Chapters 2 and 3 effectively described a vast array of overall drug biotransformation reactions mediated by enzymes. In the first part of the present chapter, the basic structural and dynamic features associated with enzyme activity are discussed. This includes the essential concept of specificity, the hallmark of enzymatic action, as well as the roles of coenzymes and effectors in the catalytic process. Dependence of the rate of enzyme-catalysis on various factors is discussed and the relevant basic kinetic expressions are presented. Mechanistic aspects at the molecular level are briefly explored. Finally, the main strategies by which cells regulate enzyme activities are described. The second part of the chapter focuses on the nature and role of specific enzyme systems and is logically divided into those mediating Phase I and Phase II biotransformations. In the former category, we meet the cytochrome P450-dependent MFO system, the microsomal flavin-containing monooxygenase system and several other key enzyme systems. Classification of cytochrome P450 isoforms and the nomenclature used to describe them are also presented. Several examples of specific cytochrome P450 subfamilies are chosen to illustrate their particular metabolic functions as well as typical drugs that serve as substrates for them. Enzyme systems that mediate Phase II reactions are exemplified in the final part of this chapter by the UDP-glucuronosyl transferases and glutathione-S-transferase. 171 172 Chapter 4 4.2 INTERACTION BETWEEN A DRUG SUBSTRATE AND AN ENZYME All of the thousands of drug biotransformation reactions (as well as all normal metabolic processes) are catalysed by enzymes [1-6]. The drug substance that is acted on by an enzyme is called the substrate of that enzyme. On the other hand, the enzyme, representing a compound that increases the rate, or velocity of a biochemical reaction is called the catalyst. Several aspects should be emphasised from the outset: • most (but not all) biological catalysts are proteins (these are called enzymes); • a catalyst, though it participates in the reaction process, is unchanged by it, at the end of reaction being found again in exactly the same state as before, ready for another cycle of biotransformation; • catalysts change rates of processes but do not affect the position of equilibrium of a reaction. This means that a thermodynamically favourable process is not made more favourable, nor is an unfavourable process made favourable, by the presence of a catalyst. Instead, the equilibrium state is simply approached more rapidly. It being generally accepted that for a reaction to take place energy is needed, we present an explanation of how enzymes function. The barrier preventing a chemical reaction from occurring is called the activation energy and refers to a high-energy transition state that a reactant molecule has to pass through in order to form products. Catalysts function by lowering this activation energy, binding the substrate in an intermediate conformation that resembles the transition state but which has a lower energy. In enzyme catalysis, one or more substrates are bound at the active site of an enzyme to form the enzyme-substrate complex, which is a highly reactive species that promotes the reaction and releases the product(s) (Figure 4.1). It is important to stress the fact that the active site portion of the enzyme molecule is not one continuous sequence of the protein. Because of the coiling of the molecule, portions of the amino acid sequence that are far removed from one another if the protein were to be stretched linearly come into close proximity when the molecule folds into its proper conformation. Enzymatic systems involved in drug biotransformation 173 R1 R2 E E P E E Fig.4.1 General mechanism of enzyme action; two reactants are bound to the same enzyme, which ensures their correct mutual orientation and proximity and binds them strongly The simplest equation to describe a one-substrate, one-product reaction catalysed by an enzyme is the following: k k E + S 1 ES 2 E+ P 4.1 As implied by Eq.4.1, the process involves a molecule of substrate binding to an enzyme molecule, the substrate being subsequently converted to product, and the latter being released from the enzyme. If we assume that conditions are such that the reverse reaction between E and P is negligible, then the catalytic formation of the product (with enzyme regeneration) will be a simple first-order process. Consequently, the rate will be determined only by the concentration of [ES] and the corresponding value of k2. However, [ES] is usually not a measurable concentration; what is measurable is either the substrate or 174 Chapter 4 product concentration as well as the total concentration of enzyme, represented by the sum of the concentrations of free and occupied enzyme: [E]t = [E] + [ES] 4.2 where [E]t represents the total enzyme concentration, [E] the free enzyme concentration, and [ES] enzyme concentration in the ES complex (occupied enzyme). Another aspect merits emphasis, namely that not all of the substrate molecules instantaneously change to product. There is a certain time required for each molecule to bind, be catalytically converted, and finally released from the enzyme. The necessary time for each transformation is influenced by a number of factors that are amenable to experimental determination. The reaction rate of the enzyme and (indirectly) the amount of enzyme present in the biological material under study can be estimated. Usually, in measuring the rate of reaction, one determines the amount of product formed and divides by the time required to form that amount of material. Inspection of Eq.4.1 shows that each molecule of substrate must combine with a molecule of enzyme to form a molecule of product. However, in measuring enzyme activity, we assume that that there are many more substrate molecules than enzyme molecules. In this situation, each enzyme molecule will bind substrate and convert it, then accept another substrate molecule for further reaction, and so on. It follows that the substrate cannot be converted any faster than the number of enzyme molecules present allows. The enzyme level is therefore said to be ‘rate-limiting’. However, when an enzyme reaction is studied, several parameters are involved: the time of contact between enzyme and substrate, the concentrations of substrate and enzyme, type of buffer, pH, temperature, necessary co-factors, presence of enzyme effectors – all of which affect the rate of the reaction. Time of contact between enzyme and substrate The rate of an enzyme-catalysed reaction (v) evolves as a function of time. According to Figure 4.2, the reaction rate is initially high (the steep linear segment corresponding to the initial rate) and decreases as equilibrium is attained, when v = 0. Enzymatic systems involved in drug biotransformation 175 [P] V=0 time Fig.4.2 The rate evolution of an enzyme catalysed reaction In the evolution of such a reaction rate several factors are involved; among them it is assumed that reduction in the substrate concentration concomitant with an increase in product concentration could favour the reverse reaction, PS; the same effect is caused by enzyme denaturation. In view of the above, it is important to stress the recommendation that the rate of such a reaction be determined before any of the phenomena mentioned above intervenes. In other words, the initial rate represents the most correct experimental datum relating to the amount of active enzyme present in the reaction environment. Substrate concentration If the enzyme itself is present in sufficiently high amounts, the rate of the reaction is determined by the concentration of the substrate present. As the substrate level increases, the enzyme reaction rate also increases. The reaction velocity, v0 is a function of the substrate concentration [S] for the enzyme-catalysed reaction. At high substrate concentrations the reaction velocity reaches a limiting value, Vm; Km is the substrate concentration at which the rate is at the half-maximum value (Figure 4.3). initial rate 176 Chapter 4 v0 Vm Vm 2 Km [S] Fig.4.3 Effect of substrate concentration on reaction rate where v0 = the reaction rate for a certain substrate concentration (the initial rate) Vm = the maximum limiting rate [S] = the substrate concentration Km = Michaelis constant, representing in a reversed relation the affinity of the enzyme for that substrate. It is assumed that when the rate attains the value Vm, the enzyme is ‘saturated’ with substrate. The active site concept provides a simple explanation of what is taking place. A certain number of available active sites are present. When adding a low concentration of substrate, each substrate molecule can eventually bind to the active site of an enzyme molecule. If the substrate concentration is increased, the probability of substrate molecules colliding with enzyme molecules yielding the [ES] complex also increases, thus increasing reaction rate. The parameters in the figure are related in the Michaelis-Menten equation: v = V S m ⋅ [ ] 4.3 [S]+ Km For the purpose of graphical representation of experimental data, it is convenient to rearrange equation 4.3. Taking the reciprocal of both sides of equation 4.3 gives: Enzymatic systems involved in drug biotransformation 177 1 = 1 + Km ⋅ 1 4.4 v Vm Vm [S] The plot of the reciprocal of the rate (1/v0) as a function of the reciprocal of the substrate concentration (1/[S]) gives a straight line, known as the Lineweaver-Burk, or double-reciprocal plot (Figure 4.4): 1/v0 α 1/Vm 1/[S] -1/Km Fig.4.4 Lineweaver-Burk plot Vm and Km can be readily determined from the graph. The slope of the graph is given by: tan Į = Km 4.5 Vm The maximum number of molecules of substrate that can be converted to product each second per active site is known as the ‘turnover number’ of the particular enzyme involved, designated kcat (the catalytic constant). Because the maximum rate is obtained at high substrate concentrations, when all the active sites are occupied with substrate, the turnover number is a measure of how rapidly an enzyme can operate once the active site is filled ( kcat = Vm / [E]t). However, since

enzymes usually do not operate at saturating substrate concentrations under physiological conditions, another parameter needs to be introduced, namely the specificity constant. This is the kcat/Km ratio, representing a measure of how rapidly an enzyme can function at low 178 Chapter 4 substrate concentrations. The specificity constant is useful for comparing the relative abilities of different compounds to serve as substrates for the same enzyme. Enzyme concentration If the substrate is present in sufficiently high amounts, the rate of reaction will become a function of the enzyme concentration. As the enzyme level increases, for a defined volume of body fluid, the rate will increase as well. In some specific situations, other reaction conditions should also be taken into account. For instance, dilution could lead to a false increase in the amount of enzyme supposedly present in the system. Effect of temperature For most enzymes, the turnover number increases with temperature; however, beyond a certain point, further increase in temperature does not lead to further increase in rate, but to loss of enzyme activity (Figure 4.5): activation inactivation Temperature Fig.4.5 Effect of temperature upon enzymatic activity The effect of increasing temperature is to increase the kinetic energies of molecules. This in turn results in higher frequencies of collision between enzyme and substrate molecules and thus higher reaction rate. Within a narrowly defined range of temperature, enzyme activity approximately Enzymatic activity (catalytic lability) Enzymatic systems involved in drug biotransformation 179 doubles for every 10°C rise in temperature (which translates into an activation energy of ~50 kJ mol-1, according to the well-known Arrhenius equation). However, a point is reached where another factor comes into play: the increase in temperature leads to an increase in the rate of unfolding of the enzyme molecule. Consequently, from its initial, rather tight globular structure, the protein begins to spread out into a more linear configuration, leading to loss of enzyme activity. This is the ‘denaturation’ process and its characteristics vary from one enzyme to another. At lower temperatures, the temperature dependence of kcat can be related to the activation energy of the slowest (rate-limiting) step in the catalytic pathway. Effect of pH Enzymes, like other proteins, are stable only over a limited range of pH. A change in the hydrogen ion concentration of the reaction medium can have profound effects on the rate of an enzyme reaction. Changes in the charges on ionisable amino acid residues result in modifications in the tertiary structure of the protein and eventually lead to denaturation. At the pH extremes, the reaction rate is rather low and gradually increases to a pH optimum, the point at which the reaction rate is greatest for the conditions. Several factors determine the pH optimum. If the substrate can be ionised at a certain pH, the degree of ionisation may affect binding to the active site and the resultant activity. At the same time, the active site may be able to exist in an ionised or unionised form, the presence or absence of charge on the active site affecting substrate binding and reactivity as well. 180 Chapter 4 The pH optimum for different enzymes is quite variable (Figure 4.6): v Alkaline phosphatase Acid phosphatase pH Fig.4. 6 The difference in pH optimum for the two physiological phosphatases Coenzymes Many enzymes require additional partners called co-factors for their activity. These act in concert with the enzymes to catalyse biochemical reactions. Co-factors may be simple inorganic ions (such as Mg2+) or complex organic molecules known as coenzymes. Many of these organic molecules are modified forms of vitamins, with the modifications taking place in the organism after their ingestion. The co-factor usually binds tightly to a special site on the enzyme, sometimes referred to as prosthetic group. An enzyme lacking an essential co-factor is called an apoenzyme, while the intact enzyme with the bound co-factor is called the holoenzyme. The most common enzymatic system in mammals and humans is known to be the M.F.O., the array of catalysed reactions including also oxidative reactions of drug biotransformations. For this type of reaction to proceed, the enzyme (the CYTP450) requires reducing equivalents (NADPH +H+) and molecular oxygen. During the reaction, reducing equivalents are consumed and one atom of molecular oxygen is incorporated into the substrate, whereas the other oxygen atom is reduced to water. Another component of the same system is represented by a flavin- containing enzyme, consisting of one mole of flavin adenine dinucleotide Enzymatic systems involved in drug biotransformation 181 (FAD) and one mole of flavin mononucleotide (FMN) per mole of apoprotein. There is strong evidence to support the role of FAD as the acceptor flavin from NADPH +H+ and FMN as the donating flavin to CYTP450 in the electron transfer events. An NADH +H+ dependent system is also required in reductive drug metabolism. Effectors Effectors can be either inhibitors or activators. Molecules which decrease the rate of an enzyme reaction (if they are present in the reaction system) are called inhibitors. Most enzymes are sensitive to inhibition by specific agents that interfere either with the binding of a substrate at the active site, or with the conversion of the enzyme- substrate complex into products. If substrate is present in too high a concentration (substrate inhibition), a decrease in the rate may be seen. In other instances, the product concentration could become sufficiently high to provide product inhibition of the enzyme. A feature that warrants emphasis is that there is increasing evidence to show that the human body has endogenous inhibitors for some enzymes. These substances, produced by the organism, exert regulatory control as a part of normal biochemical processes. In many cases, an inhibitor resembles the substrate structure and binds reversibly at the same site on the enzyme. These are the ‘competitive inhibitors’, because both the inhibitor and the substrate compete for the same binding site on the enzyme. Competitive inhibition may be prevented if the active site is already occupied by the substrate. Inhibitors that bind at sites other than the active site of the enzyme but do not compete directly with binding of the substrate are the ‘non- competitive inhibitors’. Instead, these act by interfering with the reaction of the enzyme-substrate complex. Still another possibility is the binding of the inhibitor only to the enzyme-substrate complex and not to the free enzyme, the effect being called ‘uncompetitive inhibition’. Inhibition may be reversible or irreversible, altering the enzyme structure temporarily or permanently. 182 Chapter 4 The kinetic parameters are also modified, as presented in Figure 4.7: 1/V Non-competitive inhibition Competitive inhibition Without 1/V/ inhibitor max 1/Vmax -1/Km -1/K’m 1/[S] Fig.4.7 The plot of the double reciprocal relation between [S] and the rate of an enzymatic reaction in the absence and presence of some inhibitors The slopes of the plots and the intercepts on the abscissa are simple, linear functions of [I]/Ki, where Ki is the dissociation constant of the inhibitor-enzyme complex. In the case of a competitive inhibitor, the Michaelis-Menten equation becomes: v = V [ ] m ⋅ S 4.6 [S]+ K §1 I · m ¨ + ¸ © Ki ¹ where v, Vm, [S] and Km have the same significance as before, and Ki represents the inhibition constant. Vm is constant, while Km increases. In the case of a non-competitive inhibitor, the relationship can be written as: v = Vm [S] 4.7 1+ [I] ⋅ [S]+ Km Ki Km remains constant, in contrast to Vm which is much reduced. Enzymatic systems involved in drug biotransformation 183 Other enzyme effectors act like activators. Indeed, many enzymes require for full activity, the presence of metal activators, inorganic entities that facilitate binding of the substrate to the active site, by forming ionic bridges. The metal is also effective in orientating the substrate so it can attach to the protein at the proper point and in the correct configuration. Some of the metal activators are tightly bound to the enzyme while others are more loosely attached. In the latter case, a supplemental activator must be part of the reaction mixture in order to obtain full enzyme activity. Mechanisms of enyzme action at the molecular level Any enzyme binds a molecule of substrate (or in some cases several substrates) into a special region of the enzyme called the active site (catalytic site). The active site is usually represented by a pocket surrounded by amino acid side chains that help bind the substrate and by other side chains that play a role in catalysis. The pocket fits the substrate quite closely because of the complex tertiary structure of the enzyme. This feature explains the extraordinary specificity of enzyme catalysis. The general themes frequently occurring in enzymatic reaction mechanisms include: • proximity effects • general-acid and general-base catalysis • electrostatic effects • nucleophilic or electrophilic catalysis by functional groups, and • structural flexibility. The idea underlying proximity effects is that an enzyme can accelerate a reaction between two species simply by holding the two reactants closely together in appropriate mutual orientation. The general-acid and general-base catalyses avoid the need for extreme pH values. The reactive chemical groups function either as electrophiles or as nucleophiles, their task often being to make a potentially reactive group more reactive by increasing its electrophilic or nucleophilic character, primarily by adding or removing a proton. The electrostatic interactions can promote the formation of the transition state, by stabilising the distribution of electrical charge in transition states. Electrostatic interactions can be significant even between groups whose net charge is zero. Nucleophilic catalysis by enzymes involves the formation of an intermediate state in which the substrate is covalently attached to a nucleophilic group of the enzyme. Nucleophilic groups participate in reactions of hydrolysis (of an ester or amide) and addition. 184 Chapter 4 Although precise positioning of the reactants is a fundamental aspect of enzyme catalysis, most enzymes undergo some change in their structure when they bind substrates. A first hypothesis for enzyme action was proposed by Fischer (1894) and it is the well-known lock-and-key model (Figure 4.8): S P1 P2 AS S E E E Fig.4.8 The lock-and-key model (the active site of the enzyme fits the substrate as a lock does a key) According to this model, the enzyme accommodates the specific substrate as a lock does its specific key. However, although this model explained enzyme specificity, it could not explain the catalytic process itself. Thus arose the need for an extension of Fischer’s idea: both the enzyme and the substrate must mutually adjust to take up a configuration that stabilises the transition state. In practice, the enzyme does not simply accept the substrate but instead there is mutual distortion of enzyme and substrate to produce and fit conformations close to the transition state. This model represents Koshland’s induced fit hypothesis. As indicated in Figure 4.9, induced fit implies distortion of the enzyme as well as the substrate; this distortion may be local, or it may involve a major change in enzyme conformation. S P1 P2 AS AS E E E transition state conformation Fig.4.9 The induced fit model, in which both enzyme and substrate are distorted on binding (the enzyme keeps the substrate under stress) Enzymatic systems involved in drug biotransformation 185 Summarising, we may say that an enzyme: • binds the substrate(s), • lowers the energy of the transition state, and • directly promotes the catalytic event. When the catalytic process has been completed, the enzyme must be able to release the product(s) and return to its original state, ready for another round of catalysis. Structural changes also contribute to the high specificity of the enzymatic reactions, concerning either substrate, or reaction specificity. Recently, the dynamics of these processes, approached through a variety of kinetic methods, were discussed in support of the potential roles of conformational changes in the catalytic process and in terms of dynamic coupling within the enzyme-substrate complex [7]. Specificity of enzymes Although a fundamental aspect of enzyme catalysis relies on the precise positioning of the reactants, due to their structural flexibility, when binding different substrates, most enzymes undergo some changes in their structure. Commonly such a structural change is referred to as an induced fit, and contributes to the high specificity of some enzymatic reactions. Practically, when an enzyme binds a substrate, its structure changes in a manner that brings together the elements of the active site, the

enzyme closing like a net around the substrate. Moreover, it also allows the enzyme to control the electrostatic effects that promote the formation of the transition state; in this way, the substrate is forced to respond to the directed electrostatic fields generated by the enzyme’s functional groups, instead of the disordered fields from the solvent. A particular case of enzyme specificity is stereospecificity, which occurs in so-called prochiral substrates. In some specific situations, even for a symmetrical molecule, when bound by three points to an asymmetric object, two of its previously identical atoms will no longer be equivalent; consequently only one of the two initially equivalent (now, prochiral) atoms would be able to contact the surface properly. An example is the substrate ethanol, CH3CH2OH, whose methylene hydrogen atoms become distinguishable when the molecule attaches itself to an asymmetric template. Regulation of enzyme activities Cells use two basic strategies for regulating their enzyme activities. The first strategy refers to adjusting the amount and location of key enzymes, consequently requiring mechanisms for the control of synthesis, degradation, and transport of proteins, while the second strategy, is to regulate the activities of the enzymes. We shall focus in the present material exclusively with the second strategy. 186 Chapter 4 In principle, the activities of many enzymes can be altered by changes in pH. However, this is not a very practical solution especially for intracellular enzymes, because most cells maintain their pH within narrow limits. Therefore, two other strategies are more widely applicable and efficient. • The first refers to the covalent modification of the enzyme structure, in such a way as to alter either Km or kcat. • The second is to use an effector (inhibitor or activator) that binds reversibly to the enzyme and again, alters either the Km or kcat. Such effectors may bind either at the active site itself, or at some distance from it. In this latter case, we are referring to an ‘allosteric effector’ (from Greek: allos = ‘other’, and stereos = ‘space’). • Within the first strategy, the most common regulatory mechanism is phosphorylation. It refers to some specific amino acid residues, such as serine, threonine and tyrosine and uses two separate enzymes: the introduction of the phosphate is catalysed by a protein kinase, while dephosphorylation is effected by a phosphatase (both enzymes themselves usually being under metabolic regulation). Phosphorylation involves consumption of ATP (Figure 4.10): ADP P ADP kinase O _ Enz CH2OH Enz CH2O P O _ phosphatase O Pi H2O Fig.4.10 Phosphorylation and dephosphorylation mechanism In eukaryotic organisms, phosphorylation is used to control the activities of hundreds of enzymes, in response to extracellular signals, such as hormones or growth factors. Sometimes phosphorylation can also modify an enzyme’s sensitivity to allosteric effectors. For example, phosphorylation of glycogen phosphorylase reduces its sensitivity to the allosteric activator adenosine monophosphate (AMP). Other modifying groups (acting by covalent attachment) include fatty acids, isoprenoid alcohols and carbohydrates. Their regulating enzymatic activity is, however, fragmentary. Enzymatic systems involved in drug biotransformation 187 Covalent modifications of enzymes allow a cell to regulate its metabolic activities more rapidly and in much more intricate ways than is possible by changing the absolute concentrations of the same enzymes. • Another mechanism of response to extracellular signals both in eukaryotic and prokaryotic organisms is allosteric regulation. A compound that binds at an allosteric site can serve as either an inhibitor or an activator, depending on the structure of the enzyme and does not need to have any structural relationship to the substrate. Such effectors are, for example, ATP, ADP, AMP, or Pi, often chemically unrelated to the substrate of the enzyme that must be regulated. They usually bind to an allosteric site (rather than to the active site) and their concentrations provide the cell with an indication of the available energy. However, there also, combined control systems for enzymatic activity exist, a good example being provided by glycogen phosphorylase. This enzyme, which catalyses the removal of a terminal glucose residue from glycogen, exists in two forms, ‘a’ and ‘b’, which differ greatly in their catalytic activities. Phosphorylase ‘b’, virtually inactive, can be activated by low concentrations of AMP; nonetheless, activation may be inhibited competitively by ATP. On the other hand, phosphorylase ‘a’ becomes fully active at low concentrations of AMP, being also relatively insensitive to inhibition by ATP. The structural basis for the difference between the two forms of phosphorylase is given by a punctiform modification: phosphorylase ‘a’ has a phosphate residue on serine 14 (which is absent in phosphorylase ‘b’). The interconversion of the two forms is catalysed by another enzyme, namely a cAMP-dependent protein kinase, the process being under hormonal control. In keeping with the complexity of its covalent and allosteric regulation, phosphorylase is a large enzyme, consisting of a dimer of two identical subunits and having the catalytic site buried near the centre of each subunit. Located near the catalytic site is a covalently bound molecule of PALPO – the coenzyme pyridoxal phosphate, derived from vitamin B6, which probably participates as a general acid in the catalytic mechanism. The binding site for the allosteric effectors (such as AMP, ATP) is about 30 Å from the catalytic site, at one of the interfaces of the two subunits. As final conclusions, the following aspects should be emphasised: • cells regulate their metabolic activities by controlling rates of enzyme synthesis and degradation and by adjusting the activities of specific enzymes; • enzyme activities vary in response to changes in pH, temperature, and t he concentratio ns of substra tes and products; 188 Chapter 4 • enzyme activities can also be controlled by covalent modifications of the protein or by interactions with activators or inhibitors; • the most common type of reversible covalent modification is represented by phosphorylation; • allosteric effectors (which can act as either activators or inhibitors) bind to enzymes at sites distinct from the active site; • allosteric regulation allows cells to adjust their enzyme activities rapidly and reversibly, in response to changes in the concentrations of substances that are structurally unrelated to the substrates or products; • the allosteric enzymes usually have multiple subunits and their kinetics show a sigmoidal dependence on substrate concentration. Modified enzymes and non-protein catalysts Despite the variety of enzymatic functions available in nature, modern biotechnology continually faces needs either for substances with new catalytic abilities, or enzymes capable of functioning under unusual conditions, or displaying different specificities. We are addressing here the topic of enzyme design and engineering. Essentially, three approaches exist: site-directed mutagenesis, hybrid enzymes and catalytic antibodies: • site-directed mutagenesis is focused especially on developing tolerance to extreme environmental conditions (e.g. enzymes functioning at temperatures as high as 100°C); • hybrid enzymes are fusioned proteins recombining catalytic and binding sites in novel ways; • catalytic antibodies (also known as abzymes) are antibodies that can act like enzymes; they are attaining considerable importance, especially in synthetic organic chemistry and are characterized by a remarkable stereospecificity. Until recently, it was assumed that all biochemical catalysis was carried out by proteins. Recent research has however revealed that some other molecules can also act as enzymes: these are RNA molecules, called ribozymes. Ribonuclease P, an enzyme that cleaves pre-tRNAs (yielding the active, functional RNAs) was known for some time to contain both a proteic portion and a specific RNA co-factor, and it was widely assumed that the active site resided on the protein portion. However, later studies revealed that while the protein alone was wholly inactive, in contrast, the RNA itself, under certain conditions, displayed catalytic abilities. These special conditions referred to either a sufficiently high concentration of magnesium, or a small amount of magnesium plus the presence of a small basic molecule, which was proven to be spermine. Under those circumstances, the RNA is capable of catalysing the specific cleavage of pre-tRNAs, acting like Enzymatic systems involved in drug biotransformation 189 a true enzyme, being unchanged in the process and obeying Michaelis- Menten kinetics [8]. 4.3 ENZYME SYSTEMS WITH SPECIFIC ROLES 4.3.1 Phase I enzyme systems Oxidative enzyme systems Cytochrome P450-dependent mixed-function oxidation reactions Oxidation is probably the most common reaction in xenobiotic metabolism. This is catalysed by a group of membrane-bound mixed function oxidases (M.F.O.) located in the smooth endoplasmic reticulum of the liver and other extrahepatic tissues, and called the cytochrome P450 monooxygenase enzyme system or, microsomal hydroxylase [9-11]. The subcellular fraction containing the smooth endoplasmic reticulum is called the microsomal fraction. It should, however, be noted that these membrane-bound enzymes may in fact be present in all membranes and cells [12]. The overall catalytic reaction conforms to the following stoichiometry: NAD(P)H + H+ + O2 + RH NAD(P)+ + H2O + ROH 4.8. CYT where RH represents an oxidisable drug substance, ROH, the corresponding hydroxylated metabolite NAD(P)H + H+, reducing equivalents. As evident from the above equation, during the reaction the reducing equivalents are consumed and one atom of molecular oxygen is incorporated into the substrate (as a hydroxyl group) whereas the other is reduced to water [11]. As regards cytochrome P450 and its multiple forms and catalytic cycle, some introductory details have already been given in Chapter 2. Cytochrome P450 enzymes play critical roles in the biogenesis of sterols and other physiological intermediates, the catabolism of endogenous substrates (such as fatty acids, sterols and prostaglandins), and in exogenous metabolism by catalysing the biotransformation of a wide variety of xenobiotics, including drugs, carcinogens, insecticides, plant toxins, environmental pollutants (such as pesticides, herbicides and aliphatic and aromatic hydrocarbons), and many other foreign chemicals [12]. In practice, these enzymes catalyse the monooxidation of a wide variety of structurally 190 Chapter 4 unrelated compounds, whose only common feature appears to be a reasonably high degree of lipophilicity. Understanding these processes is vital for predicting the reactivity of chemicals and reducing toxic side effects of drugs (see Chapter 8). Cytochrome P450 is the terminal oxidase component of the electron transfer system present in the smooth endoplasmic reticulum and represents a superfamily of heme-thiolate proteins with molecular weights of approximately 50 000 Da. CYTP450 consists of at least two protein components: a heme protein called cytochrome P450 and a flavoprotein called NADPH-cytochrome P450 reductase, containing both FAD and FMN. A third component essential for electron transport is a lipid, phosphatidylcholine. The haem-containing component, with iron protoporphyrin IX as the prosthetic group [13] (Figure 4.11) is the substrate and oxygen-binding site of the enzyme system, whereas the reductase serves as an electron carrier. H3C CH CH2 N H3C CH N Fe3+ 3 N HOOCCH2CH2 CH CH N 2 HOOCCH2CH2 CH3 Fig.4.11 Structure of ferric protoporphyrin IX [11] Crucial to the functioning of this unique superfamily of heme proteins is the coordination of the iron-protoporphyrin to the sulphur atom of the cysteine residue of the apoprotein [14] (Figure 4.12). The ability of CYTP450 to form a biologically inactive ferrous carbonyl complex with carbon monoxide, with a major absorption band at 450 nm, led both to its discovery and its name. As already known from the overall oxidation reaction, CYTP450 has an absolute requirement for NADPH and molecular oxygen, the rate at which various compounds are biotransformed being dependent on many factors including species, strain, sex, age, nutritional status, physiological or pathological condition, and so on. Enzymatic systems involved in drug biotransformation 191 H3C CH CH2 Cytochrome P450CysCH2S N H3C CH N Fe3+ 3 N HOOCCH2CH2 CH CH N 2 H2O HOOCCH2CH2 CH3 Fig.4.12 Ferric heme thiolate catalytic centre of cytochrome P450 The most important function of CYTP450 is its ability to ‘activate’ molecular oxygen [15], permitting the incorporation of an oxygen atom into a substrate molecule, with simultaneous reduction of the other oxygen atom, yielding a molecule of water (Figure 4.13): ROH RH 3+ NADPH Fe H NADPH 2O (6) (1) 3+ Fe RH 3+ NADPH- NADPH-cytochrome - Fe RH cytochrome O2 P-450 reductase 2 P-450 reductase (5) (2) e- e- 3+ Fe RH O- 2 2+ Fe RH (4) (3) 2+ Fe RH O O 2 2 Fig.4.13 Catalytic cycle of CYTP450 (Fig. 2, p.325 from R.E. White and M.J. Coon, 1980. Ann. Rev. Biochem., 49: 315-356. Reprinted by permission of the journal) 192 Chapter 4 From the outset, we should stress that all proteins that directly interact with molecular oxygen have a common characteristic at the most fundamental level,

namely the ability to provide either low-energy d-orbitals (e.g. metal ions such as iron and copper) for stabilising unpaired electrons, or extensively delocalized molecular orbitals (e.g. organic co-factors such as flavin, pterin, or porphyrin). The current view illustrating the cyclic pattern of the reduction and oxygenation of CYTP450 as it interacts with substrate molecules, electron donors, and oxygen (presented in the above figure) can be summarised as follows: • the ferric cytochrome P450 binds reversibly to a molecule of substrate (RH), with consequent formation of a complex (FeIII-RH) analogous to an enzyme-substrate complex. This binding of the substrate facilitates the first one-electron reduction step; • the ferric complex formed undergoes reduction to a ferrous complex (FeII- RH), by an electron originating from NADPH and transferred by the NADPH-cytochrome P450 reductase; • the reduced cytochrome P450 complex readily binds dioxygen, as the ferrous iron sixth ligand, yielding the oxycytochrome P450 complex (FeII-O2-RH); • further, the oxycytochrome P450 undergoes auto-oxidation, with the subsequent formation of a superoxide anion (FeIII-O — 2 RH); • by accepting a second electron from the flavoprotein, the ferric superoxide anion undergoes further reduction and forms the equivalent of a two-electron reduced complex, peroxycytochrome P450 (FeIII-O 2- 2 -RH); • finally, the ferric peroxycytochrome P450 complex undergoes heterolytic cleavage of peroxide anion, yielding a water molecule and the hydroxylated metabolite [9-11,15-17]. For the first electron reduction, the following mechanism is proposed (Figure 4.14) [9-11]: Fe2+ RH Fe3+ RH . FADH / FMNH2 NADPH H+ . . FADH / FMNH FAD / FMNH2 Fig.4.14 Role of flavins in the first electron reduction step Enzymatic systems involved in drug biotransformation 193 The role of the flavins in the second electron reduction of cytochrome P450 is suggested in Figure 4.15 [9-11]: Fe3+ RH 2- O2 Fe3+ RH . FAD / FMNH O- 2 NADPH H+ FAD/ FMNH2 NADP+ . FADH2 / FMNH . FADH / FMNH2 Fig.4.15 Second electron reduction involving binding of molecular oxygen Cytochrome P450 multiple forms More then 300 cytochrome P450 isoforms have been characterised to date, with respect to their sequences and corresponding encoding genes. The P450s are grouped into families and subfamilies, according to their probable structural and functional similarities. The family is denoted by a number and the subfamily by a letter. Generally, enzymes with 40% identity of the sequence are assigned to the same family and with more then 55% to the same subfamily [18]. Another number indicates the isoform’s order in the subfamily. For example, P4501B5 indicates that this specific isoform is the fifth member of subfamily B of family 1. The reader is alerted to the current use of an abbreviated nomenclature obtained by replacing cytochrome P450 with the three letters CYP. The many years of human drug metabolism study have proven that most of the biotransformation reactions occurring are mediated primarily by enzymes of the CYP1, 2, 3 and 4 families, with CYP3A4 as the most abundant isoform (from the spectroscopically detectable CYTP450 in the liver [19], this isoform is assumed to represent about 30% of the total). Nonetheless, it must be stressed that the relative importance of different isoforms in biotransformation reactions and their resulting products (more reactive, less reactive, with higher toxicity) is strongly dependent on 194 Chapter 4 the genetic idiosyncrasies of the individual, as well as on exposure to different environmental factors (including drugs, for example). Moreover, certain P450 isoforms are polymorphically distributed in the human population (details are presented in Chapter 7, Pharmacogenetics). As mentioned earlier, CytP450 has a characteristic absorption spectrum at about 450 nm, determined by the presence of the ferrous-CO complex (the value being indicative of the thiolate-ligated haemoprotein); on denaturation, a shift of the absorption maximum to approximately 420 nm is observed, characteristic of the ferrous-CO complex in imidazole-ligated proteins (as in myoglobin) [20]. Selected examples of various cytochrome P450 families Family 1 The CYP1A subfamily plays an integral role in the metabolism of two important classes of environmental carcinogens, polycyclic aromatic hydrocarbons (PAHs) and aryl amines. The PAHs are commonly present in the environment, either as a result of industrial combustion processes, or in tobacco smoke. Several potent carcinogenic aryl amines result from pyrolysis of amino acids in cooked meats. It is an inducible isoenzyme, present mainly in extrahepatic tissues where it is very well expressed [21]. CYP1A2 (also known as phenacetin O-deethylase, caffeine demethylase, or antipyrine N-demethylase) metabolises aryl amines, nitrosoamines, and aromatic hydrocarbons and is considered primarily responsible for the activation of the carcinogen aflatoxin B1 under ordinary conditions in human exposure [10]. CYP1A2 is also an important determinant in the metabolism of certain drugs, being involved in their general metabolic disposition and possible drug-drug interactions [22,23]. An interesting study has been performed fairly recently on zolmitriptan (a highly selective 5-HT receptor agonist used in the acute oral treatment of migraine) as substrate for CYP1A2, concurrently administered with other drugs, with the aim of establishing the enzyme kinetics, the metabolism of zolmitriptan and possible drug-drug interactions. Investigations were made on rat hepatic microsomes induced with different inducers [24]. Family 2 The CYP2B subfamily is represented by several isoforms including CYP2B6, CYP2D6, CYP2C8, CYP2C9, CYP2C10, CYP2C18 and CYP2C19, metabolising a range of drugs and other compounds [25]. CYP2B6 has been intensively investigated with respect to its extensive genetic polymorphism [26], its role in the biotransformation of Enzymatic systems involved in drug biotransformation 195 certain drugs [26-29], possible inhibitors [30,31] and inducers [32] and their mechanism of action. Another isoform of particular importance, because it metabolises a wide range of commonly prescribed drugs, is CYP2D6. Numerous compounds including psychotropic, cardiovascular and many miscellaneous agents, have been demonstrated to undergo CYP2D6- mediated biotransformations, although this may not be the only or the main pathway of their oxidative metabolism [25]. Possible implications of certain family 2 cytochromes in chemical toxicity and oxidative stress have recently been investigated, particularly 2E1, an alcohol-inducible enzyme [33]. Family 3 The CYP3A subfamily includes the most abundantly expressed CYTP450s in humans, about two thirds of the CYP450 in the liver belonging to the CYP3A subfamily [25], but with only one having been recently characterized, namely CYP3A4 [34]. The CYP3A subfamily metabolises a range of clinically important drugs, including nifedipine, cyclosporine, erythromycin, midazolam, diazepam, dextromethorphan, lidocaine, diltiazem, tamoxifen, verapamil, cocaine, dapsone, terfenadine, imipramine, rifampicin, valproic acid, carbamazepine and theophylline. CYP3A5 has been detected to a greater extent in adolescents than in adults and does not appear to be inducible. In contrast, it is polymorphically expressed. CYP3A4 is glucocorticoid-inducible and CYP3A7, present only in foetal livers, is involved in the hydroxylations of allylic and benzylic carbon atoms [25]. In addition to oxidative reactions, which are quite numerous (see the review in Chapter 2), cytochrome P450 is known to catalyse reductive reactions as well [35]. These reactions are usually most important under anaerobic conditions, but can, in some instances, compete with oxidative reactions under aerobic conditions. The principal reductive reaction specifically catalysed by CYTP450 is the dehalogenation of alkyl halides, but cytochrome P450, or at least, cytochrome P450 reductase, has been shown to participate in other reactions, such as reduction of azo- and nitro- compounds. In these types of reactions, the important catalytic species is the ferrous deoxy intermediate, in which the iron is not coordinated to oxygen [25]. Microsomal flavin-containing monooxygenase The microsomal flavin-containing monooxygenase (F.M.O.) system is the second most important monooxygenase system in xenobiotic metabolism. 196 Chapter 4 It is also known as ‘Ziegler’s’ enzyme in the older literature. These enzymes belong to the large class of flavoproteins, polymeric proteins exhibiting an apparent molecular weight in the range 52 000 to 65 000 Da, containing a single molecule of FAD as the prosthetic group and being at the same time NADPH- and oxygen-dependent [36]. The F.M.O. system catalyses the oxygenation of many nucleophilic organic nitrogen and sulphur compounds (including many drugs, such as phenothiazines, ephedrine, N-methylamphetamine and norcocaine) and uses as a source of reducing equivalents either NADH or NADPH (although Km for NADH is about ten times higher than that for NADPH; the higher the value, the smaller the affinity for substrate). The proposed mechanism for the F.M.O. system is presented in Figure 4.16: E FAD NADPH + H+ (6) (1) E FAD E FADH2 NADP+ NADP+ H2O2 RO + H O 2O (5) (2) 2 E FAD OOH R E FADH2 O2 NADP+ NADP+ (7) (3) (4) E FAD OOH (a) R NADP+ NADPH + H+ O R RO NADP+ 2 E NADP-ER NADP-ER-O2 NADP-E(OOH) NADP-E-RO NADP-E E (b) Fig.4.16 Proposed mechanism for the microsomal flavin-containing monooxygenase (Reproduced with the permission of Nelson Thornes from ‘Introduction to Drug Metabolism’, 2001, 3rd Ed., isbn 0 7487 6011 3 - Gibson & Skett - first published in 1986) Enzymatic systems involved in drug biotransformation 197 where, in part (a) and in part (b), E-FAD, represents the oxidized enzyme, E, represents the oxidised E-FADH2, the reduced enzyme, enzyme and R, the oxidisable substrate, ER, the reduced enzyme. RO, the monooxygenated product, The reaction mechanism, as presented in the above figure, involves flavin reduction, followed by oxygen binding and, by an internal electron transfer to oxygen, the formation of a peroxy-flavin complex. Substrate nucleophiles attack the distal oxygen of this hydroperoxide, with resultant oxygen transfer to the substrate. Finally, the enzyme complex dissociates, yielding the oxidised enzyme. As shown in the figure, in the absence of an oxidisable substrate, the peroxy-flavin intermediate may slowly decompose, yielding H2O2 (step 7). The reaction sequence is summarised in part (b) of the figure [9,11,36]. Extensive experimental studies focused on structure-metabolism relationships, and established that the best substrates were the lipophilic amines [37]. Unlike the CYTP450s, the F.M.O. system is not induced by exogenously administered xenobiotics, being however under dietary and hormonal control [25]. Prostaglandin endoperoxide synthase and prostaglandin-dependent co-oxidation of drugs Prostaglandin endoperoxide synthetase (PES) is an enzyme present in almost all mammalian cell types and catalyses the oxidation of arachidonic acid to prostaglandin H2, the precursor of other important prostaglandins, thromboxanes and prostacyclins. The enzyme, involved primarily in endogenous metabolism has two distinct catalytic functions [38], namely fatty acid cyclooxygenase activity, forming prostaglandin G2, and hydroperoxydase activity , reducing the resulting prostaglandin to prostaglandin H2 (Figure 4.17). The enzyme has been proven to exist as a dimer with the amino acids sequence established [39], and also to display enantioselectivity [40]. An important aspect to mention, and one that is described in Figure 4.18, is that several drugs display the ability to undergo co-oxidation. Among them are aminopyrine, benzphetamine, oxyphenbutazone as well as chemical carcinogens, including benzidine, benzo[a]pyrene and derivatives. 198 Chapter 4 COOH O Arachidonic acid 2 Prostaglandin synthetase = cyclooxygenase O COOH O OOH Prostaglandin G2 (PGG2) Prostaglandin synthetase Drug =hydroperoxydase O COOH Oxidized drug O OH Prostaglandin H2 (PGH2) Fig.4.17 Co-oxidation of drugs by the prostaglandin synthetase system (role in drug oxidations) (Reprinted from Biochemical Pharmacology, Vol. 31, P Moldeus et al. “Prostaglandin synthetase catalyzed activation of paracetamol”, p.1367, 1982, with permission from Elsevier) In the case of certain drugs (e.g. paracetamol [11]), the biotransformation process involves a radical-mediated mechanism, resulting in glutathione conjugation of the drug, or reduction back to acetaminophen [41] (Figure 4.18). The mechanism of acetaminophen oxidation has been the subject of numerous experimental studies [42]. In the first step (i), the reaction most probably involves a one-electron oxidation, resulting in hydrogen abstraction, to yield the phenoxy radical of paracetamol. The product phenoxy radical may undergo two pathways of further biotransformation: it can either tautomerise, or be reduced with glutathione. In the first case, a carbon-centred quinone radical is formed, which can Enzymatic systems involved in drug biotransformation 199 further react with cellular glutathione, forming the corresponding conjugate of paracetamol (ii). HN COCH3 OH Paracetamol PGG2 G S S (i) Prostaglandin synthetase PGH2 (iii) HN COCH3 HN COCH3 GSH . . O O (ii) GSH HN COCH3 SG OH Fig.4.18 Postulated mechanism for the prostaglandin synthetase mediated metabolism of paracetamol (Reprinted from Biochemical Pharmacology, Vol. 31, P Moldeus et al. “Prostaglandin synthetase catalyzed activation of paracetamol”, p.1367, 1982, with permission from Elsevier) Alternatively, the phenoxy radical may be directly reduced with glutathione, reforming paracetamol (iii). A very interesting aspect that merits highlighting is that acetaminophen oxidation displays a marked isoenzyme selectivity, with the 200 Chapter 4 most selective being the CYP1A1 isoform, which binds acetaminophen

so that its phenolic group comes into close proximity of the central iron ion [43]. As regards the phenomenon of co-oxidation we should mention that quite a variety of xenobiotics have been demonstrated to act as cofactors for the enzymatic reduction of PGG2 to PGH2, therefore being called reducing substrates [44]. In addition, an increasing number of drugs, differing significantly in their structures, have been reported to be co-oxidised by PES, although the resulting metabolites are not known in every case [45]. Therefore, the prostaglandin synthetase-dependent co-oxidation of certain drugs could very well be assumed to play a major role in drug biotransformations, particularly in those tissues low in F.M.O. activity, and naturally, rich in prostaglandin synthetase. Monoamine oxidase This is a FAD-containing enzyme widely distributed in most tissues of mammals [46]. MAO is a membrane-bound enzyme, present in two different forms, MAO-A and MAO-B, as protein sequencing, cloning and sequencing cDNA coding for humans have proven [47]. Its most common physiological substrates are primary amines, which are oxidatively deaminated, as follows [48]: RCH2NR’NR’’ + O2 + H2O → RCHO + NHR’R’’ +H2O2 4.9 The detailed, intimate mechanism is partly understood based on studies performed with both substrates and mechanism-based inactivators [49]. MAO is an enzyme of particular medical interest: on the one hand, it represents a target for selective, reversible inhibitors used therapeutically [50] and on the other, displays a considerable capacity to activate exogenous neurotoxins [51]. As an example, we mention the activation of MPTP (a tetrahydropyridine) to a neurotoxin causing Parkinsonism in monkeys and humans. Apparently, the activation of MPTP is mainly due to MAO-B, and follows several steps, with the final formation of a particularly reactive radical intermediate and of MPDP+; finally, MPDP+ is further oxidised (by unidentified membrane-bound enzymes) to MPP+ (N-methyl-4- phenylpyridinium), which represents the ultimate neurotoxin causing cell death [52]. Xanthine dehydrogenase-xanthine oxidase (XD-XO) These are two forms of the same homodimeric, cytosolic enzyme, with relatively high levels being localised in the liver and small intestine, tissues Enzymatic systems involved in drug biotransformation 201 known to be implicated in the first-pass metabolism of a variety of agents. Each subunit of XD/XO contains one atom of molybdenum, in the form of a molybdopterin cofactor [MoVI(=S)(=O)]+2, one FAD molecule, and two Fe2-S2 centers [53]. The general reaction catalysed by these enzymes can be represented by the following equation: SH + H2O → SOH + 2e- + 2H+ 4.10 where SH represents the reduced substrate and SOH is the hydroxylated metabolite. The proposed mechanism of action involves an oxygen insertion step; most probably, addition of the substrate across the Mo==S double bond takes place with simultaneous incorporation of a hydroxide, yielding a three- centre Mo-C-O bond. Finally, electron transfer and rearrangement would then yield the hydroxylated metabolite, and regenerate the molybdopterin cofactor [54]. The molybdenum hydroxylases generally catalyse oxidation of electron-deficient sp2-hybridized carbon atoms, most commonly found in aromatic heterocycles, aromatic or non-aromatic charged azaheterocycles and aldehydes [55]. In addition, XO plays a role in the oxidation of several chemotherapeutic agents [56] and purine derivatives (6-mercaptopurine, 2, 6-dithiopurine, and 2’-fluoroarabinosyldeoxypurine) [57]. Unfortunately, xanthine oxidase is also implicated in several toxic responses, the most important being the generation of reactive oxygen species, which can cause lipid peroxidation [58]. Aldehyde oxidase (AO) AO is also a cytosolic molybdozyme, existing only in the oxidised form, displaying a mechanism of action very similar to that described for XO, and fulfilling roles complementary to those of the monooxygenases in the biotransformation of both endogenous and exogenous compounds [59]. Common substrates are represented by nitrogen-containing heterocylic compounds, including several therapeutic agents such as tamoxifen-4- aldehyde [60], pyrimidone [61], and sulindac [62]. Epoxide hydrolase This is a widely distributed enzyme, that in addition to metabolising epoxides of drugs and xenobiotics, also catalyses the hydration of endogenous epoxides, which suggests a substantial role for this enzyme in endogenous metabolic reactions [63]. 202 Chapter 4 In general, epoxide hydrolase is of minor importance in normal drug metabolism, but is significant in the formation of chemical carcinogens from otherwise innocuous xenobiotics and in the metabolism of toxic intermediates formed from certain drugs by cytochrome P450-mediated reactions [64]. Four different isoforms of the enzyme have been demonstrated in humans, two of them displaying specific metabolic roles, and the other two, hydrolysing a range of alkene and arene oxides [65]. Esterases These enzymes represent a multigene family, involved in the hydrolysis of carboxylesters, carboxyamides, and carboxythioesters (see the review in Chapter 3). Several chemicals have been shown to be detoxified by liver carboxylesterase, including insecticides, herbicides, and drugs in several classes (anaesthetics, analgesics, and antibiotics). Polymorphism has been detected for cholinesterase, which is important in the hydrolysis of the muscle relaxant succinylcholine and, possibly, diacetylmorphine. From several different allelic variants, most significant to mention are the so-called atypical enzyme, found in 2% of the population and showing defective binding of anionic substrates (such as succinylcholine), and the less common ‘silent’ variant, for which no enzyme is produced [66]. Dehydrogenases The most representative is alcohol dehydrogenase, a cytoplasmic NAD+ dependent zinc metalloenzyme that catalyses the oxidation of an alcohol to an aldehyde; NAD+ is simultaneously reduced to the corresponding NADH. It is assumed that the human ADH family consists of seven genes, encoding proteins belonging to one of five classes of ADH isoenzymes based on structural and kinetic features [67]. Although of importance in determining susceptibility to alcoholism and alcohol liver disease, they are not of great importance in the metabolism of commonly prescribed drugs. 4.3.2. Phase II enzymes UDP-glucuronosyltransferases The enzyme UDP-glucuronosyltransferase is found in almost all mammalian species, present in many tissues, mostly in the liver, but also in kidney, small intestine, lung, skin, adrenals and spleen. It is mainly localised in the membrane of hepatic endoplasmic reticulum fractions, and therefore ideally positioned to glucuronidate the products of the mixed function oxidase reactions. Enzymatic systems involved in drug biotransformation 203 This enzyme has no prosthetic group, and its catalytic activity is substantially influenced by the presence of lipids [68]. As already discussed in Chapter 3, this family of enzymes catalyses the transfer of glucuronic acid to a multitude of endobiotic and xenobiotic compounds, including drugs, pesticides, and carcinogens. Indeed, some key UGTs have evolved to prevent accumulation of potentially toxic endogenous compounds, such as bilirubin (the end product of heme catabolism, excreted from the body as biliary mono- and diglucuronides), bile acids and steroid hormones. Other UGTs are concerned with maintenance of physiological levels of certain hormones, such as thyroxine and tri-iodothyronine, which are also excreted as glucuronic conjugates in the liver and bile [69]. UGT isoforms in humans have also been reported [70], but the importance of pharmacogenetic variation in the UDP-glucuronosyl transferases is still unclear. Of interest and relevance in relation to drug metabolism, we should mention an inborn error of metabolism, termed Gilbert’s syndrome, which is characterized by mild hyperbilirubinemia affecting an average of 10% of the population. In addition to this impaired bilirubin metabolism, decreased clearance of several drugs (e.g. tolbutamide, acetaminophen, rifampicin) has been reported in patients with this syndrome [71]. Sulphotransferases Sulphotransferase enzymes conjugate exogenous and endogenous compounds (including neurotransmitters with sulphate derived from PAPS – 3’-phosphoadenosine-5’-phosphosulphate) and play important roles in the metabolism of a range of drugs (phenols, alcohols and hydroxylamines), forming the readily excretable sulphate esters. The sulphotransferase enzymes are soluble enzymes found in many tissues including liver, kidney, gut and platelets, and apparently they do exist in multiple enzyme forms, with the steroid sulphating enzymes being distinct from the sulphotransferases responsible for drug conjugation reactions [72]. Glutathione-S-transferase The glutathione-S-transferase family of enzymes comprises soluble proteins predominantly found in hepatocytes that play important roles in the conjugation of a variety of hydrophobic and electrophilic compounds. The latter include epoxides, haloalkanes, nitroalkanes, alkenes, and aromatic halo- and nitro- compounds. It is, generally, a detoxifying metabolic pathway, with most glutathione conjugates undergoing further metabolism to mercapturic acids before excretion [73]. 204 Chapter 4 In addition to their ability to catalyse conjugation reactions, certain glutathione-S-transferases have the ability to bind a variety of endogenous and exogenous substrates without effecting biotransformation. Examples include bilirubin, oestradiol, testosterone, tetracycline and penicillin. The glutathione-S-transferase enzymes exist in multiple forms (at least 20 isoenzymes) as homo- or heterodimers of two subunits and they are inducible by various xenobiotics, including phenobarbital and polycyclic aromatic hydrocarbons. 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Chapter 5 INDUCTION AND INHIBITION OF DRUG- METABOLISING ENZYMES 5.1 INTRODUCTION The state of enzymatic systems involved in drug biotransformation represents an important factor in pharmacokinetic and/or pharmacodynamic variability. The changes in the state of enzymatic systems may be qualitative and quantitative. Qualitative changes are commonly due to impairments in the state of the enzymatic systems, genetically pre-determined (enzymopathies). Quantitative changes may evolve in two directions: either towards the stimulation of enzyme activity (enzyme induction) or the reverse, a reduction in enzyme activity (enzyme inhibition). The pharmacological consequences of both phenomena are quantitative and refer to the modification of intensity and/or duration of the pharmacological effect of the drug, the modification of t1/2 and biotransformation rate, the appearance of adverse reactions of overdosing, and therapeutic inefficacy. In this chapter, both enzyme induction and enzyme inhibition are examined closely, with an emphasis on the cytochrome P450 system. Many examples are quoted to illustrate these effects and their general impact. An extensive discussion of the role of other factors affecting drug biotransformation follows. Recognizing that there is much interaction between them, these factors are systematically treated under the categories of internal and external factors. The present chapter deals with some of the internal factors that have direct implications for the cytochrome P450 levels/activity, namely the dietary factors, comprising macro- and micro- nutrients, as well as tobacco smoking (considered also as adietary component since it is inhaled deliberately). Under these factors are also 209 210 Chapter 5 discussed the so-called non-dietary factors, such as pyrolysis products (compounds normally formed during cooking) and various food additives. 5.2 INDUCTION 5.2.1 Induction of the Cytochrome P450 system Induction is defined as an increase in enzyme activity associated with an increase in intracellular enzyme concentration [1-5]. From a genetic point of view, this increase in enzymic protein is usually caused by an increase in transcription of the associated gene. The stimulation of enzyme activity represents a process of temporal, adaptive increase in the concentration of a specific enzyme, due either to an increase in its rate of synthesis, or in a decrease of its degradation rate. The direct consequence is an accelerated rate of biotransformation of both endogenous compounds and xenobiotics, by co-administration of another compound, designated as an inducer. The inducer will modify drug metabolism in different ways (either qualitatively or quantitatively) and it is therefore expected to alter the pharmacological effects of drugs (increase in the metabolism of the drug involved in interaction, and decrease in the quantity of drug available for pharmacological activity). In order for this to take place, 1-2 weeks are usually needed, the process being an adaptive, temporal one, as indicated above. Inductive properties may be displayed by compounds with quite different chemical structures, pharmacological actions, or even different toxicities. Drugs of abuse are also known to induce gene expression [6]. The pharmacodynamic and pharmacotherapeutic consequences are reflected by a decrease in pharmacodynamic activity, and hence inefficacy at the usual therapeutic doses. As a first conclusion, we may therefore emphasise that enzyme induction contributes to inter- and intraindividual variation in drug efficacy and potential toxicity associated with drug-drug interactions. On the other hand, we note that alterations in drug efficacy are directly dependent on several factors, including the extent of enzyme induction (in a particular individual), the relative importance of the enzyme in multiple pathways of metabolism, and on the therapeutic ratio of substrate and metabolite(s). In this context, we may add that potential toxicity will depend on changes in metabolic pathways associated with an alteration in the balance between drug activation and detoxication [7]. In an attempt to localise the site of induction of drug metabolism, significant advances have been made in considering the role of the liver. As is already known, the major organ responsible for drug metabolism in most species (including man) is the liver. It then becomes evident that the main enzymatic system involved will be that of the cytochromes P450, the Induction and inhibition of drug-metabolising enzymes 211 well-known family of oxidative hemoproteins responsible for a wide variety of oxidative transformations in a variety of organisms (see Chapter 4). The extent of induction of hepatic metabolism can reasonably be expected to be reflected in experimentally accessible indicators such as increased drug clearance, decreased drug plasma half-life, increased plasma bilirubin levels and others. On the other hand, it should be borne in mind that the wide range of drugs and chemicals that act as inducers has been investigated for the most part on hepatocytes in vitro. Kinetic data obtained with isolated hepatocytes in vitro were then extrapolated to laboratory animals. However, although enzyme induction commonly results in increased rate of xenobiotic metabolism in vitro, the effects of enzyme induction

may be dampened by physiological constraints in vivo. Furthermore, animal experiments can give only an indication of possible human response, necessitating very cautious extrapolation [8]. One of the most common types of induction is that which is substrate-dependent. A well-known example of this phenomenon is the influence of phenobarbitone on the metabolism and duration of action of several drugs. Drugs affected include oral anticoagulants (anticoagulant effect decreased due to increased metabolism) [9], tricyclic antidepressants (antidepressant effect decreased, by the same mechanism) [10], corticosteroids (corticosteroid effect decreased, by the same mechanism) [11], narcotics (increased CNS depression with meperidine, increased meperidine metabolites) [12], theophyllines (theophylline effect decreased due to increased metabolism) [13] and the muscle relaxant zoxazolamine (substantial metabolism increase, and consequently a significant decrease in the paralysis time) [2]. Pre-treatment with phenobarbitone has also been shown to markedly increase the metabolism of felodipine and its pyridine analogue [14]. Moreover, the inducing action of phenobarbitone may affect the expression of several specific CYP450 isoforms, as revealed in a recent study [15]. The same phenomenon was observed even more recently in pregnant rat and foetal livers and placenta, impacting on different cytochrome P450 isoforms [16]. From the above examples it is obvious that phenobarbitone induces the metabolism of different drugs, thus affecting the intensity and duration of their pharmacological action. The assumed molecular mechanism is a substantial increase in intranuclear RNAs that represent precursors to cytochrome P450 and mRNA. The immediate consequence will be a substantial increase in the hepatic levels of certain P450 isoforms, particularly CYP2B1and CYP2B2, with the former considered as the major phenobarbitone-inducible cytochrome P450 [17]. Accordingly, we may conclude that the major inductive effect of phenobarbitone in the liver is to increase specific mRNA levels by augmenting transcription, rather than 212 Chapter 5 translational efficiency, or by stabilising the pre-existing levels of protein precursor. The other main type of induction response is receptor-mediated, by interactions with important regulatory pathways. For example, many drug- metabolising enzymes that are also involved in metabolism of endogenous cellular regulators (steroids, eicosanoids) have been proven to be inducible by hormones [18]. Moreover, there is evidence now that other hormones, such as the growth hormone, are capable of altering human cytochrome P450 expression [19]. In the case of the CYP1 family, this type of induction response is mediated by a specific cytosolic aryl hydrocarbon (Ah) receptor [20]. The best-known example is that of induction of cytochrome P450s by polycyclic aromatic hydrocarbons (PAHs) [21], which combine with this specific receptor (in a similar manner to hormone response), resulting an inducer- receptor complex. Furthermore, this complex is translocated to the nucleus of the hepatocyte whereupon induction-specific mRNA is transcribed from DNA. In the nucleus, the translocated Ah receptor forms a heterodimer (with a second nuclear protein), which will then bind to a common response element, known as the XRE (xenobiotic responsive element) that functions as a transcriptional enhancer, resulting in stimulation of gene transcription [22]. Large amounts of newly translated, specific cytochrome P450 are then incorporated into the membrane of the hepatic endoplasmic reticulum, resulting in the observed induction of metabolism of certain drugs and xenobiotics including tamoxifen, tacrine, acetaminophen, dietary phytochemicals and carcinogens, such as the aromatic amines produced in cooking and those found in cigarette smoke. This type of response is common both for phase I and phase II metabolic reactions (details of which appear in the following subsection). A third type of induction response is that which is inhibitor-mediated interaction with the heme group of the cytochrome P450s, resulting in inhibition of endogenous function and consequent disruption of endogenous pathways catalysed by a specific cytochrome P450 isoform. Well known examples include the induction of CYP2E1 by isoniazid [23] and induction of CYP3A1 by macrolide antibiotics [24]. However, in this context we have to remind the reader that the CYP2E1 isoform is one of the most toxicologically important of the cytochrome P450 enzymes. This is borne out by numerous studies which have revealed that this specific isoform is implicated in the activation of acetaminophen and organic solvents to hepato- and nephrotoxic intermediates, as well as in the activation of nitrosamine procarcinogens and in the etiology of alcohol-induced liver damage [21,23]. Unfortunately, many xenobiotics, as well as some dietary and pathophysiological factors, increase CYP2E1 expression [23]. An interesting fact worth emphasis is that Induction and inhibition of drug-metabolising enzymes 213 the induction of the CYP2E1 isoenzyme arises through multiple mechanisms, depending on the induction stimulus [2]. Induction has been proven to occur at all regulatory levels, from transcription to mRNA stabilisation, increases in translational efficiency, and post-translational protein stabilisation [23]. Nevertheless, the predominant xenobiotic- dependent induction is assumed to be via stabilisation and inhibition of degradation of the CYP2E1 apoprotein. A well-studied case is that of ethanol, which at low concentrations has been proven to stabilise the CYP2E1 apoprotein [25]. A question of great interest in this connection is whether ethanol is an enzyme inducer or an enzyme inhibitor. It has been proven that at low concentrations ethanol acts like an inducer, whereas at high concentrations it acts as an inhibitor [26]. An interesting aspect, related to the variability in the metabolism of narcotic drugs, was reviewed relatively recently [27]. The liver P450s are primarily responsible for their biotransformation, ensuring both their oxidative or reductive metabolism, which is of crucial importance as regards the toxicity of their metabolites. These are commonly reactive intermediates or free radicals, which may either combine with macromolecular cellular components (generating an autoimmune response), or induce peroxidation of membrane lipids. However, what the study cited above revealed is that since different isoforms of CYTP450 are greatly induced by pre- or co- administration of certain drugs, so also may the metabolism of the narcotic drugs vary greatly from one patient to another, depending on previous or concurrent drug treatments. 5.2.2 Induction of other enzyme systems Although the CYTP450 is by far the dominant enzymatic system involved in drug metabolism, it should be pointed out that other enzyme systems may undergo induction as well. We refer especially to some of the phase II metabolising enzymes, such as UDP-glucuronosyl transferase (UDPGT) and glutathione-S-transferase (GST). Naturally, since the phase II enzyme systems are involved in the major routes of detoxication, there is much interest in the induction of these systems, especially for cancer chemoprevention. The induction of UDPGT and GST has been proven to be highly dependent on the nature of the inducer under consideration, as well as on the species variability. In experimental animals, both enzymes have been inducible by phenobarbitone-type inducers and Ah receptor ligands [28,29], and an ethanol-inducible UDPGT has been described in rabbit [30]. As far as endogenous compounds are concerned, the bilirubin UDPGT has been 214 Chapter 5 reported to be induced by several drugs, including phenobarbitone and rifampicin [31]. GSTs in the alpha class (GST A1 and A2) have been reported to be inducible by several xenobiotics including phenobarbitone, dithiolethiones and oltipraz [32]. 5.3 INHIBITION 5.3.1 Inhibition of the Cytochrome P450 system Inhibition of drug metabolism by pre- or co-administration of other drugs or xenobiotics is a well-recognized phenomenon, with pharmacological and toxicological effects, reflected by exacerbated pharmacodynamic activity and adverse effects of relative overdosing at the usual therapeutic doses. In the context of the common practice of polypharmacy, another topic of great interest arises, namely drug-drug interaction [33-35] (Consequences of this phenomenon are discussed in Chapter 8). The other major interest in enzyme inhibition is based on a very important sector of therapy, namely the selection of enzymes as targets for drug action. As in the case of induction, inhibition can also take place by different pathways and mechanisms. In principle, inhibition involves either the blocking of enzymatic synthesis, the destruction of pre-formed enzymes, or inactivation of the enzyme by their complexation with drug metabolites. The direct consequence of enzyme inhibition is the delay in the biotransformation of certain drugs, resulting thus in increased plasma concentrations and potentiation or prolongation of their pharmacological action. The level of drug biotransformation may be decreased to the point of complete inhibition by various compounds which can interfere either before their contact with the MMFOs or, more commonly, through direct action on this enzymatic system. It is important to note that drug binding at the level of different tissues is also a significant factor in limiting the in vivo rate of biotransformation (decreased concentration of free, unbound drug). Basic mechanisms of enzyme inhibition involve one of the following: 1) competitive inhibition 2) non-competitive inhibition 3) uncompetitive inhibition 4) product inhibition 5) transition-state analogues 6) slow, tight-binding inhibitors 7) mechanism-based enzyme inactivation Induction and inhibition of drug-metabolising enzymes 215 8) inhibitors that generate reactive intermediates that can covalently be attached to the enzyme. Overviews of some special mechanisms will be presented, followed by a few prominent examples involving various enzymes. Competitive inhibition occurs when the ‘normal’ substrate and the inhibitor share structural similarities. The inhibitor may or may not be a co- substrate and the intermediate complex [ES] may or may not be present. Screening for inhibition is a very important and routine practice in the pharmaceutical industry today and therefore new approaches have been introduced to handle the ever-increasing numbers of new drug candidates. One of the successful statistical, experimental approaches is commonly designated as virtual kinetics [36]. Competitive inhibition is a relatively common phenomenon in drug metabolism, being of significant relevance especially in the field of drug-drug interactions (see Chapter 8), since we acknowledge that many enzymes have multiple drug substrates that can compete with each other. In classical non-competitive inhibition, the inhibitor will usually bind to a site distinct from that of the substrate, resulting in a decrease of Vmax without a change in Km, according to the Michaelis-Menten equation (or the linearised Lineweaver-Burk representation). Un-competitive inhibition, although well defined, is seldom observed. It presumes binding of the inhibitor only to the [ES] complex and affects both the Vmax and the Km values, which decrease, but still maintaining the Vmax/Km ratio constant. In this context, it is evident that the enzyme efficiency would not really change. ‘Product inhibition’ occurs when the metabolic product generated by the enzyme inhibits the reaction on the substrate (‘feedback inhibition’). This usually occurs when the product has physical characteristics very similar to those of the substrate. A well-known example is that of benzene, which is oxidised to phenol by a specific P450 isoform. It has been proven that both the initial substrate (benzene) and the product (phenol) compete with each other [37]. Transition-state analogues are compounds that are non-covalently bound to the enzyme, resembling the transition state of the enzymatic reaction. The complex formed is [EI], leading to inactivation of the enzyme. However, it is important to note that enzyme activity may be restored by removal of the inhibitor using different methods, the most common being gel-filtration and dialysis. Slow, tight-binding inhibitors bind to the enzyme at a slow rate, inhibit competitively, and practically inactivate the enzyme irreversibly [38]. Possible causes of inactivation are associated with different mechanisms: conformational change of the three-dimensional structure of the enzyme (including therefore alteration of activity), a change in the protonation state 216 Chapter 5 of the enzyme, reversible formation of a covalent bond, or displacement of a water molecule at the active site. Mechanism-based enzyme inactivators are special, unreactive compounds, with structures similar either to the substrate or to the product of the enzyme; due to this similarity these compounds undergo a catalytic transformation by the enzyme. The characteristic feature here is that the resulting species inactivates the enzyme before leaving the active site [39]. Some characteristic patterns of mechanism-based inhibition include first- order kinetics, irreversibility, and covalent binding to either the protein moiety, or the prosthetic group of the enzyme. Inhibitors that generate reactive intermediates that can covalently be attached to the enzyme are not particularly effective themselves, but following their oxidative biotrans- formation, they bind tightly to the heme of the CYT, preventing in this way further involvement of the iron atom in catalysis. As far as the CYTP450 enzyme superfamily is concerned, several types of inhibition have been described. An interesting inhibition mechanism, and one with profound pharmacological implications, is the destruction of hepatic cytochrome P450. Compounds having the ability to effect this include xenobiotics containing either an olefinic, or an acetylenic function, such

as acetylene, allobarbital, ethylene, fluoroxene, vinyl chloride and norethindrone. The majority of these compounds are relatively inert per se, requiring metabolic activation by cytochrome P450, after which they form substrate-haem adducts, thus destroying the enzyme. Because of this pattern of action, these compounds are commonly designated as ‘suicide substrates’ of the haemoprotein [40]. The immediate and major consequence of the formation of these adducts (involving haem modifications), will obviously be a significant and sustained decrease in the levels of functional CYTP450, leading to a reduction in drug-metabolising capacity of the liver. A significant point to note is that since the target of the olefinic-induced inhibition is the haem, administration of exogenous haem would be very helpful in restoring both the liver CYTP450 content and the drug-metabolising activity. Another important group of inhibitors of CYTP450 activity, though acting through other mechanisms, comprises metal ions [41]. It is generally accepted that they do not modify the existing CYTP450-haem, but in contrast, act by modulating both the synthesis and the degradation of the haem prosthetic group of the cytochrome. Studies in the 1970s established lead as an inhibitor of a number of CYTP450 related oxidations. Further research revealed that the inhibitory effect may be a combined one, involving both the protein synthesis and the haem cofactor [42]. Other metals of interest as regards their inhibitory potential include copper, cobalt, cadmium, arsenic and mercury. Induction and inhibition of drug-metabolising enzymes 217 Cobalt, for instance, in the form of cobalt-haem (substituting the iron from the prosthetic group of CYTs) has a pronounced influence on both the biosynthesis and biodegradation of hepatic haem, and consequently on drug metabolism in general. Because of the substantial decrease in both hepatic microsomal content of CYTP450 and total haem, following cobalt pre- treatment, a substantial decrease in the metabolising activity of liver enzymes has often been reported [2]. In the 1980s, Abbas revealed the inhibition of CYTP450 by mercury [43]; several years later, it was established that the molecular mechanism involves loss of cytochrome P450 content and impairment in the formation of the whole cytochrome [44]; in addition, enhanced rates of haem degradation were established [45]. Interestingly, recent studies reported contrary results, namely specific induction of a particular CYTP450 isoform, 1A1, in in vitro cultured cells [46]. Cadmium is another toxic metal having a long history of investigation. It has been proven to inhibit drug biotransformation in particular species (rats, for example), most probably by inducing haem oxygenase (like lead), and resulting in decreased levels of the whole cytochrome contents [47]. More recent studies have revealed a broad specificity of inhibitory effects of cadmium on particular CYTP450 isoforms, such as 2E and 3A [48], and CYP 4A11 from human kidney [49]. Two other interesting aspects that warrant mention are the age and sex dependence of cadmium inhibitory effects: inhibition has been shown to increase with age [50], and to differ with sex, being for example greater in male rats than in females [51]. Being responsible for so many, severe toxicological consequences (including renal dysfunction and cardiovascular effects accompanied by hypertension), cadmium continued to be a focus of attention for toxicologists who established a dose-dependent relationship between the effects of cadmium (according to its burden in different tissues) and the expression of several specific CYTP450 isoforms. One of these isoforms has been proven to be the same CYP 4A11 from human kidney; with respect to its enzymatic activity, this isoform is involved in the hydroxylation of unsaturated fatty acids, which in turn are involved in the regulation of the salt balance in the kidney. The decrease in its enzymatic activity, as a consequence of the inhibitory effect of cadmium, was associated with the development of high blood pressure [52]. As in the case of mercury, more recent studies revealed that in some instances, cadmium may display an inductive effect in vitro as well, acting on specific CYTP450 isoforms such as CYP1A1 and CYP2C9; no species differences have been observed thus far [53,54]. Unfortunately, ‘cadmium poisoning’ is relatively frequent, if we acknowledge that food crops grown 218 Chapter 5 in cadmium-containing soils are the major source of cadmium exposure to humans. The inevitable consequence of such exposure will definitely be adverse effects on specific organ systems, with the most severe impact on the kidney. Cadmium-induced renal dysfunction, associated with polluted areas, such as those in Japan for example, has recently been proven to increase the risk of mortality [55]. Another well-known and potent toxic metal is arsenic. It exerts its inhibitory effects in a relatively specific manner that differs from those above, in the sense that its action involves two steps: after an initial increase in cytosolic ‘free’ haem, there follows a dramatic decrease both in cytosolic ‘free’ haem and the general content of CYTP450. In this situation, it has also been proven that there is a several-fold increase in haem oxygenase, resulting in a significant decrease of CYTs haem content, and consequently, the general content of cytochrome [56]. Numerous other studies revealed both the importance of the involvement of arsenic species in the inhibition of the cytochrome P450 system, as well as the fact that significant decrease in P450 is largely associated with the 1A1 isoform [57]. As far as the various species of arsenicals have been shown to display different effects on the CYTP450 system, studies revealed that the most significant inhibition is associated with the arsenite species, As3+ [58]. Very interesting on-going research associates the genetic polymorphisms identified in humans with the role of different isoforms in inducing cancer, in populations exposed to arsenic [59]. As a concluding remark concerning the impact of toxic metals on drug metabolism, we may affirm that it concerns either haem biosynthesis or haem degradation, with resulting changes in the synthesis of the global P450 cytochromes. An interesting inhibition mechanism, well understood at the molecular level, involves compounds that, though not being inhibitors per se, are capable of forming inactive complexes with hepatic cytochrome P450. These compounds require, as do the olefinic or acetylenic compounds, a pre- activation, being in fact common substrates for the P450CYTs. After the production of the metabolic intermediate, the latter will bind tightly to the haemoprotein, forming spectrally detectable, inactive complexes, which are thus prevented from further participation in drug metabolism. This mechanism is supported by experiments on laboratory animals, which, following pre-treatment with such compounds, showed delayed drug biotransformation, resulting for instance in a significant increase in both hexobarbital narcosis and zoxazolamine paralysis times. Compounds with such properties include amphetamine, cimetidine, dapsone, isoniazid, sulphanilamide, piperonal and piperonyl butoxide [2]. A very recent example refers to the inhibitory effect of N,N’,N’’- triethylenethiophosphoramide (thioTEPA) on the CYP2D6 isoform, involved in the biotransformation (by 4-hydroxylation) of cyclophosphamide Induction and inhibition of drug-metabolising enzymes 219 [60]. ThioTEPA is frequently used in chemotherapy regimens that include cyclophosphamide, being required for its activation. The detailed mechanism of this inhibition, studied on human liver microsomes using recombinant P450 enzymes and bupropion as a substrate, revealed a time- and concentration-dependence of the inhibition. The inhibitory action of two compounds and the pharmacokinetic consequences of another irreversible inhibition, on the CYP 2B6 isoform have also been investigated [61]. An evaluation of the potential inhibitory or inductive action of daptomycin was recently performed on human hepatocytes [62]. Human P450 cytochromes can also be inhibited by nicotinic acid and nicotinamide [63]. Recent spectrophotometric analysis indicates, as expected for nitrogen-containing heteroatomic molecules, that in this case the inhibition occurs via coordination of the pyridine nitrogen atom to the heme iron. As mentioned earlier, either the inductive or inhibitive action of different compounds may affect both the phase I as well as phase II enzymes. A study was recently performed on some of the most important hepatic and extrahepatic (kidney, lung and gut) phase II enzymes, including UDPGTs, GSTs and NAT, with propofol in various concentrations, as the chosen substrate. The study was performed on microsomal and cytosolic preparations from both human and other species. As regards the human conjugation enzymes involved, the study revealed that propofol displayed a concentration-dependent inhibition, with the activity of UDPGT significantly decreased, that of GST affected insignificantly, and with NAT activity practically unaffected. Inter-species differences have been demonstrated [64]. 5.4 CONSEQUENCES OF THE ABOVE PHENOMENA As final conclusions, we should mention the following: • the human cytochrome P450 enzyme system, and to some extent phase II metabolising enzymes, are susceptible either to induction or inhibition mechanisms; • sometimes, because of a too rapid biotransformation (direct consequence of increased enzyme activity), megadoses of drug are required, which may not always be possible (e.g. for drugs with broad therapeutic index); plasma levels are too rapidly achieved and the clearance is also too rapid, so the drug has insufficient time to display its pharmacological action; • induction, by increasing enzyme activity, will result in decreased metabolism of certain drugs, contributing to significant inter- and intra- individual variations in drug efficacy and potential toxicity, associated with drug-drug interactions; 220 Chapter 5 • as shown in the corresponding subsection, it is important to mention that the clinical effects following alterations in drug efficacy are dependent on several factors, including the extent of enzyme induction, the relative importance of the enzyme in the multiple pathways of metabolism, and on the therapeutic ratio of substrate and metabolite; • different inducers (drugs, or other compounds from food and the environment) display substrate specificity for CYTP450 isoforms; • prevalent inducing conditions in humans include smoking, alcohol consumption and diet; • elevated levels of specific CYP450 isoforms (1A and 2E1) may contribute to increased risk of cancer; in particular, CYP2E1 may contribute to alcohol-induced liver damage and acetaminophen toxicity in alcohol consumers. • inhibition of drug metabolism represents a subject of great interest for several reasons. It can give rise to a decrease in drug biotransformation, low plasma levels, decreased clearance (possibility of overdosing at common, therapeutic doses) and increased risk in the occurrence of drug- drug interactions. Besides decreasing the therapeutic effect on one drug by concurrent administration of another, it is unfortunately proven that some drug-drug interactions may be even fatal; • practical aspects of inhibition include an understanding of the phenomenon at the molecular level, especially as it relates both to such drug- drug interactions (prediction, avoidance or minimisation of the risks), as well as the utilisation of enzymes as therapeutic targets. In this context we recommend that the reader consult available general reviews dealing with the clinical implications and modern procedures involved in metabolic screening in vitro [65,66]. 5.5 DIETARY AND NON-DIETARY FACTORS IN ENZYME INDUCTION AND INHIBITION Numerous studies and clinical observations have revealed that drug biotransformation can also be significantly increased by some exogenous compounds present in the diet [67-70] or in the environment [71]. Before discussing the main dietary factors, we should emphasise from the outset that generally, food intake has been proven to have a considerable influence on the bioavailability of drugs with extensive pre-systemic metabolic clearance. This phenomenon commonly occurs with lipophilic bases, but very rarely, if ever, with lipophilic acids. Thus, concurrent food intake with compounds acting like lipophilic bases will significantly reduce their pre-systemic clearance, consequently enhancing their bioavailability. In contrast, pre-systemic clearance of acidic drugs is commonly unaffected by Induction and inhibition of drug-metabolising enzymes 221 food. In addition, studies have revealed that even among lipophilic bases, concurrent food intake will act variably, in direct correlation with the type of biotransformation involved; this is usually a decrease in pre-systemic clearance in drugs undergoing hydroxylations, glucuronidations and acetylations, while in contrast, the bioavailability of lipophilic bases that undergo pre- systemic dealkylation usually remains unaffected [69]. Another general consideration is that nutritional deficiencies usually result in decreased rates of drug metabolism, with some notable exceptions of certain vitamins (B1 and B2) that enhance the rates of metabolism of some xenobiotics. At the same time, a deficient diet may, in certain instances, favour occurrence of drug-induced toxicity and carcinogenity [70]. In discussing the dietary factors, two distinct major groups have to be considered: the macro- and the micro-nutrients. It should be emphasised as well, that under dietary factors we shall discuss also alcohol consumption and the components of tobacco smoke. The group of macronutrients includes proteins, lipids and carbohydrates. The impact of a correct protein diet is obvious, if we consider that all enzymes

are proteins [72]. Consequently, we may affirm that if there is a general decrease in overall microsomal protein, the extent of drug metabolism will decrease as well. This may affect not only the pharmacological response, by decreasing it, but also result in toxicological effects, generally because of delayed clearance. Protein deficiency in rats, for example, decreased the total liver cytochrome P450 content, with concomitant alterations in its composition, namely increased Ile and Leu levels, decreased Asp, Glu and Phe levels [73]. In constrast, in some specific cases, low protein diet can be beneficial; one example is that of aflatoxin-induced hepatotoxicity, which may be reduced either by low protein diet (acting like an inhibitor of phase I metabolism), or by phenobarbitone treatment (induction of phase I metabolism), in both cases the production of the epoxide intermediate being reduced. An interesting study focused on the effects on theophylline metabolism accompanying a change from a high-protein to a high- carbohydrate diet [74]. The results indicated a decrease in drug biotransformation by almost one-third (very similar to the effect exerted by cimetidine). Lipids are necessary as well for normal drug metabolism for several reasons: they are required by the drug metabolising enzymes as membrane components and possibly also for specific interactions. Experiments showed that the quantity and quality of dietary fat affect lipid composition, and consequently, physical characteristics of biological membranes, including their stability and drug passage into the membrane [75]. In addition, they 222 Chapter 5 may affect the enzymatic activity of several phospholipid-dependent enzymes associated with these membranes. These changes will accordingly affect the inducibility of these enzymes significantly, resulting in alterations of the pharmacological response to certain drugs. The most important components of a correct lipid diet are assumed to be linoleic acid and arachidonic acid. Therefore, treatment with polyunsaturated fatty acids is considered to be beneficial, because it increases the microsomal content of these fatty acids and consequently increases drug metabolising capacity. Essential fatty acids are known to be required for the interaction of different substrates with the active site of cytochrome P450. (See Chapter 4 for a discussion of the importance of the lipid component in some enzymatic systems). The content of dietary lipids on the activities of different hepatic microsomal drug-metabolising enzymes such as demethylases, hydroxylases and cytochrome P450 was proven to be of utmost significance. For example, experimental studies on rats revealed that in some instances, a high-fat diet might produce more hepatotoxic effects [76]. From the class of macronutrients, the carbohydrates seem to have less significant effects. However, a well-known example is that of glucose, which, at high intake levels, can inhibit certain drugs (e.g. phenobarbital), resulting in specific secondary effects and lengthening the sleeping time caused by the drug. At the same time, excess of glucose has been shown to decrease the general hepatic cytochrome P450 content, and consequently, the enzymatic activity [2]. Interestingly, a high-carbohydrate diet, in a comparative study with a high-protein diet, has been shown to display quite the opposite effect. While the increased protein content, as expected, increased the hepatic biotransformation rate by increasing the total content of CYTP450, experiments based on pharmacokinetic studies with a specific substrate revealed that increased carbohydrate content in the diet produced the opposite effect on the activity of the same enzymatic system [77]. Similar effects were evident in a comparative study of long-term feeding with high-fat (FAT) diet versus high-carbohydrate (CHO) diet. The study was performed on rats, and the control substrate used was pentobarbital. The results suggested that the FAT-diet increased the activity of hepatic metabolising enzymes, while the CHO did not. The results also revealed sex differences, only the females being affected in this way [78]. On the other hand, lack of carbohydrate in the diet was associated with a two- to threefold increase in the biotransformation of various xenobiotics in rats [79]. The experiments proved that, contrary to general belief, the CHO play an important role in regulating hepatic drug-metabolising enzyme activity, acting like enhancers when in low concentrations and as repressors when in excess. The control substrate was ethanol and the results showed Induction and inhibition of drug-metabolising enzymes 223 that a decrease in CHO intake may significantly increase the action of ethanol, while an opposite effect was observed at high-CHO content in the diet. Similar effects on rats were evident from studies that employed orally administered or intraperitoneally injected phenobarbital, polychlorinated biphenyl and 3-methylcholanthrene as control substrates [80]. Having a much greater impact on the drug-metabolising capacity of certain enzymatic systems by far, is a special group of micro-nutrients, namely, the vitamins. Vitamins are biochemical effectors indispensable for life and are essential constituents (or at least, should be) of a normal, correct diet. Apart from other functions (specific, or as enzyme cofactors), vitamins have been proven to be required also for the biosynthesis of proteins and lipids. We have already presented the role of the macro-nutrients as vital components of drug-metabolising enzymatic systems; thus it is obvious that changes in vitamin levels (particularly, deficiencies) will have an important impact on drug-metabolising activity in general. Vitamins influence enzymatic activity predominantly as inhibitors. In different vitamin deficiencies, the enzymatic activity is generally decreased through various mechanisms, involving either (and more commonly) a direct decrease in cytochrome P450 levels, or a reduction in other CYTP450 system components, such as NADPH-cytochrome P450 reductase. Sometimes the inhibitory action is very specific; for example, vitamin A deficiency has enzyme-selective effects on drug metabolism. Studies have proven that a vitamin A-free diet (in rats for example) will result in lower levels of some specific enzymes relative to control animals. A relatively recent study showed that after four days of retinol administration to BALB/c mice, the activity of only CYP3A was decreased, while the catalytic activity of other enzymes (both phase I and phase II enzymes) remained relatively unchanged [81]. The control substrate was paracetamol and further observations of the study were that vitamin A potentiates paracetamol-induced hepatotoxicty, without involving changes in the corresponding biotransformation enzymes. Other interesting aspects have been revealed experimentally. For example, the potentiation of paracetamol-induced hepatotoxicty was proven not to occur in the kidney; the suggested conclusion indicated an organ- specific response [82]. An interesting study on the impact of vitamin A-deficient, or supplemented diet, on the expression of different CYTP450 isoforms was performed on Syrian hamsters [83]. After a six-week observation period, the vitamin A-deficient diet resulted in a decrease in the total CYTP450 content, implicit in the catalytic activities of different CYT isoenzymes. The opposite effect was observed with the vitamin A-supplemented diet, which resulted in 224 Chapter 5 a marked increase in the activity of testosterone 7α-hydroxylase. The authors thus suggested that dietary vitamin A deficiency or supplementation displays not only organ-specific response, but enzyme specificity as well. A point worth highlighting is that in certain instances, vitamin A supplementation can display an inhibitory effect on drug-induced hepatocarcinogenesis in the rat [84]. A dietary supplementation with β-carotene (the most common vitamin A precursor) proved to be effective in increasing certain microsomal and cytosolic enzymes such as cytochrome b5, NADPH cyt c reductase, and aryl hydrocarbon hydroxylase. It was therefore suggested that β-carotene is particularly protective in limiting the initiation phase of the toxic process. Another important vitamin displaying opposite effects in deficiency and excess is vitamin B1 (thiamine). The mechanisms by which thiamine deficiency can affect hepatic microsomal enzyme activities have been elucidated by investigating the activities of two constitutive cytochrome P450 isoforms, namely P 450IIE1 and P450IIC11 [85]. The experiment used male Sprague-Dawley rats and was performed for a period of three weeks. The results showed an increase in the IIE1 isoform, while the activity of the other enzyme remained unchanged. In addition, an elevation in the activity of cytosolic glutathione S-transferase was also observed. The overall conclusion of the study was that thiamine deficiency displays enzyme specificity. Supplementation of vitamin B1, either in the diet, or by direct parenteral administration has been proven to result in significant effects on the hydroxylation function of rat liver [86]. Experiments on rats showed an increase in the general CYTP450 content, as well as in some other specific microsomal enzymes, namely demethylases and hydroxylases. An interesting aspect revealed during the same experiment was that when given in excess (e.g. by repeating parenteral doses), thiamine caused an opposite effect. A diet deficient in vitamin B1 results in organ-specific effects, the tendency being to increase some specific hepatic and pulmonary microsomal enzyme activities, while for renal drug metabolism the consequences are quite the opposite [87]. Commonly, these effects – similarly to vitamin A - are attributed to changes in the microsomal content of cytochrome P450 or its NADPH- reductase component. However, more recent studies have found that thiamine can also act by changing the type of cytochrome present [2]. Vitamin B2 (riboflavine) likewise displays specific effects on drug- metabolising enzymes, initial studies having been made about 30 years ago [88]. They refer to the induced decrease in certain enzyme activities (particularly demethylation and hydroxylation enzymes) by riboflavine Induction and inhibition of drug-metabolising enzymes 225 deficiency. The experiments revealed decreased levels of both hepatic cytochrome P450 and cytochrome b5. Further experimental studies have proven that the activities of both drug-metabolising enzymes and lipid peroxidation were decreased in low- or deficient-riboflavine groups. Experiments involving supplementary administration of vitamin B2 resulted in increased activities of drug enzymes [89]. Other experiments proved that NADPH-dependent lipid peroxidation was markedly decreased in the liver microsomes of groups with riboflavin- deficient diet [90]. Experiments comparing the effects of a standard diet and a riboflavin- deficient diet proved that concurrent consumption of ethanol enhanced the intestinal phospholipid concentration in the deficient diet group, whilst no significant effects on the concentration of proteins or phospholipids was observed in the standard diet group. Riboflavin deficiency decreased both intestinal phase I and phase II enzyme levels [91]. A more recent, related study followed variations of the drug- metabolising enzyme activities mediated by vitamin B2 deficiency. In this study, the substrates were polychlorinated biphenyls (PCBs), known to induce liver lipid peroxide formation in rats. These components are well- known inducers of the liver microsomal cytochrome P450, and vitamin B2 deficiency has been proven to promote this induction [92]. Vitamin B2 being a component of NADPH-cytochrome P450 reductase (which is itself a component of the MMFO system), it is not surprising that a deficiency in riboflavin will result in a general decrease in activity of the enzymatic system. Vitamin C (ascorbic acid) has special status among the vitamins, being the only one that cannot be synthesised in some organisms, including man, monkey and guinea-pig, due to an inherited enzymopathy. Therefore, these species in particular will show a special requirement for this vitamin. Studies on guinea-pig species proved that individuals with vitamin C deficiency are more sensitive to the effects of particular drugs (e.g. pentobarbitone, procaine) [93]. This increased sensitivity was attributed to a marked decrease in drug-metabolising capacity. Studies on experimental animals suggest that the vitamin C deficiency may interfere with heme biosynthesis, consequently directly affecting cytochrome P450 levels. An interesting experiment concerning the effects of vitamin C on hepatic drug metabolising function in hypoxia/re-oxygenation was relatively recently performed [94]. Results revealed that increase in total and oxidised glutathione levels were attenuated by vitamin C by itself, or in combination with vitamin E. Total and oxidised glutathione levels were markedly increased during hypoxia, but markedly decreased in the presence of vitamin C, E, or their combination. By increased oxidation and glucuronidation, 226 Chapter 5 vitamins C and E synergistically improve the hypoxia/reoxygenation hepatocellular damage as indicated by abnormalities in drug metabolising function. This protection appears to be mediated by decrease in oxidative stress. Vitamin E has been under the scrutiny of researchers since 1976, when attempts were made to correlate drug metabolism and hepatic heme proteins in a vitamin E-deficient diet in rats [95]. Hepatic homogenates have been analysed for CYTP450 content and specific drug metabolizing enzyme activities. While no difference could be detected between the vitamin E- deficient population and the control group, decreased levels have been noticed for some microsomal hydroxylases and demethylases. No relevant association could be made between heme protein synthesis and a vitamin E- deficient diet. Interesting studies performed in the same period revealed another important aspect for a drug to

display its pharmacological action, through examination of the effect of vitamin E deficiency on intestinal transport of passively absorbed drugs. Sodium barbital was used as the control compound. In vitamin E-deficient animals, its absorption rate was enhanced compared with the control group. This increase in the absorption rate was attributed to vitamin E-deficiency-induced alterations of the intestinal membrane structure, and was confirmed by using other control compounds passively absorbed through the intestinal membrane. On the other hand, it was observed that the transport rate for drugs normally rapidly transported (e.g. salicylates) was not modified by the deficiency state [96]. As far as hepatic drug metabolism is directly involved, a comparative study tried to elucidate whether there are significant differences in normal and vitamin E-deficient animals [97]. The general conclusion of the study, which monitored several specific enzymes as marker parameters, was that vitamin E-deficiency did not influence these parameters significantly, only a slight increase in NADPH oxidase activity being noted. Another study proved that in the presence of polychlorinated biphenyls, vitamin E displayed an inductive effect on various microsomal enzymes [98]. This observed activity enhancement was associated with increased liver weight and the amount of liver microsomal protein after application of Delor 103 (a specific polychlorinated biphenyl preparation). Vitamin E has been proven to stimulate catalytic activity of certain microsomal hydroxylases and demethylases. Concerning the microsomal hydroxylations of certain drugs, after oral doses of vitamin E, the result was a clear stimulatory effect [99]. However, important observations were that this inductive effect is relatively specific and can be reversed (or prevented) by pre-administration of actinomycin D. It is well recognized that by manipulating dietary levels of vitamin E, lipid peroxidisability (especially of bio-membranes) can be altered. Experimental studies tried to associate the increasing lipid peroxidation Induction and inhibition of drug-metabolising enzymes 227 induced by a vitamin E-deficient diet and the adriamycin-induced inhibition of hepatic drug metabolism [100]. The results proved that vitamin E deficiency produced a significant elevation in hepatic lipid peroxidation, but without any considerable alterations in the activities of specific microsomal hydroxylases and demethylases. In contrast, pre-treatment with adriamycin displayed a significant inhibitory effect, decreasing the same enzyme activities by up to 63%. Nonetheless, the final conclusion of the study was that decreases in drug metabolism were independent of dietary vitamin E and did not correlate with lipid peroxidisability. It was thus suggested that adriamycin-induced depression of hepatic drug-metabolising enzymes was not mediated by elevated lipid peroxidation. In vitamin E-supplemented diets, studies revealed a significant increase both in the total cytochrome P450 content and NADPH-cytochrome P 450 reductase activity, in rat liver. On the other hand, further experiments proved that vitamin E protected protein sulphhydryl groups and lipids against peroxidation, which can induce apoprotein loss. Interestingly, observations established that the protective effect against –SH and lipid peroxidation extend to protection of the CYTP450 apoprotein, but not to the enzyme activity, which was only partially protected. The most significant conclusion was that, at least in vitro peroxidation-dependent loss of P450 is not directly related to lipid/SH oxidation, but is instead mediated by heme degradation from the P450 holoenzyme [101]. The same protective effect was observed in experimental animals infected with influenza virus, which resulted in depression of different monooxygenase enzyme activities. In a dose-dependent relation, vitamin E was demonstrated to both decrease lipid peroxidation enhanced by the viral infection, and to increase the enzyme activities depressed by the same cause [102]. Based on its protective anti-peroxidation effects, an association between a retinoic acid metabolism blocking agent (RAMBA) and vitamin E was relative recently proposed [103]. Examples of compounds in this category include 2-benzothiazolamine derivatives. Combined formulations with vitamin E are administered as capsules or injectable solutions. Vitamin E is metabolised by CYTP450-mediated side-chain oxidations. Often, these enzymes are regulated by their substrates themselves. However, tocopherols are able to activate gene expression of different CYP450 isoforms via the pregnane X receptor (PXR), a receptor with nuclear localisation capable of regulating a variety of drug metabolising enzymes [104]. Decreased activity in a number of enzymatic systems involved in the biotransformation of drugs has also been reported for vitamin K deficiency. A vitamin K-poor diet, as well as vitamin deficiency, have been proven to be accompanied by a decreased activity of various microsomal enzymes, 228 Chapter 5 including demethylases, hydroxylases, NADH- and NADPH-reductases. A very interesting hypothesis aimed at explaining the above effects correlates changes in the enzymatic activity with the weakening of both hydrophobic and polar interactions in the microsomal membranes [105]. A very recent study focused on possible effects of a synthetic vitamin K analogue (menadione) on enzyme activity [106]. It was demonstrated that depending on dose and duration of treatment, menadione displays an inductive effect on both phase I and phase II drug metabolising enzymes. Dietary vitamin K, among other factors such as age or genetic polymorphism in the CYTP4502C9 isoform, also plays an important role in the inter-individual variability in responses to warfarin. The impact of these findings in clinical practice is still being assessed [107]. Based on the discussion in the above examples, it is evident that we can highlight the fact that vitamins are essential not only for good health, but also for maintaining normal levels of drug metabolism. The impact of deficiencies is reflected in decreased enzyme activity, with the consequences described in the previous subsection. Minerals are likewise required in very small amounts in the diet, both for maintenance of good health and for normal physiological function, including normal functioning of the enzymatic systems involved in xenobiotic metabolism. Some of the most important minerals which have been proven to influence drug metabolism in one way or another by affecting enzyme activity, include iron, magnesium, calcium, zinc, copper and selenium. Depending on the enzyme affected, the effects may manifest as an increase, or instead as a decrease, in enzyme activity, with consequent impact on drug biotransformations. In addition, it should be mentioned that in some instances, no changes have been reflected as effects on drug metabolism, depending also on the enzyme or enzymatic system involved. Usually, we refer to mineral deficiencies, which as expected, will generally result in decreased metabolism. The only mineral deficiency resulting in increased metabolism involves iron. This seems anomalous, given that iron is essential in the haem group of cytochrome P450s. Experimental studies on rats revealed that iron deficiency is sex-dependent, occurring only in the male, in which a significant increase in the CYP3A2 isoform (a male-specific isoenzyme) was observed. The activities of drug-metabolising enzymes in female rats were not increased by iron deficiency (because of lack of the CYP3A2 isoform) [108]. However, the opposite effect was observed during a long-term study focused on the effects of iron-deficiency on different drug metabolising enzymes in both hepatic and extra-hepatic tissues including lung, kidneys and intestinal mucosa [109]. An important aspect involving iron-deficiency is the observed marked decrease in intestinal drug metabolism, which may result in significant toxicological consequences, if we refer particularly to Induction and inhibition of drug-metabolising enzymes 229 the protective role of the intestinal enzymes against certain procarcinogenics, such as PAHs, for example. Another experimental system was employed to study the effect of iron-deficiency on both phase I (activating) and phase II (conjugating) xenobiotic metabolising enzymes [110]. Microsomes and cytosolic preparations were made from various tissues including liver, lungs, kidneys and intestinal mucosa at the end of the experiment. Activities of many phase I and phase II enzymes were investigated and the results showed that in most cases, their activities were significantly decreased by iron deficiency; it was concluded that this may result in the persistence of some ingested compounds in the body, without appropriate elimination, which might prove to be harmful to the host. The effects of copper deficiency are variable, but generally result in decreasing the metabolism of certain drugs by affecting the corresponding enzyme activity. However, important consequences of copper deficiency have been observed during pregnancy, resulting in both structural and biochemical abnormalities of the foetus. The aim of one study was to establish the mechanisms of copper deficiency-induced teratogenesis, available data suggesting that more mechanisms may be involved in the associated dysmorphogenesis, including especially the free radical defence mechanism [111]. An interesting aspect to mention is that copper excess has the same effect as copper deficiency, most commonly a decreased ability of enzymatic systems to metabolise drugs or other xenobiotics (-see also the discussion on copper enzymes in Chapter 4). As an example we may mention a significant decrease in the metabolism of aniline in rats pre-treated over one month with an excess of copper [112]. Another important aspect to highlight is that copper excess (as well as an excess of other transition metals such as molybdenum and zinc) can be toxic, so most organisms have developed defence mechanisms to form detoxification pathways. These mechanisms commonly act by reducing uptake, sequestration or enhancing elimination, and are controlled at different levels (transcriptional, translational and enzymatic) by inducing specific conformational changes which will affect the metal binding [113]. In zinc deficiency, both phase I and phase II biotransformation reactions are influenced, namely decreased, the effects being related to reduced levels of cytochrome P450s and reduced activities of certain phase II enzymes, namely UDPGTs and GSTs [114]. Recent studies have revealed that zinc deficiency may result in a decrease in the activities of specific demethylases and hydroxylases and even in the inhibition of the synthesis of a specific P450 isoform (CYP2D11) [115]. However there are also examples of enzyme activities that remain unchanged in zinc deficiency, for example that of the microsomal epoxide hydrolase [116]. As for the phase II 230 Chapter 5 enzymes, the activity of glutathione-S-transferase (important in conjugation reactions and involved in detoxication) has been proven to be significantly decreased in zinc deficiency [116]. On the other hand, as in the case of copper, zinc excess has also been proven to display an inhibitory action. Studies on experimental animals showed that high intake of zinc for a longer period inhibited NADPH- cytochrome C reductase, benzphetamine-N-demethylase and glutathione S-transferase activity, while the cytochrome P450 and cytochrome b5 activities were not obviously changed [117]. Magnesium deficiency, often found to correlate with calcium deficiency, has been proven to influence in the same manner the action of certain enzymatic systems, particularly that of the cytochrome P450s. However, experiments on rats proved that in magnesium deficient (‘MgD’) diet, no significant alterations were observed as far as different phase I enzymes were studied, with only aniline metabolism reduced by 30%. A fourfold decrease in the MgD group was identified for a phase II enzyme, a UDP-glucuronosyltransferase [118]. An interesting explanation to support especially the action of magnesium is based on the interaction of this mineral with the phospholipids and thyroid hormones. In magnesium deficiency, both thyroid hormone levels and microsomal content of phospholipids are depleted, thus resulting in decreased drug-metabolising capacity [2]. Calcium deficiency was shown to decrease the rates of metabolism of various drugs (prolonging their activity, e.g. for hexobarbital), by both oxidative and reductive pathways in liver microsomes, decreasing specific enzyme activities [119]. An essential trace element, closely related in biochemical action to vitamin E, is selenium. This element is particularly important because it was demonstrated to be a novel regulator of cellular heme metabolism, displaying an enhancement of two essential microsomal and mitochondrial enzymes involved in heme synthesis. However, at high concentrations in vitro selenium acted as an inhibitor [120]. As with copper, excessive selenium levels can also be inhibitory, as can be the deficiency. A role of selenium in the biosynthesis of microsomal components and phase II enzyme activities has been suggested [121]. From the above considerations we may conclude that the impact of dietary components and their interaction on drug metabolism can be extremely complex; on the other hand, the effects tend to occur predominantly under conditions of deficiencies. Malnutrition, unfortunately still prevalent in Third World countries, must be addressed in order to prevent or counteract the effects mentioned, since they often have complex and s ometimes unp redictable consequences. Induction and inhibition of drug-metabolising enzymes 231 However, when discussing effects of diet on enzyme activities and their impact on drug metabolism, we have to consider also the so-called non- nutritional factors as still being part of diet; these would include ingestion of pyrolysis products

(formed during cooking) and tobacco smoking. Considering the pharmaco- and toxicological consequences, particular attention should be paid to the products formed in meat (or fish) when fried. These so-called pyrolysis products, commonly breakdown products of tryptophan, are enzyme inducers, displaying specificity for the P4501A1 isoform. At the same time, as they resemble closely the pyrolysis products from tobacco, they are likewise potential carcinogens/mutagens. The inducing effect will be reflected by increasing rate of biotransformation and consequently, reduced bioavailability of the drug. It is assumed that a polycyclic hydrocarbon inducer of CYTP450 is responsible for this effect. It is formed as a pyrolysis product in fried or charcoal-broiled meat. Interesting studies revealed not only a species difference, but also an organ and an enzyme specificity, in the action exerted by compounds of this type [122- 123]. For example, in the case of the pyrolysis product from fried or charcoal-broiled meat, the target organ is commonly the liver, the main detoxication organ in the body. In animals treated with masheri (a form of roasted tobacco paste) the activities of enzymes occurring mostly in extrahepatic tissues was determined. The GI tract has been proved to be principally affected and so could become a predispositional factor in determining susceptibility to carcinogen exposure. As for enzyme specificity, an increase in the activity of phase I and a marked decrease in activity of phase II detoxication enzymes were observed. The species difference was determined on mice, rats and hamsters [122]. The activity of specific enzymes, including CYTP450, benzo[Į]pyrene hydroxylase and glutathione-S-transferase, decreased in the order: hamsters, rats and mice. Because of chemical similarity with the pyrolysis products of tryptophan, some other groups of components should be considered in this context as well. We refer particularly to the indole type group of compounds found in cabbages and Brussels sprouts. The inductive effect has been proven to be species-dependent. This is supported by the different metabolisms affected in various species. If, for example in rats, these compounds induce the biotransformation of barbiturates, in humans they increase caffeine metabolism [2]. Administered to healthy volunteers, both Brussels sprouts and cabbage displayed stimulatory effects on antipyrine and phenacetin metabolism, by decreasing mean plasma half-life, increasing clearance rate and enhancing phase II conjugation reaction [124]. On other drugs, the effects were different, with more influence on phase II conjugative reactions. The most frequently quoted example is that of acetaminophen; its glucuronidation is enhanced, as is the amount of 232 Chapter 5 urinary recovery of the corresponding glucuronide, for which a mean increase of 8% was observed. In contrast, no comparable changes have been noticed in the biotansformation of acetaminophen to its sulphate conjugate. Also, no changes in the plasma glucuronide/oxazepam ratio were observed, suggesting a substrate specificity of the inductive effect exerted by cabbage or Brussels sprout [125]. An interesting and worthwhile aspect to stress is the potential of cabbage (and other Brassica species) as potent dietary cancer-inhibitors. The idea is supported by the fact that dietary cabbage has been reported to increase the aromatic hydrocarbon hydroxylase (AHH) microsomal enzyme system, and consequently to enhance the rate of metabolism of certain procarcinogenic drugs and carcinogens. Bacterial studies have also suggested that cabbage may additionally display a demutagenic activity [126]. Other non-nutrient components (but still categorised in the class of dietary factors) are food additives, flavourings, colourings and the like. These have usually been shown to act either as inducers or inhibitors of the metabolism of particular drugs. As an example, we should mention that di-t- butyl hydroxytoluene significantly increases the activity of some enzymes, including demethylases and hydroxylases in rat microsomes. Because of increased metabolism, duration of pentobarbital narcosis is significantly decreased. Final observations suggested that lipid-soluble compounds that are metabolised in liver microsomes, such as di-t-butyl hydroxytoluene, may generally increase the activities of drug-metabolising enzymes in liver microsomes [127]. A commonly used colouring agent for foods (as well as for some pharmaceutical preparations) is erythrosine. Examining its action in rat liver homogenates on labelled T4 and T3, experiments proved that in a dose- dependent manner, erythrosine inhibited the de-iodination of T4, and consequently the formation of T3. Further experiments revealed that other pathways of T4 metabolism were inhibited as well [128]. Being inhaled deliberately, tobacco smoke is still considered a ‘dietary’ component, which can affect drug therapy by both pharmacokinetic and pharmacodynamic mechanisms. It usually displays intense inducing effects [129], in a way quite similar to that observed for ingestion of charcoal-broiled meat. The related factor is identified as the polycyclic hydrocarbon benzo[a]pyrene. The inducing effect will be reflected in low plasma levels of certain drugs, due to their increased biotransformation. A well-known and commonly quoted example is that of phenacetin metabolism in smokers and non-smokers [130]. Another example, considered as a marker for drug metabolism is antipyrine; tobacco smoke (which contains at least 3000 components) was found to increase the drug’s clearance, lowering its bioavailability [130]. Induction and inhibition of drug-metabolising enzymes 233 At the same time other drugs have shown no alterations in their biotransformation, thus indicating that tobacco smoke acts as a selective inducer [131]. A significant aspect is that enzymes induced by tobacco smoking may also increase the risk of cancer by enhancing the metabolic activation of carcinogens. Compounds believed to be implicated here are the polycyclic aromatic hydrocarbons, which are potent inducers of various CYTP450 isoforms. The most significantly affected has been proven to be an extrahepatic enzyme, the CYP1A1 isoform present in the lung. There is some evidence that high inducibility of this enzyme is more frequent in patients with lung cancer. Drugs for which induced metabolism due to cigarette smoking may have clinical consequence include theophylline, caffeine, tacrine, imipramine, haloperidol, pentazocine, propranolol, flecainide and estradiol. At the same time, clinical trials suggested that cigarette smoking leads to other pharmacological consequences, such as a faster clearance of heparin, a decrease in the rate of insulin absorption (after s.c. administration, due to the cutaneous vasoconstriction produced), heart-rate lowering during treatment with β-blockers, less analgesia from some opioids and less sedation from benzodiazepines. All these associated effects are attributed to the stimulant action of nicotine. However, some of the tobacco smoke components have proven to act like enzyme inhibitors; examples include cadmium and carbon monoxide. As studies have thus far been confined to animal studies and the in vitro situation, the relevance for human drug metabolism has not been established. In all cases, from all actual data, it is the inductive effects of tobacco smoke that are prevalent [132]. Another extremely important aspect of tobacco smoking is that it can affect drug therapy via different mechanisms [133]. In an extended experimental study, both pharmacokinetic and pharmacodynamic drug interactions are described. As a direct consequence, cigarette smoking can reduce the efficacy of certain drugs or make drug therapy quite unpredictable. Pharmacokinetic interactions are presented for various drugs including theophylline, diazepam, propranolol and flecainide. This type of interaction causes enhanced plasma clearance, decrease in absorption, and induction of CYTP450 enzymes. Therefore, patients who are smokers would be in the situation of requiring larger doses of a certain drug for obtaining the desired therapeutic effect. The pharmacodynamic interactions, described in the study for antianginal and antihypertensive agents, oral contraceptives and histamine-2-receptor antagonists have an important impact in increasing the risk of adverse reactions, especially in smoker patients with cardiovascular or peptic ulcer disease, or in women smokers using oral contraceptives. 234 Chapter 5 In the next chapter, we encounter other internal factors that influence drug biotransformation through their direct impact on the cytochrome P450 system. These include species, sex, age, disease state, hormonal control, as well as some external, environmental factors (excepting the heavy metals treated in the present chapter). A separate chapter discusses the impact of genetic factors. References 1. Ronis MJJ, Ingelman-Sundberg M. 1999. Induction of Human Drug -Metabolising Enzymes: Mechanisms and Implications. In: Woolf TF, editor. Handbook of Drug Metabolism. New York, Marcel Dekker Inc, pp 239-262. 2. 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Stimulatory effect of Brussels sprouts and cabbage on human drug metabolism. Clin Pharmacol Ther 25:88-95. 125. Pantuck EJ, Pantuck CB, Anderson KE, Wattenberg LW, Conney AH, Kappas A. 1984. Effects of Brussels sprouts and cabbage on drug conjugation. Clin Pharmacol Ther 35:161-169. 126. Alberto-Puleo M. 1983. Physiological effects of cabbage with reference to its potential as a dietary cancer-inhibitor and its use in ancient medicine. J Ethnopharmacol 9:261- 272. 127. Takanaka A, Kato R, Omori Y. Effect of food additives and colors on microsomal drug- metabolizing enzymes of rat liver. Shokuhin Eiseigaku Zasshi 10:260-265. 128. Ruiz M, Ingbar SH, Charles A. 1982. Effect of erythrosine (2’,4’,5’, 7’-tetraiodofluorescein) on the metabolism of thyroxine in rat liver. Endocrinology 110:1613-1217. 242 Chapter 5 129. Jusko JW. 1979. Influence of cigarette smoking on drug metabolism in Man. Drug Metab Rev 9:221-236. 130. Scavone JM, Joseph M, Greenblatt DJ, LeDuc BW, Blyden GT, Luna BG, Harmatz, Herold S. 1990. Differential effect of cigarette smoking on antipyrine oxidation and acetaminophen conjugation. Pharmacology 40:77-84. 131. Barau M, Flotats I, Massot M. 2001. Interactions between tobacco and drugs. Circ Farm 59:10-16. 132. Zevin S, Benowitz NL. 1999. Drug interactions with tobacco smoking: an update. Clin Pharmacokinet 36:425-438. 133. Schein JR. 1995. Cigarette smoking and clinically significant drug interactions. Ann Pharmacother 29:1139-1148. Chapter 6 FACTORS THAT INFLUENCE DRUG BIOTRANSFORMATION 6.1 INTRODUCTION An account of the influence of species, sex, age, hormonal status and disease state on drug biotransformation forms the major part of this chapter, these parameters collectively being referred to as ‘intrinsic factors’. Reported results of several recent studies of these factors are reviewed, with the aim of indicating their variable nature as well as their interdependence. Thus, for example, we will encounter cases where for certain drugs, there are only subtle differences in the biotransformation routes in different species, while for others, dramatically different pathways are adopted, leading to the formation of vastly different metabolic products. Interdependence of most of these factors is a natural expectation, given that the status of an individual’s metabolising activity and pathological status vary over a lifetime. Thus, for example, the effects of natural attrition of the metabolising activity in an aged patient and a specific disease state can interact in a way that results in a unique mode of metabolic clearance of a drug. One significant point emerges from the discussion, namely the variability in the outcomes of drug metabolism observed for different species. This unpredictable element reminds us of the caution that should be exercised in extrapolating from animal studies to humans and the implications that this has in the evaluation of new drugs in the pharmaceutical industry. The final section of this chapter summarises the effects of external (environmental) factors on drug biotransformation, examples of which were encountered in earlier chapters. Continual introduction of new chemical substances into the environment through waste production and industrial activity remains a major international issue, necessitating inter alia on-going studies of their effects on human drug metabolism. 243 244 Chapter 6 6.2 INTRINSIC FACTORS Drugs, as well as other xenobiotics are metabolised via various pathways, including phase I and phase II reactions, which involve participation of numerous enzyme systems. Therefore, it is reasonable to assume that there are many factors that can determine or influence along which pathway a particular drug will undergo biotransformation and the extent to which this will proceed. These factors are usually arbitrarily divided into internal and external factors, with nevertheless considerable interaction between them [1,2]. 6.2.1 Species Examples of species differences in drug biotransformation are numerous, continuously investigated, and encountered in both phases of biotransformation [3,4]. An interesting observation is that they may involve the same route, but differ in the rate along that particular pathway (i.e. quantitatively different) or they may adopt different pathways (i.e. differing qualitatively) [5,6]. It should be noted as well that there is not always a direct relationship between metabolism, half-life and action of a drug [7]. Selected examples An interesting quantitative species difference in phase I metabolism is known for caffeine, both in terms of total metabolism and metabolite production [8]. Thus, the total metabolism is highest in humans, decreasing in the order - monkey, rat and rabbit. While there are no significant differences in the formation of theobromine, marked differences have been recorded for the other two metabolites, paraxanthine and theophylline, with paraxanthine formation highest in humans and lowest in monkey, whereas the reverse obtains for theophylline [8]. An interesting aspect is the way caffeine biotransformation reactions proceed in higher plants, the variability of caffeine catabolism again being dependent on species and to a greater extent, on the age of different tissues investigated. As an example, it was reported that in young tea leaves, theophylline is re-utilised for caffeine biosynthesis, while in aged leaves of Coffea arabica, it undergoes further metabolism resulting in 7-methylxanthine accumulation. Other species of Coffea have been proven to convert caffeine to methyluric acids. Obviously, these cases exemplify qualitative differences, as well as species- and age-dependence [9]. A well-known quantitative example is that of species variation in hexobarbitone metabolism, affecting half-life and sleeping time. Investigations have been made on man, dog, mice and the rat [10]. The Factors that influence drug biotransformation 245 longest half-time was registered for man (~360 min). The sleeping time increased in the following order: mice, rats, dogs and man. The main conclusion of the experiment, apart from demonstrating that the oxidative metabolism of hexobarbitone is markedly influenced by species, was that the biotransformation is inversely related to the half-time and duration of action of the investigated drug, the highest metabolism being registered for mice and decreasing in the opposite order as for the sleeping time for example. A recent example refers to the variation in the metabolism of selegiline (structure in Figure 6.1) ((-)-form of deprenyl) in seven different species [11]. From literature data, it is known that selegiline undergoes N-dealkylation, yielding several metabolites, namely N-desmethylselegiline, methamphetamine and amphetamine. CH3 N CH2 C CH CH3 Fig.6.1 Selegiline The investigations made during the study referred to, and performed on liver microsomes of different species, in addition to characterizing the potential metabolic variations, also proved the existence of another metabolite, the N-oxide. The rate and extent of formation of this metabolite was found to be markedly influenced by species, the highest rate of production occurring in dog and hamster, being much lower in humans, and zero in the rat. Another example of quantitative variation was revealed from experimental studies investigating the metabolic profile of a relatively novel diuretic. A comparative approach was adopted, aimed at demonstrating its metabolism in experimental animals and human liver microsomes [12]. Increased rates of metabolism were observed in rats and monkeys, and six metabolites, designated RU1, RU2, RU3 and MU1, MU2, MU3 for the respective species, were identified in their urine. Quantitatively, only three of these were considered to be major metabolites in rat and monkey urine, namely RU3, RU1 and MU3 respectively, whereas in the dog, the unchanged drug was observed as the major urinary component. This indicated a net difference between the rat and the monkey, both displaying extensive biotransformation, and the dog, in which only little metabolism occurred. In contrast with dogs, humans showed similarities with rats, suggesting a common metabolic pathway. 246 Chapter 6 Six species have been investigated in connection with the psychoactive drug of abuse 4-bromo-2,5-dimethoxyphenethylamine (2C-B) (street names ‘Venus’, ‘Bromo’, ‘Erox’, ‘XTC’ or ‘Nexus’) (Figure 6.2). H3C O 6 5 1 CH2 CH2 NH2 Br 4 2 3 O CH3 Fig.6.2 Structure of the psychoactive drug of abuse, 4-bromo-2,5-dimethoxyphenethylamine Hepatocytes from human, monkey, dog, rabbit, rat and mouse were incubated with 2C-B in an attempt to identify the resulting metabolites and to monitor possible toxic effects [13]. Investigations established that the drug under study undergoes oxidative deamination with successive formation of two metabolites, which may or not undergo further metabolism by demethylation. Marked differences were noticed with two other, less common metabolites identified, one of these occurring only in mouse hepatocytes, the other in human, monkey and rabbit, but not in dog, rat and mouse, supporting the idea of qualitative interspecies variations. Another aim of the study, as mentioned above, was to compare the toxic effects exerted by 2C-B on hepatocytes of the six investigated species: the differences observed were only minor. However, another important aspect was revealed, namely that large differences in susceptibility of hepatocytes may occur between different individuals. The biotransformation pathways of a relatively novel drug used as an acute oral treatment for migraine, namely zolmitriptan (Figure 6.3), were comparatively investigated in human and rat liver microsomes [14]. O H O NH N N(CH3)2 Fig.6.3 Structure of zolmitriptan Factors that influence drug biotransformation 247 Although the reports indicated that the drug was metabolised by the same CYP isoform in both types of microsome, the numbers of metabolites nevertheless differed. This suggests that the report presents a reasonable and economical in vitro model for comprehensive studies of zolmitriptan metabolism, including biotransformation pathways, enzyme kinetics, induction and inhibition phenomena, interspecies differences and the possible occurrence of drug interactions. An interesting study, involving both phase I and phase II biotransformations, has been performed in an approach using comparative interspecies data for both prospective design and extrapolations from animal findings to humans [15]. The aim was to reduce the potential for human risk and increase therapeutic benefit. For paclitaxel (Figure 6.4) for instance, markedly different metabolites were observed to occur in rats and humans, which renders metabolic drug-drug interaction investigations in rats practically irrelevant for humans (thus, qualitative differences). In contrast, for zidovudine (AZT), the variations were quantitative, with a high rate of glucuronidation in humans, resulting in a much shorter half-life than that observed in animals, which display negligible glucuronidation. This study revealed more significant features: qualitative differences in phase I biotransformation and quantitative variations

in phase II, with no relevant similarities to allow extrapolations and drug-drug interaction predictions from animals to humans. O O O OH H C CH H3 H 3 3C C 3 O NH O CH3 H O O OH OH O O CH O O 3 Fig.6.4 Paclitaxel Advanced analytical procedures (e.g. LC/MS, high field NMR spectroscopy) have been used to examine the potential differences in the biotransformation of efavirenz [16], a potent and specific inhibitor of reverse transcriptase commonly recommended in the treatment of HIV infections. Metabolites produced by humans, rats, guinea pigs, hamsters and monkeys were investigated. Observations confirmed that efavirenz (Figure 6.5) is 248 Chapter 6 extensively metabolised by all species, with marked species differences in the metabolites isolated and structurally determined. Although the major metabolite, namely the O-glucuronide conjugate, proved to be common to all five species studied, other metabolites displayed species specificities as follows: the sulphate conjugate was found in rats’ and monkeys’ urine, but not in that of humans, while GSH-related metabolites were identified only in the urine of rats and guinea pigs. 16 15 13 12 14 F3C Cl 5 6 O 7 8 N O H Fig.6.5 Efavirenz Differences in the production of reactive metabolites may sometimes result in species-selective nephrotoxicity. For example, efavirenz was reported to produce renal injury (necrosis of the renal tubular epithelial cells) in rats, but not in monkeys or humans. Here, a species-specific glutathione adduct, produced only by rats, was deemed responsible for this nephrotoxic effect [17]. Species differences involve, as mentioned above, both phases of biotransformation. An interesting study was performed to investigate the maintenance of drug-metabolising capacities in collagen gel sandwich and immobilisation cultures of human and rat hepatocytes [18]. L-proline was added to the medium to improve albumin secretion. As far as most important phase I enzyme systems are concerned, namely the cytochrome P450- dependent monooxygenase (CYP) and microsomal epoxide hydrase (mEH) systems, comparative measurements of enzyme activities in the absence and presence of L-proline, revealed that their biotransformation enzyme activities were not affected by the addition of L-proline. Instead, the activity of an important phase II enzyme, GST, was decreased in rat hepatocytes, whereas in humans it remained almost unchanged. As human hepatocytes showed a better maintenance of GST activities than the rats in the presence of L-proline, species differences were again demonstrated. Another study investigated whether there are also species variations in maintaining certain phase I and phase II enzyme activities after cryopre- Factors that influence drug biotransformation 249 servation of liver slices prepared from five different species, namely mouse, rat, dog, monkey and human [19]. The conclusion of the study was that although the metabolic patterns and rates of biotransformation varied among these species, the phase I and phase II metabolic capacities of the liver slices were well maintained after cryopreservation. For certain drugs, and depending upon the species investigated, variations have not proven to be very significant. For example, an experiment concerning orbifloxacin metabolism in two species, pigs and calves, aimed at establishing possible species differences, proved that in both species the metabolic pathway of the drug was the same, differing only in the amount of the excreted metabolite [20]. Indeed the final, common metabolite was the glucuronide, excreted in average amounts of 3% and 1% in pigs and calves respectively. In addition, the remainder of the drug was excreted unchanged in both species. However, a qualitative difference was noted, namely that calf urine contained also a product of oxidative metabolism. Quantitative species differences were established for the immunosuppressive drug cyclosporine A (CSA) (Figure 6.6). [21]. Investigations were performed on liver and small intestinal microsomes from rat, hamster, rabbit, dog, baboon and man. H3C CH3 HO CH3 CH3 CH CH2 CH3 O O H3C H N N CH3 N N N O CH CH3 O O CH3 H3C CH3 CH CH 3 3 CH3 CH CH CH 2 2 CH CH3 O O O N CH CH H 3 N 3 H N N H3C N N CH2 CH CH H 3 O CH3 O CH3 CH O CH3 H3C CH3 Fig.6.6 The structure of cyclosporine A 250 Chapter 6 The metabolic pathways of CSA are known to result in two principal metabolites, the hydroxylated and N-demethylated CSA, which accounted for most of the CSA metabolised in all tested species. However, marked variations occurred in the biotransformation rate, measuring only 2-8% over 30 min in rats, in contrast to dogs, whose liver microsomes proved to be very efficient, yielding a 70-100% change in the same period. Investigations having been performed on both liver and small intestinal microsomes, another objective of the study was to determine possible differences determined by different tissues of the same organism. Measurements of the formation of the principal metabolites in the two investigated organs indicated a similar metabolic profile, but with differences in the rate of metabolism, that in the small intestine being slightly slower. Differences in the metabolic profiles were the subject of investigation for panomifene (Figure 6.7), an analogue of tamoxifen, an anti-estrogen for hormone-dependent tumors [22]. Liver microsomes from mouse, rat, dog and human were used. The observed routes of biotransformation were hydroxylation and side chain modifications. Although seven metabolites were detected in the incubated mixtures, there was only one produced by all species that had lost the side chain. Interspecies differences concerned the metabolites with the truncated side chain, as follows: in the case of rodents, the microsomal system led to loss of the hydroxyethylamino group, while for incubated mixtures containing microsomes of all three other investigated species, only the loss of the hydroxyethyl group was detected. Other important observations made during the experiment were (a) that of the seven metabolites detected, three were produced exclusively by the dog and (b) that human liver microsomes produced an oxidised form of the metabolite containing a double bond in the side chain, this compound not being detectable in the other species investigated. F3C C C CH2 CH2 OH O CH2 CH2 N H Fig.6.7 Panomifene (analogue of tamoxifen) Factors that influence drug biotransformation 251 Different profiles, as well as quantitative species differences, were observed in the metabolism of L-775,606, a selective 5-HT1D receptor agonist, developed for the acute treatment of migraine [23]. Species investigated included human, dog, monkey and rat. For three of these (human, monkey and rat), the main metabolites were the hydroxylated M1 and the N-dealkylated M2. In contrast, in the dog the N-oxide metabolite (M3) was prevalent, representing an average of about 40%, whereas in the other investigated species, its formation represented a minor pathway, with the excreted metabolite corresponding to less then 5%. In an interesting experiment accomplished both in vitro and in vivo, the metabolic fate and the toxicity of dapsone (Figure 6.8) were comparatively investigated in rat, mouse and man [24]. The metabolites were determined by HPLC/MS and metHb formation was used as toxic endpoint. The investigations focused especially on the toxic aspects and possible consequences during dapsone administration. As for the in vitro investigations, the results revealed that the greatest toxicity occurred in rats, with a significant difference between sexes: ∼36% metHb formed in males and only 8.2% in females. In humans, the metHb toxic metabolite was found in an amount of ~11%, while in the mouse, only 4% under the same conditions. The rank order of toxicity was in direct relation to the formation of the hydroxylamine metabolite in vitro. However, experiments proved that the microsomes from all tested species were able to reverse the reaction, reducing the hydroxylamine back to dapsone. In contrast, under in vivo conditions the species most susceptible to dapsone toxicity proved to be the human, the sensitivity to toxic effects decreasing in the order: human, mouse, rat. Interspecies and sex differences also occurred in the biotransformation of the drug, in that the hydroxylamine and its glucuronide were detected only in male rats and humans, but not in female rats or mice. H2N SO2 NH2 Fig.6.8 Dapsone Species differences may also account for stereoselective reactions. Experiments were performed with fifteen O-acyl propranolol (PL) prodrugs, using rat and dog plasma and liver subfractions [25]. The aim of the study was to investigate both species differences and substrate specificities for the stereoselective hydrolysis of the tested prodrugs. As far as species was concerned, significant differences in the hydrolytic activities of prodrugs 252 Chapter 6 were established, in rat plasma being in the range of 5-119-fold greater than those in dog plasma. In contrast, dogs displayed a higher hepatic hydrolytic activity, especially in cytosolic fractions. The significant differences in the hydrolytic rates therefore represent quantitative species differences. As for stereoselectivity, the study also revealed important interspecies differences: hydrolysis in dogs generally showed a preference for the (R)-enantiomer, whereas in the rat, for all of the prodrugs containing substituents of low carbon number, the (S)-enantiomer was preferentially hydrolysed. Following a previous report of species differences in the tolerability of rhein (a constituent of rhubarb), with rabbits displaying the highest susceptibility to kidney disturbances, a complex phase I and phase II metabolic investigation was performed in an attempt to elucidate species differences in the biotransformation of this compound [26]. Experiments were performed in vivo, with 14C-labelled rhein; tested species included the rat, rabbit, dog and man. The common major metabolites determined in all tested species were the phenolic monoglucuronide and monosulphate. The urine samples of rabbits showed an additional hydrophilic metabolite fraction. The in vitro experiments performed on subcellular liver fractions of rabbits revealed the presence of several metabolites, including three monohydroxylated metabolites, their corresponding quinoid oxidation products and a bis-hydroxylated derivative. The hydroxylated phase I metabolites were further detected as glucuronides in all tested species, whereas the quinoid product was found only in rabbit urine. It is assumed that this metabolite displays a potential reactivity with endogenous macromolecules and generates that species-dependent susceptibility. Species differences can also impact on inhibition phenomena. Investigations of the inhibition of pentobarbital biotransformation in the presence of empenthrin (Figure 6.9) support this idea [27]. Empenthrin (a synthetic pyrethroid) has been reported to display an inhibitory effect on pentobarbital metabolism, resulting in prolongation of the sleeping time. CH3 H3C C O CH HC C O C HC H C C H3C CH3 H3C CH2 CH3 Fig.6.9 Empenthrin (a synthetic pyrethroid) Factors that influence drug biotransformation 253 This phenomenon was observed for mice (the inhibitory effect being determined as dose-dependent), but not for other species investigated, namely rats, dogs, guinea pigs or hamsters. Further experiments using microsomal fractions expressing human CYPs were performed to determine the possible inhibitory effect of empenthrin on pentobarbital metabolism in humans. The final results revealed that the inhibition of pentobarbital by empenthrin occurred only in mice and not in any other of the other species investigated, including humans. As previously mentioned, species differences may be implicated in several aspects, including qualitative and quantitative differences in drug biotransformation route, influences on stereoselective biotransformations and even on inhibition phenomena. An interesting and relatively recent study revealed the impact of species differences also on the distribution of drug metabolising enzymes. Complex investigations followed the expression of nine CYP450 isoenzymes and three GSTs in the pancreas of several species including humans [28]. The seven species tested in comparison to humans, were mice, hamsters, rabbits, rats, dogs, pigs and monkeys. A first finding was the large variation in the cellular localisation of the enzymes among the eight investigated species, with most of the enzymes expressed only in the pancreas of hamster, mouse, monkey and man. The other tested species were lacking several enzyme isoforms. However, in human tissue, four enzymes were lacking in almost half the cases. All of these observations concerning interspecies differences in the distribution of some of the most important drug-metabolising enzymes support the notion that great caution needs to be exercised when attempting to predict or extrapolate from animal data to humans. This last observation confirms the importance of species differences, especially for drug-design in the pharmaceutical industry, where a suitable model reflecting human patterns of biotransformation and toxicity is desirable. 6.2.2 Sex As already indicated in some of the above examples, qualitative and quantitative differences in both phases of drug metabolism are related to sex as well [29]. Initial observations of this feature were made in the early 1930s, when researchers noticed that female rats required

only half the dose of a barbiturate compared to male rats to induce sleep. Later investigations indicated that this was due to the reduced capacity of the female to m etabolise the barbiturates [30]. 254 Chapter 6 Sex differences have been intensively studied, not only in relation to sex-dependent metabolism of various xenobiotics [31], but also with the aim of correlating sex-dependent pharmacokinetics, pharmacodynamics, efficacy, and the possible occurrence of adverse reactions [32]. Sex differences, sometimes related to species or age, are now being observed for a wide range of substrates, including commonly prescribed drugs or even endogenous compounds, including steroid sex compounds [30]. Like other factors that influence drug metabolism, sex differences are considered to determine also biotransformation variations. Therefore, before introducing a new drug into therapy, combined studies investigate both species and sex differences on the metabolic profile of the candidate. As an example, we refer to such a combined study for the in vitro investigation of sex and species differences in the metabolism of BOF-4272, a drug intended for the treatment of hyperuricaemia [33]. Rats, mice and monkeys of both sexes were used in the study. The results of the investigations made on various incubation mixtures revealed that both the pathways involved (i.e. types of metabolites resulting) as well as the rates of biotransformation of the tested drug were significantly influenced by both sex and species differences. On the other hand, results of other investigations examining the influence of sex and age on different enzyme activities showed no significant differences [34]. 6.2.3 Age It has long been recognized that the newborn, young and elderly display marked differences in drug biotransformation and are more susceptible to drug action. These differences are chiefly due to the enzymatic systems involved in drug biotransformation and the development of their metabolising capacity. Thus, the increased sensitivity of neonates may be related to their very low, undeveloped metabolising capacity, until adult levels of enzyme activity are achieved. On the other hand, in the elderly, the decrease in drug-metabolising capacity also appears to be dependent on these factors, important changes in the overall metabolism occurring with ageing. An important aspect to be borne in mind is that the factors influencing drug metabolism are split arbitrarily and that they are interrelated. Examples have been given so far regarding species, sex and age. We should also highlight the fact that the status of enzymatic systems and their metabolising capacity may develop in many different ways, the patterns varying and being dependent on the species and sex [35-41]. Thus, a very recent study in fact reviewed the influence of age and sex on CYP enzymes in relation to drug bioequivalence [42]. Factors that influence drug biotransformation 255 The concern for controlling drug therapy, especially in the elderly to provide desired pharmaceutical effects at lower risks, continues to be a principal aim of research. Specific aims include efforts to try and prevent adverse reactions and to optimise therapy for the individual patient [43]. Unfortunately, as already mentioned, important changes in drug metabolism do indeed occur with ageing. For example, the significant reduction in liver volume accompanying ageing will be reflected in a reduction in the total amount of cytochrome P450 produced, and this could be associated with reduced ability of these enzymes to function. Other problems occurring with ageing, still not very well understood and needing to be revisited in view of recent advances, include the following: the effect of age on extrahepatic enzymes (especially CYPs), the impact of induction and inhibition phenomena on enzymatic systems in the elderly, the effect of the environment on drug metabolism in the aged given the increasing complexity of the CYPs involved in human metabolism, pharmacology and function of transporters, the decline in general metabolic capacity, and general frailty of older people [44]. Taking cognisance of the above, it is understandable and expected that all these conditions will result in altered drug handling and especially, altered pharmacodynamic responses. Recognizing the central role of the liver in the general metabolism of both drugs and other xenobiotics, we should also mention, besides the reduction of hepatocyte mass (with corresponding effects on the hepatic enzyme system activity), the reduction of hepatic blood flow and changes in sinusoidal endothelium. These changes will affect drug transfer and oxygen delivery, resulting in reduced hepatic drug clearance. Another current problem in the elderly is related to renal clearance reduction, which is generally disease-related. Altered pharmacokinetics and pharmacodynamics are expected also in patients with cardiovascular diseases. Also worth remembering is the effect of age on pancreatic secretion [45]. But perhaps one of the major problems resulting in adverse reactions and drug-drug interactions is the still very common practice of polypharmacy, responsible for increased morbidity and mortality in the elderly. This is another aspect that is peculiar to elderly patients, who consume a disproportionate amount of prescription and non-prescription medications. Such practice can obviously lead to many negative consequences, primarily placing the elderly at risk of developing significant drug-drug interactions, which often go unrecognized clinically and which are responsible for increased morbidity in this sector of the population. Drugs can interact to mutually alter absorption, distribution, metabolism or excretion characteristics, or interact in a synergistic or antagonistic fashion 256 Chapter 6 altering their pharmacodynamics. In addition, one must be aware that co-administered drugs, foods and nutritional supplements can also alter the pharmacological actions of a medication. These alterations may cause the action of a drug to be diminished or enhanced. Another major issue is that drugs may also interact with diseases, potentially worsening disease symptoms. Therefore, prudent use of medications and vigilant monitoring are essential for preventing the elderly from the high risk of adverse reactions and drug-drug interactions, whose unfortunate consequences have been noted above [46-52]. Considering the physiological changes in main organ functions in the elderly as well as the pharmacokinetic parameters of various drugs, accumulations of drug metabolites presents another important problem. In this context, particular attention should be paid to an adequate treatment scheme designed to ensure the optimum therapeutic effect with a minimum risk of toxic effects. In fact, a starting dose which is 30-40% less than the average dose used in adults is generally recommended, not only for renally excreted drugs, but also for compounds metabolised and excreted by the liver [53-54]. Ageing is directly related to ovarian hormonal activity, and progesterone metabolites, specifically, have been proven to affect the response to various psychotherapeutic agents, resulting in increased risk of adverse effects. Studies on benzodiazepines, for example, demonstrated that their metabolism is altered, either resulting in a decrease in their clearance or an alteration of the effect-concentration relationship. These effects may result in increased risk of adverse reactions, particularly in older patients with anxiety disorders. Therefore, establishing the appropriate low dose for optimal treatment will minimise adverse effects. The intimate mechanisms involved are not completely understood, but it has been suggested that they could be related to modulation of the GABA-antagonist receptor by neurosteroids [55]. Other drugs that were investigated with respect to the role of drug- metabolising enzymes and the effects of age included different alkylphenoxazone derivatives, benzodiazepines and neuroleptics, bisphosphonates (BPs) as therapeutic drugs for osteoporosis, anxiolytics and others [56-59]. A special category includes ‘ultra-aged’ patients. Aspects concerning decreased drug absorption, metabolism and excretion, decline of protein binding, lower blood flow, disturbance of blood brain barrier, adverse reactions and drug interactions for this category of patients have been reviewed, with the purpose of establishing proper therapeutic management [60]. Factors that influence drug biotransformation 257 Two other important aspects of age-related changes are sensitivity to environmental factors and nutritional effects on hepatic drug metabolism in the elderly [61,62]. The cited works review pharmacodynamic and toxicokinetic changes in absorption, distribution, metabolism, excretion and sensitivity, as well as age-associated differences in hepatic drug metabolism, and the effects of nutrition on drug bioavailability, distribution and hepatic metabolism. An important issue in improving the quality of life of the elderly has recently been reviewed and concerns CoQ10 implications in energetic metabolism, a well-known anti-oxidant effect with relevance to health food and medical drugs [63]. At the other end of the scale, special attention is paid to neonates and children, as regards the development of their enzymatic systems. Unpredictable developmental changes in drug biotransformation have been proven to play a role not only in the pharmacokinetic profile, but also in the pathogenesis of adverse drug reactions in children. Most of these developmental changes have a genetic determinant, which causes variations in different metabolising enzymes, whereby normal, therapeutic drug doses can result in functional overdoses due to drug accumulation. This relative overdosing is determined by inefficient elimination via the affected pathways. Furthermore, idiosyncratic forms of toxicity may occur when a relative increase in reactive metabolite formation is due to imbalances in bioactivation and detoxification processes. Phenotyping and genotyping would be very helpful under such circumstances to prevent these effects [64]. Extra-hepatic metabolism has to be considered as well, the renal clearance and volume of distribution being at least as important as hepatic metabolism [65]. Typically, drug metabolism is significantly reduced in the neonatal period because of lack of enzymatic activity. A recent investigation reviewed the effect of age on the biotransformation of four drugs [66]. The subjects were infants and children, and the tested drugs included caffeine, midazolam, morphine and paracetamol. The first observation was that in the neonatal period, for all four tested drugs, clearance was markedly reduced. Further observations confirmed that (with the exception of paracetamol) this reduced clearance is maintained in infants and children under the age of two years, and that there is considerable inter-individual variation in clearance values for all ages and for all tested drugs, appearing to be the greatest for midazolam. The third important observation suggests that for children older than two years, the mean plasma clearance values for all four drugs are more or less similar to those in adolescents and even adults. 258 Chapter 6 6.2.4 Pathological status The way in which the body clears drugs is affected by many disease states. Among them, those of primary concern are considered to be diseases affecting the liver: cirrhosis, alcoholic liver disease, cholestatic jaundice, and liver carcinoma [67]. Other factors responsible for variation in drug metabolism are the endocrine disorders, such as diabetes mellitus [68], hypo-and hyperthyroidism [69], pituitary disorders [70], and various types of infections (bacterial, viral, malaria) [71]. In cirrhosis for example, replacement of parts of the liver by fibrous tissue leads to a reduction in the number of functional hepatocytes. In this situation, it seems absolutely reasonable that drug metabolism should be impaired. It is known for example that human cytochromes P450, particularly the CYP2A6 isoform, catalyse the bioactivation of various drugs and even carcinogens. Recent studies proved that in cases of liver disease, including cirrhosis (but also viral hepatitis or parasitic infestation), this isoform is over-expressed, and as such may therefore be considered a major liver catalyst in pathological conditions [72]. An important consequence of such liver disease (or other organ impairment) arises during transplantation processes; it is well known that prior to transplantation, organ dysfunction may occur because of stress and anxiety, and this may result in altered pharmacokinetic behaviour of some psychotropic agents. In case of cirrhotic patients, an increased drug bioavailability due to portosystemic shunting was noted, which therefore therefore required drug dosage adjustment. Studies on different psychotropic agents suggest that a selection of these, concurrently administered with an appropriate dosage adjustment, could ensure the lowering of risk of drug accumulation [73]. Another recent article reviews the implications of oxidative stress and the role of cytochrome P450s and cytokines in drug-induced liver diseases, which according to some recent studies can be also induced by immunological mechanisms [74,75]. In this context, we should mention that especially in the last few years, great importance has been attributed to antioxidants in the treatment of drug- induced liver oxidative stress, due to the central role of this organ in the general metabolism. Effects of natural antioxidants have been investigated in vitro on liver redox status by biochemical, analytical and histological methods, in order to assess the overall free radical-antioxidant balance. Factors that influence drug biotransformation 259 Studies have also been performed in animal models and in humans with Gilbert’s disease and alcohol liver disease. The results confirmed the

role of free radicals in alcoholic patients, stressing the greater vulnerability of women to alcohol toxicity. As regards Gilbert’s disease, investigations found no alterations of free radical-antioxidant balance, but in contrast, an improvement in the non-enzymatic antioxidant defense system [76]. The impact and consequences of drug-induced liver diseases on drug pharmacokinetics and toxicity in the case of pathogenesis are continuously investigated. Recently, the role of polymorphism of drug metabolising enzyme systems has been reviewed [77]. A comparative study was performed on normal mice to investigate the effects of drug-induced liver injury using prednisolone (PSL) versus Angelica sinensis Polysaccharides (ASP), on hepatic metabolising enzyme activities of both phases. ASP was shown to increase content and catalytic activity of several enzymes viz. CYTP450, different demethylases and hydroxylases, and GSH-related enzymes. In contrast, PSL significantly decreased the liver mitochondrial glutathione content, whereas all other enzyme activities were increased. An important observation was that treatment with ASP could restore the GSH content, which is important for detoxication (by glutathione conjugation) of certain xenobiotics, including drugs [78]. An interesting aspect recently investigated concerns the CYTP450 superfamily. The multiple CYP450 isoforms (CYPs) are well known as being involved in the biotransformation of numerous drugs, other chemicals, as well as endogenous substrates. Unfortunately, the hepatic CYPs may also be involved in the pathogenesis of several liver diseases, due to their catalytic activity mediating activation of certain drugs to toxic metabolites (see Chapter 8). Incidences of drug-induced hepatotoxicity, as well as nephrotoxicity and cardiac failure are well known and unfortunately relatively frequent. The most frequently cited examples of hepatotoxicity refer to halothane and acetaminophen (see Chapter 8). There are usually several mechanisms involved in drug-induced liver disease. One of them is an immunological one (see ref. 75), presumably determined by the covalent binding of the metabolite to CYP, which will result in formation of anti-CYP antibodies, leading to so-called ‘immune-mediated hepatotoxicity’. Another mechanism, related to the CYP2E1 isoform, is associated with lipid peroxidation and production of reactive oxygen species, resulting in damage to hepatocytes and mitochondrial membranes. The explanation for involvement of this particular CYP isoform relies on the observation that in alcoholic patients, its levels are significantly increased. Thus, it was first associated with alcohol-liver disease and non-alcoholic steatohepatitis. 260 Chapter 6 However, due to its ability to activate carcinogens, investigations also suggested a possible role of this isoform in hepatocellular carcinoma. Considering the liver as the main location for the most important enzymatic systems, it is expected on the other hand that in patients with liver diseases, drug metabolism should be impaired. Particularly vulnerable isoforms have been proven to be different CYPs such as 1A, 2C19 and 3A, while others (2D6, 2C9, 2E1) appeared to be affected to a lesser extent. An interesting feature is that the pattern of CYP isoenzyme alterations differs with the etiology of the liver disease, with the most severe modifications occurring in cirrhosis [79]. Other liver diseases have also been proven to alter drug metabolism by altering the activities of metabolising enzymes. A prime example is alcohol-induced disease, unfortunately the most common type of chronic liver disease in many countries. An important aspect revealed by one study [80] is that alcohol can interact with other factors of risk for hepatic disease, especially hepatitis C infection and also concurrent consumption of hepatotoxic drugs (acetaminophen, for example), resulting in more severe disease and increased risk of adverse reactions and drug-drug interaction occurrence, than occurs when alcohol alone is the risk factor present. Another interesting aspect to mention, demonstrated in a recent investigation on rats, is that hepatic and extrahepatic (e.g. intestinal) metabolic activities involving especially the cytochrome P450 system are influenced by surgery and/or drug-induced renal dysfunction [81]. The most marked decreases (of about 66%) were observed for the hepatic CYP3A metabolic activities, in the case of nephrectomy. Less marked, but nonetheless significant decreases were observed also in drug-induced renal dysfunction following i.m. injection of glycerol (about 60%), and i.p. injection of cisplatin (about 49%) (Figure 6.10). In contrast, the intestinal metabolic CYP3A activity was weakly increased in rats injected with glycerol, and remained practically unchanged in the case of injected cisplatin or surgery (nephrectomy). Cl NH3 Pt2+ Cl NH3 Fig.6.10 Cisplatin Factors that influence drug biotransformation 261 These results suggest a dependence of the extent of lowering of hepatic P450 activities on the etiology of renal failure. In addition, the experimental observations led to the conclusion that alteration of the same enzyme activity in extrahepatic tissues (particularly in intestine, where this tissue was examined experimentally) cannot always be correlated with that in the liver. 6.2.5 Hormonal control of drug metabolism – selected examples Hormones, known to play a major role in the general metabolism, have similarly been proven to control the biotransformation of drugs, in direct connection with other factors such as age, sex, or in particular physiological states, such as pregnancy. An example is the apparent connection between certain sex-specific drug- and steroid-metabolising enzyme activities in rats and the sex- dependent expression of those specific enzymes, under gonadal steroid and growth hormone control [82]. Another sex and age connection with the control of the growth hormone (GH) was the focus of interesting cDNA cloning investigations [83,84]. The study examined especially cytochrome P450, it being established that GH is involved in the control of rat hepatic drug- and steroid-metabolism, particularly through the action of this enzymatic system. The results showed low levels of CYTP450 in neonates, and an increase after one month, both in male and female rats. At adult stage, important sex differences were recorded, in female rats the content being about three times higher than in male rats. Thyroid status contributes to differences for several drugs administered in equi-active doses on several forms of UDPGTs [85]. As experimental animals, rats having different thyroid hormonal status were employed, namely normal (control group), hypothyroid and hyperthyroid. The drugs tested were ciprofibrate, bezafibrate, fenofibrate and clofibrate (Figure 6.11). The responses were markedly modulated by the thyroid status, with an average increase of about 5% in hyperthyroid animals. The results confirmed the role of hormonal control upon the enzyme induction displayed by certain drugs (or other xenobiotics). The hypothalamo-pituitary-liver axis has also been proven to function as a hormonal control system in the metabolism of drugs and endogenous compounds [86]. 262 Chapter 6 CH3 O C COOH O CH3 C NH CH2 CH2 Cl benzafibrate CH3 CH3 O C C O CH O H3C O CH3 C Cl fenofibrate CH3 O C C O CH2 CH3 H3C O Cl clofibrate Fig.6.11 Structures of some of the cited fibrates 6.3 ENVIRONMENTAL FACTORS These are usually considered to be those influences in our surroundings that can affect (sometimes markedly) drug metabolism. Of course, there are a large number of environmental chemicals that potentially could affect drug biotransformations, usually grouped into heavy metals (already discussed, see previous chapter), industrial pollutants and pesticides. The most important industrial pollutants are typically aromatic or aromatic polycyclic compounds and polychlorinated biphenyls (Figure 6.12). Many of these have been already discussed under different circumstances (inductive enzyme effects, procarcinogenic effects). Factors that influence drug biotransformation 263 Cl Cl Cl Cl Cl Cl 3,3',4,4',5,5'-hexachlorobiphenyl Cl Cl Cl Cl Cl Cl 2,2',4,4',6,6'-hexachlorobiphenyl Fig.6.12 Polychlorinated biphenyls (common industrial pollutants) Pesticides are also of various types (insecticides, herbicides), and are considered environmental contaminants in air, soil, water and food. They will not be discussed further in the present monograph. 6.4 FURTHER OBSERVATIONS As has been discussed in the last two chapters, there are numerous factors (some of them interactive) that can affect drug metabolism, therefore making its control an extremely complex problem. 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Chapter 7 IMPACT OF GENE VARIABILITY ON DRUG METABOLISM 7.1 INTRODUCTION Dispensing of medicines is already strongly influenced by considerations of genetic factors that play a role in drug response. With the extremely rapid accumulation of knowledge of the genetic make-up of the human species and the recent technological advances accompanying it, this tendency is set to increase significantly in the future and therefore a basic knowledge of the principles of pharmacogenetics is essential to the health professional. This chapter sets out to provide a basic introduction to these disciplines, beginning with principles and nomenclature. Several specialised sub-disciplines (e.g. toxicogenomics, proteomics) are also outlined. A discussion of species-dependent biotransformations and their genetic control, illustrated with recent examples, follows. The discipline of pharmaco- informatics is briefly described and discussed, and finally the implications of genetics in the future dispensing of drugs are outlined. 7.2 BASIC PRINCIPLES OF PHARMACOGENETICS Pharmacogenetics, still considered a relatively new field of clinical investigation, is the study of genetically determined variations in drug response; practically, it reflects the linkage between an individual’s genotype and that individual’s ability to metabolise a foreign compound [1-6]. The term was first proposed in 1959 [7]. Large inter-individual differences that may occur in the disposition of many drugs (or other xenobiotics) are controlled, at least in part, by genetic 269 270 Chapter 7 factors. In this context it is important to mention that while environmental factors including smoking, alcohol consumption and drug use, diet, occupational exposure to chemicals, and disease can vary during the course of drug therapy, genetic factors are constant throughout life. Most commonly, genetic variations reflect themselves in different rates and extents of drug elimination from the body. This explains, in the first place, the possibly marked differences in dosage requirements for many patients, resulting in the need to individualise doses and, in general, the therapeutic treatment. On the other hand, it is assumed that differences in metabolism of various therapeutic compounds as a consequence of genetic polymorphism, can lead to severe toxicity or even therapeutic failure, by altering the relation between dose and blood concentration of the pharmacologically active drug. This is determined by the absence, insufficiency or alteration of metabolising enzymatic systems, due to the genetic aberrations. Under these circumstances, it is evident that understanding the mechanisms of genetic variation in drug effects could be the key to applying pharmacogenetic principles to improve the therapeutic strategies, by ensuring greater efficacy and decreased risk of adverse reactions or toxicity. Thus, genetic variations will be important firstly for those genes encoding drug-response proteins that are expressed in a monogenic fashion. If a single locus determines the expression of a drug-response gene, then it is assumed that the genetic variation has the potential to contribute to inter-individual variation in drug response. At the same time it should be noted that these inter-individual variations help to explain also the inter-individual differences and susceptibilities observed in disease states such as cancer, hypercholesterolaemia, alcoholism, and toxicity to environmental pollutants or industrial chemicals. Therefore, we mention here a relatively recent addition to the discipline of pharmacogenetics, generally known as ‘ecogenetics’, which deals with the dynamic interactions between an individual’s genotype and environmental agents, including industrial chemicals, pollutants, plant and food components, pesticides, and other chemicals. In this context, we should mention also the related field of toxicogenetics, dealing with an individual’s predisposition to different toxic effects of drugs, including carcinogenesis and teratogenesis, for example. Population (interethnic) differences in response to drugs give rise to the terms ethnopharmacology or pharmacoanthropology, that represent another area of relatively recent interest, having obvious implications for drug therapy especially in multiracial societies. In keeping with very recent developments, we should introduce also the following topics [8]: Impact of gene variability on drug

metabolism 271 - toxicogenomics: Here, investigation based on the use of whole- genome and specialty microarrays yields information concerning the response to xenobiotics at the genomics level (mainly gene expression). In these studies, toxicity is classified on the basis of gene transcriptional patterns. The intention is to extrapolate the toxicities of new chemicals by comparing their patterns with databases of responses and well-documented toxicological endpoints. Toxicogenomics profiling is however limited by the fact that unless the structure of the new compound bears a strong resemblance to those existing in the database, it will not yield reliable predictions of toxicity. This is a definite drawback since the essence of modern drug design is the incorporation of novel structural features into potential drug molecules. - proteomics: This may be defined as ‘high-throughput separation, display and identification of proteins and their interactions’ [8]. Analytically, the methodology used to achieve this includes 2D-PAGE electrophoresis separation, mass spectrometry and NMR spectroscopic detection. The method finds use both in the study of new targets for toxicants as well as in predictive profiling. An essential idea that underlies the application of proteomics to toxicogenomics is that specific groups of xenobiotics should induce specific patterns of protein expression. As proteomics permits the assaying of body fluids with rapid detection of biomarkers, there is some advantage over mRNA expression. - metabonomics: This is the study of metabolic profiles at the organism (i.e. large-scale) level, where human metabolism is considered the basis of cellular organization and responsible for responses to stimuli through control of cellular signalling. Consequently, a measure as drastic as administration of a drug will determine the expression of metabolic enzymes qualitatively and quantitatively, these modifications being interrelated to the organism’s responses to gene mutation, drug intervention and disease state. A distinct advantage of metabonomics technology is its treatment of changes in metabolite concentration [8]. - chrono-pharmacogenetics: This is based on a fairly recent concept, namely that changes in the expression level of genes vary with the time of day [9]. It is well known that biochemical, physiological and behavioural patterns vary with the time of day, such variation being at the basis of biological organization. In a recent study [9] expression levels in the liver of 3906 genes in Fischer 344 rats were determined as a function of time of day. While the maximum estimated changes observed for most genes were less than 1.5-fold, statistical tests revealed that 67 genes displayed significant alterations in expression as a function of time of day. Interestingly, these turned out to be genes playing important roles in key cellular pathways including drug metabolism and other major processes. 272 Chapter 7 From all of the above considerations, we may conclude that even inter-individual variability in drug metabolism can be determined by several factors. However, the one still considered to be most important is the existence of genetic polymorphism in the genes encoding the metabolising enzymes. It should be borne in mind that protein structure, three-dimensional configuration and concentration – may also alter the action of drugs in various qualitative and quantitative ways. It should be stressed that successful predictions of effects of genetic variations and their consequent pharmacological and clinical implications are due to changes in the encoded amino acid sequences, with consequent modifications in the three- dimensional structure of the newly synthesised protein, and its subsequently modified function and properties [10]. It is already known from previous chapters that part of the fate of a drug entering the body involves (besides interaction with enzymes, a requirement for biotransformation), interactions with proteins and lipids as well. After passing through membranes (lipoprotein structures) by various mechanisms (see Chapter 1), they react with plasma and/or tissue proteins and interact with their specific receptors. Therefore, it is obvious that genetic mutations that alter even in a punctiform manner, the quality or quantity of these proteins, or characteristics of membranes or receptors, will result in disturbances of the pharmacokinetics of a drug or drug-cell interactions. Consequences of pharmacogenetic variations in drug metabolising enzymes may include: a. alteration in the kinetics and duration of action of certain drugs; these phenomena are due either to inherited deficiencies in metabolising enzymes, or to the reverse, an over-expression of them. In the case of deficiencies in metabolising enzymes, the altered kinetics result in retarded inactivation, increased blood concentrations and decreased clearance, leading to overdose with possible adverse reactions or even toxicity; on the other hand, with over-expression of metabolising enzymes, the consequence will be a decreased drug blood concentration, and subsequently, therapeutic inefficacy (sometimes resulting in the need for administering megadoses); b. drug-drug interactions and c. idiosyncratic adverse drug reactions (see Chapter 8). Therefore, the objectives of research in pharmacogenetics are multiple and involve the following: 1. identification of genetically controlled variations in an individual’s ability to metabolise a foreign compound (drug or other xenobiotic); 2. study of the molecular mechanisms causing these variations; 3. evaluation of clinical relevance and 4. development of simple methods to identify those individuals who may be susceptible to variable and abnormal responses to drugs administered in normal doses. Impact of gene variability on drug metabolism 273 Some important, general principles to mention in support of the detailed aspects for the discussion that follows are: (a) if in some patients the desired, expected effect is not obtained (or even worse, adverse reactions or toxicity appears) with standard, safe doses of a drug, then the most likely cause may be a genetic variability or an inherited metabolic effect; (b) therefore, any unexpected or unusual (qualitative, but in particular quantitative) response of an individual to a drug should be a warning signal to investigate the genetic source of such variation. Before detailing some modern procedures generally used in pharmacogenetics, it would probably be useful to define some of the terms commonly used in this area: - allele: one of two or more alternative forms of a gene at the same site in a chromosome that determines alternative characteristics in inheritance; - autosome: one of 22 pairs of chromosomes not connected with the determination of the sex of the individual; - autosomal dominant: a trait that is expressed in the heterozygous (see below) state; - autosomal recessive: a trait expressed only in the homozygous state; - gene: a DNA segment (in a chromosome) that carries the encoded genetic information necessary for protein synthesis; - genotype: a gene combination at one specific locus or any specified combination of loci; - heterozygous: having different alleles at the genetic locus determining a given character; - homozygous: having identical alleles at the genetic locus determining a given character; - isoenzymes: electrophoretically distinct forms of an enzyme displaying the same catalytic role; - phenotype: the visible expression of a gene; - polymorphism: the coexistence of individuals with distinct qualities as normal members of a population [5]. Genetic polymorphisms with functional effects on drug metabolism are usually detected on the basis of discontinuous variation in phenotype, where phenotype represents either levels of enzymes or rate of metabolism. Pharmacogenetic polymorphisms in genes encoding xenobiotic-metabolising enzymes may have a variety of effects, depending on both the reaction catalysed and the type of substrate. That is the reason why, nowadays, in pharmacogenetic studies, one applies genotyping of polymorphic alleles encoding drug-metabolising enzymes to the identification of an individual’s 274 Chapter 7 drug metabolism phenotype. This knowledge, when applied to drug selection or dosing, can avoid adverse reactions or therapeutic failure. Phenotyping is accomplished by administration of a drug test, followed by measurement of the metabolic ratio. The main condition is that the metabolism of the drug should be solely dependent on the function of a specific drug-metabolising enzyme. Furthermore, defining the individual’s phenotype, relative to a reference substrate, will allow the drug metabolism phenotype for other substrates of that enzyme to be predicted. Hence arises the clinical importance in predicting adverse or inadequate response to certain therapeutic agents. It is emphasised that in pharmacokinetic studies, phenotyping has the advantage over genotyping, in revealing drug-drug interactions or defects in the overall process of drug metabolism. Genotyping involves identification of defined genetic mutations that will give rise to the specific drug metabolism phenotype. Included in these mutations, we may mention: the genetic alterations that lead to over- expression, the absence of an active protein product (also known as a “null allele”), or the production of a mutant protein with diminished catalytic activity. As already mentioned, but important to recall here, is the fact that genetic polymorphism (such as genetic mutation or gene deletion) is a permanent cause of variation in drug metabolism phenotypes, while others are considered transient causes (enzyme inhibition or induction). With drugs, the consequences of a polymorphism may be either toxic plasma concentrations or lack of pharmacological response. Toxic plasma concentrations, associated with accumulation of specific drug substances, are autosomal recessive traits and characterise the so-called ‘poor-metabolisers’ (PM). In contrast, lack of response, characteristic for ‘extensive’ or, ‘ultra- extensive metabolisers’ (EM, UEM), is a consequence of increased drug metabolism, resulting in too rapid a rate of elimination. The UEM is an autosomal dominant trait arising from gene amplification [11]. For certain classes of therapeutic agents as well as environmental carcinogens, there is strong evidence that genetic polymorphism of drug- metabolising enzymes plays a significant role in adverse effects of therapeutic agents or incidence of exposure–linked cancer [12-14]. 7.2.1 Species-dependent biotransformations and their genetic control We have, in Chapters 2 and 3, already classified enzymes involved in drug metabolism either as phase I (non-synthetic) or phase II (synthetic or conjugative). The corresponding two reaction types often complement one another in function, in the sense that through catalysis of oxygenation, Impact of gene variability on drug metabolism 275 oxidation, reduction, and hydrolysis reactions, phase I enzymes generate functional groups that subsequently serve as a site for different conjugation reactions, catalysed by phase II enzymes. As a result, we find it opportune to classify the polymorphisms as well. However, before proceeding to a fairly detailed presentation of these polymorphisms, a very important aspect that needs highlighting in this context concerns the pharmacogenomics in the newborn [15]. It is well established that deficiency in hepatic and renal drug metabolism and disposition are characteristics of the human newborn. Superimposed on genetic polymorphisms that determine drug metabolism and transport, the immaturity of drug-handling ability in the newborn could result in significant interpatient variability, both as regards dosage requirements and responses to medications. Hence, the role of pharmacogenomics in this situation is to individualise drug therapy for the newborn to minimise adverse effects and optimise drug efficacy. A. Phase I polymorphisms As already presented, the major route of phase I metabolism is oxidation by cytochrome P450 mixed-function monooxygenases (see Chapter 2). Owing to the diversity of this heme thiolate protein, quite a number of forms have been characterised in humans, with reference to their specificity and unique regulation. A relatively recent article reviews the impact of the cytochrome P450 enzyme system genetic polymorphism upon drug biotransformation and (most probably) incidence of drug-drug interactions [16]. It is well recognized that the pharmacokinetics of many drugs often vary considerably among individuals, precisely because of variations in the expression of different cytochrome P450 (CYP) enzymes. In this subsection we shall focus on some of the various polymorphic CYP enzymes, with emphasis on clinical implications and testing strategies. Subfamily CYP2 [17] CYP2D6 is an isoenzyme of particular importance because it metabolises a wide range of commonly prescribed drugs including antiarrhythmics, ȕ-adrenergic blockers, antidepressants and antipsychotics. It is also, by far, the best characterised P450 enzyme demonstrating polymorphic expression in humans. The best-known polymorphism is the debrisoquine/sparteine polymorphism, which involves mutations in the CYP2D6 gene. This was first recognized following adverse reactions in sub-populations of patients receiving the antihypertensive debrisoquine or the oxytocic sparteine. Until recently, more than 50 mutations and 70 alleles have been described for this isoform, many of these resulting in an inactive protein. This isoform comprises 2 to 6% of the total hepatic cytochrome P450 content, but is responsible for the biotransformation of many important drugs 276 Chapter 7 [2]. As mentioned above, the earliest evidence of polymorphic expression was identified during clinical trials on the antihypertensive drug debrisoquine. Since then, several additional drugs have been identified for use in phenotyping studies, including dextromethorphan, and more recently, propafenone [5] and antidepressants [18].

There are interethnic differences in the prevalence of the phenotype of debrisoquine hyroxylase. The clinical significance of this drug metabolism polymorphism owes to the fact that about 5-10% of Europeans and 1% of Asians lack CYP2D6 activity, and these individuals are classified as ‘poor metabolisers’ (PMs). It is assumed that in the case of the Caucasian population, the most common mutated allele generating the PM phenotype is CYP2D6B, which in fact is almost absent in Orientals. The prevalence of ‘extensive’ (EM) and ‘ultra extensive’ metaboliser (UEM) phenotype in Caucasians is also relatively high (about 7%), and is the result of a partially deficient allele CYP2D causing the exchange of a proline (position 34) with a serine [19]. As for the interracial differences in CYP2D6 genotypes, two situations have been revealed: • mutations giving decreased activity and, • mutations giving increased activity. In the case of mutations giving decreased activity, studies showed that a specific fragment of 11.5 kDa contains a deletion of the entire CYP2D6 gene, while another fragment (of 44 kDa) contains an inserted pseudogene [5,19]. The allele-specific polymerase chain reaction technique could distinguish a ‘splicing mutation’, present in most of the CYP2D6 genes of the 44 kDa fragments of Caucasians, this being in fact the reason why they are non-functional. This mutation, known also as the B mutation, accounts for about 75% of the mutant CYP2D6 alleles. Unlike the Caucasian population, among the Chinese people, the B mutation has not been detected and is in fact reflected in the low frequency (<1%) of PMs in this population [5,19]. In contrast, the gene deletion allele has been found to be similar in Caucasians, Chinese and Black people [20,21] indicating, in fact, that this gene deletion occurred before the evolutionary separation of the three races. Mutations giving increased activity appeared in individuals having 10- 12 extra copies of the CYP2D6 gene, this resulting in an ultra-rapid metabolism of substrates of CYP2D6 [22]. Extra genes seem to be present, with more or less the same frequency, within all three major races. As mentioned earlier, in this case what obtains is an increased rate of biotransformation, resulting in rates of elimination that are too rapid, and therefore possibly yielding no therapeutic effect from normal therapeutic doses. An interesting recent study dealt with different allele and genotype Impact of gene variability on drug metabolism 277 frequencies, including CYP2D6 in a random Italian population [23]. Here it was found that volunteers could be divided into four CYP2D6 genotype groups comprising 53.5% with no mutated alleles (homozygous EMs), 35.0% with one mutated allele (heterozygous EMs), 3.4% with two mutated alleles (PMs) and 8.3% with extra copies of a functional gene (UMs). Frequencies of CYP2D6 detrimental alleles in these subjects were similar to those of other Caucasian populations. In contrast, the prevalence of CYP3D6 gene duplication among Italians was very high, confirming the tendency for the higher frequency of CYP2D6 UMs in the Mediterranean area relative to Northern Europe. We may thus conclude that a pronounced variation in the activity of CYP2D6 may be seen both within and between the three major races. This variation, basically caused by mutations in CYP2D6 locus, may manifest differently, as follows: a) no encoded enzyme, b) unstable enzyme, or, c) enzyme with increased activity (gene duplication, triplication, or amplification). Modified phenotypes may have important consequences both as regards the therapeutic efficiency, and/or exposure to various xenobiotic toxicities [24]. That is why genotyping could play a major role in preventing adverse reactions. A large number of drugs (the average estimate is 25-30%) have been shown to be metabolised by CYP2D6, all of them being lipophilic bases, and the binding between drug and enzyme being of an ion-pair type. Some selected examples will be given in subsection C. Recent studies revealed another important aspect that is very significant (given the large variety of drugs metabolised by this isoform), namely that its activity may be inhibited by concurrent administration of various chemicals (drugs or other xenobiotics). The consequences should be as expected viz. an increase in the metabolism of the co-administered drug [25]. As an example, we refer to effects of co-administration of drugs on the pharmacokinetics of metoprolol [25]. Celecoxib significantly increased the AUC of metoprolol and the extent of this interaction was more pronounced in individuals having two fully functional alleles relative to those with a single fully functional allele. Rofecoxib, on the other hand, had no significant effect on the pharmacokinetics of metoprolol. Thus, celecoxib evidently inhibits the metabolism of the CYP2D6 substrate metoprolol in this situation, whereas rofecoxib does not. Clinically, relevant interaction may occur between celecoxib and CYP2D6 substrates, particularly those with a narrow therapeutic index. Another relevant example, involving another isoform, but based on the same principle, is the following which involves thioTEPA (N,Nƍ, 278 Chapter 7 NƎ-triethylenethiophosphoramide), an agent commonly administered in high-dose chemotherapy including cyclophosphamide. Following previous studies which concluded that thioTEPA partially inhibits cytochrome P4502B6 (CYP2B6)-catalysed 4-hydroxylation of cyclophosphamide, a study probing the detailed mechanism of this CYP2B6 inhibition was undertaken [26]. Potent inhibition of CYP2B6 activity was confirmed with bupropion as substrate. The inhibition of the isoform CYP2B6 by thioTEPA was established as being time- and concentration-dependent. Furthermore, the loss of CYP2B6 enzymatic activity was shown to be NADPH-dependent and could not be restored. One conclusion of the study was that the pharmacokinetic consequences of irreversible inactivation are more complex than those of reversible inactivation, since the metabolism of the drug itself can be affected; drug interactions will be determined not only by dose, but also by the duration and frequency of application. A final, but very important aspect involving variation in CYP2D6 genotype, is its impact on non-response or even appearance of adverse reactions during treatment with various drugs. A well-documented example involves antidepressants [18]. Adverse effects or inadequate clinical response often accompany treatment with antidepressants, several of which are substrates for cytochrome P450 (CYP) 2D6. Depending on the polymorphism of the CYP3D6 gene, enzyme activity for individuals can span the range from PMs to UMs. In the study referred to, CYP2D6 genotyping was undertaken using a panel of polymerase chain reaction techniques. The study identified both poor and intermediate metaboliser alleles, as well as allelic duplications of the CYP2D6 isoform. Patients displaying adverse effects had two inactive alleles (PMs). For 19% of the non-responders, amplification of fully functional alleles was established. For psychiatric patients treated with CYP2D6-dependent antidepressants, the conclusion was that the CYP2D6 genotype is associated with adverse effects and non-response. CYP2C19 The next best-characterised CYP-related drug metabolism polymorphism in humans is associated with the metabolism of the (S)-enantiomer of the anticonvulsivant mephenytoin [27]. As in the case of CYP2D6, specific genetic mutations lead to a PM phenotype, with respect to several common therapeutic drugs. The phenotype is inherited in an autosomal recessive manner [28] and, in contrast to the previously described polymorphism, no ‘ultra extensive metaboliser’ phenotype has been reported for this polymorphic enzyme. As in the case of CYP2D6 polymorphisms, significant interethnic differences are characteristic for the PM type; approximately 3% of Caucasians and some black populations (e.g. Zimbabwean Shona) are poor Impact of gene variability on drug metabolism 279 metabolisers, while in the Oriental population the estimate is around 20% [29,30]. The (S)-mephenytoin hydroxylase reaction is catalysed by CYP2C19, and two mutant alleles associated with the defect have been identified [31]. The principal genetic defect in PMs of mephenytoin is a punctiform exchange of a guanine residue with an adenine residue in exon 5, resulting in an aberrantly spliced CYP2C19 mRNA. The direct consequence is that translation of this mRNA will lead to the production of a truncated, and consequently inactive protein. This is considered a null allele and is designated m1 (or CYP2C19*2, after other authors [5]). Further evaluation of PM subjects revealed a second mutant allele, designated CYP2C19 m2 (CYP2C19*3, after [5]), resulting from a G636 to A mutation, consequently leading to a premature stop codon. This mutation has been proven to be unique to Japanese individuals [31]. All Japanese PMs whose phenotype could not be explained by the m1 mutation, were found to be either homozygous or heterozygous (m1m2) for the mutant allele [2]. Nevertheless, we must mention the existence of an EM phenotype, which comprises both the homozygous dominant and heterozygous recessive genotypes. A noteworthy aspect is that individuals of the PM phenotype, due to decreased metabolism of specific drugs such as mephenytoin, are predisposed to CNS adverse effects [3]. Other drugs known to be CYP2C19 substrates include omeprazole [32], propranolol [33] and diazepam [34]. While substrates for CYP2D6 are all lipophilic bases, substrates for CYP2C19 could be bases (propranolol), acids (mephenytoin) or even neutral drugs (diazepam). The clinical consequence of the CYP2C19 polymorphism has not been fully described. Yet, in about 20% of persons of certain ethnic origins that lack the isoenzyme, the consequences could be of considerable clinical importance. Also important to stress is that one of the CYP2C19 substrates, omeprazole, is also a CYP1A2 inducer. Consequently, high serum levels of omeprazole (such as might appear in persons deficient in CYP2C19) may result in increased CYP1A2 activity [35]. CYP2C19 also appears to be the major enzyme that activates the antimalarial chloroguanide (proguanil) [36] by cyclization; therefore, in deficient individuals, this compound may be ineffective. The CYP2D6 and CYP2C19 polymorphisms have been studied less extensively in Black than in Caucasian and Oriental populations. Nevertheless, such interracial differences should be considered during drug development. If the metabolising enzymes of a novel drug have been thoroughly investigated in, for example, a European country, the disposition 280 Chapter 7 might then be predicted for an Asian population (and further confirmed in a small phenotyped or genotyped population). An important aspect to consider, as revealed by recent clinical observations, is that drug-induced hepatitis may be related to the consumption of Atrium – a combination preparation of phenobarbital, febarbamate and difebarbamate – in the PM phenotype of mephenytoin hydroxylase [37]. A decrease in the oral clearance of diazepam was described in Caucasian PMs after a single dose [34]. About 14 years ago, another important aspect was revealed: CYP2C19 polymorphism can be induced by different drugs, for example by rifampicin treatment [38]. More recently, it has been shown that CYP2C19 polymorphism is subject to interactions not only with co-substrates but also with a number of drugs that can inhibit its activity both in vitro and in vivo [39]. CYP2C9 This is an important CYP450 isoform, involved in the biotransformation of quite a range of therapeutically important drugs, including tolbutamide, (S)-warfarin, as well as a range of non-steroidal anti-inflammatory drugs, including diclofenac and ibuprofen [40]. The frequency of the various CYP2C9 allelic variants also varies among ethnic groups, as follows: in whites, an average of 0.06-0.10%; lower frequencies among African Americans, averaging 0.005-0.01%, and in the Chinese population, about 0.02% [41]. All of them are PMs, with the consequences already stated. Selected examples appear in subsection C. CYP2E1 This is an ethanol-inducible isoenzyme, responsible for the metabolism and bioactivation of many procarcinogens [42] and certain drugs, including ethanol and acetaminophen [43,44]. Actually, it metabolises mainly low molecular weight compounds, such as acetone, ethanol, benzene and nitrosamines. CYP2E1 is encoded by a single gene in humans, located on chromosome 10 [45]. Two alleles of this gene, C and c2, have been identified in humans. For each location on the gene where polymorphic mutations have been observed, there is a designation for the wt allele as well as for the mutant allele. For example, the common wt allele with respect to the C mutant allele designating a simple point mutation located in intron 6 of CYP2E1, is designated D. Interestingly, the absence of this allele (C) has been associated with lung cancer in a Japanese control-study [46]. Mutation c2, more rare, may potentially result in increased expression of functional Impact of gene variability on drug metabolism 281 protein, consequently leading to increased metabolism of CYP2E1 substrates. A marker of CYP2E1 activity in vivo is provided by the skeletal muscle relaxant, chlorzoxazone [47]. However, the non-bimodal distribution of oral and fractional clearance values suggested that a single CYP2E1 allele is predominant in the population studied. A great limitation of this cohort study was that no individuals homozygous for the c2 variant were identified in Caucasian subjects [48]. Apparently, the lack

of c2 alleles identified in this study is due to the interracial differences in the prevalence of the c2 allele, first described in the Japanese [49]. Subfamily A CYP3A In humans, this family comprises the 3A3, 3A4, and 3A5 isoenzymes – in adults, and the 3A7 isoenzyme in foetal liver. The most abundant isoenzyme in the adult is 3A4, accounting for 20- 40% of the total hepatic CYP in humans, this being also the one with the widest range of drug substrates. The latter include benzodiazepines, erythromycin, cyclosporine and dihydropyridines [50]. Although levels of CYP3A4 activity vary considerably among individuals, no genetic basis for this polymorphic expression has been defined to date. However, the closely related gene, CYP3A5 has been proven to show a polymorphism in its expression, detectable in only 10-20% of adult livers [51], but with the molecular basis still unclear. It shows a similar, but not identical, substrate specificity for CYP3A4. In addition to the potential for genetic variability in expression or activity, CYP3A activity is also known to be induced on exposure to barbiturates and glucocorticoids and to be inhibited by macrolide antibiotics such as erythromycin [2]. Interestingly, extrahepatic expression of CYP3A can influence phenotyping approaches, depending on the route of test drug administration [52]. The cytochromes P450 CYP1A1 and 1A2, have also been suggested as showing polymorphism, with the molecular basis however not being identified. CYP1A1 is less important for drug metabolism, but of considerable importance in the activation of certain procarcinogens, such as benzo[Į]pyrene. In contrast, the closely related CYP1A2 is of greater importance in drug metabolism, being involved in the biotransformation of important drug substrates like theophylline, imipramine, clozapine, phenacetin and acetaminophen [53]. The level of induction of CYP1A2 by aromatic hydrocarbons is less than for CYP1A1; still, some of the variation seen in CYP1A2 levels in non-smokers might reflect polymorphism in induction owing to passive smoking, diet, or even environmental factors. 282 Chapter 7 Recent studies reveal the important role of some CYTP450 isoforms in the metabolism of certain drugs, as well as an incidence of drug-drug interactions. Based on previous studies indicating that CYP1A2 is the principal isoform responsible for lidocaine metabolism, a study was preformed to assess the effect of a cytochrome P450 (CYP) 1A2 inhibitor, fluvoxamine, on the pharmacokinetics of intravenous lidocaine and its pharmacologically active metabolites MEGX and GX [53]. A second aim of the investigation described was to establish whether fluvoxamine-lidocaine interaction was dependent on liver function. A randomised, double-blind, two-phase, crossover design was employed in the study, details of which appear in reference 53. The authors concluded that liver function did modify the effects of fluvoxamine co-administration, with lidocaine clearance reduced by 60% on average in patients with mild liver dysfunction, but practically unaffected in cases of severe liver dysfunction. The kinetics of formation of the metabolites MEGX and GX were affected in an analogous manner i.e. severely impaired in cases of healthy patients and those with mild cirrhosis, but there was practically no change for subjects with severe liver cirrhosis. Conclusions drawn from this study were (a) that CYP1A2 is the enzyme that is chiefly responsible for the metabolism of lidocaine in patients with normal liver function, and (b) that there is a reduction in fluvoxamine-lidocaine interaction as liver function gets worse. The latter effect was attributed to the likely decrease in the hepatic level of CYP1A2 accompanying this condition. In an analogous study, the interaction between ciprofloxacin and pentoxifylline was examined, as was the possible role of CYP1A2 in this interaction. Furafylline was employed as a selective CYP1A2 inhibitor here [55]. Other phase I polymorphisms are either relatively common, but not of great importance in drug metabolism, or else rare, but important in the biotransformation of a limited range of drugs. Examples include polymorphisms detected in some esterases (paraoxonase and cholinesterase) [56-58], epoxide hydrolases [59], and dehydrogenases [53]. Examples and details are presented in subsection C. A very recent example describes the characterisation of a new CYTP450 isoform, 4F11, and its role in the metabolism of some endogenous compounds and drugs [60]. Its catalytic properties with respect to endogenous eicosanoids were examined. CYP4F11 was found to have a considerably different profile from that of CYP4F3A and was a better catalyst for many drugs including benzphetamine, ethylmorphine, chlorpromazine, imipramine and erythromycin, the latter being the most efficient substrate. Modelling of the structural homology led to the Impact of gene variability on drug metabolism 283 conclusion that for CYP4F11, a more open access channel exists than in CYP453A, this being a possible reason for its capacity to act on large substrate molecules such as erythromycin. B. Phase II polymorphisms For most commonly prescribed therapeutics, the major phase II-metabolising enzymes are, in general, the UDP-glucuronosyltransferases and the sulphotransferases. Although there is some evidence for the existence of polymorphisms in certain isoforms of both enzyme families, the molecular and genetic basis are not still very well understood. In contrast, two most common polymorphisms in genes encoding some phase II enzymes are well known for N-acetyltransferase 2 (NAT2) and glutathione S-transferase M1 (GSTM1). UDP-glucuronosyltransferases The importance of pharmacogenetic variation in the UDP- glucuronosyltransferases is still not very clear. However, few cases of inter- subject variation in activity in the general population have been reported. Both in Caucasian and Oriental populations, 5% of subjects show very low levels of glucuronide excretion [33]. A particular inborn error of metabolism will be presented in subsection C. A very recent study investigated the possible involvement of two UGT isoforms, 1A9 and 1A8, in metabolism of particular drugs and the possible appearance of drug-drug interactions [61]. Inhibitory properties of a novel gastroprokinetic agent, Z-338, were examined and compared with those of cisapride to assess its potential for drug-drug interactions. In in vitro studies using human liver microsomes, no significant inhibition of terfenadine metabolism or of any of the isoforms of cytochrome P450 (CYP1A1/2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) by Z-338 was evident. It was established that Z-338 was primarily metabolised to its glucuronide by UGT1A9 and UGT1A8, while showing no significant inhibition of P-glycoprotein activity. Cisapride, however, ‘strongly’ inhibited CYP3A4 and ‘markedly’ inhibited CYP2C9. It was further concluded that drug-drug interactions are unlikely to arise upon co-administration of the agent Z-338 with CYP substrates at clinically effective doses. Sulphotransferases These are phase II enzymes conjugating both endogenous and exogenous compounds, thus playing an important role in the biotransformation of a range of compounds. Five isoforms have been identified, but the molecular basis and the pharmacological effects of this variation are still unclear [63]. Nonetheless, the human sulphotransferase family is a complex one, this statement being supported by at least two facts: 284 Chapter 7 - there are two separate genes (STP1 and STP2) encoding proteins, that show 96% homology and appear to be both phenol sulphotransferases [64]; - there is evidence for the existence of allelic variants of each of the phenol sulphotransferases and for the occurrence of two alternative promoters in STP1 [65]. N-Acetyltransferases Acetylation reactions of different chemical groups are catalysed in humans by two N-acetyltransferases, designated as NAT1 and NAT2. Polymorphism has been detected in both of them, with a more significant impact on NAT2. A variation in the ability of certain patients to metabolise different drugs, including isoniazid, sulphamethoxazole, hydralazine, caffeine, nitrazepam, sulphamethazine, procainamide and dapsone, is well known; in addition, the acetylation polymorphism probably is the best-known classic example of a genetic defect in drug metabolism. On the basis of the ability to acetylate these drugs, individuals are classified into two phenotypes, namely ‘slow’ and ‘rapid’ (or ‘extensive’) metabolisers. Family studies established that this ability is determined by two alleles at a single autosomal gene locus. Slow acetylators have a deficiency of hepatic acetyltransferase and are homozygous for a recessive allele [66]. They maintain higher concentrations of un-acetylated drugs for longer periods in body fluids, thus resulting in a greater incidence of adverse drug reactions, due to accumulation of the administered drug (or its phase I metabolites). The precise percentage of slow acetylators in the population varies with ethnic origin: among most European and North American populations, the prevalence of slow acetylators is between 40-70%, whereas, among certain Asian populations, it is only about 10-30% [67]. Thus far, four variant alleles with low activity have been identified; it is assumed that these variations are due to amino acid substitutions, and their frequency varies also between ethnic groups, with NAT2*7A (in which the 857G is exchanged with A) most common among Japanese, and NAT2*14A (where the 191G is exchanged with A), common in individuals of African origin, but not in other ethnic groups [53]. Rapid acetylators presumably have a cytosolic N-acetyltransferase; a second cytosolic N-acetyltransferase, NAT1, has been proven to present selectivity for the metabolism of other types of compounds such as p-aminosalicylic acid and p-aminobenzoic acid, and to be strictly independent of the NAT2 polymorphism. The acetylator polymorphism is important from the standpoints of both clinical responses to drugs and disease susceptibility, affecting both the efficacy and the occurrence of adverse effects for a number of drugs. An interesting fact to note is the observation that the phenotypic expression of NAT2 may be influenced by AIDS [5]; patients afflicted with AIDS have been demonstrated to be slow acetylators, which may offer a Impact of gene variability on drug metabolism 285 good explanation for the high incidence of adverse drug reactions to sulphonamides among these patients. Selected examples will be given in subsection C. Glutathione S-transferases These are phase II enzymes that catalyse the glutathione conjugation of both endogenous and exogenous compounds, generally having a detoxifying action (see Chapter 3). Several polymorphisms have been identified. The most significant and well-characterised GST polymorphisms have been reported for the class µ-enzyme GSTM1 in the class Φ-enzyme GSTT1; there are also reports of polymorphisms in GSTM3, GSTP1 and GSTA2 [6]. The GSTM1 and GSTT1 polymorphisms are of more importance in toxicology than in drug metabolism, with the GSTM1 having a possible role in the metabolism of nitrogen mustard [68,69]. Methyltransferases These enzymes catalyse methylation of both endogenous molecules, such as neurotransmitters, and of xenobiotics, using S-adenosylmethionine as a methyl donor group (see Chapter 3). Methylation may occur at different heteroatoms (S, N, O), and it is assumed that at least four separate enzymes carry out these reactions. However, for relevance to drug therapy, polymorphism has been clearly established only for thiopurine S-methyltransferase. The interindividual differences are significant: approximately 0.3% of Europeans have undetectable activity, while about 11% present intermediate levels [70]. Examples appear in a subsection below. C. Consequences of monogenic variability – selected examples Monogenic variability takes place on a single specific gene and is due either to deletions or point mutations resulting in splicing defects. In the case of CYP2D6, an isoenzyme of particular importance because it metabolises a wide range of commonly prescribed drugs, the most common deficient alleles, giving over 98% of the PMs, are represented by CYP2D6*3, *4, *5 and *6. In the *4 allele for example, a guanine is replaced by an adenine at the 3’-ending of intron 3. In the 5* allele, there is a complete deletion of the gene. PMs show, as already mentioned, higher plasma levels of several drugs, which consequently puts them at increased risk of adverse reactions. However, this also depends very much on the particular drug in question and the overall contribution of CYP2D6 to its metabolism. Mutant alleles of the CYP2D6 gene related to the PM phenotype have been studied in numerous laboratories over the last 20 years. 286 Chapter 7 These studies identified the primary mutations (some of them already mentioned) that cause either null alleles or decreased-function alleles, resulting either in total loss of activity, or partial decrease in enzyme function. Knowing that for some of the numerous substrates of this cytochrome isoform, polymorphic oxidation may have important therapeutic consequences, it is obvious that for these specific drugs knowledge of the phenotype could be of utmost help in individualising the dose range required for optimal therapy. For example, pronounced differences in plasma half-life and metabolic clearance have been reported between EM and PM individuals in the biotransformation of flecainide [71]. The implications are that in the case of PMs the plasma steady-state concentrations are achieved only after 4 days of therapy, whereas in

the case of EMs, the required time is halved. Moreover, PMs with impaired renal function will be at greater risk of developing flecainide toxicity because of the decreased renal clearance, resulting in potentially dangerous accumulation of the drug. In the case of CYP2C19, two new allelic variants that contribute to the PM phenotype in Caucasians have been isolated: CYP2C19*2 and CYP2C19*3. The more common, accounting for about 80% of mutant alleles both in Europeans and Orientals is the first mentioned, namely, the CYP2C19*2. Both inactivating mutations are single-base-pair substitutions, with aberrant splice site created in CYP2C19*2 and a premature stop codon in CYP2C19*3. The most important consequence (already mentioned above) is the predisposition of the PM phenotype individuals to CNS adverse effects after administration of even a single 100 mg dose of mephenytoin. CYP2C9 is another important drug-metabolising enzyme, with a large number of widely used drug substrates. Several single-base-pair substitutions that obviously result in amino acid changes account for the CYP2C9 polymorphism. The most common are Arg144Cys (CYP2C9*2) and Ile359Leu (CYP2C9*3) [72]. A particular example is that of tolbutamide biotransformation in homozygous for the recessive Leu allele (CYP2C9*3), who are PMs [73]. However, perhaps some of the most important and clinically relevant examples involving the existence of polymorphism are reflected by the N-acetylation polymorphism. For example, PMs of isoniazid are more likely to accumulate the drug to toxic concentrations and so are at risk of developing peripheral neuropathy [74]. In contrast, EMs might have to be given unusually high doses to attain efficacy. An important issue in this context is the increased occurrence of severe phenytoin toxicity in PMs of isoniazid, when both drugs are given simultaneously. The assumed mechanism is a non-competitive inhibition of the p-hydroxylation of phenytoin displayed by isoniazid [75]. A relatively recent example involves paraoxonase (PON1), a Ca2+- dependent glycoprotein that is associated with high-density lipoprotein Impact of gene variability on drug metabolism 287 (HDL). Two genetic polymorphisms, determined by punctual amino acid substitutions at positions 55 and 192, have been reported. The major determinant of the PON1 activity polymorphism is assumed to be the position 192 [76]. One of the most important consequences of PON1 polymorphisms is that they are important in determining the capacity of HDL to protect low-density lipoproteins against oxidative modifications, which may explain the relation between the PON1 alleles and coronary heart disease. In the same context, by protecting lipoproteins against oxidative modifications (most probably by hydrolysing phospholipid hydroperoxides), PON1 may also be a determinant of resistance to the development of atherosclerosis. Finally, PON1 polymorphs, by hydrolysing organophosphate insecticides may be responsible for determining the selective toxicity of these compounds in mammals. 7.3 PHARMACO-INFORMATICS An active biological compound introduced into the body will generate a sequence of events. According to the informational causality laws (principles), it has been proven that both the desired therapeutic effects and the adverse reactions are of informational nature. It was definitively revealed that a specific drug may be toxic not necessarily due to the dose, rhythm of administration, variations in the metabolism profile determined especially by the genetic polymorphism of the enzymatic systems involved, but precisely because of the information transmitted, especially in correlation with the receptor substrate and the whole body. It is assumed that the impact of the pharmacological information depends not only on the quality and quantity of the signal, but especially on the significance conferred by a specific type or subtype of receptor system. Thus, from the essential characteristics of the receptors involved, we can mention: - selectivity (meaning the strict preference for a specific molecular type of ligand); - saturability (given by the identical number of sites in the receptor molecule which can attach the ligand); - the cellular location (the receptor being generally located on the cells that will generate the biological response). Consequently, a rational conception of a new therapeutic entity should primarily take into account the three-dimensional structural details of the receptor. Nowadays, there are several more or less routine procedures, such as X-ray diffraction, NMR spectroscopy and MS that can elucidate such structure. In a subsequent stage, computational techniques are used for the 288 Chapter 7 theoretical evaluation of possible interactions between these receptors and ligands capable of eliciting useful responses. The most important conclusion from the above brief considerations is that a medicinal substance represents nothing outside the body. The drug in question may become a pharmacological signal only if its chemical structure allows its integration into the network of the receptor substrates of the body [77]. 7.4 IMPLICATIONS FOR THIRD MILLENNIUM MEDICINE The main conclusion that arises from all the above considerations is that both phenotyping and genotyping techniques can nowadays be successfully applied by the clinical laboratory for linking human genetics to therapeutic treatment. In the last few years, through the methods of molecular genetics, and more recently with the discovery of the human genome map, many clinical observations concerning the therapeutic response to a particular treatment, the incidence of adverse reactions, or the toxicity of different drugs or their metabolites, can now be understood at the molecular level. This could be very helpful in more effective prescribing, particularly for compounds with narrow therapeutic index, so that the ratio of therapeutic effect/toxic risk may be increased to the benefit of the patient. Genotyping in particular could represent a good alternative to predicting the appearance of some undesirable secondary effects (or even of some pathologies following a treatment), allowing in this way the selection of the most efficient drug for the specified profile, in other words, the individualisation of the treatment. In fact, the announced perspective for the third millennium is precisely such ‘personalised medicine’, possibly through creation of an ‘ID genetic card’ for every patient, completed even in the first years of life; such a contingency would assist medical personnel to find the formula for indicating a therapeutic treatment as close as possible to the ideal i.e. one which is both efficient and devoid of adverse effects. Finally, we should stress that both pharmacogenetics and pharmacogenomics have become rapidly emerging fields with implications not only for efficient and safe drug therapy, but also for drug discovery and development and for the assessment of the risk for developing certain diseases. It should be borne in mind too that in the future, physicians should be more aware of these inherited variations of drug responsiveness, because as already highlighted, they are a constant factor throughout a patient’s life. Impact of gene variability on drug metabolism 289 Under the circumstances, new diagnostic procedures should be developed and appropriate dosage adjustments carefully made. In the next (penultimate) chapter of this book, two special topics with significant clinical implications are considered, namely drug-drug interactions and adverse reactions. 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Diphenylhydantoin intoxication: a complication of isoniazid therapy. Am Rev Respir Dis 101:377-384. 76. Mackness B, Durrington PN, Mackness MI. 1998. Human serum paraoxonase. Gen Pharmacol 31:329-336. 77. Hiroyuki O. 2002. Pharmaco informatics. Farumashia 38:120-124. Chapter 8 DRUG INTERACTIONS AND ADVERSE REACTIONS 8.1 INTRODUCTION Two principal aspects of drug metabolism are addressed in this chapter, namely drug-drug interactions and adverse reactions. Since drug-drug interactions can occur at various stages following drug administration, these are systematically subdivided into interactions associated with the pharmacodynamic phase, pharmacokinetic interactions, and interactions occurring during the biotransformation phase. Known interactions between drugs and food, alcohol and tobacco smoke are treated separately. A special feature of the present chapter is an extensive tabulation of drug-drug interactions which serves as a useful reference to those occurring most frequently, together with their biological consequences. In the treatment of adverse reactions that follows, these are first defined and an attempt to classify them according to various criteria is presented. A significant emphasis is given to allergic reactions and associated toxicity in the extensive discussion that follows. The latter is supported by a wide range of examples. Finally, a brief outline of some of the modern approaches to predicting drug metabolism is presented. 8.2 DRUG-DRUG INTERACTIONS 8.2.1 Definitions, concepts, general aspects Today, with the increasing complexity of therapeutic agents available, and widespread polypharmacy (a particular problem especially in the elderly, who receive more medications than younger individuals), the potential for drug interactions is enormous. Drugs can interact to alter the absorption, 295 296 Chapter 8 distribution, metabolism or excretion of a drug, or interact in a synergistic or antagonistic fashion altering their pharmacodynamics. Generally, the outcome of an interaction can be harmful, beneficial or clinically insignificant. Although clinically often unrecognized, many of the drug interactions are responsible for increased morbidity. Drug interactions are of utmost importance in clinical practice, since they account for 6-30% of all adverse reactions (ADRs). In some cases, drug interactions can be useful, and it is already a relatively current practice for prescribers to use known interactions to enhance efficacy in the treatment of several conditions such as epilepsy, hypertension or cancer [1]. An example illustrating beneficial effects rather than ADRs, involves the co- administration of carbidopa (an extracerebral dopadecarboxylase inhibitor), together with levodopa to prevent its peripheral degradation to dopamine [2]. On the other hand, association of theophylline with ciprofloxacin, for instance, causes a two- to threefold increase in theophylline serum level, resulting in theophylline toxicity [3]. A drug interaction is a measurable modification in magnitude or duration of the pharmacological response of one drug, due to the presence of another drug that is pre- or co-administered. Many drug interactions involve an effect of one drug on the action or disposition of another, with no recognizable reciprocal effects [4]. Usually, this modification of the action of one drug by another is a result of one or more of four principal mechanisms: a) pharmaceutical, b) pharmacodynamic, c) pharmacokinetic, and d) metabolic [5]. It should be stressed that usually the term ‘drug interactions’ refers to drug-drug interactions, although it can be taken to include interactions between drugs and food constituents, alcohol, or environmental factors. In addition, the term may include even interferences by drugs in clinical laboratory tests, with important consequences for diagnoses [6]. Drugs may also interact with diseases, potentially worsening their symptoms [7]. A definition with important implications was given a decade ago by Thomas [8], according to which a drug interaction is “considered to occur when the effects of giving two or more drugs are qualitatively and quantitatively different from the simple sum of the observed effects when the same doses of the same drugs are given separately”. The implications mentioned above may involve different aspects: either increased or decreased activity of two drugs given concurrently (in a purely quantitative manner), qualitative change in the effect of a drug, antagonism of the effects of one drug by another, resulting in annulment of beneficial effects of therapy, and potentiation of an unwanted effect. Especially for this last possible effect, as has often been observed, patients are in many cases exposed to unnecessary risk, by pre- or co-administration of therapeutic agents that are assumed to interact adversely. Fortunately, many interactions Drug interactions and adverse reactions 297 are predictable, and avoidance of unwanted effects or therapeutic ineffectiveness is thus possible [9,10]. Therefore, a priority of the clinical pharmacist today, to increase the likelihood of identifying or preventing an adverse drug reaction, primarily involves knowing or predicting those situations in which a potential drug interaction is likely to have clinically significant consequences, recognizing these clinical settings, and understanding the mechanisms by which they occur. In this case it is important either to recommend steps that may be taken to avoid them (e.g. altering sequence of administration and time interval between administration of two drugs), or preferably alternative treatments. 8.2.2 Interactions associated with the pharmacodynamic phase Pharmacodynamic interactions, assumed to be the most common drug interactions in clinical practice, are those for which the effects of one drug are altered by the presence of another drug at its site of action [3]. At the same time, some of the most clinically important ADRs also result from pharmacodynamic interactions [6]. Most of these interactions have a simple mechanism, consisting either of summation or opposition of the effects, and therefore being either synergistic or antagonistic. Most of these interactions are intuitively evident; thus, it is not surprising for instance, that two drugs with sedative properties (e.g. alcohol and benzodiazepines) can potentiate each other’s sedative action. Other synergistic interactions

include those between a diverse range of drugs, such as tetracyclines, clofibrate, and estrogens, with warfarin, leading to increased anticoagulation [6]. When two drugs concomitantly administered share similar adverse effects, their association can produce additive side effects. For example, hydrocortisone and hydrochlorothiazide together can produce additive side effects of hyperglycaemia or hypokalemia [6,8]. Another example is the increased risk of bleeding in anticoagulant patients taking salicylates [3]. In some instances, important interactions may occur between drugs acting at a common receptor. When used deliberately, many of these interactions can generally be useful. For example, we may mention the use of naloxone to reverse opiate intoxication. Less directly, by the local increase in acetylcholine (caused by cholinesterase inhibition), muscular relaxation by tubocurarine can be reversed [4]. The antagonistic interactions can be partial, when the global antagonistic effect is smaller than that of the sum of the individuals, or total, when the global effect is null [12]. A well-known example is that of the antiparkinsonian levodopa, whose action can be antagonised by certain 298 Chapter 8 dopamine-blocking drugs, such as haloperidol and chlorpropamide [11]. Usually, such interactions are due to direct effects at the receptors (the same or different), but more often can also occur by indirect mechanisms, due either to an interplay of receptor effects or combined interferences with biochemical or physiological mechanisms [2-6]. If generally, both drugs compete directly for the same receptor, it has to be stressed that often such interactions involve a more complex interference with physiological mechanisms [11]. Often, through a pharmacodynamic mechanism, the risk of certain toxic effects can be potentiated. A general example is that of diuretic- induced hypokalemia and hypomagnesemia that may act to increase the risk of dysrhythmias caused by digoxin [5,8,11]. Besides the additive (or synergistic) and/or antagonistic interactions, in the category of pharmacodynamic interactions, could be also included interactions due to changes in drug transport mechanisms [2,11]. For example, the tricyclic antidepressants are able to potentiate the action of epinephrine and norepinephrine through their blocking the neuronal re-uptake of amines. In contrast, the antihypertensive effects of certain adrenergic neurone blocking drugs (e.g. debrisoquine, bethanidine) are prevented or even reversed by tricyclic antidepressants, most probably by the same mechanism as above. Other drugs, such as aminoglycosides and especially, local anaesthetics, may exert weak inhibiting effects on neuromuscular transmission. In patients with normal neuromuscular transmission, the effect is more or less evident, unlike in those who received e.g. neuromuscular blocking drugs, or patients with myasthenia gravis. In such circumstances, these drugs may produce apnoea or even neuromuscular paralysis. Also noteworthy are the indirect pharmacodynamic interactions, several of them with potential clinical significance [2,6]. Well-known examples involve co-administration of aspirin and NSAIDs with anticoagulants, such as warfarin. Because these drugs are known to cause gastrointestinal lesions, including ulcerations, it is obvious that such concomitant administration may provide a focus for bleeding. In co- administration with anticoagulants, salicylates may also lead to enhanced tendency to bleeding, through inhibition of platelet aggregation. Another interesting pharmacodynamic interaction through an indirect mechanism, worth mentioning in this context, is that between propranolol and glycogen: propranolol reduces the breakdown of glycogen (the major energy- storage polysaccharide in mammals), subsequently delaying the elevation in blood glucose levels after hypoglycaemia. Finally, it is necessary to include here the situation when pharmacodynamic interactions involve unknown and multiple mechanisms, Drug interactions and adverse reactions 299 for instance by involving different sites of action, or by inhibiting the P-glycoprotein efflux transporter [11]. To illustrate how pharmacodynamic drug interactions may arise, some examples in more detail follow. • NSAIDs and corticosteroids: both are known to cause gastrointestinal irritation, subsequently leading to bleeding and ulceration. Under these circumstances, it is easily predicted that in association, the incidence and risk of bleeding will rise significantly (presumably due to a simple additive effect). Therefore, during concomitant use, close monitoring of patients is strongly recommended. Ultimately, concomitant treatment for gastrointestinal damage should be advisable [11,12]. • NSAIDs, loop diuretics and antihypertensive agents: reducing renal sodium excretion, NSAIDs increase renal prostaglandins that accompany administration of certain diuretics, such as furosemide. Through the same mechanism, the antihypertensive efficacy of ACE inhibitors and ȕ-adreno receptor blockers may be reduced as well [2,5]. • neuromuscular blockers and/or aminoglycoside antibiotics and anaesthetics: • the aminoglycoside antibiotics are known to display, as an additional pharmacological action, that of potent neuromuscular blockers; therefore, their concomitant administration with neuromuscular blockers may lead to prolonged, or even fatal respiratory depression. It is assumed that these effects are additive to the conventional neuromuscular blockers that act on the post-synaptic membrane. Consequently, concurrent use must be avoided. In the case of anaesthetics that may cause prolonged neuromuscular blockade, it is recommended that the postoperative period be closely monitored [3,8,11,12]. • phenothiazines and antihypertensives: certain phenothiazines, such as promazine and chlorpromazine, have been shown to cause postural hypotension. Under these circumstances, if the patient is also taking an antihypertensive drug, the reaction may be exaggerated. Such cases have been reported following co-administration of a phenothiazine with various antihypertensive agents, including captopril, nadolol, clonidine and nifedipine [3,12]. • corticosteroids and digitalis glycosides: administered systemically, corticosteroids (particularly cortisone, deoxycortone and hydrocortisone, occurring naturally as well) have been proven to increase potassium loss, concomitant with sodium and water retention. Subsequently, oedema and hypertension result, which can lead to cardiac failure in some individuals. Under these circumstances, if these drugs are co-administered with digitalis glycosides, it is advisable to monitor the patient well [11]. 300 Chapter 8 8.2.3 Pharmacokinetic interactions: incidence and prediction Pharmacokinetic drug interactions can occur during any of the processes assumed to represent the fate of a drug in the body and contributing to the drug’s pharmacokinetic profile. The positive aspect is that for a new drug candidate, thorough preliminary studies can be undertaken before it appears on the market. This would confirm either the presence or absence of possible pharmacokinetic interactions that such a drug could cause. Such interactions may affect: a) absorption of orally administered drugs, through different mechanisms such as: chemical interactions (chelation and complexation), alteration of gastrointestinal motility, changes in gastrointestinal pH, perturbation of gastrointestinal flora; b) distribution; c) drug metabolism (epecially through enzyme induction or inhibition effects, discussed separately in subchapter 8.2.4); and d) excretion [2,3,5,11]. a) Absorption Generally, as already outlined in Chapter 1, the process of absorption from the gastrointestinal tract in the case of orally administered drugs is variable and complex. Consequently, drug interactions of this type are difficult to predict. However, the significant advances made in this area, especially during the last decade, permit (through mathematical approaches to prediction based on competitive enzyme inhibition) an early assessment of potential drug-drug interactions in patients that are taking concurrent medications [3,9,10]. Most of these interactions refer to the rate of absorption, although, in some instances the extent of absorption may be affected as well. Changes in the rate of absorption – in most cases, delay of the process, can be of real clinical significance when referring either to drugs having a short half-life, or when achievement of rapid and high plasma levels may be critical (as may be the case with analgesics or hypnotics). Usually, this phenomenon is expected to appear if inappropriate combinations are administered without sufficient separation in time; therefore, most of these interactions can be avoided by simply allowing a two or three hour interval between the administration of the interacting drugs [3,6,12]. The mechanisms of generating such interactions are various: • certain drugs given orally can sometimes react directly within the gastrointestinal tract, leading either to chelates or complexes, forms which are not readily absorbed. Examples include: - interaction of tetracyclines or fluoroquinolone antibiotics with metal ions (e.g. aluminium and magnesium in antacids, or iron salts), resulting in reduced drug absorption due to formation of a chelate Drug interactions and adverse reactions 301 complex within the gut; this chelation with divalent or trivalent ions, leading to insoluble complexes, may result in severely reduced plasma levels of the administered drugs and thus, therapeutic inefficacy. - interaction of digoxin, warfarin or thyroxine with cholestyramine and related anion exchange resins, with the same consequence of reduced absorption due to binding/complexation in the gut (in fact, the adsorption of the former onto cholestyramine). Nevertheless, such effects may sometimes be used to therapeutic advantage: - activated charcoal, which acts as an adsorbent agent within the gut (although it can affect the absorption of certain drugs), may be used with good efficacy in the management of poisoning; - cholestyramine and related anion exchange resins, binding cholesterol metabolites and bile acids, prevent their re-absorption in the intestinal lumen, thus lowering plasma levels of total cholesterol. • altering the rate of gastric emptying is assumed to generally alter the rate of drug absorption as well; drugs that retard gastric emptying may delay or attenuate the rate of absorption of other co-administered drugs. For example, drugs with anticholinergic effects (anticholinergic agents, antihistamines, and phenothiazines), tricyclic antidepressants, and opioids, that decrease the rate of gastric emptying will consequently increase the necessary time to achieve the therapeutic plasma levels of drugs administered concurrently. In some instances, bioavailability of the affected agent may be reduced as well [2]. On the other hand, attention is drawn to drugs that increase the rate of gastric emptying, resulting in an accelerated absorption of certain co-administered drugs. For example, metoclopramide has been shown to accelerate the absorption of diazepam, propranolol, paracetamol, and conversely, to reduce that of digoxin. Other drugs that enhance gastric emptying include cisapride and domperidone, and as a consequence of their effects, may cause earlier and higher peak concentrations, which could be dangerous especially in the case of index drugs. The rate of gastric emptying is especially important when a rapid onset of effect of the drug is desired: rapid relief pain or onset of sedation, and in instances where parenteral administration is not feasible. Among the factors that slow gastric emptying, apart from the concurrently administered drugs already referred to above, we should also mention food, heavy exercise, and autonomic neuropathy [3]. • changes in bacterial flora, generally caused by broad-spectrum antibiotics, may affect the absorption of any drugs subject to metabolism by bacterial enzymes. As a clinically relevant example (although the mechanism has not been fully elucidated), we should mention the reduction in oestrogen levels resulting from diminished bacterial flora that results in an increased risk of contraceptive failure [2]. 302 Chapter 8 • changes in gastrointestinal pH. As already discussed in Chapter 1, the gastrointestinal mucosa having an essentially lipid-based structure, drugs will usually pass through them by simple diffusion, if they are in a lipid- soluble form. However, it is known that drugs vary in their lipid solubility, and many of them may act as weak acids or bases; in the latter case, a proportion of the dose exists in dissolved (ionised) form, some still remaining unionised, in a dynamic equilibrium. Therefore, in such circumstances, changes in gastric pH can affect the solubility and absorption of ionisable drugs, shifting the balance of this equilibrium very significantly. Drugs such H2 antagonists, proton pump inhibitors and antacids, by increasing gastric pH, will markedly reduce the bioavailability of certain drugs such as ketoconazole, for example, which requires an acidic medium for adequate absorption. Some more detailed examples follow: • digoxin and metoclopramide: on concurrent administration, the serum levels of digoxin have been shown to be reduced by about a third [3]. Apparently, metoclopramide increases the mobility of the gut to such an extent, that both full dissolution and absorption of digoxin remain incomplete by the time it is eliminated in the faeces. Under these circumstances, two options exist: either to increase the digoxin dose, which would not be advisable since digoxin is an index drug, or to administer them with a sufficient time interval between doses. On the other hand, propantheline seems to exert quite the opposite effect, increasing digoxin plasma levels, through reduction in gut motility. In either case, the patient could be placed outside the desired range for plasma levels, either for obtaining the expected therapeutic effect, or instead being subject to increased risk of toxic effects [12]. Since digoxin has a narrow therapeutic index, its levels require very close monitoring. • ketoconazole and antacids, H2 blockers and proton pump inhibitors: for adequate absorption, ketoconazole, being a poorly soluble base

needs to be converted into a soluble salt; usually, this is mediated by the acid in the stomach, resulting in the corresponding hydrochloride salt. In this situation, it is obvious that co-administration of antacids (which raise the pH in the stomach), or H2 blockers, agents that reduce gastric acid secretion, will cause a reduction in both the dissolution and absorption of ketoconazole. Clinical observations confirmed dramatic reductions in ketoconazole plasma levels upon its co-administration with ranitidine or cimetidine [2]. For managing this interaction, two methods may be suggested: either to administer ketoconazole when the stomach contents are most acidic, or to ensure a suitable temporal separation between ketoconazole and H2 blockers or antacids. In both situations, to ensure ketoconazole efficacy, it is advisable to monitor the effects of treatment. Drug interactions and adverse reactions 303 • regarding fluoroquinolone antibiotics and divalent/trivalent metallic ions: on concurrent administration with antacids containing calcium, magnesium or aluminium, clinical observations indicated reduced absorption of these antibiotics, reflected in their reduced plasma levels. The most probable mechanism involves the interaction of certain functional groups on the antibiotics with the metallic di- or trivalent ions, forming insoluble chelates within the gut, that are not absorbed to any great extent, and in addition, appear to be relatively inactive as antibacterials. Iti is appropriate to mention here that a relatively new product, iron- ovotransferrin, through its ability to combine directly with the transferrin receptors of intestinal cells, will consequently release little ionic iron into the gut. This would presumably reduce the incidence of combination with quinolones, as was confirmed for iron-ovotransferrin on co-administration with ciprofloxacin [3]. b) Distribution The major distributional process that may contribute to drug interactions is binding to plasma proteins [13]. Following absorption, and after passing through the liver, a drug reaches the systemic circulation and is distributed throughout the body, including its site of action. This phase of distribution depends on several factors, including the ionic composition, lipid-solubility, and protein-binding characteristics of the drug. Protein binding may refer to either plasma albumin binding, or, outside the bloodstream, to tissue proteins, and directly influences the pharmacokinetics of a drug. It is well known that only free drug can exert a pharmacologic effect. Drugs that are generally highly bound to plasma proteins are also potentially subject to displacement from their specific carrier proteins by a concurrently administered drug that might display a higher affinity for the same protein. Such a displacement interaction, involving reduction in the extent of plasma protein binding of one drug by the presence of a co-administered one, consequently results in an increased unbound fraction of the displaced drug [2,3,8,13,14]. The unbound (i.e. free in solution) molecules are pharmacologically active, while the bound ones form a circulating, but pharmacologically inactive reservoir. Since the two forms exist in a dynamic equilibrium, biotransformation and excretion of free, active molecules, results in their immediate replacement by molecules from the inactive reservoir. There are several examples of clinically important interactions that are attributed entirely to protein-binding displacement, the most frequently cited example probably being that between warfarin and NSAIDs. The anticoagulant effect of warfarin is potentiated in co-administration with different NSAIDs, most probably because of displacement of the former from its protein-binding sites [2]. Another example is the marked diuresis 304 Chapter 8 observed in patients with nephrotic syndrome when they were given clofibrate [3]. c) Interactions due to altered biotransformations (see following subchapter) d) Excretion (elimination interactions) The renal excretion of drugs (or their metabolites) may be affected by a co-administered drug in various ways [1-3,6,11]. A change in glomerular filtration rate, tubular secretion or urinary pH can alter the elimination of some drugs. Selected examples: • some acidic drugs lower the urinary pH, whereas some antacids (or bases) cause an increase in the pH of urine. Therefore, the excretion of other ionisable compounds that display appreciable renal clearance is expected to be influenced in some way. Aciduria, for example, will increase the renal clearance of certain basic drugs, such as amphetamine, antihistamines and tricyclic antidepressants. Conversely, for acidic drugs, including salicylic acid, phenobarbital, and nitrofurantoin, the renal clearance will increase with increasing urine pH. • many drugs share a common transport mechanism in the proximal tubules, and consequently can reduce one another’s excretion by competition. In practice, the clearance of drugs actively secreted into the tubular lumen can be significantly inhibited by other drugs. Examples include the reduction in renal clearances of penicillins and indomethacin by co-administration of probenecid, and of methothrexate by salicylates and NSAIDs. However, it is important to stress that in certain situations this type of interaction can be used to advantage: for example, by decreasing the clearance of penicillin, probenecid actually prolongs its duration of action. On the other hand, the opposite consequence may be reported as well: methotrexate toxicity can be caused by inhibition of its tubular secretion, by some of the drugs mentioned above. • of course, diuretics are certainly expected to exert such effects. These compounds reduce sodium absorption, a phenomenon leading indirectly to increased proximal tubular re-absorption of monovalent cations. In certain instances, this increased re-absorption can cause accumulation and potentially fatal toxicity (e.g. in patients treated with lithium salts) [5]. • digoxin excretion can be reduced by several drugs including amiodarone, quinidine, spironolactone and verapamil, and this will increase its toxicity. Drug interactions and adverse reactions 305 8.2.4 Interaction during the biotransformation phase Because of significant inter-patient variation, the biotransformation of one drug can be dramatically affected by other pre- or co-administered drugs. Actually, it is assumed that most clinically important drug-drug interactions result from perturbations of drug metabolism, involving either induction or inhibition of metabolising enzymes. When two drugs are involved with the same range of enzymes, this can lead to changes in the extent of metabolism of either or both, either increasing or decreasing, with consequent changes in plasma levels. a) Enzyme inhibition Decreasing enzyme activity, which is an extremely common mechanism underlying the interaction of two drugs, often results in high drug plasma concentrations, exaggerated and prolonged responses, and subsequently, an increased risk of toxicity [1-3,6,10-12,14]. The direct consequences of inhibitory interactions may be more severe than those from induction, which often lead to only diminished efficacy. Clinically significant interactions of this type generally involve the most common enzyme system, namely the hepatic microsomal mixed function oxidases, the most representative being the cytochrome P450 isozymes. Several different mechanisms mediate inhibition-based interactions. Among these, probably the most common and significant is substrate competitive inhibition. Other recognized mechanisms include interference with drug transport, alteration of the conformation (or expression) of the P450 enzyme, as well as interfering either with the energy or cofactor supply [6]. Sometimes, competition can even result in irreversible inactivation, a mechanism that leads to the most enduring effects [3]. Drugs that are able to inhibit the MMFOs, by competitive binding to cytochrome P450, usually form a stable complex with it, which obviously will prevent access of other agents to the P450 enzyme system [2,11,12,14]. Drugs commonly involved in such types of interactions (due to enzyme inhibition) include amiodarone, azapropazone, chloramphenicol, cimetidine, ciprofloxacin, diltiazem, disulfiram, enoxacyn, erythromycin, ethanol, fluconazole, fluoxetine, fluvoxamine, isoniazid, itraconazole, ketoconazole, metronidazole, miconazole, nefazodone, omeprazole, oral contraceptives, paroxetine, phenylbutazone, propoxyphene, quinidine, sulphinpyrazone, sulphonamides, valproate and verapamil. The clinical significance of this type of interaction depends on various factors; these refer either to the drugs involved (e.g. dosage, alteration in pharmacokinetic properties of the affected drug), or to patient characteristics, 306 Chapter 8 such as disease state. Interactions of this type are again most likely to affect drugs with a narrow thepeutic range. Representative examples: • the association of cimetidine or ciprofloxacin – both enzyme inhibitors, with theophylline, which could result in a doubling in plasma concentration of the latter [2,3,8,9,12]. • a severe interaction occurred following co-administration of the enzyme inhibitors erythromycin, ketoconazole and terfenadine, as first described by Honig et al. [15]. Further studies demonstrated that terfenadine is converted by a specific P450 enzyme, namely CYP3A4, to an active metabolite. On the other hand, ketoconazole being a potent inhibitor of CYP3A4 isoform, on concurrent administration with terfenadine will dramatically reduce the latter’s metabolism, resulting in increased concentration of the parent drug; in this situation a quinidine-like action may result, leading to ventricular arrhythmias and prolongation of QT interval [2,12]. • an interesting example that involves a stereoselective inhibition is the association warfarin/enoxacyn [12]. Warfarin exists in two enantiomeric forms, (R)-warfarin and (S)-warfarin, the (S)-enantiomer being more active then the (R)-enantiomer. In humans, the (S)-enantiomer is almost totally eliminated as the (S)-7-hydroxylated-metabolite, while the (R)-enantiomer is predominantly biotransformed to the (R)-6-hydroxylated metabolite. Co-administration of enoxacyn inhibits metabolism of the less potent (R)-enantiomer, causing a reduction in its clearance. On the other hand, the co-administration of phenylbutazone inhibits the metabolism of the more potent (S)-warfarin predominantly, resulting in a greater proportion of it in plasma, and subsequently, in increased anticoagulant effects (the anticoagulant potency of the (S)-enantiomer being five times greater than that of the (R)-enantiomer) [2]. In the same context, though with less significant clinical consequences, we should mention the association warfarin/cimetidine. This is also a stereoselective inhibition involving the (R)-enantiomer. This form is, however, less active then the other enantiomer, so it is assumed that interaction will produce only a weak effect upon the anticoagulant effect of warfarin [12]. • indinavir and ketoconazole: in vitro studies on rat hepatic microsomes indicated that ketoconazole inhibits the biotransformation of indinavir by a competitive mechanism, with a Ki value of about 2.5µM. As a result, on pre-administration of ketoconazole, both the bioavailability and AUC value of indinavir increased significantly [12]. • fluoxetine and imipramine: both being co-substrates for the same P450 isoform, CYP2D6, on co-administration of fluoxetine, the plasma Drug interactions and adverse reactions 307 concentration of imipramine increases several fold, due to the same competitive inhibitory mechanism, as above [12]. • terfenadine and erythromycin: similar mechanism of action as above; terfenadine is metabolised by participation of another P450 isoform, namely, CYP3A4. On concurrent administration of erythromycin, the plasmatic levels of terfenadine increase, both drugs being co-substrates for the same enzyme isoform. b) Enzyme induction The phenomenon of induction of cytochrome P450, as a mediator of metabolic drug interactions, has also been recognized for sometime[1-3, 6, 8,11,14]. Enzyme induction may occur by a number of different mechanisms, but generally results in increased amounts of enzyme, and thus, in increased rate of biotransformation reaction [16]. In general, two major consequences arise with induction-based interactions: either increased metabolic clearance, leading to reduced therapeutic efficacy, or the opposite, namely metabolic activation, yielding a toxic metabolite, resulting in increased toxicity. As an example we quote the increasing risk of acetaminophen-induced hepatotoxicity on co-administration of isoniazid, due to an increase in the formation of the toxic metabolite of the former [17]. It is useful to note that the phenomenon of enzyme induction primarily affects phase I metabolism, although there is evidence that some phase II reactions may also be affected [2]. The effects of enzyme induction vary considerably between individuals, depending on various factors such as age, concurrent drug treatment, genetic factors and disease state. Enzyme induction is generally dose-dependent and represents the process of temporary adaptative increase of a specific enzyme concentration. The process is essentially attributable either to the increase in the rate at which the enzyme is synthesised, or to a decrease in its degradation rate. The enzyme inducers encountered most commonly in clinical practice include barbiturates, carbamazepine, griseofulvin, phenytoin and rifampicin. Some of the best recognized examples and most widely studied drug interactions of this type include: • warfarin and phenobarbital: an interaction that is well-documented and often cited. Phenobarbital is known as a potent inducer of many P450 isoforms, including those involved in warfarin’s biotransformation. As a consequence of enzyme induction, the plasma levels of warfarin will decrease; in order to maintain the therapeutic effect, a substantial increase in the therapeutic dose will be needed. Under these circumstances, close monitoring of the patient is strongly recommended [12]. 308 Chapter 8 • antiepileptic drugs, frequently administered in combination: some combinations involve true interactions by reciprocal effects [18], and some of the consequences are therefore quite complex. Close monitoring of plasma levels of co-administered drugs, should however enable the consequences of these interactions to be recognized and, if not avoidable, at least

minimised. • nelfinavir and rifampin: both are used in HIV-patients. Nelfinavir, a non-nucleoside reverse transcriptase inhibitor, is partially metabolised by the P450 isoenzyme CYP3A. The antitubercular drug rifampin is a very potent inducer of this isoform, consequently increasing nelfinavir’s biotransformation, which results in a greater clearance from the body. The AUC is dramatically reduced (by about 80%) and avoidance of this combination is therefore strongly recommended [3]. An alternative could be the co-administration of rifabutin, also an enzyme inducer, but far less potent than rifampin. The AUC of nelfinavir in this case is reduced by about 30% only. Other inducers of the isoform CYP3A, such as carbamazepine, phenobarbital, and phenytoin are expected to produce similar reduction phenomena, and such combinations are best avoided. 8.2.5 Other selected, miscellaneous recent examples The constant interest in possibly undesirable effects that might arise from drug-drug interactions is reflected in the numerous studies and clinical observations that aim to reveal, predict and minimise such effects. Under the circumstances, attention has focused on new possible co-administrations, the potential interactions, and consequences of therapeutic or toxicological significance [19]. Examples of these follow: • a recent article reviewed pharmacokinetic herb-drug interactions, taking in account that in recent years, the number of such interactions has increased [20]. Assuming that most herbal medicines have a broad therapeutic range, in order to identify, and predict such interactions in practice, systematic in vitro screenings as well as more clinical studies have been proposed. • some interactions between statins and various drugs or even foods have also been reviewed recently [21]. Statins, medicines currently used for the treatment of hyperlipidemias, competitively inhibit HMG CoA reductase – an enzyme found in the liver; statins also display affinities for various P450 isoenzymes (CYP3A4, 2C8, 2C9). Because of this, they might be expected to be involved in metabolism-type drug interactions, and more. Recent studies confirm that all statins are absorbed orally, so the impact of food present in the stomach could be extremely important in achieving the Drug interactions and adverse reactions 309 desired therapeutic effect. Usually, an individualization of the treatment is recommended to avoid interactions and generally to improve this form of treatment of hyperlipidemia. A limited number of clinical observations showed that the anticoagulant effects of warfarin can be increased in some patients on concurrent administration of lovastatin. The asumed mechanism is one of enzyme inhibition, resulting in increased anticoagulant effects of warfarin, with subsequent bleeding and increased prothrombin times reported. Itraconazole, a potent inhibitor of the CYP3A4 isoform, acts more predictably. Some of the statins commercially available and in therapeutic use, such as lovastatin and simvastatin, are metabolised by CYP3A4; on co-administration of itraconazole, the serum levels of the former are dramatically increased due to itraconazole’s enzyme inhibiting action. The most common recommendation in the case of co-administration is dosage reduction if either is given concurrently with itraconazole [3]. • an interesting study concerned possible pharmacokinetic and pharmacodynamic interactions of drugs for internal diseases, such as analgesics, antiallergics antibiotics, anticoagulants, anticonvulsants, antihypertensives, ȕ-blockers, gastroenterologic drugs, nonsteroidal antirheumatics and a series of new antidepressants, in an attempt to evaluate their clinical relevance [22]. Being co-substrates of the same P450 isoform, several other drugs were also shown to give potential interactions with antidepressants [23]. • many studies in recent years have been motivated by the need to individualize therapeutic schemes and avoid interactions and dangerous adverse effects as well. These studies had their origin in inter-individual differences in the activities of metabolising enzymes, as a consequence of pharmacogenetic factors, which in fact have been proven to play an important role in the response of individuals receiving the same, specific treatment (same dose, intervals of administration, and so on). A particular study focused on the large inter-individual variability in the human biotransformation of risperidone, a drug mainly metabolised to the corresponding 9-hydroxylated metabolite by specific P450 isoforms, and in particular CYP2D6. Because a large number of drugs have been described to be biotransformed by the same isoform, evaluation of the possible drug interactions on the enzyme appeared as an important issue, as did the consequent clinical significance of this phenomenon [24]. • the use of immunosuppresants prescribed to prevent rejection of transplanted organs or tissues, as well as in the treatment of autoimmune disorders, is on the increase. Therefore, a sound knowledge of the pharmacokinetics of these drugs is helpful in avoiding different drug-drug 310 Chapter 8 interactions that might occur on co-administration of other drugs, such as tacrolimus, sirolimus, monoclonal antibodies and glucocorticoids [25]. • a mechanistic approach to drug interactions involving antiepileptics, frequently administered in combination and many of them involving interactions by reciprocal effects, has been revisited recently [26]. The study focused on the most common antiepileptic drug interactions, which are pharmacokinetic in nature. Interactions involving various antiepileptic drugs are expected to appear either by enzyme induction or inhibition, or displacement of protein binding. Such interactions are discussed in detail. • an interesting relatively recent study, revealed interactions between NSAIDs and angiotensin converting enzyme inhibitors (ACEI) on concurrent administration [27]; the latter are indicated in the treatment of hypertension, myocardial infarction and congestive heart failure. • oral contraceptives have been shown to be involved in many drug- drug interactions, that consequently will reduce their efficacy. An extensive study focused on oral contraceptive interactions with various drugs: e.g. anticonvulsants, antibiotics, adsorbents, analgesics and corticosteroids [28]. • the neuromuscular blocker succinylcholine hydrochloride (used as adjunct in surgery) is biotransformed not typically in the liver, but in the serum, by the circulating enzyme pseudocholinesterase. On co-adminis- tration of cyclophosphamide, the latter irreversibly inhibits this enzyme, reducing the biotransformation of succinylcholine. As a result, respiratory insufficiency and prolonged apnoea may appear [11]. • significant clinical interactions have been shown to appear on co-administration of lithium salts with a large range of medicines, including antidepressants, neuroleptics, anticonvulsants, antibiotics, muscle relaxants, chemotherapeutics and hormones [19]. The clinical observations have been reviewed and the conclusions summarised in a relatively recent account [29]. • some methotrexate interactions; being excreted mainly unchanged in the urine, methotrexate is a likely candidate for displaying excretion-type interactions. Some of the drugs that have been seen to interact with methotrexate include anion exchange resins, NSAIDs, penicillins, uricosurics and urinary alkalinisers and the interactions presumably involve excretion mechanisms. For some of these, detailed discussions are offered [11]: the NSAIDs are known to inhibit prostaglandin (PGE2) synthesis, which will result in a fall in renal perfusion. As a consequence, a rise in methotrexate serum levels is observed, consequently leading to increased toxicity. It has also been suggested that protein-binding displacement may play a part. In attempting to avoid these interactions, if the concomitant use Drug interactions and adverse reactions 311 of NSAIDs is thought appropriate, it is strongly recommended that treatment be monitored closely, and folinic acid rescue therapy should be available. Another interesting example with severe clinical consequences is afforded by the co-administration of methotrexate and penicillins. These, acting like weak acids, will compete with methotrexate in the kidney tubules for excretion. Since penicillins have been proven to cause marked reductions in the clearance of methotrexate from the body, severe toxicity and even death have occurred as a result of such interactions. To avoid or minimise these unwanted effects, the same recommendations as above are suggested. When excreted in bile, methotrexate is then re-absorbed through the enterohepatic cycle, in the gut. Marked falls in methotrexate plasma levels have been reported in patients given concomitantly cholestyramine orally. The assumed mechanism involves binding of methotrexate to the cholestyramine in the gut, thereby preventing re-absorption. Concurrent use should be monitored and dosage adjustments made as necessary [11]. • a recent study has focused on methadone interactions [30]. Methadone is biotransformed almost exclusively by the liver, the main biotransformation of both methadone enantiomers being N-demethylation. In methadone metabolism, more P450 isoforms are involved viz. CYP3A4 and CYP1A2 (being inducible isoforms) and CYP2D6 (not inducible, but subject to genetic polymorphism). Often, the main metabolic substrates of the same CYP are administered concurrently; consequently, the drug that has a higher affinity for that CYT isoform partly prevents the biotransformation of the other drugs. Since most drugs are substrates for the CYP isoforms involved in the metabolism of methadone, interactions are expected to take place readily. Drugs that could be co-administered during methadone maintenance treatment and that are assumed to produce drug-drug interactions of the kinetic type include anticonvulsants, antidepressants, antifungals, benzodiazepines and macrolide antibiotics. Some of these drugs are inhibitors, inducers or substrates of CYP3A4 or CYP2D6. Specific examples show that generally the effects on methadone are either increased or decreased plasma levels, usually moderate in severity, delayed or rapid in onset, and involving different mechanisms on the CYP isoforms implicated (most of them are inducers or co-substrates, competing with methadone). Particular mention is made of the interaction with the antidepressant fluvoxamine, which inhibits both CYP3A4 and 2D6; consequently, although administered in therapeutic doses, plasma concentrations of methadone will correspond to those that are inhibitory in vitro. Another noteworthy point in this context is that maintenance treatment with methadone remains the best choice in HIV-positive heroin addicts; therefore, the most frequent interactions that can take place and that are of 312 Chapter 8 utmost clinical significance are those between methadone and antiretroviral drugs. The antiretrovirals are usually metabolic inducers of the liver CYP3A4 isoform, implying an increase in enzymatic activity, with consequent decrease in the amount of methadone available. The antiretrovirals, including abacavir, amprenavir, didancosine, efavirenz, indinavir, nelfinavir, nevirapine, ritonavir, stavudine and zidovudine, generally determine, as already mentioned, a decrease (minor or moderate) in methadone plasma concentrations, a few with rapid onset (didanosine, stavudine), and the majority with delayed effect (even up to 8-10 days, e.g. efavirenz). Interesting co-administrations involve association of methadone with different combinations of antiretroviral drugs, such as: ritonavir + lopinavir, ritonavir + nelfinavir, ritonavir + nelfinavir + nevirapine, nevirapine + efavirenz etc. The effects on methadone are, just as in the previous case, minor or moderate decreases in its plasma levels. The selected examples presented above illustrate that clinically important interactions may occur when methadone is taken concomitantly with other drugs. Because these pharmacokinetic interactions are generally extremely variable among patients, it is recommended that in the course of long-term treatments, the daily dose should be personalised. • the interaction between terfenadine and ketoconazole, and the corresponding clinical consequences [15] were outlined above. More recently, another interesting interaction has been communicated [31]. The inhibitory properties of a novel gastroprokinetic agent (Z-338) were investigated and compared with those of cisapride, to evaluate its potential for drug-drug interactions. While there was no notable inhibition of terfenadine metabolism or of any of the P450 isoforms involved in biotransformation, the study showed that, on the other hand, cisapride markedly inhibited both of the main CYP isoforms involved in metabolisation, namely CYP3A4 and CYP2C9. From the prediction method used (based on Ki and PK parameters), it was concluded that this novel gastroprokinetic agent is considered unlikely to cause significant drug-drug interactions when co-administered with CYP substrates at clinically effective doses. • recent studies demonstrated the stimulative action of acetaminophen on the peroxidative metabolism of anthracyclines by a common effect of enzyme induction [32]. Frequently administered concurrently with various anthracyclines, such as daunorubicin and doxorubicin, acetaminophen has been proven to stimulate their oxidation by lacto- and myeloperoxidase systems strongly, resulting in irreversibly altered products. The phenomenon has considerable clinical significance because the biological properties of transformed anthracyclines are quite different Drug interactions and adverse reactions 313 from those of the corresponding parent drugs. It is possible that this enhanced acetaminophen induced degradation might interfere with the therapeutic effects of these drugs (viz. anticancer and/or cardiotoxic). • omeprazole and clarithromycin is an association commonly used in the treatment of Helicobacter pylori associated gastroduodenal ulcer. It has been demonstrated that a pharmacokinetic interaction occurs between these two co-administered drugs, with consequences on omeprazole’s biotransformation: the combination resulted in a significantly reduced value (almost one-half) of the 5-hydroxylated metabolite and increased levels of unchanged omeprazole, with mean value of AUC increased about twofold [33]. The clearance and volume of distribution of omeprazole were dramatically reduced on co-administration with clarithromycin. The conclusion of the study was that the concurrent administration of clarithromycin and the proton pump inhibitor omeprazole, resulting as it does in markedly increased levels

of omeprazole, can consequently improve the therapeutic response to this drug. Interestingly, no significant changes in the pharmacokinetics of pantoprazole and corresponding metabolites were observed. • by an inhibitor mechanism, fluvoxamine was demonstrated to modify the pharmacokinetics of lidocaine and its two pharmacologically active metabolites [34]. The study also revealed (confirming in vitro studies) that the main isozyme involved in the biotransformation of lidocaine is a P450 isoform, CYP1A2. Inhibiting the isoform responsible for the metabolism of lidocaine, concurrently administered fluvoxamine resulted in significant decreases in lidocaine clearance, depending also on the state of health of the liver of the subjects. The main conclusion of the study was that the extent of fluvoxamine-lidocaine interaction decreases in patients with liver dysfunction, most likely because of the concomitant decrease in the hepatic level of CYP1A2. • possible interaction between ciprofloxacin and pentoxifylline was recently investigated [35]. In the murine hepatic microsomes, previously incubated with ciprofloxacin, the metabolism of pentoxifylline was found to decrease significantly, suggesting a possible inhibitory effect of the former. • an interesting study revealed the induced biotransformation of zolmitriptan in rats, as well as the interaction between six drugs and this highly selective 5-HT receptor agonist used in acute oral treatment for migraine [36]. Studies were carried out in rat hepatic microsomes treated with different inducers. Earlier clinical observations revealed that potential drug interactions can take place on co-administration with diazepam, propranolol, and moclobemide; thus this study continued investigations with other drugs that might possibly interact with zolmitriptan, namely 314 Chapter 8 fluvoxamine, cimetidine and diphenytriazol. The in vitro model approach employed is increasingly used in drug development to enable early predictions of possible clinically significant drug interactions during co-medication. Fluvoxamine showed a potent inhibitory effect on CYP1A2, the main P450 isoform involved in the biotransformation (by N- demethylation) of zolmitriptan, resulting in increased plasma levels and reduced clearance. Diphenytriazol appeared to display the same effect. Propranolol, metabolised by the same CYP1A2, competes for the active site of the enzyme when administered with zolmitriptan, displaying a competitive inhibitory effect with the same consequences (increased mean Cmax and AUC and prolonged mean t1/2 for the former). Moclobemide, a MAO-A inhibitor, decreased the clearance of zolmitriptan, subsequently elevating plasma concentration indirectly. • a profound interaction between tacrolimus and a combination of lopinavir and ritonavir in three liver-transplanted patients has recently been described [37]. The clinical observations based on tacrolimus blood concentrations and half-life revealed that the combination of antiretroviral agents led to a much greater increase in tacrolimus blood concentrations than did the use of a single protease inhibitor, such as nelfinavir for example. From the clinical observations, it was concluded that, depending on liver function, when therapy with the combination of antiretroviral agents is initiated, a dose of 1 mg/wk or less of tacrolimus may be sufficient to maintain adequate blood tacrolimus concentrations, and patients may not need a further dose for 3 to 5 weeks. • an interesting example of one drug inhibiting the metabolism of a specific CYP2D6 substrate is represented by the effect of celecoxib on the pharmacokinetics of metoprolol [38]. Because celecoxib inhibits the metabolism of metoprolol, it is expected to increase the area under the plasma time-concentration curve of metoprolol, which in fact is almost doubled. In contrast, a comparative study revealed that this effect is not observed with rofecoxib. The interactions that may occur between celecoxib and different CYP2D6 substrates can be of important clinical relevance, especially with drugs having a narrow therapeutic index. 8.2.6 Other frequent and relevant interactions In the following subsection we present in tabulated format, a selection of the most frequent and important drug-drug interactions and the consequent biological effects (Table 8.1): Drug interactions and adverse reactions 315 Tab.8.1 Selected examples of frequent drug-drug interactions and consequent biological effects Drug Drug(s) of Interaction consequences interaction Acetaminophen alcohol severe hepatotoxicity with (Paracetamol) therapeutic doses of acetaminophen in chronic alcoholism (proposed mechanism: increased formation of hepatotoxic acetaminophen metabolites and glutathione depletion) [39] anticoagulants, increased anticoagulant effect oral (mechanism not established) [40] barbiturates acetaminophen hepatic toxicity (mechanism not established) [41] benzodiazepines possible diazepam toxicity (mechanism not established) [42] cholestyramine decreased acetaminophen effect (decreased absorption) [43] isoniazid acetaminophen toxicity (increase in toxic metabolites) [44] probenecid possible acetaminophen toxicity (decreased metabolism and renal excretion) [45] zidovudine granulocytopenia (mechanism not established) [46] Acyclovir narcotics: possible meperidine toxicity meperidine (decreased renal excretion) [47] probenecid possible acyclovir toxicity (decreased renal excretion) [48] 316 Chapter 8 zidovudine lethargy (unknown mechanism) corticosteroids [49] Alkylating agents azathioprine liver necrosis (mechanism not established) [50] corticosteroids decreased effect (increased metabolism) [51] cyclosporins nephrotoxicity (mechanism not established) [52] Aminoglycoside antifungals nephrotoxicity (synergism) [53] antibiotics cephalosporins nephrotoxicity (mechanism not established) [54] cisplatin nephrotoxicity (mechanism not established) [55] cyclosporins renal toxicity (possibly additive or synergism) [56] digoxin decreased digoxin effect (possible decreased absorption) [57] furosemide ototoxicity and nephrotoxicity (additive) [58] polymyxins nephrotoxicity; increased neuromuscular blockade (possibly additive) [59] vancomycin possible nephrotoxicity and ototoxicity (possibly additive) [60] Antacids antihistamine, H2- decreased cimetidine, ranitidine blockers and nizatidine effect (decreased absorption) [61] benzodiazepines decreased oral clorazepate effect (decreased absorption) [62] ȕ-adrenergic decreased oral effect (decreased blockers absorption) [63] Drug interactions and adverse reactions 317 cephalosporins possible decreased effect (decreased absorption) [64] corticosteroids decreased oral corticosteroid effect (decreased absorption) [65] digoxin decreased digoxin effect (possible decreased absorption) [66] hypoglycaemics possible hypoglycaemia (increased absorption and accelerated insulin response) [67] quinidine possible quinidine toxicity (decreased renal excretion) [68] quinine possible quinine toxicity (decreased renal excretion) [69] tetracyclines decreased oral tetracycline effect (decreased absorption) [70] vitamin C possible aluminium toxicity (possibly increased absorption) [71] vitamin D possible bone toxicity with aluminium compounds (increased deposition of aluminium in bone, possibly due to increased absorption) [72] Anticoagulants, Antifungals decreased anticoagulant effect oral (griseofulvin) (mechanism not established) [73] antihistamine, H2- increased anticoagulant effect blockers (decreased metabolism) [74] barbiturates decreased anticoagulant effect (increased metabolism) [75] carbamazepine decreased anticoagulant effect (increased metabolism) [76] 318 Chapter 8 chloral hydrate increased anticoagulant effect (displacement from binding) [77] cholestyramine decreased anticoagulant effect (binding of drug in intestine) [78] disulfiram increased anticoagulant effect (decreased metabolism) [79] fluoroquinolones increased anticoagulant effect (mechanism not established) [80] glutethimide decreased anticoagulant effect (increased metabolism) [81] macrolide increased anticoagulant effect antibiotics (possibly decreased metabolism) [82] nalidixic acid increased anticoagulant effect (displacement from binding) [83] NSAIDs increased bleeding risk (inhibition of platelets, other mechanisms) [84] phenytoin phenytoin toxicity (decreased metabolism) [85] propafenone increased warfarin effect (probably decreased metabolism) [86] spironolactone decreased anticoagulant effect (hemoconcentration) [87] sulphonamides increased anticoagulant effect (decreased metabolism and displacement from binding sites) [88] tetracyclines increased anticoagulant effect (mechanism not established) [89] Drug interactions and adverse reactions 319 thyroid hormones increased anticoagulant effect (increased clotting factor catabolism) [90] valproate increased anticoagulant effect (probably displacement from binding sites) [91] vitamin A increased anticoagulant effect with large doses (mechanism not established) [92] vitamin C decreased anticoagulant effect (mechanism not established) [93] vitamin E increased anticoagulant effect with large doses (mechanism not established) [94] Barbiturates chloramphenicol possible barbiturate toxicity (decreased metabolism) [95] contraceptives, decreased contraceptive effect oral (increased metabolism) [96] corticosteroids decreased corticosteroid effect (increased metabolism) [97] narcotics: increased CNS depression with meperidine meperidine (increased meperidine metabolites) [98] quinine possible phenobarbital toxicity (probably decreased metabolism) [99] valproate phenobarbital toxicity (decreased metabolism) [100] Chloramphenicol hypoglycaemics increased hypoglycaemic effect (mechanism not established) [101] phenytoin phenytoin toxicity (decreased metabolism) [102] metronidazole dystonic reactions (mechanism not established) [103] 320 Chapter 8 pheno-thiazines possible chlorpromazine toxicity (decreased metabolism) [104] Digitoxin diltiazem possible digitoxin toxicity (mechanism not established) [105] ethacrynic acid digitoxin toxicity (potassium and magnesium depletion) [106] furosemide digitoxin toxicity (potassium and magnesium depletion) [107] neuromuscular increased incidence of blocking agents arrhythmias (mechanism not established) [108] thiazide diuretics digitoxin toxicity (potassium and magnesium depletion) [109] Estrogens phenytoin decreased estrogen effect (increased metabolism) [110] vitamin C increased serum concentration and possible toxicity of estrogens with 1 gram/day of vitamin C (decreased metabolism) [111] Fluoroquinolones antacids decreased fluoroquinolone effect (decreased absorption) [112] iron decreased fluoroquinolone effect (decreased absorption) [113] penicillins possible ciprofloxacin toxicity with azlocillin (decreased metabolism) [114] theophyllines theophylline toxicity (decreased metabolism) [115] Drug interactions and adverse reactions 321 zinc decreased ciprofloxacin or norfloxacin effect (decreased absorption) [116] Haloperidol barbiturates decreased haloperidol effect (increased metabolism) [117] isoniazid possible haloperidol toxicity (probably decreased metabolism) [118] lithium encephalopathy, lethargy, fever, confusion, extrapyramidal symptoms [119] methyldopa dementia (mechanism not established) [120] phenothiazines agranulocitosis (mechanism not established) [121] tacrine possible parkinsonian symptoms (possibly additive) [122] Insulin angiotensin- increased hypoglycaemic effect converting (probable increased insulin enzyme inhibitors effect) [123] naltrexone possible increase in insulin requirements (mechanism not established) [124] NSAIDs possible increased hypoglycemic effect with large doses of salicylates (mechanism not established) [125] Methotrexate azathioprine azathioprine toxicity (fever, rash, muscle pain) (mechanism not established) [126] cisplatin methotrexate toxicity (decreased renal clearance) [127] cyclosporin toxicity of both drugs (decreased elimination) [128] 322 Chapter 8 NSAIDS methotrexate toxicity (decreased renal excretion) [129] penicillins possible methotrexate toxicity (decreased excretion) [130] sulponamides possible methotrexate toxicity (displacement from binding sites and decreased renal excretion) [131] tetracyclines possible methotrexate toxicity (displacement from binding) [132] trimethoprim pancytopenia (probably additive inhibition of folate metabolism) [133] Nifedipine NSAIDs possible decreased antihypertensive effect with indomethacin (mechanism not established) [134] phenytoin phenytoin toxicity (mechanism not established) [135] rifampin decreased antihypertensive effect of nifedipine (possibly increased metabolism) [136] selective serotonin nifedipine toxicity with reuptake fluoxetine (probably decreased inhibitors metabolism) [137] Omeprazole digoxin possible digoxin toxicity (increased absorption) [138] disulfiram confusion, catatonic reaction, and disorientation (mechanism not established) [139] phenytoin possible oral phenytoin toxicity (decreased metabolism) [140] Drug interactions and adverse reactions 323 Penicillins anti-coagulants, decreased anticoagulant effect oral with nafcillin (increased metabolism) [141] cephalosporins possible cefotaxime toxicity with mezlocillin in patients with renal impairment (decreased excretion) [142] lithium hypernatremia with ticarcillin (decreased renal excretion) [143] neuromuscular recurrent neuromuscular blocking agents blockade with IV piperacillin (mechanism not established) [144] Rifampin barbiturates decreased barbiturate effect (increased metabolism) [145] corticosteroids marked decrease in corticosteroid effect (increased metabolism) [146] haloperidol decreased haloperidol effect (increased metabolism) [147] isoniazid hepatotoxicity (possibly increased toxic metabolites) [148] trimethoprim- possible rifampin toxicity sulfamethoxazole (possibly decreased metabolism) [149] Sulphonamides barbiturates increased thiopental effect (decreased albumin binding) [150] cyclosporins decreased cyclosporin effect with sulphadiazine (possibly increased metabolism) [151] hypoglycaemics increased hypoglycaemic effect (mechanism not established) [152] 324 Chapter 8 phenytoin possible phenytoin toxicity (decreased metabolism) [153] Tetracyclines digoxin possible digoxin toxicity (decreased gut metabolism and increased absorption) [154] lithium lithium toxicity (decreased renal excretion) [155] phenytoin decreased doxycycline effect (increased metabolism) [156] theophyllines possible theophylline toxicity (mechanism not established) [157] zinc decreased tetracycline effect (decreased absorption) [158] Trimethoprim azathioprine possible azathioprine toxicity with sulfasalazine (decreased metabolism) [159] cyclosporin nephrotoxicity (synergism) [160] dapsone dapsone toxicity, methemoglobinemia (probably decreased metabolism) [161] digoxin possible digoxin toxicity (decreased renal excretion and possibly decreased metabolism) [162] Verapamil carbamazepine carbamazepine toxicity (decreased metabolism) [163] clonidine A-V Block (possible synergy) [164] digoxin digoxin toxicity (probably decreased biliary excretion) [165] Drug interactions and adverse reactions 325 Conclusions: • From the above examples it can be observed that drug-drug interactions may occur due to different mechanisms and in most cases these are known at the molecular level; these variations may have as targets the absorption, protein binding or excretion processes, but most frequently the biotransformation process. For some interactions, the mechanisms are not yet established, but the interactions were revealed by clinical observations. • At each level, the modifications are an increase or a decrease in the effect by different mechanisms, most of them described in the Table. • In most cases, the interaction modifies the effect of only one of the co-administered drugs. Unfortunately, there are cases when such interactions may cause different pathologies as well, including for example nephrotoxicity, hepatotoxicity, ototoxicity, A-V block, methemoglobinemia, leucopenia, recurrent neuromuscular blockade, pancytopenia, confusion, catatonic reaction, and disorientation. • In this context, besides limiting the polytherapy, it is strongly recommended that patients known to suffer from e.g. hepatic or renal impairments, or hypersensitivities, be monitored. • The importance of knowing the mechanism of such interactions is obvious for predictions and limitations of the phenomena. As already mentioned in different chapters or subsections of the present work, the essential role is played by the enzymatic systems involved in drug biotransformation, the two primary mechanisms being enzyme induction and enzyme inhibition. • Both in vitro and in vivo methods are available for evaluating the potential interactions between different drugs (or drugs and other entities) administered concurrently. 8.3 INTERACTIONS BETWEEN DRUGS AND OTHER ENTITIES

8.3.1 Drug-food interactions The presence of food in the stomach is very important especially for the absorption process, causing irregularities in absorption or lowering the stomach pH. It is important as well to highlight the importance of the emptying rate of the stomach and the avoidance of certain nutrients during a specific treatment. Recent studies reviewed the pharmacokinetic drug-food interactions influencing drug plasma levels, as well as the bioavailability changes resulting from concomitant intake of drugs and meals [166]. Thus, 326 Chapter 8 following the normally administered dose of a drug, a decrease in the desired and expected effect should occur. Conversely, in a very few instances, concomitant food intake can be beneficial. Liedholm and Melander [166], in 1986, concluded that “concomitant food intake can increase the bioavailability of propranolol by transient inhibition of its presystemic primary conjugation”. New knowledge relating to interactions between drugs and diet is steadily accumulating. In this context, pharmacokinetic interactions encompass not only drug-drug interactions, or those mediated by herbal medicines, but also interactions involving several foods and even beverages [20]. Recently, possible interactions between food and statins (medicines used for the treatment of hyperlipidemias) have been investigated [21]. This is of utmost importance since diet plays an essential role and exerts considerable influence on the prevention and/or treatment of these pathologies. As a consequence, therapy is commonly begun in combination with dietary advice. This is a very important aspect for several reasons: first, all statins are orally absorbed, so food intake is extremely important in achieving the appropriate therapeutic effect; avoidance of interactions between statins and foodstuff, and consequent alterations in the therapeutic benefits, is necessary; and last, but not least, statins are substrates for different CYTP450 isoforms, thus making them possible candidates for interactions with different components of foodstuff that are co-substrates for the same enzymes. A well-known example is that of grapefruit juice. This beverage, commonly consumed by the general population, is an inhibitor of the intestinal CYP3A4 isoform responsible for the first-pass biotransformation of many drugs. The possible interactions that might occur would lead to increased serum levels and/or decreased clearance, increasing the risk of overdosing. Some of its most notable effects concern the cyclosporins and some calcium anatagonists [167]. More recently, the risk of grapefruit juice interactions has been reviewed, with emphasis on aspects of pharmacokinetics and mechanisms of elimination, which could play a critical role when this beverage is consumed with certain drugs, especially those that are substrates for the CYP3A4 isoform [168]. Although these interactions may not necessarily alter the drug response in most instances, the recommendations are either an alternative medication (one that evidently does not interact with grapefruit juice), or avoidance of the combination to prevent toxicity [168]. Other drug-nutrient interactions were the subject of study in the case of patients receiving enteral nutrition (EN). A recent review [169] discusses problems in extrapolating available data to current practice and provides recommendations for managing interactions of this type. Drug interactions and adverse reactions 327 Finally, it is interesting to note that sometimes the concurrent consumption of food and certain drugs may be beneficial, the drug-nutrient interactions (DNI) resulting either in an increase of drug effect or reduced toxicity. A recent review focuses on specific nutrients that enhance drug effects or reduce their toxicity [170]. 8.3.2 Interactions with alcohol The effects interactions between alcohol and different drugs have been the subject of numerous studies and clinical observations for many years. The first point to highlight is that the subsequent effects depend on whether the consumption of alcohol is chronic or acute. For example, with chronic alcohol abuse, the effect of anticoagulants may be dramatically decreased, because of the increased rate of their biotransformation; in this situation, alcohol is an enzyme inducer. This phenomenon was first reported in the early 1970s [171]. On the other hand, in acute alcohol intoxication, increased anticoagulant effects of both oral anticoagulants and heparin have been reported. The assumed mechanism is that of decreased metabolism; under these circumstances alcohol acts like an enzyme inhibitor [172]. Important interactions involve barbiturates, benzodiazepines, beta- adrenergic blockers, chloral hydrate, cycloserine, cyclosporin, felodipine, hypoglycaemics, isoniazid, nifedipine, NSAIDs, phenothiazines, phenytoin, tetracyclines and verapamil. In some instances the interaction may involve only the effect of the drug e.g. decreased sedative effect of barbiturates with chronic alcohol abuse (due to increased barbiturate metabolism) [173], or increased CNS depression in case of concurrent consumption with benzodiazepines [174]. Sometimes, however the consequences are more serious, either increasing the toxicity of certain drugs (e.g. cyclosporin) [175] or even causing adverse reactions e.g. orthostatic hypotension (with felodipine) [176], increased incidence of hepatitis (with isoniazid) [177] or hepatotoxicity (with methotrexate) [178], bleeding (with aspirin) [179], and impaired motor coordination (with phenothiazines and phenylbutazone) [180]. A recent cohort study focused on patients with schizophrenia and related psychoses, who frequently use, abuse and become dependent on psychoactive substances. The most frequently abused substances were nicotine, alcohol and cannabis. Among the results of interest in this subsection is the reported situation that patients with psychotic disorder and current substance abuse (dual diagnosis, DD) are of enhanced risk for alcohol abuse [181]. 328 Chapter 8 Interactions of toxicological significance between alcohol and psychiatric drugs have been reviewed recently, with a focus on antidepressants and antipsychotics [182]. The study revealed that either acute or chronic consumption of alcohol, when combined with psychiatric drugs, may result in clinically significant toxicological interactions, including those leading to fatal poisoning. It is assumed that these toxicological effects characterise, in fact, overdosing due to decreased biotransformation, delayed by acute alcohol ingestion. More updated information on this topic can be found in the Handbook of Drug Interactions [183]. 8.3.3 Influence of tobacco smoke Cigarette smoking remains highly prevalent in most countries. Tobacco smoke, deliberated inhaled, is considered a self-inflicted effector of drug metabolism. Inhalation of tobacco smoke, with its more than 3000 chemical components, may be considered a different way of ingesting pyrolisis products. It affects drug therapy by both pharmacokinetic and pharmaco dynamic mechanisms. Pharmacokinetic drug interactions are assumed for example for theophylline, tacrine, insulin, imipramine, haloperidol, pentazocine, flecainide, estradiol, propranolol, diazepam, chlordiazepoxide, while pharmacodynamic interactions have been described for antihypertensive and anti-anginal agents, antilipidemics, oral contraceptives and histamine- 2-receptor antagonists [184]. Commonly, pharmacokinetic interactions may call for larger doses of certain drugs due to an increase in plasma clearance, although other accepted mechanisms involve a decrease in absorption, an induction of main drug-metabolising enzyme systems, or a combination of all three factors. In contrast, pharmacodynamic interactions may increase the risk of adverse events in smokers with certain pathologies, such as cardiovascular or peptic ulcer disease [184]. However, the most common effect of tobacco smoke is assumed to be an increase in drug biotransformation through induction of specific enzyme activities. More, or less marked, effects on plasma levels of different therapeutics can be seen following tobacco smoking. Measurements of plasma levels of certain drugs, due to increased metabolism either by the intestinal mucosa or first-pass through the liver, confirm this. Examples include phenacetin (much lower plasma levels in smokers compared with non-smokers, due to increased metabolism) [185], antipyrine (increase in drug clearance) [186], estrogens (possible decreased estrogen Drug interactions and adverse reactions 329 effect, increased metabolism) [187], tricyclic antidepressants (decreased antidepressant effect, increased metabolism) [188], propranolol (decreased propranolol effect, increased metabolism) [189], phenylbutazone (decreased phenylbutazone effect, increased metabolism) [190], mexiletine (decreased mexiletine effect, increased metabolism) [191]. More recent studies provided novel evidence that cigarette smoking accelerates chlorzoxazone and caffeine metabolism, by markedly enhancing oral clearance [192]. Other studies focused on the influence of tobacco smoke on specific isoforms of CYTP450. Owing to its inducer effect, tobacco smoke may increase the risk of cancer by enhancing the metabolic activation of carcinogens. From the numerous compounds present in tobacco smoke, the polycyclic aromatic hydrocarbons (PAHs) are believed to be responsible for the induction of CYP1A1, CYP1A2, and CYP2E1. Conversely, and still with no evidence in humans yet, other components such as carbon monoxide and cadmium displayed inhibitory effects on CYP enzymes [193]. The same studies revealed that due to nicotine, which is known to display a stimulant action, cigarette smoking may cause heart-rate and blood pressure lowering. Furthermore, nicotine, due to the cutaneous vasoconstriction induced, may slow the rate of insulin absorption after i.v. administration. 8.4 ADVERSE REACTIONS 8.4.1 Classification criteria Adverse drug reactions are unwanted effects caused by normal therapeutic doses [194-197]. From the total reported adverse reactions, about 7% are severe, with an average of 0.32%, being fatal. Adverse reactions share some characteristics such as: • they may be induced by the majority of drugs, • they may appear immediately (the allergic reactions), or after a certain period of time (carcinogenity, mutagenity, teratogenity), • they may appear more frequently in certain situations, such as self- medication, during co-administration of several drugs, in children (immaturity of the enzymatic systems), in the elderly (decrease in enzymatic activity), in particular physiological states (pregnancy), in pathological states pre-existing or co-existing with drug administration including renal, digestive, hepatic dysfunctions, cardiovascular diseases, dysfunctions in the routes of biotransformation, malnutrition, excessive consumption of alcohol, tobacco, coffee, greater individual sensitivity and reactivity (usually caused by genetic enzymatic deficiencies). 330 Chapter 8 Several criteria have been proposed for a classification of the adverse reactions, but this remains a difficult task due to the complexity of the mechanisms involved, and the incidence and/or variable severity. In principle, the following criteria may be used: • predictibality, • some clinical and experimental characteristics, • the producing mechanism, and • the location criteria [194,196,197]. According to the first mentioned criterion, adverse reactions may be grouped into: • predictable and, • unpredictable adverse reactions. The first group covers the so-called type A adverse reactions, which in fact constitute the great majority of adverse drug reactions and are usually a consequence of the drug’s main pharmacological effect. They are dose- related and usually mild, although they may sometimes be severe, or even fatal. A term often applied to this type of adverse reaction is that of ‘side-’ or ‘collateral effect’. Such a reaction may be either a consequence of incorrect dosage or of impaired drug elimination. In contrast, the so-called type B adverse reactions, are not predictable from the drug’s main pharmacological action, are not dose-related, and generally are severe, with a considerable incidence of mortality. These types of adverse reactions, also called ‘idiosyncratic’, occur rarely and usually have either a genetic or an immunological basis [194,197]. Based on the second criterion, the adverse reactions have similarly been classified into two types: • experimentally reproducible, • irreproducible adverse reactions. In fact, they correspond to the first established groups, as follows: - the first of these, being experimentally reproducible, are of course predictable; as predictable adverse reactions, according to the previous characterization, they are dose-related; the main consequence (with potential benefit for the patient) is the possibility of reducing the unwanted effects of such reactions by simply reducing the administered doses; - as for the second type, corresponding obviously to the unpredictable ones, with no dose-relation and severe manifestations, the strong recommendation would be to stop the therapy. As for the third criterion, which is more didactic, three classes are distinguished: • adverse reactions of toxic type, • ‘idiosyncratic’ type adverse reactions and • adverse reactions of allergic type. Drug interactions and adverse reactions 331 The first group refers to functional and morphological unwanted disturbances, which may appear in some patients under similar conditions of administration and usual doses. Determinant factors include the different individual reactivities, the drug-drug interactions, the pathological state of the organism, the state of the enzymatic systems involved in drug biotransformation and the small therapeutic index of certain drugs. The most severe adverse reactions of this type are assumed to be the mutagenic, teratogenic and carcinogenic effects. The ‘idiosyncratic’ type adverse reactions are unusual reactions, qualitatively and quantitatively different from the common effects of a drug in the majority of a population, and most commonly are determined by genetically inherited enzymopathies. Many of them are strain-dependent. Included here are also the so-called type D reactions – delayed reactions, such as carcinogenesis induced by alkylating agents, or retinoid-associated teratogenesis. Other important examples are the blood dyscrasias, including thrombocytopenia, anaemia and agranulocytosis [197]. As for the adverse reactions of allergic type, we should stress that drugs may cause a

variety of allergic responses, and moreover, a single drug can sometimes be responsible for more than one type of allergic response. It is assumed that this type of adverse reaction involves immune mechanisms, in the sense that most drugs, which are in general of low molecular weight, can however combine with substances of high molecular weight (usually proteins), forming an antigenic haptene conjugate. Most commonly, after the reaction Ag-Ac (antigen-antibody) takes place, serotonin, histamine as well as other chemical mediators are liberated, causing an allergic response. According to the immune mechanism involved, this type may be subdivided into the following subtypes: - subtype I: anaphylactic reactions, due to the production of reaginic IgE antibodies. They commonly occur with foreign serum or penicillin, but may also occur with some local anaesthetics and streptomycin; - subtype II: cytotoxic reactions, due to antibodies of class IgG and IgM, which (on contact with antibodies on the cell surface) are able to fix complement, causing cell lysis; - subtype III: immune complex arthus reactions; these soluble, circulating complexes can fix in the small vessels and basal membranes, activating the complement, and subsequently determining various inflammatory phenomena. - subtype IV: delayed hypersensitivity reactions, due to the drug forming an antigenic conjugate with dermal proteins and sensitised T-cells reacting to drug , causi ng a rash [194,197]. 332 Chapter 8 Other proposed categories include: • continuous reactions due to long-term drug use (e.g. analgesic neuropathy) and • end-of-use reactions, such as withdrawal syndromes following discontinuation of a treatment (with e.g. benzodiazepines, tricyclic antidepressants or ȕ-adrenoreceptor antagonists) [194,197]. Summarising the factors involved in adverse drug reactions, we should classify them also according to so-called ‘patient’ factors, ‘prescriber’ factors and ‘drug’ factors. The patient factors may be intrinsic (age, sex, genetic abnormalities, presence of organ dysfunction etc.) or extrinsic (environment, malnutrition, xenobiotics). The prescriber factors refer generally to incorrect dosage or drug combination, duration of therapy etc., while the drug factors refer mostly to drug-drug interactions. Although it is probably not possible to avoid allergic drug reactions altogether, the following measures can decrease their incidence: • the drug history is essential whenever treatment is anticipated; • drugs given orally are less likely to cause severe allergic reactions than those given by injection; • prophylactic skin testing should become more routinely practised because it could probably reduce the risk of anaphylaxis or other less severe reactions [194,196,197]. As indicated in the first three chapters, and illustrated by numerous examples in Chapters 2 and 3, the processes of drug metabolism result in biotransformation of the drug to metabolites that differ chemically from the parent drug, consequently displaying altered affinities for the drug receptor. This change in the structure of the drug may be beneficial or detrimental. For example, when ‘inactive’ drugs, such as prodrugs (inert species whose pharmacological effect depends entirely on metabolism) are biotrans formed yielding active metabolites, the process is obviously beneficial and is called pharmacological activation. However, in general, biotransformation of a drug prepares it for excretion and in this case, the process of metabolism results in pharmacological deactivation. When certain toxins or potentially toxic drugs are involved, such metabolism leading to ‘detoxication’ is obviously of invaluable benefit. On the other hand, when a drug (or other xenobiotic) is transformed into a toxic metabolite, the reaction is called ‘toxicological activation’ or, ‘toxication’, and this is obviously detrimental to health. Such a metabolite may act or react in a number of ways to elicit a variety of toxic effects at different levels, as will be evident from examples cited in the next section. Drug interactions and adverse reactions 333 It is essential to stress that the occurrence of a toxication reaction at the molecular level does not necessarily imply toxicity at the levels of organs and organisms. On the other hand, when metabolic toxication reactions occur, they are always accompanied by competitive and/or sequential reactions of detoxication that compete with the formation of the toxic metabolite. This may lead to its inactivation. The existence of essential survival mechanisms should also be borne in mind. These act to repair molecular lesions by removing them immunologically and/or by regenerating lesioned areas. From the above discussion, it is evident that the process of drug metabolism may either decrease or increase toxicity of a given drug compound depending on the biological potencies of the drug and its metabolites; for this reason we focus in the present subchapter on a more detailed examination of the toxicological aspects of drug metabolism. Classification criteria refer to the adverse reactions and toxicological consequences, the most severe of them including hepatotoxicity and nephrotoxicity, pulmonary toxicity, carcinogenesis and teratogenesis. 8.4.2 Selected examples Oxidation of some secondary hydroxylamines may yield nitroxide and other reactive metabolites, possibly accounting for the hepatotoxicity of these chemicals. If the metabolic intermediates of such compounds undergo N-oxygenation they may possibly form complexes with the cytochrome P450 enzymes, inhibiting them reversibly. A well-known, medically relevant example is given by norbenzphetamine, which undergoes a two-step N-oxidation (Figure 8.1). In the first step, it is hydroxylated to the corresponding hydroxylamine and in the second, the product is oxidised to a nitrone intermediate [198]. The reaction is catalysed mainly by a FAD-containing monooxygenase. The nitrone intermediate is susceptible to further oxidation to the corresponding nitroso derivative under CYTP450 catalysis. It is assumed that this nitroso derivative is the metabolic intermediate (MI) responsible for formation of a complex with the CYTP450, resulting in a (usually) reversible inhibition of the enzyme [199]. The binding of such metabolic intermediates to CYTP450 involves the presence of the enzyme in reduced form; under such conditions, the nitrogen atom will interact with the iron cation [200] (Figure 8.2): 334 Chapter 8 CH3 CH2 CH N CH2 FAD-containing H monooxygenase norbenzphetamine CH3 CH2 CH N CH2 OH secondary hydroxylamine CH3 CH2 CH N CH2 O nitrone CH3 CH2 CH N O nitroso derivative Fig.8.1 Oxidation of norbenzphetamine yielding a nitrone and a nitroso species P450 Fe2+ N R O Fig.8.2 Formation of the complex with the CYTP450 enzyme Many more studies have focused on the biotransformation of primary arylamines, given their toxicological significance. An interesting and representative example in this context is procainamide, studied in the early 1980s (Figure 8.3). In human liver microsomes, procainamide was shown to be metabolised to a hydroxylamine intermediate, which further undergoes Drug interactions and adverse reactions 335 non-enzymatic oxidation to the corresponding nitroso-compound; this is assumed to covalently react with glutathione (and thiol groups in proteins), forming sulphinamide adducts [200]. Two other possibilities exist for the intermediate nitroso derivatives: they can either bind to a hydroxylamine, yielding an azoxy derivative, or even to the parent primary amine forming an azo compound (Figure 8.3). As in the previous case of secondary hydroxylamines, the nitroso metabolites of primary arylamines can also form complexes with reduced cytochrome P450 [201]. H2N C NH (CH2)2 N(CH2 CH3)2 O procainamide O C N N C C N N C azoxy azo Fig.8.3 Structure of procainamide and other intermediary metabolic groups responsible for the toxicity of the drug The toxicological significance of primary arylamines, and polycyclic arylamines in particular, is very considerable due to the carcinogenic and mutagenic potential of their intermediates, involving highly reactive species, namely, nitrenium ions (aryl-N+-H) [202]. There is evidence that such nitrenium ions may be responsible for the covalent binding to DNA of certain drugs [203]. These highly reactive ions are known to exist in two states, namely singlet and triplet [204]. This has turned out to be of fundamental importance, since the nitrenium ions of non- toxic amines exist preferentially in the triplet state whereas singlet states have been attributed to nitrenium ions of mutagenic/carcinogenic amines. Therefore, it was concluded that for the initiation of either carcinogenic or mutagenic process, the nitrenium ions must exist in the singlet state. An important conclusion drawn from the above examples is that hydroxylamine formation may generally be considered as a route of toxication. Among the compounds known to be N-hydroxylated (rather than forming other intermediates, such as e.g. N-oxides) much interest is focused 336 Chapter 8 on carcinogens occurring as amino acid pyrolysates, in cooked or charred foods. A representative example is the mutagenic and carcinogenic compound IQ (2-amino-3-methylimidazo[4,5-f]quinoline) [205] (Figure 8.4): NH2 N N CH3 N Fig.8.4 Structure of the carcinogenic and mutagenic compound IQ Another interesting example involves the N-hydroxylation of the endogenous purine base, adenine; it is assumed that the 6-N-hydroxylated derivative is genotoxic and carcinogenic [206] (Figure 8.5): NH2 NH OH 6 N N N N N N N N H Fig.8.5 6-N-hydroxylation of adenine It is again assumed that highly reactive intermediate nitrenium ions are implicated in mutagenic and carcinogenic effects associated with such heterocyclic hydroxylamines. 1,2-disubstituted hydrazines can also be N-oxygenated, yielding first the corresponding azo intermediates, which may either rearrange to hydrazones, or be further oxygenated to azoxy derivatives. The product hydrazones are reversibly hydrolysable, forming primary amines and aldehydes [207]. For some alkyl azo- and azoxy-derivatives, toxicity results from further activation by Į-carbon hydroxylation, occurring after the initial hydrogen abstraction [208]. In contrast, certain aromatic azo-compounds with a para-amino group are potentially carcinogenic due to the activation of the amino group, while the azo-group has been shown to undergo reduction Drug interactions and adverse reactions 337 [209]. A compound of particular significance and medicinal interest (first studied some twenty years ago) is the anticancer drug cyclophosphamide. Here, toxication is determined by the N-C oxidative ring cleavage and takes place mainly in hypoxic tumor cells. According to Borch [210] the first step is a preferential oxidation at the 4-position, yielding the 4- hydroxycyclophosphamide. This carbinolamine intermediate is in equilibrium with aldophosphamide, its open-ring tautomer. Subsequent dehydrogenation of these intermediates deactivates the drug and yields the corresponding urinary metabolites 4-oxocyclophosphamide and carboxyphosphamide. The undehydrogenated aldophosphamide remains in a keto-enol equilibrium with aldophosphamide, another urinary metabolite [211]. Under the relatively anaerobic conditions within tumor cells, both the biologically active metabolite phosphoramide mustard and the toxic metabolite acrolein are generated from the aldophosphamide. N-nitroso derivatives (nitrosamines) comprise a special group of xenobiotics, intensively studied and comprehensively reviewed, whose biotransformation can lead to highly reactive metabolites. This accounts for their potential hepatotoxicity and carcinogenicity. A much studied and potent mutagen and carcinogen, representative for the toxication of dialkyl- and alkylarylnitrosamines, is dimethylnitrosamine, a substrate of CYP2E1 [212]. The biotransformation is complex, toxication beginning with an N-dealkylation that produces a C-centred radical and an Į-nitrosamino alcohol. The latter is a highly reactive intermediate, readily decomposing to the N-dealkylated species, formaldehyde and diazomethane. Following elimination of dinitrogen, the diazo intermediate decomposes to give a carbonium ion [213] (the methyl cation, in the given example), which may react as a strongly electrophilic species at different nucleophilic sites of biomolecules. If the biomolecule happens to be e.g. DNA, a ‘molecular injury’ is taking place, simultaneously initiating a sequence of events that possibly may lead to hepatotoxicity, carcinogenicity, or other toxic effects. As regards the C-centred radical formed initially, it breaks down spontaneously to nitric oxide and N-methylformamidine. This latter intermediate hydrolyses to form methylamine and formaldehyde, while the nitric oxide is oxidised to nitrite [214]. Another important mechanism of toxication involves the cytochrome P450-catalyzed oxidation of sulphur-containing compounds, yielding as reactive, electrophilic species, the corresponding sulphenic acids. An interesting example of metabolic toxication is that of the sulphur- containing steroidal drug, spironolactone (Figure 8.6): 338 Chapter 8 O O O S C CH3 O Fig.8.6 Structure of spironolactone Following a sequence of metabolic reactions, this aldosterone antagonist will finally yield sulphenic acid. In the course of the biotransformation, cytochrome P450 is destroyed, the intermediates accounting for this toxication being assumed to be the thiyl radical and/or the sulphenic acid [215]. The proposed mechanism involves thiol oxidation to disulphides, sulphinic and sulphonic acids. Another example of biological and toxicological interest is the oxidation of certain 4-alkylphenols to the corresponding quinone methides. These intermediates, seen in hepatic and pulmonary microsomes of some species, act like strongly alkylating agents which may undergo additions at the exocyclic methylene carbon, thereby binding covalently to macromolecular, soluble nucleophiles. This reactivity with nucleophiles was shown to correlate with hepatotoxicty [216]. Benzyl S-haloalkenyl sulphides having the general structure presented in Figure 8.7 are substrates of CYTP450-catalysed S-dealkylation that yields an unstable thiol [217]. The latter easily rearranges to mutagenic thioacylating intermediates,

such as thioketenes and/or thioacyl chlorides [218]. R' Cl Cl S R Cl Cl Cl S CH2 R' = Cl R = CH2 Fig.8.7 General structure of benzyl S-haloalkenyl sulphides Drug interactions and adverse reactions 339 S-haloalkenyl-L-cysteine conjugates can be activated to the same unstable thiols by the action of cysteine-conjugate ȕ-lyase, a pyridoxal phosphate-dependent enzyme, found mainly in the kidney. It cleaves L-cysteine conjugates to thiols, NH3 and pyruvic acid [219], being of interest from the toxicological point of view in the context of kidney-selective delivery of thiol-containing drugs. Such an example of renal activation by S-C cleavage, is given by S- (6-purinyl)-L-cysteine, to the corresponding 6-mercaptopurine (Figure 8.8): NH2 S H S COOH N N N N N N N N H H Fig.8.8 ȕ-lyase catalysed S-C cleavage of S-(6-purinyl)-L-cysteine Usually, the S-haloalkenyl-L-cysteine conjugates are formed from glutathione in the liver, but the high reactivity of ȕ-lyase accounts for their nephrotoxicity by activating them to thiols in the kidney. The sulphoxidation of thioamides is also of considerable interest due to the potential toxicity of some metabolites, in particular their hepatotoxicity and carcinogenicity. Studies have been made on different good substrates for the FAD-containing monooxygenase [220], such as those presented in Figure 8.9: S S CH3 C NH2 C NH2 thioacetamide thiobenzamide Fig.8.9 Substrates for FAD-containing monooxygenase catalysed sulphoxidation, yielding potentially toxic metabolites The reason for such thioamides being good substrates of this enzyme is their resonance, which increases the nucleophilic character of the sulphur atom [221] (Figure 8.10): 340 Chapter 8 S S + R C NH2 R C NH2 Fig.8.10 Resonance structures for thioamides, increasing the nucleophilic character of the sulphur atom The monooxygenase-mediated oxygenation of thioamides yields different intermediates such as sulphines and sulphenes, and as end-products, acetamide, other polar compounds, and microsomal-bound material. Very recently the mechanisms of covalent binding of reactive species and examples of bioactivation were updated [222]. The role and implications in pharmacological interactions of one of the most important drug-metabolising enzyme systems, CYTP450, were highlighted in a recent review [223]. Metabolic induction develops following repeated administration of a drug, with the synthesis of new enzyme and with the increase of its activity. The result is an increase in the metabolism of the drug involved in the interaction and a decrease in the quantity of drug available for pharmacological activity. In order for this to take place, one or two weeks are usually needed. On the other hand, enzymatic inhibition develops quickly since it takes a short time for the drug to bind to the enzyme. Inhibition of activity of the enzyme decreases the metabolism of the drug and therefore increases its pharmacological activity. Pharmacokinetic interaction can also occur when two or more drugs that are metabolic substrates of the same CYP are administered concurrently. In this case the drug that has the greatest affinity for that cytochrome can prevent in part the metabolism of the other drugs. Most drugs are substrates of only five isoenzymes (CYP3A4, 1A2, 2C9,2C19,2D6); therefore, interactions can take place readily. The drugs that during absorption undergo a considerable first-pass effect or that have a low therapeutic index are the ones most often subject to significant interactions. Many interactions are not clinically apparent because plasma concentrations with therapeutic doses are lower than those used to cause the interaction in vitro. A very recent review refers to the role of the same enzymatic system in chemical toxicity and oxidative stress, based on studies with the CYP2E1 isoform [224]. As already stressed at the beginning of the chapter, drug allergies, known also as hypersensitivities, are reactions with a special nature. Clinical manifestations are very different and of various severities and include agranulocitosis, anaphylaxis, bronchospasm, dermatitis, fever, Drug interactions and adverse reactions 341 granulocytopenia, haemolytic anaemia, lupus erythematosus, nephritis and thrombocytopenia. Commonly, the pathophysiology of such adverse reactions involves the presence of an organic molecule, generally larger than most drug molecules, recognized as non-self, and thus, inducing an immune response. Sometimes however, even small, non-immunogenic organic molecules, covalently bound to an endogenous macromolecular carrier, may form a conjugate that will elicit an immune response. It is important to note that such drug-carrier conjugates may be formed if the drug or its decomposition products that might arise during manufacturing are chemically reactive, or if the drug is biotransformed into reactive intermediates. This is exemplified by carbamazepine. As this anticonvulsant is known to be associated with frequent incidence of hypersensitivity, it would obviously be of interest to understand the molecular basis of such reactions. This question has been investigated [225], the authors postulating that reactive metabolites are responsible in many cases, including incidences of agranulocytosis and lupus. It is postulated that many drug hypersensitivity reactions, especially agranulocytosis and lupus, are due to reactive metabolites generated by the myeloperoxidase (MPO) (EC 1.11.1.7) system of neutrophils and monocytes. This led to a study of the metabolism and covalent binding of carbamazepine with MPO/H2O2/Cl- and neutrophils. Metabolism and covalent binding were observed in both systems and the same pathway appeared to be involved; however, the metabolism observed with the MPO system was approximately 500-fold greater than that observed with neutrophils. The metabolites identified were an intermediate aldehyde, 9- acridine carboxaldehyde, acridine, acridone, choloroacridone, and dichloroacridone. It was postulated that the first intermediate in the metabolism of carbamazepine is a carbonium ion formed by reaction of hypochlorous acid (HOCl) with the 10,11-double bond. Though there was no direct proof for the proposed carbonium ion, its presence was considered to be consistent with the likely mechanism for the observed ring contraction. Iminostilbene, a known metabolite of carbamazepine, was metabolised by a similar pathway leading to ring contraction; however, the rate was much faster and the first step possibly involves N-chlorination and a nitrenium ion intermediate. The data confirmed that carbamazepine is metabolised to reactive intermediates by activated leukocytes. Such metabolites could be responsible for some of the adverse reactions associated with carbamazepine, especially reactions such as agranulocytosis and lupus which involve leukocytes [226]. One of the best-defined models of hypersensitivity reactions is penicillin allergy. The hypersensitivity reactions, with an apparent prevalence of about 2%, may be divided into: 342 Chapter 8 - immediate (anaphylaxis, asthma, urticaria); - accelerated (urticaria, laryngeal oedema, asthma, local inflammatory reactions); - late (commonly with urticaria, fever, haemolysis, granulocytopenia, eosinophilia and rarely, with acute renal insufficiency and thrombocytopenia) [194,197]. An interesting and useful approach to adverse reactions might be that of following the specific organ systems involved: • probably the most common is dermatologic toxicity, and it may be mentioned that drug-induced cutaneous reactions may occur as solitary manifestations, or be part of a more severe systemic involvement. Most frequently associated with allergic skin reactions are the penicillins, sulphonamides, and blood products [197]. • many drugs as well as other xenobiotics (industrial chemicals and solvents) are associated with impaired auditory or vestibular function. Beginning with streptomycin in the 1940s, ototoxicity has become a major clinical problem. Indeed, in the last twenty years it has been suggested that more than 130 drugs and chemicals are associated with this type of adverse reaction [227]. Major classes include aminoglycosides, antimalarials, anti- inflammatory drugs, diuretics and some topical agents [228-231]. • other organs responsive to influences from both topical and systemic medications are the eyes. Ocular toxicity involves blurred vision, disturbances of colour vision, degeneration of the retina and other untoward effects on the cornea, sclera, or optic nerve [232]. It is important to note that the untoward responses are sometimes genetically determined [233]. • many drugs have been shown to cause renal dysfunction, either through reactive intermediates, or because of drug-drug interactions. While this point was mentioned earlier, it is worth emphasising the importance of close monitoring of patients with impaired renal function, or in case of administration of certain drugs known to cause renal injury. Noteworthy in this context, because of their wide therapeutic utility, are the NSAIDs, which are often associated with fluid retention, hyperkalemia, deterioration of renal function, interstitial nephritis, papillary necrosis and even chronic renal failure (especially with prolonged use of high doses) [234]. Interstitial nephritis, for instance, can be produced by numerous therapeutic agents, most frequently by penicillins, cephalosporins, sulphonamides, rifampicin, cimetidine, allopurinol, diuretics and, as mentioned, NSAIDs. A lower incidence of nephrotoxicity seems to attend the use of aminoglycosides [235]; in this case the adverse reaction appears to be more closely related to the length of time rather than to concentrations. However, with other drugs, a solution for reducing nephrotoxicity was found. For example, alternative formulations with amphotericin B have been produced, in which the drug is Drug interactions and adverse reactions 343 encapsulated into liposomes or other lipid carriers [236]. Special attention should be given to cyclosporins because of their narrow therapeutic index, marked variability in clearance, variable bioavailability and extensive drug interactions [237]. In view of these features, the need for therapeutic drug monitoring is obvious. • hemopoietic toxicity is a very important type of adverse reaction, since the hemopoietic system is notably vulnerable to the toxic effects of drugs. Adverse effects may involve platelet and coagulation defects, aplastic anaemia, thrombocytopenia and agranulocytosis. Unfortunately, there are a great number of drugs associated with (or presumed to be associated with) haematologic disorders; almost 20 years ago, they were summarised by Verstraete and Boogaerts [238]. They included in this category aspirin, carbenicillin, ticarcillin, cephalosporins, chloramphenicol, phenylbutazone, sulphonamides, heparin, dipyrone, mianserin, sulfasalazine, the group of penicillins, cimetidine and the thiouracil derivatives. As a severe adverse reaction we mention, with more detail, the thrombocytopenia, caused by inceased platelet destruction or by bone marrow suppression. Such hypersensitivity can be caused by a large number of drugs and commonly these patients presumably have drug-related antibodies of both IgG and IgM classes, known to be involved in the destruction of platelets. Clinical observations revealed that transient, mild thrombocytopenia occurs in about one-quarter of patients receiving heparin, probably due to heparin-induced platelet aggregation. The severe form follows the formation of heparin- dependent antibodies. • hepatotoxicity can also be caused by numerous drugs in common use [239]. It is noteworthy that while many agents cause asymptomatic liver injury, chronic and acute hepatic injury may develop as well. Usually, drug- induced hepatotoxicity may be either predictable, causing hepatocellular necrosis, or idiosyncratic. In the first case, the injury is due to intrinsic toxicity of the drug or its metabolite(s) and the injury is dose-related and commonly reproducible in animals. As a well-known example we mention acetaminophen. Idiosyncratic reactions are generally unpredictable, do not relate to drug dose and usually occur because of hypersensitivity (with the usual clinical implications). A very interesting and important aspect that should be mentioned in this context is that even dietary supplements can result in hepatotoxicity, developing with cirrhosis or even fulminant hepatic failure [240]. • pulmonary toxicity; commonly, adverse pulmonary reactions to drugs are considered likely when the cause of a respiratory illness is not clear. Clinical syndromes are heterogeneous, including hypersensitive lung disease, drug-induced lupus with potential for lung involvement, 344 Chapter 8 bronchiolitis obliterans, pneumotitis-fibrosis and noncardiogenic pulmonary oedema. Causative agents are numerous and include ampicillin, carbamazepine, hydralazine, imipramine, isoniazid, nitrofurantoin, penicillin, phenytoin, sulphadiazine, methotrexate, griseofulvin, oral contraceptives, phenylbutazone, procainamide, quinidine, sulphonamides, sedatives or opioid overdose. The most frequent, severe pulmonary toxicity reactions are associated with amiodarone (Amiodarone Trials Meta-Analysis Investigators 1997). Severe pulmonary diseases may also be caused by cytotoxic drugs, through different mechanisms. For example, bleomycin may cause adverse effects by generating reactive oxygen metabolites, while for mitomycin, appearance of adverse effects is associated with the alkylating properties of the drug [241]. Main classes of drugs that induce pulmonary parenchymal disease include cytotoxic antibiotics, nitrosoureas, alkylating agents, cyclophosphamide, chlorambucil, methotrexate, azathioprine, 6-mercaptopurine, cytosine-arabinoside, procarbazine, and vinca alkaloids [197]. Of course, particular attention should be paid to special categories of patients, such as pregnant and breast-feeding women, infants and children, and the elderly [194-197]. • Differences in drug effects in pregnancy are usually explained by altered pharmacokinetics [242]: increased volume of distribution, hepatic metabolism and renal excretion all tend to reduce drug concentration, while decreased plasma albumin levels increase the ratio of free drug in plasma. Under these circumstances, it is obvious that prescription of drugs to a pregnant woman warrants cautious consideration in order to strike a balance between possible adverse drug effects on the foetus and the risk

of leaving maternal disease inadequately treated. Therefore, usual recommendations stipulate the following: minimise prescribing; use ‘tried and tested’ drugs whenever possible in preference to new agents; use the smallest effective dose; remember that the foetus is most sensitive in the first trimester. It is well known that the most severe drug–induced consequence, especially in the first trimester (period of organogenesis), is teratogenity (foetal malformation). Commonly used drugs that have demonstrated teratogenity in humans include anticonvulsants, lithium, warfarin, phenytoin, sodium valproate, carbamazepine, sex hormones and retinoic acids. For some of them, the mechanism is known at the molecular level e.g. carbamazepine and phenytoin are metabolised to arene oxides; these are reactive, electrophilic compounds that may bind to foetal macromolecules, which consequently may be implicated in the production of malformations [243]. Moreover, arene oxides are known to be metabolised by epoxide hydrolase, and therefore a genetic defect in epoxide hydrolase activity may also be associated with phenytoin-induced teratogenity [244]. After the first trimester, the risk of anatomic defects decreases, the impact of drugs Drug interactions and adverse reactions 345 simultaneously moving from structural to physiological effects. From the numerous drugs that might possibly be required to be administered during pregnancy, antibiotics are the most common. Those that are considered safe include penicillin, ampicillin, amoxicillin, erythromycin and cephalosporins. On the other hand, certain antibiotics should be avoided; these include chloramphenicol, tetracyclines, aminoglycosides, sulphonamides, metronidazole and ciprofloxacin [245]. Another problem in this context is that drugs given to a mother who is breast-feeding her infant may pass into the breast milk and consequently into the baby. Most drugs enter breast milk by passive diffusion; therefore, small molecules are expected to cross more easily than large ones. Nonetheless, it should noted that there are several factors that influence the transfer of drugs from mother to infant in breast milk. Some of them affect the concentration of drug in the mother (drug dose, frequency, route, clearance rate, plasma protein binding); others affect the transfer across the breast (breast flow rate, metabolism of drug within the breast, molecular weight, degree of ionization, water/lipid solubility of the drug, relative binding affinity to plasma and milk protein). Finally, others affect drug concentration in the infant (frequency and duration of feeds, volume of milk consumed, ability of the infant to metabolise the drug – directly dependent on the development of drug-metabolising enzymatic systems involved). Among drugs absolutely contraindicated during breast-feeding because of their negative effects on the infant, should be mentioned ciprofloxacin, chloramphenicol, doxepine, cyclophosphamide, cytotoxic drugs, iodine-containing compounds, androgens, ergotamine and laxatives. The corresponding effects are arthropathy, bone marrow suppression, respiratory suppression, neutropenia, cytotoxicity, effect on thyroid, androgenisation of the infant, vomiting, convulsions and diarrhoea. As final conclusions and recommendations, the following are noted: - drug concentrations should be monitored monthly: - women taking medication during pregnancy should have a detailed ultrasound scan at 20 weeks’ gestation in order to identify any foetal abnormality [197]; - women receiving phenobarbitone should avoid breast-feeding. • Infants and children. Pediatric patients represent a condition of unstable pharmacokinetics [246]. A knowledge of age-related changes in drug absorption, distribution, and clearance is essential to optimise drug efficacy and minimise or even avoid the risk of toxicity. Under these circumstances, special attention must be paid to the pharmacokinetic variable. For example, concerning absorption, diminished intestinal motility and delayed gastric emptying in neonates and infants will result in a longer period of time for a drug to reach appropriate therapeutic plasma 346 Chapter 8 concentration. Drug distribution and protein binding in neonates and children are also influenced by changes in body composition that accompany development. For instance, the extracellular water compartment of body weight is almost double in the neonate compared with the adult; this may have clinically important consequences, especially with water-soluble drugs that are distributed throughout the extracellular water compartment [247]. The other determinant of drug distribution is its protein binding. Since the free (unbound) drug concentration is responsible for drug effects, age-related changes in protein binding may exert important influences on drug efficacy and toxicity, especially in drugs with a narrow therapeutic index [248]. Under these conditions, drug biotransformation is strictly dependent on the development of the enzymatic systems involved. Usually, the decreased ability of neonates to metabolise drugs, due to the immaturity of their enzymatic systems, results in prolonged elimination half-lives. As a consequence, this can predispose neonates to adverse drug reactions, caused by relative overdosing. Another aspect that merits emphasis is that many of the drugs prescribed for neonates or children can potentially inhibit or enhance the metabolism of other drugs. The clinical significance of these interactions is dictated by the magnitude of the increase or decrease in the clearance of the index drug. Also worth stressing are the possible consequences of co-administration of drugs, especially those with inhibitory effects on hepatic drug metabolism together with a hepatically metabolised drug having a narrow therapeutic range, resulting in increased serum concentrations, overdosing and even toxicity. Finally, we refer to possible drug interactions due to altered renal function. Most of them are undesirable - for instance enhancing methotrexate toxicity by inhibition of its tubular secretion in co-administration with salicylates. However, some of these interactions can, on the other hand, be beneficial e.g. probenecid reduces renal penicillin excretion. In conclusion, in pediatric patients drug concentrations are directly and strongly dependent on various factors such as drug dosage, the pharmacokinetic properties dictated by the liver and kidney functions, and genetic variability in drug metabolism. • Drugs in the elderly. The elderly constitute a particularly heterogenous patient group, who are at increased risk of appearance of adverse reactions, for several reasons: - elderly people take more drugs (at least three to four different drugs daily). The most commonly prescribed are diuretics, analgesics, tranquillisers, and antidepressants, hypnotics and digoxin. As already mentioned, all of these are associated with a high incidence of important adverse reactions: Drug interactions and adverse reactions 347 - pharmacokinetics change with increasing age (and often, concomitant disease), leading generally to higher plasma concentrations of drugs, and consequently, increased susceptibility to side-effects; - with advancing age, homeostatic mechanisms become less effective, so these individuals are less able to compensate for adverse effects; - increasing age produces changes in the immune response (increased risk of allergic reactions); also, the central nervous system becomes more sensitive to the actions of sedative drugs. Of the main reasons listed above that determine increased risk of drug toxicity in the elderly, the most important by far is considered to be the pharmacokinetics, potentially modified by ageing. By influencing drug disposition, these age-related changes might be expected to alter the response to drugs, which consequently may explain why older patients seem to be more susceptible to both the therapeutic and the toxic effects of many drugs. As far as absorption is concerned, it is well known that the elderly exhibit several alterations in GI function that might result in impaired or delayed absorption of a drug. However, relatively recently it was demonstrated that very few drugs displayed delayed or reduced absorption after oral administration in the elderly [249]. In contrast, the active transport of calcium, iron, thiamine and vitamin B12 declines with age, due either to decreased intestinal blood flow rate (up to 50%), or to increased gastric motility. However, unless GI pathology is present, it appears that age per se does not affect drug absorption to a significant extent. Body composition is one of the most important factors that may produce altered distribution of drugs in elderly patients, ageing being generally associated with loss of weight and lean body mass, increased ratio of fat to muscle, and decreased body water [250]. Therefore, hydrophilic drugs that are commonly distributed mainly in body water or lean body mass should have higher concentrations in blood in the elderly, especially when the dose is based either on the total weight or surface area. Conversely, highly lipophilic drugs tend to have larger volumes of distribution in older persons due to increased proportion of body fat. This may be partly responsible for the age-related increase in the volume of distribution of thiopental and some of the benzodiazepines [251]. As far as hepatic metabolism is concerned, a decrease in the rate of hepatic clearance of some but not all drugs was noted with advancing age. This is first determined by age-related decreases in liver size and blood flow [252]. On the other hand, it is well known that hepatic clearance of drugs is strongly dependent on the enzymatic activity, both microsomal and non- microsomal enzymes being involved in both phases of drug biotransformation. It was established that the activities of phase I pathways are often reduced in the elderly, whereas phase II pathways are generally 348 Chapter 8 unaffected [253]. Obviously, the reduced rate of clearance of certain drugs may lead to important clinical consequences, such as accumulation of drug (relative overdosing), leading to adverse reactions [254, 255, 256]. The most consistent effect of age on pharmacokinetics is the age- related reduction in renal excretion, with both glomerular and tubular functions being affected. As a consequence, it is generally assumed that drugs that are significantly excreted by the kidney will display diminished clearance of plasma from the elderly. Drugs with decreased renal excretion in old age, and consequently with potentially severe toxic effects, include amantadine, ampicillin, atenolol, captopril, chlorpropamide, cimetidine, digoxin, doxycycline, enalapril, furosemide, lithium carbonate, penicillin, procainamide, phenobarbital, ranitidine and tetracycline [257]. These alterations in pharmacokinetics in the elderly (especially impaired renal elimination and hepatic metabolism of drugs) may also contribute to exacerbated consequences of drug-drug interactions. A well- known example is the effect of dexamethasone on phenytoin metabolism, when these drugs are concurrently administered. Both are substrates for the same metabolising enzymes, resulting in increased serum phenytoin levels, and even toxicity [258]. Similarly, a drug-drug interaction causing a decrease in renal drug excretion (in addition to an already poor renal elimination capacity) could result in increased toxicity in a vulnerable elderly patient. 8.5 SUMMARY From all the above considerations and examples, it emerges, as a first major conclusion, that a significantly important step in assessing the potential toxicity of a drug and its metabolites is the prediction of entry and fate of the compound in human body. With this in mind, three distinct approaches should be stressed. The first refers to the predictions that can possibly be made based on both in vitro and in vivo data concerning metabolic transformations of a particular drug. The second implies knowing and understanding the enzymatic systems involved in these biotransformations, while the third concerns the possibility of extrapolating data from in vitro or in vivo results on the one hand, and interspecies results, on the other. There are predictions of in vivo drug-drug interactions based on in vitro data gathered from the literature [9]. They are based, in principle, on mathematical models using measurable, specific parameters, introduced for the calculation of the hepatic intrinsic in vivo clearance for a particular drug. Some of them are successful and some are not. Nowadays, in vitro experiments use human microsomes, hepatocytes, liver slices and isoform-specific microsomes from expressed systems. It is important to note that if studies are based on human microsomes, some accounting should be Drug interactions and adverse reactions 349 made for the interindividual variability in the expression of the target isoform [10]. As a successful prediction, we may mention the well-known tolbutamide-sulfaphenazole case, which concurrently administered may cause severe side effects, such as hypoglycaemic shock. An approximately five-fold increase in both AUC and t1/2 of tolbutamide in co-administration with sulphaphenazole was reported [259]. Both drugs being co-substrates for the same CYTP450 isoform (CYP2D9), on concurrent administration of sulphaphenazole, the biotransformation of tolbutamide is inhibited. The main metabolisation pathway of tolbutamide in vitro is a CYP2D9-mediated hydroxylation. In vivo, the hydroxylated metabolite follows sequential biotransformations, resulting in a carboxylated metabolite. The Ki value for sulphaphenazole, a specific inhibitor of the isoenzyme involved in the main biotransformation pathway is extremely small (~0.1-0.2 µM). As a consequence, the inhibitor’s affinity as co-substrate for the same enzyme will be very strong, as will its inhibitory action. The inhibition of this metabolic pathway results in a reduction of the total clearance of tolbutamide of about 80% (relative overdosing induced by co-administration of sulphaphenazole). If a substantial inhibition of an isoform-specific

probe is observed, it is strongly recommended (as a matter of genuine practical interest) that the magnitude of the same effect for other substrates of that isoform be assessed. The above interaction is species-dependent, following more or less the same pattern in rats, but the opposite in rabbits (total clearance increased by 15-30%) [259]. A well-studied and previously mentioned interaction in this context is that between terfenadine and ketoconazole [260]. Terfenadine is extensively and rapidly metabolised by CYP3A4 isoenzymes. On the other hand, ketoconazole is known to be a potent CYP3A4 inhibitor; consequently, co- administration of ketoconazole results in a dramatic decrease in the biotransformation of terfenadine, with consequent increase in plasmatic levels. Other examples based on the same mechanism (competitive inhibition of one drug as co-substrate for the same enzyme) include caffeine-ciprofloxacin [261] and cyclosporin-erythromycin [262]. An interesting mechanism, different from the ‘classic’ ones presented above, is that of mechanism-based inhibition, in which the inhibitor is biotransformed into a metabolite that covalently binds to the enzyme, resulting in its irreversible inactivation [263]. However, a special case, when the inhibition is not called ‘mechanism-based inhibition’, is that when the inhibitor is metabolically activated by one enzyme and inactivates another. Such an example, which determined 5-fluorouracil (5-FU) toxicity caused by high blood concentrations, is its interaction with sorivudine (an antiviral drug). Sorivudine is sequentially biotransformed into a metabolite that is rate-limiting in the metabolism of 5-FU. More attention to this type of 350 Chapter 8 interaction is needed because the inhibitory effect remains after the elimination of sorivudine from blood and tissues, possibly leading to serious side-effects [264]. In this context, it should also be mentioned that many drugs (other than sorivudine) are reported to be mechanism-based inhibitors e.g. macrolide antibiotics (erythromycin, troleandomycin – against CYP3A4) [265], orphenadrine (against CYP2B1) [266], and furafylline (against CYP1A2) [267]. Another important problem that warrants mention is that some enzymes act not only in the liver but in the gut as well, therefore playing an important role in the first-pass metabolism following oral administration. Such an important isoform is CYP3A4, an enzyme that metabolises many drugs, including cyclosporins [268]. As examples we can quote the decreased bioavailability of cyclosporin after co-administration of rifampicin – an inducer of CYP3A4, and its increased bioavailability by co-administration of ketoconazole, an inhibitor of the same isoform [269, 270]. A new and fashionable approach in drug discovery is predictive ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) [271]. Here, candidate drugs may be designed and their structures optimised by constructing computational models that associate structural variations with changes in response [272]. The process relies on large databases containing ADMET data for known structures. An offshoot of this approach is the possibility of predicting human ADMET properties from human in vitro and animal in vivo ADMET data. The success or otherwise of this approach is limited by the quality of the database and the level of sophistication of the modelling methods. The status of in-silico prediction of drug metabolism and related toxicity has been reviewed [273]. Problems that are encountered include prediction of the nominal metabolic transformations for a given molecule and gaining an understanding of the nature of the enzymes that might be involved as well as possible alternative routes of transformation. In so-called ‘rule-based’ metabolism prediction studies, the aim is to predict both metabolic pathways as well as metabolites that might be generated [272]. Following formation of the first predicted metabolite, the possibility of its being metabolised to multiple products must be considered, as must the subsequent metabolism of those products. This could rapidly result in an unmanageable number of metabolites and therefore limitations on this number can be imposed, based on e.g. the probability of a particular metabolite being formed, the stability of the metabolite, and so on. Ideally, such predictions should be implemented at an early stage of drug discovery to eliminate ‘junk’ leads. Nonetheless, the problem remains a challenging one due to the complexity of the human body, including such factors as differences in enzymatic phenotype that could alter drug metabolism. Input Drug interactions and adverse reactions 351 from other areas is necessary to gain a comprehensive picture of the metabolic fate of a given compound. QSAR modelling usually examines the interactions between small drug molecules and macromolecules such as their metabolising enzymes [273]. Recent developments in this area include QSAR models for substrates of the major drug-metabolising enzymes, including human cytochrome P450s. QSAR modelling of metabolic stages following that above is also an important area for investigation. Concluding remarks A considerably detailed treatment of drug-drug interactions and adverse reactions has been presented above, together with older and more recent examples of each. Much of this material relates to known, well-documented cases that are of general interest. However, a crucial aspect of drug-drug interactions and adverse reactions is the possibility of predicting their occurrence for new drug candidates. 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Chapter 9 STRATEGIES FOR DRUG DESIGN 9.1 INTRODUCTION The previous chapters in this book dealt with the major aspects of the metabolism of drugs as well as some basic pharmacokinetic principles. This chapter, aimed primarily at the aspiring medicinal chemist, attempts to illustrate how considerations of pharmacokinetics and metabolism serve as invaluable input to the process of drug design and optimisation of drug in vivo activity. There is a vast literature on this subject and the treatment below is necessarily selective. However, the aim here is to highlight the main principles and popular strategies that are applied to overcome pharmacokinetic problems and to use metabolism to advantage in the discovery and development of drugs, as well as indicate useful recent literature sources for further study. 9.2 PHARMACOKINETICS AND METABOLISM IN DRUG RESEARCH 9.2.1 General overview It is now widely accepted that while structure-activity relationships (SAR) have an important place in drug discovery and design, in particular to identify ligands with optimum affinities for their receptors, the most effective way to increase the therapeutic index of a new drug candidate intended for a specific application is to complement SAR-based approaches with additional data on its metabolites, its pharmacodynamic and pharmacokinetic properties and toxicological implications [1]. In other words, optimisation of in vitro activity through the employment of SAR- guided synthesis alone is no assurance of favourable in vivo activity, since 369 370 Chapter 9 the latter is subject to pharmacokinetics and metabolism that determine e.g. the drug bioavailability, duration of action, biotransformation into active/inactive/toxic metabolites, and so on [2]. An earlier survey [3] indicated that some 40% of a sample of ~300 new drug candidates investigated in humans were subsequently withdrawn due to serious shortcomings in their pharmacokinetics, as reflected in e.g. poor oral absorption, extensive first-pass metabolism, unfavourable distribution or clearance, or a combination of these. This emphasises the need for understanding the principal factors affecting pharmacokinetics viz. drug lipophilicity and solubility (see Chapter 1). These properties can be manipulated by chemical modification of the active compound or via formulation approaches so as to overcome the above problems, ideally without compromising the intrinsic pharmacological activity of the pharmacophore. From a historical perspective, the rational use of metabolism input to the drug discovery process is a relatively recent innovation [4]. Frequently in the past, such information has mainly been used to explain the failure of a molecule to achieve its expected performance. During the last two decades however, the explosive growth of knowledge in the area of drug- metabolising enzymes coupled with technological advances in analytical instrumentation has allowed medicinal chemists to acquire valuable information on the metabolic fates of new drug candidates at an early stage of their development [2]. In addition, as shown in Chapters 1-3, based on a wealth of accumulated data, rules exist for predicting both the pharmacokinetic behaviour of a compound as well as its likely major routes of metabolism from a knowledge of its molecular structure and physicochemical properties [4]. During the last decade, there has been a growing emphasis on rapid metabolism assessment in the discovery phase [5] and numerous in silico tools have been developed to predict the metabolic properties of candidate drugs, e.g. their metabolic stability, likely sites of metabolism and ensuing metabolites, rates of metabolism, drug-drug interactions, clearance and toxicology. The status of such computational models has recently been reviewed [6,7]. Exploitation of the existing knowledge bases and responsible use of computerised resources can aid the medicinal chemist in optimising drug in vivo activity. As is evident from earlier chapters, nature has evolved a formidable array of metabolic mechanisms to handle both endogenous and xenobiotic substances in humans. One feature of the metabolism of xenobiotics is the prevalence of oxidative processes, which may not only detoxify them, but also generate toxic, reactive intermediates such as epoxides and radicals. Mention has been made earlier of the possible negative consequences that can ensue from reaction of such intermediates with endogenous macromolecules. Therefore, as regards drug design, one principle that serves Strategies for drug design 371 as a guideline is that oxidative pathways for the biotransformation of candidate drugs should generally be avoided. One way to achieve this is to rely on inactivation of designed drugs via hydrolytic mechanisms such as those effected by esterases that are widespread in the body. In summary, consideration of pharmacokinetic and metabolic factors indicates that, in principle: (a) rational synthetic modifications can be made to a drug candidate to ensure its favourable absorption, distribution and clearance; (b) at the same time, appropriate functional groups, or other moieties such as carrier groups that undergo predictable metabolism can be attached to the pharmacophore to direct the specific routes of activation or deactivation as needed [8]. These guiding principles should lead to the development of drugs with high therapeutic indices. An elegant and current application of (b) above is represented by metabolism-based drug-targeting, whereby advantage is taken of the prevalence of specific, known enzymes in an organ, body compartment or diseased tissue, to design a molecule that is metabolised only at that site, where it subsequently releases the active drug. Such an approach (see section 9.2.5 below) has in recent years led to the development of safer, more effective drugs with site-specificity and hence displaying fewer side effects. In the sections that follow, an attempt is made to describe some of the main approaches to chemical modification that may result in improved drug pharmacokinetics, favourable metabolism or both. In addition, some developments in drug targeting are described, where they might depend on predictable metabolism. In each case, the design concept is briefly explained and illustrated with one or more pertinent examples. In keeping with the title of this book, we have attempted to include primarily recent case studies, though for didactic purposes reference is occasionally made to older examples in the literature. Subsections 9.2.2-9.2.4 focus

on chemical manipulation of drugs, highlighting the rationale behind the discovery of prodrugs, hard drugs and soft drugs respectively, with examples. Strategies based on more sophisticated ‘chemical delivery systems’ that deliberately include drug- targeting as their goal are described in section 9.2.5. For completeness, section 9.3 includes a brief interlude on aspects of formulation approaches that are mainly aimed at improving oral absorption of poorly soluble drugs. Some of these approaches involve physical modification of new chemical entities and may not rely on the creation of covalent bonds between the active drug and the matrix or carrier moiety. Nevertheless, the authors believe that medicinal chemists should be aware that alternatives to synthesis may sometimes be the route to meeting their objectives. Finally, in section 9.4 we underscore the crucial roles of pharmacokinetics and metabolism in drug design in the hope that their consideration by aspiring medicinal chemists will result in the development of safer and more effective drugs in the future. 372 Chapter 9 9.2.2 The prodrug approach According to the original definition by Albert [9], a prodrug is a chemical with little or no pharmacological activity that undergoes biotransformation to the therapeutically active metabolite. Actually, the ‘activation’ of the prodrug i.e. its conversion to the pharmacologically active form, may proceed under enzyme control, by non-enzymatic reaction, or by each of these in sequence. In general, the intention of the prodrug approach is to improve the efficacy of an established drug [10]. Prodrugs are developed to address numerous shortcomings, but probably most frequently to improve oral bioavailability, either by enhancing oral absorption or by reducing pre-systemic metabolism. As indicated in Chapter 1, transport of a drug through membranes, and hence its absorption, depends critically on the balance between the drug’s aqueous solubility and its lipophilicity. Optimisation of this balance is often achieved by attaching a ‘carrier moiety’ to a polar group such as an acidic, alcoholic, phenolic or amino-function of the active species to yield the prodrug, which should then undergo predictable metabolism to release the active form in the body. Chemical derivatisation used to improve lipophilicity often involves conversion of acidic, phenolic and alcoholic functions into appropriate esters that are metabolised to the corresponding active drugs by esterases, which are ubiquitous. Aldehydes and ketones may be converted into acetals, and amines into quaternary ammonium species, amino acid peptides and imines [11]. More recent developments involving prodrugs relate to their activation in two-step targeting therapies such as ADEPT (antibody-directed enzyme prodrug therapy) and GDEPT (gene-directed enzyme prodrug therapy). These approaches hold particular promise in the area of cancer treatment through selective liberation of anticancer drugs at the surface of tumour cells. In contrast to the prodrugs described above, which are developed primarily to overcome pharmacokinetic problems, those used in ADEPT and GDEPT therapies are associated with site-specific drug delivery. Prodrug activation again relies on specific enzymes but in this case these are ‘pre- delivered’ to the desired sites of action [12]. Several examples of relatively simple prodrugs are described first in this section. This is followed by a description of more complex systems that utilise prodrugs with the specific intention of site-specific delivery. A recent example of a successful prodrug is ximelagatran (Figure 9.1), which upon absorption is converted into its active metabolite melagatran [13], a potent competitive inhibitor of human α-thrombin. Strategies for drug design 373 O H O O HO N N N H NH Melagatran 2 NH O H O O CH N N 3CH O N 2 H NH Ximelagatran 2 N OH Fig.9.1 The active drug melagatran and its prodrug ximelagatran [13,14] Melagatran was developed in the search for a new generation of oral anticoagulants with more predictable pharmacokinetic and pharmacodynamic properties than those of drugs in previous use, such as dicoumarol and warfarin. But despite having the necessary pharmacodynamic properties of a new antithrombotic agent, the oral bioavailability of melagatran was found to be only ~5%, which precluded its oral administration. This led to the development of its prodrug ximelagatran, produced by ethylation of the –COOH group and hydroxylation of the amidine group of the active compound. Poor bioavailability was attributed to the strong basic amidine functionality, originally selected in the design phase to fit the arginine side-pocket of thrombin [14]. Hence this was replaced by the less basic N-hydroxylated amidine. In addition, an ethyl ester protecting group was introduced. Biotransformation of the prodrug to melagatran, involving ester cleavage and reduction of the amidoxime function, was demonstrated in vitro using microsomes and mitochondria from liver and kidney of pig and human. These chemical modifications resulted in significant reduction in the hydrophilicity of the active molecule, the prodrug having an apparent in vitro permeability coefficient around 80-fold higher than that of melagatran. Following oral administration of ximelagatran, it is thus rapidly absorbed and converted to melagatran which has a bioavailability of ~20% i.e. significantly greater than that following oral administration of the active 374 Chapter 9 drug. The successful performance of the prodrug ximelagatran led to its full- scale clinical evaluation in 2003. Further details of the pharmacodynamics, pharmacokinetics and metabolism of ximelagatran and melagatran have been published [13,14]. A similar approach to improving oral absorption has been applied for some time to many antibiotics. Ampicillin is poorly absorbed when administered orally. Large doses are thus required to achieve the necessary therapeutic level, leading to toxic effects in the GI tract. The prodrug strategy was employed to develop derivatives such as bacampicillin, pivampicillin and talampicillin (Figure 9.2), by esterification of the polar carboxylate group to yield these lipophilic, enzymatically labile prodrugs, all of which are metabolised to the active antibiotic ampicillin. Whereas the absoprtion of ampicillin is less than 50%, the above prodrugs are absorbed to the extent of 98-99% and continue to be widely used [2]. O H H H S Ph C N NH H N H 2 O O C O COCH2CH3 O CH cam 3 O Ba picillin O H H H S Ph C N O NH H H H H N S 2 O O C O COC(CH H 3)3 Ph C N O 2 NH H N 2 O OH Pivampicillin O Ampicillin O H H O H S Ph C N O NH H N 2 O O O Talampicillin Fig.9.2 Prodrugs yielding ampicillin as the common active metabolite [11] Strategies for drug design 375 These compounds fall into the class of ‘tripartate carrier-linked prodrugs’ [10,11,15], characterised by the presence of three distinct moieties, namely the active drug, a linking structure and a carrier group. In the case of bacampicillin, these moieties are respectively the ampicillin ‘core’, -OCH(CH3)O- and –COEt. In the first phase of metabolism of bacampicillin, the latter moieties are enzymatically hydrolysed to yield carbon dioxide and ethanol (Figure 9.3), and in the second phase, spontaneous loss of acetaldehyde from the resulting intermediate releases the active drug, ampicillin [11]. H H RC O C O COCH2CH3 RC O C OH + CO2 + CH3CH2OH O CH3 O O CH3 Bacampicillin O H H H S ( R = Ph C N N H N RCOOH CH3CHO H ) + 2 O Ampicillin Fig.9.3 Metabolism of bacampicillin [11] Analogous synthetic strategies have been employed in the development of prodrugs of biologically active phosphate esters, as detailed in a recent review [15]. Here, both improvement in bioavailability as well as some degree of site-specificity, reflected in elevation of the concentration of the active species within cells, have been achieved. At physiological pH in the range 7.0-7.4, phosphate esters, O=P(OR)(O-)2, are in the deprotonated state and therefore generally do not readily permeate cellular membranes. In order to increase bioavailability and cell permeability, various masking groups (MG) have been developed that convert the charged phosphate esters into neutral molecules, rendering the resulting prodrugs more permeable to cell membranes. As shown schematically in Figure 9.4, following diffusion of the prodrugs through cellular membranes, the masking groups are removed by hydrolysis to yield the charged phosphate ester [15]. Since the active, charged species has thus been regenerated within the cell, it is effectively ‘trapped’ there, where it can carry out its medicinal function. Within the cell, conversion of the prodrug into the phosphate ester may occur either by chemical or enzymatic hydrolysis. 376 Chapter 9 Though elegant as a design concept, this prodrug approach does nevertheless present significant synthetic challenges as regards the type of masking group to be employed to achieve appropriate stability for optimum chemical/enzymatic hydrolysis. In the case of anti-HIV nucleosides [16], successful derivatives have been based on the use of an unsymmetrical, cyclic bio-activatable protecting group [17] that undergoes a tandem reaction in its hydrolysis to yield the active drug. O diffusion O X MG X MG RO P RO P O X MG X MG O RO P prodrug O hydrolysis phosphate ester O X O RO P O cell wall Fig.9.4 Prodrug concept for biologically active phosphate esters [15] The same ‘tripartate’ principle employed in the development of ampicillin prodrugs described above has also been applied to phosphate esters [15]. Here, the protecting moiety attached to an oxygen atom of the active phosphate ester is of the form –X-Y. The residues X and Y are chosen such that enzymatic cleavage initially splits off the terminal group Y. The resulting intermediate is unstable, the linking group X leaving spontaneously to yield finally the charged phosphate group. Acyloxyalkyl ester prodrugs of this type, for example, are hydrolysed by the enzyme carboxyesterase, yielding the active phosphate, an aldehyde, a carboxylate and two protons. One concern relating to the use of prodrugs is the possible toxicity of the degradation products, in particular formaldehyde, which is associated with carcinogenicity. More recent work does, however, suggest that the human body can tolerate low levels of formaldehyde better then previously believed [18]. The medicinal chemist needs to be aware of the possibility of toxic metabolites or byproducts arising during the metabolism of the administered compound [19]. The reader is referred to the informative review [15] for further details on strategies used to synthesise analogous prodrugs including those of Strategies for drug design 377 nucleotides and inositol phosphates. Interestingly, despite intensive efforts in the area of phosphate-containing prodrug candidates, clinical success has been very limited, and therefore this endeavour poses ongoing challenges to the medicinal chemist. Another challenging area for the medicinal chemist is the delivery of peptides [9,20]. The two-step activation strategy referred to above for phosphate ester prodrugs has been employed in the case of peptide prodrugs, with the difference that, following the first step (enzymatically mediated hydrolysis of the ester), the second involves non-enzymatic intramolecular nucleophilic attack which cleaves the amide bond, releasing the peptide and a cyclic by-product (Figure 9.5a). An example of such a prodrug is shown in Figure 9.5b. H2O(a) O O X O X OH 1 N peptide CO N peptide CO H H 2H 2 (b) H2N peptide COOH O + O CO X N peptide O H O Fig.9.5 (a) Peptide prodrug undergoing two-step activation and (b) an example of a prodrug based on the design concept in (a) [9, and refs. therein] In the metabolism of the model compound, step 1 unmasks a phenolic –OH group, which in step 2 is the nucleophilic centre that attacks the amide bond. Other pharmacokinetic objectives that may be addressed by the prodrug approach include improvement in absorption via parenteral routes and extending the duration of drug action by slow metabolic release. An example of a prodrug fulfilling the second objective is bambuterol, derived from the active β2-adrenergic agonist terbutaline [21]. Chemical modification in this case involved conversion of the two phenolic groups on the terbutaline molecule into their diethylcarbamato ((CH3)2-N-CO-) esters. Activation of the prodrug involves hydrolysis in blood serum (mediated by a 378 Chapter 9 cholinesterase) as well as oxidation involving monooxygenase present in various organs and tissues. The extended duration of action in this case, however, is novel, relying on prolonged inhibition of the cholinesterase by covalent attachment of the diethylcarbamato group of bambuterol. Consequently, bambuterol produces a more sustained bronchodilating effect than the parent terbutaline and can thus be administered less frequently. Organ- or tissue-selective delivery is also possible via the prodrug approach, as exemplified recently by capecitabine, an orally active prodrug of the antineoplastic 5-fluorouracil [22]. The prodrug (Figure 9.6)

is activated sequentially by carboxylesterase present in the liver, by cytidine deaminase in liver and tumour cells, and finally by thymidine phosphorylase present in tumours. The crucial feature of this metabolism that accounts for the successful ‘targeting’ performance of the prodrug is that the final step, release of 5-fluorouracil, occurs selectively within tumour cells. O O stepwise H O N metabolism N H3C N NHCOO(CH2)4CH3 HN O HO OH F F Capecitabine 5-fluorouracil Fig.9.6 The prodrug capecitabine and its active metabolite 5-fluorouracil [22] Another topical area in which prodrugs feature prominently relates to the development of anti-HIV nucleosides and their analogues. FDA- approved drugs in this class include zidovudine (AZT), didanosine, zalcitabine, stavudine and lamivudine. These compounds are effective inhibitors of HIV reverse transcriptase (HIV-RT) and are active as antiretrovirals in the form of their triphosphate metabolites, whose sequential formation is catalysed by a variety of cellular kinases. The cellular pharmacology, structure-activity relationships and pharmacokinetics of a number of anti-HIV nucleosides and their prodrugs have been the subject of a recent review [23]. The CNS and the lymphatic system act as reservoirs for HIV, but the nucleosides as such have limited penetration into these areas. Hence, one motivation for prodrug design is to overcome this delivery problem. One example of the documented prodrug design strategies is cited here. Phospholipid prodrugs (Figure 9.7) have been designed for 2’, 3’-dideoxynucleoside analogues such as AZT (denoted X in Figure 9.7), to Strategies for drug design 379 improve their therapeutic profiles [23]. With such prodrugs, intracellular release of the nucleoside analogue or its monophosphate occurs, in the latter case bypassing the monophosphorylation step in the further metabolism to the active triphosphate. R2 R2 CO O CO O O R1COO C O P O X R H 1COO C O P P O X H O O OH Fig.9.7 Two types of phospholipid prodrugs for anti-HIV agents such as AZT [23] We mention here that another very important type of prodrug for 2’,3’-dideoxynucleoside analogues is based on the redox chemistry of dihydropyridine (DHP)-pyridinium salt interconversion, a strategy which has been employed elsewhere to design drugs that can cross the blood-brain barrier (BBB) [24,25]. However, since this type of system more appropriately falls under the classification of a chemical delivery system, a discussion of this strategy is reserved for section 9.2.5 where greater focus is given to drug targeting approaches. Simple derivatisation via ester or amide formation does not guarantee a successful prodrug. A case in point is the poorly soluble antiviral acyclovir, which though possessing a hydroxyl group that could be chemically modified to improve absorption, has not yielded successful ester prodrugs [2]. Instead, the prodrug desoxyacyclovir (Figure 9.8) does result in superior oral delivery of acyclovir. Here, advantage is taken of oxidative metabolism by the enzyme xanthine oxidase, present in the gut and the liver, for biotransformation of the prodrug to the active acyclovir. O xanthine N N oxidase N N N N N N N N CH2OCH2CH2OH CH2OCH2CH2OH Desoxyacyclovir Acyclovir Fig.9.8 The first step in the metabolic activation of the prodrug desoxyacyclovir [2] 380 Chapter 9 As with the nucleoside analogues described above, in vivo phosphorylation of acyclovir is necessary for the eventual activity to be manifested, which in this case is inhibition of the viral DNA polymerase. A conceptually different category of prodrug that warrants discussion here is that used in novel therapies such as ADEPT (antibody-directed enzyme prodrug therapy) and GDEPT (gene-directed enzyme prodrug therapy), techniques that have developed through advances in molecular biology. This type of prodrug is non-toxic and instead of being activated by an endogenous enzyme, is metabolised in ADEPT to the active agent by an engineered enzyme-antibody conjugate that has been delivered site- specifically to a tumour cell in advance [26,27]. In the practice of ADEPT, for example, the monoclonal antibody-enzyme conjugate is administered intravenously, whereupon it localises in tumour cells. Several hours later, the anticancer prodrug is administered and is thus selectively activated at the tumour cells by the delivered enzyme. Normal cells, being devoid of the enzyme, are unaffected by this treatment. If only one prodrug is administered, the delivered enzyme must obviously catalyse a scission reaction for that specific compound. In general though, a desirable requirement of the enzyme should be versatility of catalytic action that may enable it to activate several anticancer agents. In GDEPT, a foreign gene encoding for the desired enzyme is delivered to the target tumour by means of a viral or liposomal vector. Following gene expression, the enzyme converts the non-toxic prodrug into the cytotoxic agent at the desired site. Examples of enzymes that have recently been considered as suitable candidates for the activation of anticancer prodrugs in ADEPT and GDEPT approaches are β-galactosidase and β-glucuronidase [28]. The former is expressed in mammalian cells transduced with the E. coli LacZ gene. Figure 9.9 shows a designed prodrug 1 that has been shown to undergo conversion to the antitumour alkylating phosphoramide mustard 2 when incubated with E. coli β-galactosidase [29]. The first step in the proposed mechanism of activation involves enzymatic cleavage of the 4-β-D-galactopyranosyl unit to yield an intermediate phenol. The latter undergoes spontaneous 1, 6-elimination to release the cytotoxic mustard 2 as well as a quinone methide which spontaneously converts to 4-hydroxybenzylalcohol 3. On the basis of these findings, prodrug 1 was considered to have good potential in conjunction with GDEPT to increase antitumour selectivity in cancer therapy. An analogous prodrug 4 (Figure 9.9) was designed with a β-D-glucuronic acid linked to a self-immolative spacer (in this case an N-(o-hydroxyphenyl)-N-carbamate) and a phenolic mustard 5 [30] as a candidate for use in conjunction with ADEPT. The presence of a spacer separating the drug and the enzyme substrate in systems of this type has been reported to produce more effective prodrugs [31]. Strategies for drug design 381 HO CH2OH O O N(CH2CH2Cl) HO O C O P 2 H HO 2 N(CH2CH2Cl)2 1 O N(CH CH Cl) HO P 2 2 2 HO N(CH CH C 2 H 2Cl) + H O 2 2 2 3 COOH HO O O NO HO 2 HO N(CH H 2CH2Cl) O 2 MeNCOO N(CH2CH2Cl)2 4 5 O OH O OH COMe COMe OH OH OMe O OH O O OMe O OH O O Me Me CH2OH OH 7 HO HO O O CH2OCONH NH + 2 OH O2N 6 OH HO CH2OH O2N 8 Fig.9.9 Activation of prodrugs designed for use in ADEPT and GDEPT therapies [28,29] The cytotoxicities of the prodrug and the drug were compared, the former showing a reduced toxicity, which is a requirement for its application in ADEPT. In contrast to prodrug 1, compound 4 was designed to be cleaved by β-D-glucuronidase, and was indeed shown to release the drug 5 in vitro in the presence of E.Coli β-D-glucuronidase. 382 Chapter 9 Analaogous prodrug design strategy has been applied to the anthracycline antibiotic daunorubicin [28]. The prodrug 6 (Figure 9.9), incorporating a para-substituted benzyloxycarbonyl group as a bioreversible amine protective group, was shown to be degraded into the active drug daunorubicin 7 and 3-nitro-4-hydroxybenzyl alcohol 8 in the presence of E.coli β-galactosidase. The first activation step is cleavage of the galactopyranosyl unit (as for compound 1) and subsequent decarboxylation to give the products 7 and 8. Incubation of 1 in culture with LacZ-transduced human cancer cells yielded 100-300 fold cytotoxicity enhancement compared to controls, confirming that 6 is a good substrate for the enzyme and has potential for use in ADEPT or GDEPT therapy. Variations on the above methods include MDEPT (melanocyte- directed enzyme prodrug therapy) and VDEPT (virus-directed prodrug therapy). In contrast to ADEPT and GDEPT, in MDEPT, for example, the activating mechanism depends specifically on the enzyme tyrosinase, already present in melanoma cells and uniquely associated with them, thus circumventing the need for prior enzyme delivery to the tumour site. Prodrugs have been developed to treat malignant melanoma using this targeting technique. Examples are shown in Figure 9.10. Prodrug 1 [32] was designed to contain a catechol moiety to take advantage of tyrosinase oxidation, which would lead to release of the drug (a phenol mustard in this case). A carbamate linkage acts as the spacer in this prodrug. A later study by the same group [33] showed compound 2, a urea prodrug that releases aniline mustard upon exposure to mushroom tyrosinase, to be a more suitable candidate for MDEPT than 1 because of its greater stability in sera. This result highlighted the possibility of increasing the stability and half- lives of prodrugs of this type under physiological conditions by replacement of urea for carbamate linkages. HO H N O N(CH2CH2Cl)2 1 HO O HO H H N N N(CH2CH2Cl)2 2 HO O Fig.9.10 Carbamate and urea prodrugs, candidates in MDEPT [32,33] Strategies for drug design 383 In the context of prodrugs useful in anticancer treatment, we should mention also those that are hypoxia-selective. Relatively high proportions of hypoxic cells in tumours, compared with normal tissue, enable their targeting by prodrugs that can undergo bioreductive activation, releasing the cytotoxic agent intracellularly. Several classes of bioreductive drugs are known and have been reviewed recently [12]. An important example is tirapazamine (3-amino-1,2,4-benzotriazine 1,4-di-N-oxide), which is selectively toxic to hypoxic cells present in solid tumours. At low oxygen levels, intracellular metabolism converts the prodrug to a radical that is extremely toxic, causing DNA damage. The toxicity towards aerobic cells is lower because the toxic radical reverts to tirapazamine in the presence of oxygen. Evidently tirapazamine may be activated by multiple nuclear reductases [34]. Novel drug design strategies for targeted anticancer therapy were recently highlighted in the literature, with particular emphasis on effective chemical linkages between the targeting molecule and the cytotoxic agent [35]. Illustrative examples of conjugates with monoclonal antibody (mAb) include prodrugs containing calichimeacin (anti-leukemic) and paclitaxel derivatives. In the first case, conjugation is achieved by introducing a pH- sensitive bifunctional linker, which facilitates intracellular release of calicheamicin. In the second mAb conjugate, following its endocytosis, metabolic activation by non-specific cellular esterases releases free paclitaxel. The macromolecular nature of the antibody scaffold may present some problems with distribution and clearance and there is a trend towards the design of new anticancer agents with low molecular weight ligands. Other macromolecules, including soluble polymers, have been employed in prodrug design to address drug solubility as well as stability issues, and problems such as enzyme degradation, hydrolysis and oxidative reduction that can occur in sera. Design strategies aimed at improving soluble macromolecular delivery systems have been reviewed [36]. While incorporation of macromolecules as constituents of prodrugs has in recent years contributed to increasing drug biovailability and reducing toxicity, there is a need for more effective constructs with additional capacities such as drug targeting and timed delivery. Covalent coupling of drug molecules to polymers such as polyethylene glycol (PEG) or its derivatives could render them more soluble and more stable to systemic degradation. PEG is an approved material for medicinal application owing to its biocompatibility and non-toxic, non- antigenic and non-immunogenic nature. Figure 9.11 shows examples of prodrugs obtained by covalent coupling of activated PEG to the antiherpes agents valacyclovir and acyclovir [37]. In synthesising the prodrugs, appropriate derivatisation of PEG was necessary since its terminal –OH groups are not suitable for direct attachment of the drugs. The 384 Chapter 9 acyclovir-polymer conjugate was thus obtained by attaching the drug to carboxyl-PEG using an ester bond. Similarly, for the valacyclovir prodrug, the active agent was coupled to chloro-PEG using a covalent C-N bond. In vitro studies on the release of the drugs from these conjugates were performed in different media. It was concluded that PEG-valacyclovir is the more suitable prodrug for therapeutic use owing to its higher stability in various media and its larger percentage of drug release over time. In particular, this prodrug was considered suitable for oral administration, whereas the PEG-acyclovir prodrug, with the higher rate of hydrolysis, was considered suitable for other forms of administration and in cases where rapid drug release is required. O O N NH HN N N N NH H2N N N 2 NH(CH2)2O(CH2CH2O)32-CH2CH2NH C O (CH2)2OCO CH HC COO (CH2)2OCH H 2 2 CH(CH3)2 CH(CH3)2 O O N NH HN N N N NH2 H2N N N CH2O(CH2)2OCO(CH2)2NH(CH2)2O(CH2CH2O)32(CH2)2NH(CH2)2COO(CH2)2OCH2 Fig.9.11 PEG-based prodrugs of valacyclovir (top) and acyclovir (bottom) [37] Advances in the development

of a number of acid-sensitive macromolecular anticancer drug delivery systems (DDSs), spanning the range from simple to site-specific antibody-targeted polymer-drug conjugates, have been reviewed [38]. A requirement for activation of these systems is that the spacer unit (separating the active drug from the polymeric backbone in the DDS) should be susceptible to lysosomal enzyme or chemical hydrolysis at physiological pH, or under conditions of environmental pH change. The principle behind this approach has proven to be valid, but much research is needed to optimise the performance of such DDSs. Because of their structural complexity, in vitro cytotoxicity studies are generally inadequate for assessing their activities and more feedback from in vivo studies in conjunction with computer-modelling are evidently necessary for their further development [38]. A considerable variety of prodrugs has been presented above with the intention of illustrating various design strategies for achieving different Strategies for drug design 385 delivery objectives, in particular the improvement of pharmacokinetic properties and overcoming metabolic hurdles. Further examples can be found in the reviews quoted above. The study of an account of the development of fosphenytoin [39], a prodrug of the sparingly-soluble anticonvulsant phenytoin, is strongly recommended. This offers a detailed account of how practical problems associated with drug development may be addressed and is of interest not only from the chemical synthetic viewpoint, but also indicates the importance of understanding aspects of formulation, pharmacokinetics and metabolism in prodrug design. The reader is especially referred to ref. 10, which is a recent commentary on the philosophy of prodrug research. One important conclusion that the author of the review draws is that the various objectives of prodrug development are interlinked, in the sense that the development of a prodrug may lead to beneficial properties over and above those originally intended by the structural modifications implemented. So, for example, improvement in drug solubility (a pharmaceutical objective) effected by prodrug formation may yield improved oral absorption (a pharmacokinetic objective). Similarly, unintended site-specificity might also result through improved chemical stability of the prodrug. A second observation that is made in ref. 10 regarding the prodrug approach is that it should be a promising chemical strategy in cases where there is a significant gap between the structural nature of the pharmacophore and other desirable pharmaceutical, pharmacokinetic or pharmacodynamic properties, since a traditional optimisation strategy would usually fail under these circumstances. Thus, many prominent medicinal chemists advise implementation of a prodrug strategy at an early stage of lead optimisation. On the other hand, some of the weaknesses of simple prodrugs stem from the single chemical conversion involved in their metabolism to yield the active species [1]. The potential advantage of multiple conversions is one factor that has contributed to the evolution of chemical delivery systems (see section 9.2.5). 9.2.3 The hard drug approach Hard drugs are pharmacologically active compounds that undergo little or no metabolism i.e. the term ‘hard’ is synonymous with ‘metabolically stabilised’ [40,41]. The concept implies that after exerting their medicinal effect, hard drugs are excreted by the body unchanged. An important advantage is that since metabolism is absent or very limited, the risk from toxic metabolites (as might occur with prodrugs, for example) is minimised. However, the reader will appreciate that to design a hard drug, a formidable strategy would be required to ensure that the candidate drug could escape 386 Chapter 9 biotransformation by the plethora of enzymes that mediate Phase I and Phase II biotransformations of substrates of extremely wide chemical and structural diversity (Chapters 2-3). Thus, in practice, achieving a high degree of metabolic stabilisation is often fortuitous but structural modification can improve ‘hardness’, as indicated in examples that follow. Enalaprilat (Figure 9.12) is a potent, orally active ACE inhibitor, regarded as an important example of a hard drug on account of its very limited metabolism and exclusive excretion via the kidney [2,42]. In contrast to the ACE inhibitor captopril, which contains a thiol function (believed to be the origin of its adverse side-effects through in vivo disulphide formation with endogenous proteins), enalaprilat is a carboxyalkyldipeptide whose structure was designed to effect significantly stronger inhibition of ACE than that displayed by captopril [43]. This goal was indeed achieved with concomitantly reduced side effects compared to captopril. COOR CH3 N N Enalaprilat: R = H H O COOH Enalapril: R = Et CH3 Captopril HS N O COOH Fig.9.12 Structures of ACE inhibitors [42] As it happened, the poor lipophilicity of enalaprilat (octanol/water partition coefficient ~0.003 [44]) due to the presence of two carboxylic acid groups in the molecule, resulted in poor oral absorption (<10%) [2]. The prodrug strategy described in section 9.2.2 was therefore subsequently employed whereby one of the carboxylic acid groups was converted into its ethyl ester, to yield the widely prescribed drug enalapril (Figure 9.12), with significantly improved absorption (~60%) [2]. This prodrug is metabolised by hepatic esterolysis to enalaprilat as the major metabolite [45]. Strategies for drug design 387 Bisphosphonates (Figure 9.13) were developed as inhibitors of bone resorption and display a remarkable metabolic stability, warranting their description as hard drugs [2,46]. The discovery of these compounds was based on earlier observations that inorganic pyrophosphates could bind to calcium phosphate, inhibiting the formation of calcium phosphate crystals and crystal dissolution in vitro, but lacking in vivo activity on bone resorption. Pyrophosphates (containing the P-O-P bond) were found to undergo rapid in vivo hydrolysis before reaching the site of bone destruction, whereas bisphosphonate analogues (characterised by P-C-P bonds) resisted biotransformation and successfully inhibited bone resorption. The bisphosphonates have high aqueous solubilities, lacking the typical substrate properties associated with metabolisable drugs. Consequently, they display simple pharmacokinetics and are exclusively excreted by the kidneys [46]. The high aqueous solubilities, however, result in extremely low oral bioavailability in humans (e.g. ~0.7% for alendronate). O OH O OH O OH Cl P HO P HO P OH OH OH Cl P OH NH2(CH2)3 P OH H3C P OH O OH O OH O OH clodronic acid alendronic acid etidronic acid Fig.9.13 Representative parent acids of bisphosphonates with the ability to inhibit bone resorption Drugs with metabolic stabilities approaching those of the biphosphonates are exceptional. More frequently, metabolic stability must be built in to lead compounds by appropriate systematic chemical synthesis. Furthermore, chemical modification should ideally not compromise pharmacological activity or potency. In some instances, other advantageous activities may fortuitously be gained during the iterative process of synthesis and biological evaluation. An example illustrating the input of metabolism data to drug optimisation concerns the development of analogues of the potent cholesterol absorption inhibitor 1, (-) SCH 48461 (Figure 9.14) [47]. Some detail is presented here to illustrate the interplay between SAR-input and metabolism data input in the process of drug discovery/optimisation, in this case with the intention of metabolic stabilisation. 388 Chapter 9 From metabolism studies, four primary sites of biotransformation in the molecule 1 had been identified (a,b – hydroxylation sites, c, d – demethylation sites) that led to no fewer than eleven metabolites. Since previous SAR-studies had indicated that the C-3 phenylpropyl group was an essential pharmacophore but could tolerate some modification, benzylic hydroxylation (site b) could be blocked via e.g. substitution of the benzylic C atom by an oxygen atom. Unexpectedly, this led to a significant reduction in potency of the product (±) 2 relative to (±) 1 (the racemate of (-) SCH 48461). It was then reasoned that (±) 2 might have been rendered metabolically labile by enhanced hydroxylation at site a relative to (±) 1, due to the presence of the electron-rich phenoxy group. c OMe OMe b a 3 4 N N O O d 1: (-) SCH 48461 OMe rac- 1 OMe OMe OMe O O F N N O O rac- 2 rac- 3 OMe OMe OMe O F resolution N 4: (-) SCH 53079 O rac- 4 Fig.9.14 Stages in the metabolic stabilisation of a cholesterol absorption inhibitor [47] Strategies for drug design 389 Hence, (±) 3 was synthesised, site a being blocked by a fluorine atom. This did in fact restore activity comparable to that of (±) 1. SAR considerations had also indicated that the methoxy group of the N-aryl moiety of 1 was not required for activity, a prediction confirmed by in vivo evaluation. Hence the p-methoxyphenyl moiety was replaced by a phenyl group and finally, resolution of the product (±) 4 yielded the eutomer (-) 4, designated (-) SCH 53079. The latter compound was found to be equipotent to (-) SCH 48461 and yielded only three metabolites as their glucuronide conjugates in animal studies. Two examples in the more recent literature that illustrate the iterative process of synthesis and biological evaluation to improve the metabolic stability of lead compounds warrant further study. One of these relates to the metabolic stabilisation of inhibitors of TNF-α, whose over-expression is implicated in a number of diseases [48]. The other concerns the metabolic stabilisation of a benzylidene ketal M2 muscarinic receptor antagonist [49]. The challenge in the latter case was to overcome (while maintaining M2 selectivity and affinity) in vivo cytochrome P450-catalysed oxidative cleavage of the methylenedioxy group in the lead compound 1 (Figure 9.15), leading to a catechol intermediate. The latter, if further oxidised to an ortho- quinone, could induce toxic effects by DNA alkylation. In summary, the steps involved in the overall metabolic stabilisation and retention of M2 activity of 1 included: (a) replacement of the metabolically labile methylenedioxy group with a p-methoxyphenyl group to yield 2 (with poor M2 activity compared to 1, however), (b) replacement of the sulphonamide with a naphthamide moiety to yield 3 (with restored M2 activity, but with the new moiety susceptible to undesirable metabolic oxidation to an arene oxide), (c) introduction of a fluorine atom at the 4-position of the naphthamide moiety (to optimise metabolic stability). O O a O O b O O N S N O S O N O N O 1 SO2-n-Pr MeO 2 SO2-n-Pr O O c O O O S N O S N F O N O N MeO 3 MeO O 4 O Fig.9.15 Stages in the metabolic stabilisation of an M2 muscarinic antagonist [49] 390 Chapter 9 Compound 4 demonstrated excellent M2 affinity and selectivity, and human microsomal stability [49]. As a ‘bonus’, 4 displayed better bioavailability in rodents and primates than 1. As a conclusion to this section, it is appropriate to comment on the question of metabolic stability in general and to reflect its current status in drug discovery and development. Ideally, metabolic stability is an issue addressed at the preclinical stage. Metabolism studies are thus used to identify candidate drugs with favourable pharmacokinetic and safety profiles for human administration. However, since new chemical entities cannot be administered directly to humans in the early stages of drug discovery, determination of their metabolic stabilities must rely on data from in vivo animal studies as well as in vitro cellular or subcellular (e.g. liver microsome) systems, and computational models. Part of this process involves the construction of libraries of compounds (typically based on combinatorial chemistry) and filtering them for metabolic stability in order to eliminate ‘junk’ leads [1]. The detailed protocol for preclinical metabolism studies and their utility in establishing the metabolic stabilities of candidate drugs, and hence predictions for human metabolism, have been described in a recent leading article [7]. In this context, computational models include those aimed at predicting specific enzyme-substrate binding affinity, the positions of metabolic attack in candidate drug molecules, and their rates of metabolism. While the success rates for prediction of in vitro properties from computational models appear to be improving, it remains true that accurate prediction from in vitro and animal studies to humans is still a significant challenge. Thus, qualitative and quantitative predictions of metabolic stability of new chemical entities in humans are still rather limited. 9.2.4 The soft drug approach This concept was introduced in the late 1970’s [50]. A soft drug is a pharmacologically active compound that is deactivated in a predictable and controllable way after it has fulfilled its therapeutic role [14]. A soft drug, therefore, not only possesses the required pharmacological activity, but also has built-in structural features that ensure its deactivation and detoxification in a desired way after it has carried out its biological action. Various subclasses of soft drugs have been defined and described in detail

[24], the most successful of these being based on the inactive metabolite approach and the soft analogue approach [14]. In the inactive metabolite approach [24], one begins with a known inactive metabolite of the lead compound. This is used as the basis, in the so-called ‘activation stage’, for design of new molecules that are isosteric Strategies for drug design 391 and/or isoelectronic analogues of the lead that gave rise to the inactive metabolite. Chemical modification of the inactive metabolite is thus intended to yield new molecules that would be metabolised to the inactive metabolite in a single step, thus ensuring the important requirement of ‘predictable metabolism’. To ensure also ‘controllable metabolism’ (that could influence e.g. the rate and/or primary site of metabolism) appropriate chemical modification is performed during the activation stage. An example from the class of soft β-blockers is illustrative [51]. Among the various metabolites of the well-known β-blockers atenolol and metoprolol (Figure 9.16) there is a common, significant metabolite (a phenylacetic acid derivative) that is known to be pharmacologically inactive. This molecule was therefore used to design new analogues of the β- blockers with predictable and controllable metabolism. Conversion of the carboxylic acid moiety of the inactive metabolite into an ester using a variety of substituents (R’ in –COOR’) thus yielded a family of soft β-blockers with variable transport properties and degradation rates, depending on the nature of R’. Thus, for example, with R’ = -CH2SCH3, the derived compound, due to its rapid hydrolysis in vivo, displayed ultra-short antiarrhythmic activity on intravenous administration. On the other hand, the ethyl adamantanyl derivative has proven to be effective as a topical antiglaucoma agent, producing significant, prolonged reduction of intraocular pressure. At the same time, it undergoes rapid hydrolysis in human blood, a most important advantage, since this eliminates undesired systemic activity. R Atenolol NH2 R R O common R inactive metabolite OH OR' Metoprolol O O soft β-blockers OMe (R = -OCH2CH(OH)CH2NH-iPr) Fig.9.16 Design of soft β-blockers based on the inactive metabolite approach [51] 392 Chapter 9 Essentially the same approach was adopted more recently to design ultra-short acting, soft bufuralol analogues, which are also β-blockers [52]. In this case the aromatic moiety of the lead compound, bufuralol, contains an ethyl substituent, which is metabolised to an acetic acid group in the inactive metabolite. Esterification of the inactive metabolite with various reagents led to seven soft analogues of bufuralol, all of which were demonstrated to undergo extremely rapid metabolism by blood and tissue esterases to the common inactive acidic metabolite. Four members of the series displayed β-blocking potencies ranging between 25 and 50% that of the lead bufuralol. An example of a highly successful corticosteroid designed on the above principle is the anti-inflammatory and anti-allergic loteprednol etabonate derived from prednisolone [1,8,53]. This compound is topically effective in the treatment of ocular inflammation, thereafter undergoing rapid biotransformation to inactive metabolites. Figure 9.17 shows the metabolism of loteprednol etabonate to its primary inactive metabolite [1,8]. In 1999, a turnover exceeding 250 million USD was reported for the drug [54]. O OCH2Cl O OH HO OCO2C2H5 HO OCO r 2Ct s 2Hes e a e 5 O O Loteprednol etabonate ∆1-cortienic acid 17α-ethylcarbonate Fig.9.17 Metabolism of loteprednol etabonate to its primary inactive metabolite [1,8] The clinical status and the success or otherwise of recently developed soft glucocorticoid steroids, including fluocortin-21 butyl ester, tipredane, butixocort propionate, itrocinonide, GW 215864 and loteprednol etabonate, have been reviewed [55]. In the search for safer dihydrofolate reductase (DHFR) inhibitors, the soft drug approach has recently been applied to synthesise a series of compounds in which the methylamino-bridge of non-classical inhibitors (e.g. trimethoprim) was replaced with an ester function [56]. These compounds were prepared as potential soft drugs intended for inhalation to treat Pneumocystis carinii pneumonia. This strategy anticipated rapid deactivation Strategies for drug design 393 of these lipophilic esters by ubiquitously distributed esterases following their therapeutic action in the lungs. An interesting feature of this programme was the use of an automated docking and scoring procedure as well as molecular dynamics simulations to select the target compounds for synthesis. Meaningful data could be obtained from the docking routines since high- resolution X-ray structures of the human reductase with and without complexed inhibitors are available. At this point, it is worth noting that the particular strategy employed in the examples above is a special case of the more general ‘retrometabolic drug design’ (RMDD) philosophy [8,24], manifested also in the design of more elaborate chemical delivery systems (CDSs) which are aimed at targeted drug delivery (see section 9.2.5). The general aim of the RMDD approach is thus to incorporate metabolism as well as targeting into the drug design process in a systematic way so as to derive safe, locally active compounds. The second category of ‘soft drug design’ referred to earlier, namely the soft analogue approach, differs from the inactive metabolite approach in that the new soft compounds are close structural analogues of the selected lead compound into which a metabolically ‘weak spot’ has been deliberately incorporated by chemical synthesis. Again, ideally a one-step deactivation and non-toxic products are desirable, the first requirement often being achievable if the sensitive part of the molecule is susceptible to hydrolytic metabolism. One area in which this approach has been employed is in the design of certain classes of long chain ammonium antibacterial agents [24,57-59]. In the case of cetyl pyridinium analogues, the ‘hardness’ of the parent cetyl pyridinium compound (containing the fully saturated N+–(CH2)15-CH3 chain) reflects that it requires several oxidative metabolic steps for its deactivation. As indicated earlier, oxidative metabolism generally leads to toxic intermediates. This hardness has been reduced significantly in the cetyl pyridinium analogue of Figure 9.18 by incorporation of the ester function, which lends itself to the facile and predictable metabolism shown [57,59]. The parent compound and the analogue display comparable activities as antimicrobials [24]. Because conventional long-chain quaternary ammonium compounds are used in massive quantities in a wide variety of pharmaceutical, domestic and industrial applications, there is serious concern regarding their effects on the environment. This is a strong motivation for producing novel soft quaternary ammonium compounds that can undergo facile degradation [59]. 394 Chapter 9 + Cl O metabolism O N C O (CH ) N + H 2 10CH3 2 H H cetyl pyridinium soft analogue + HOOC (CH2)10CH3 Fig.9.18 Metabolism of a soft analogue of an antibacterial [24,57,59] Two categories of soft drug design were highlighted above to illustrate some of the principles involved. Other categories include controlled-release endogenous agents, activated soft compounds and active metabolite-based drugs [24]. Controlled-release endogenous agents are derived from e.g. natural hormones and neurotransmitters. Appropriate chemical modification can result in retardation of their typically rapid metabolism and thus yield soft drugs with prolonged action and/or site-specificity. To generate activated soft compounds, a pharmacophore is introduced into the structure of a non-toxic, inactive compound; the activated form loses the pharmacophore in vivo, yielding the original non-toxic species. Finally, active metabolite-based drugs are oxidative metabolites of a parent drug that still retain activity and are consequently more readily inactivated in vivo. In the last case, for drugs undergoing sequential oxidative metabolism to yield eventually an inactive metabolite, some previous metabolite (e.g. ideally that preceding deactivation) could represent a useful drug. Choosing a metabolite that appears earlier in the metabolic sequence would be counterproductive, raising complications of control due to the simultaneous presence of a number of its active metabolites. Many further instructive examples from all of the subclasses of soft drugs can be found in the references quoted in this section, as well as in a very recent review that also features the role of in silico tools in the design process [60]. 9.2.5 Strategies based on Chemical Delivery Systems As discussed briefly in the above section, soft drug design represents one extreme strategy in the overall scheme of retrometabolic drug design (RMDD) [1,8,24,61]. The complementing strategy in RMDD is based on the concept of the ‘chemical delivery system’ (CDS), which evolved from prodrugs (section 9.2.2) in the early 1980s. The essential difference between prodrugs and CDSs is that the latter rely on multi-step activation and contain targetor moieties [62]. Thus, in the nomenclature used to describe these concepts, a prodrug (as defined in section 9.2.2) consists essentially of the Strategies for drug design 395 active drug (D) attached to ‘modifier functions’ (F1 - Fn), which control the molecular properties of the prodrug (e.g. by altering its lipophilicity, acting as protecting groups) [8,62]. But whereas prodrugs are often designed to overcome problems such as poor absorption and rapid first-pass metabolism (see section 9.2.2), they are not necessarily designed to ‘target’ specific tissues, organs or other sites in the body. On the other hand, in the design of a CDS, an inactive derivative of a drug D, the intention instead is to incorporate the goal of targeting, so that ideally the drug is released from its CDS only at the intended site, but is present as an inactive species elsewhere in the body, from where it is safely eliminated. In the general case, therefore, the CDS is designed as follows: an active molecule (the drug ‘D’) is synthetically transformed into an inactive molecule by attachment of not only modifiers (F1 - Fn), but also a ‘targetor function’ (T) [8]. The role of the T function is to effect a specifically higher or sustained concentration of the drug at the site of interest and ‘lock-in’, while the F functions may control other molecular properties of the CDS (as in prodrugs). The RMDD approach should ensure that on administration of the CDS, the steps leading to drug activation are predictable, sequential metabolic reactions, generating inactive intermediates, disengaging the F functions first and eventually the T function (once the latter has performed its targeting role). As regards the targeting aspect, this may rely on the prevalence of specific enzymes at the target site (e.g. in ocular delivery), or on transport properties that are site-specific (e.g. for delivery to the brain). One classification of chemical delivery systems includes the site- specific enzyme-activated CDS, the enzymatic physical-chemical based CDS, and the receptor-based CDS, and these have been illustrated with appropriate examples [24]. Some more recent examples of CDSs are described here, beginning with the simpler varieties and progressing towards more complicated ones. Many of these systems have been successful as ocular hypotensive agents and in the treatment of brain disorders (e.g. Alzheimer’s disease). The design of CDSs for ophthalmic drugs takes advantage of the fact that the various compartments of the eye are regions having particularly high concentrations of metabolising enzymes of wide variety [8]. Bioactivation and targeting then rely on successive reduction- hydrolysis metabolic steps. In the case of brain-targeting, the ‘lock-in’ principle is based on the fact that a lipophilic precursor (e.g. T-DF) will readily cross the blood-brain barrier (BBB), but following e.g. local oxidative enzymatic conversion to a hydrophilic species (T+ -DF), the latter becomes trapped and the drug D is finally enzymatically released within the brain. The challenge of drug delivery to the brain stems from the very special nature of the BBB, as described later. Adequate delivery of a host of drugs that act on targets in the brain is an essential requirement. Such drugs include antidepressants, anaesthetics, anticonvulsants, antibiotics, anticancer 396 Chapter 9 and antiviral agents. The CDS approach represents an important recent advance in meeting this challenge [63]. These concepts are illustrated with several examples below. The design, in vitro stability and ocular hypotensive activity of t-butalone CDSs have recently been described [64]. Here, the challenge was site-specific delivery to iris-ciliary body tissues of the active drug t-butaline 1 (Figure 9.19), a selective β2-adrenoreceptor agonist, normally used to treat bronchorestrictive disorders. Two issues had to be addressed to prepare CDSs suitable for ocular delivery, namely the delivery aspect and the local bioactivation aspect. OH O HO NHC(CH H 3) O 3 NHC(CH3)3 1 2 OH OH t-Butaline t-Butalone O RO NHC(CH3)3.HCl 3. R = -COCH2CH(CH3)2 4. R = -COC(CH3)3 OR t-Butaline CDS OH O HO NHCH HO 3 NHCH3 5 6 Phenylephrine Phenylephrone O RO NHCH3 7. R = -COCH2CH(CH3)2 8. R = -COCH2Ph 9. R = -COC(CH3)Phenylephrine CDS 3 Fig.9.19 Examples of chemical delivery systems for ophthalmic application [64-67] Strategies for drug design 397 Since t-butaline is relatively

hydrophilic, its permeability across biphasic corneal membrane is impaired, with the result that topical administration of its aqueous solution at 2% dose level does not alter the intraocular pressure (IOP) in test animals (normotensive rabbits) significantly. The lipophilicity of t-butaline was thus increased by esterification of the aromatic hydroxyl groups. The incorporated diisovaleryl- and dipivalyl-substituents thus correspond to the ‘modifier’ functions, which in this case facilitate corneal permeability. The ‘targeting’ aspect was addressed by converting the remaining hydroxyl group to a keto- function [64]. The rationale for the above approach was based on the analogy with esters of adrenolone, which were shown in previous work [65] to undergo a site-specific reduction-hydrolysis metabolic sequence by reductases and esterases present only in the iris-ciliary body, to the corresponding adrenaline derivatives. This is a key feature in the design strategy for ocular delivery. In the case of t-butaline, these chemical modifications thus produced the CDSs 3 and 4 (Figure 9.19), described as bioreversible diacyl derivatives of t-butalone 2. It is important to note that, in keeping with the CDS concept, t-butalone 2 is an inactive precursor of the active drug 1 and that the strategy being employed here is the site-specific CDS approach, based on predictable multi-step metabolic activation of bioreversible, inactive compounds at the site of action. These CDSs thus fall into the category of site-specific enzyme-activated CDSs [24]. Favourable results for the IOP-lowering and in vivo disposition of the dipivalyl terbutalone 4 in rabbits were reported [66] as were details of the synthetic procedures for the CDSs 3 and 4 and their comparative biological evaluation [64]. The outcomes of primary interest here were that both CDSs 3 and 4 exhibited a significant ocular hypotensive activity and that duration of action was found to be dependent on the ‘modifier’ function i.e. it can be controlled by judicious choice of steric bulk in the esterification step. A wide variety of ester functions may be employed to alter the drug lipophilicity in CDSs of the type described above. For ocular delivery, however, strongly lipophilic functions have been employed for optimal corneal penetration. For example, in earlier studies of the design of soft β-blockers for ophthalmic use, the ester groups included the cyclohexylglycol, adamantylmethyl, adamantylethyl, endo- and exo- norbornyl, and isopinocamphyl functions [8]. The strategy described above for transforming t-butaline into a CDS has also recently been employed for phenylephrine 5 (an α1-selective adrenergic agonist used in the eye for its mydriatic effect), by synthesising esters of the inactive ketone precursor phenylephrone 6 [67] (Figure 9.19). 398 Chapter 9 In this case, esterification of the single phenolic group on the molecule was performed with the isovaleryl, phenylacetyl and pivalyl functions, and the mydriatic effect and ocular distribution/metabolism of the three resulting compounds 7-9 were investigated. Whereas phenylephrone showed no mydriatic activity whatsoever, the three esters displayed a significantly more potent mydriatic effect than phenylephrine, the phenylacetyl ester 8 being the most potent, with a short duration of action. It should be noted that for ocular delivery of the parent compound (phenylephrine hydrochloride), very high concentrations are usually employed because of the poor penetration of this hydrophilic drug into the epithelium of the cornea. This results in drainage of the drug into the nasolacrimal duct, with subsequent systemic distribution and primary systemic side effects. This is a common problem with administration of β-adrenergic antagonists directly into the eye and can result in heart-rate reduction (e.g. as found with the first widely used anti- glaucoma drug Timolol) or adverse respiratory events. A major advantage of the derived CDSs, such as that described for phenylephrine, is that the active drug is generated metabolically only in the iris-ciliary body tissues of the eye and is undetected in the systemic circulation. The site-specific chemical delivery systems described above rely on ocular bioactivation for their drug targeting. One aspect that has been neglected thus far in describing the above systems is the question of stereospecificity. This important issue is often not explicitly mentioned in reported studies. It should be noted, however, that earlier studies based on ocular bioactivation of a CDS [68-72] showed that the released drug was the active (S)-isomer. Here the CDS was of the ketoxime-type i.e. an oxime or alkoxime, derived from the ketone corresponding to the β-adrenergic antagonist (Figure 9.20). In step 1, the CDS is metabolised by an oxime hydrolase present in the eye, reverting to the ketone from which it was chemically derived. The product is then metabolised in step 2 by a ketone reductase to give the active amino alcohol, In this case, the second step turned out to be stereospecific, yielding the active (S)-isomer [2,68]. One of the potential drug candidates that resulted from this strategy was alprenoxime, a CDS for the well-known β-blocker alprenolol. A shortcoming of the oximes, however, was their chemical instability in aqueous media, which shortened their shelf-lives. Subsequent conversion of the oximes (R = H in Figure 9.20) to the methoximes (R = CH3) led to a significant increase in chemical stability and improved the overall performance of the CDS. Strategies for drug design 399 NOR O 1 ArOCH2 CH2NHR' ArOCH2 CH2NHR' 2 R = H, CH OH 3 R' = alkyl ArOCH2 * CH2NHR' Fig.9.20 Sequential metabolism of a ketoxime with a stereospecific outcome [2,68] The examples above illustrated design principles for the eye as the target organ for drug delivery. Here we discuss drug targeting to the central nervous system (CNS). Earlier, it was noted that the BBB represents a major obstacle for drug delivery to the brain. This is due to its unique lipoidal bilayer structure, which prevents the passage of hydrophilic drugs to the CNS. The use of a lipophilic prodrug may improve the level of drug uptake by the brain, but its efflux is likewise enhanced, resulting in low tissue retention. Poor selectivity (such prodrugs may enter other tissues as well) and the possibility of reactive catabolism of lipophilic prodrugs are additional factors that may not lead to an increase in the therapeutic index of a drug intended for delivery to the brain [1,63]. An essential concept in developing CDSs for brain-targeting is that of ‘lock-in’, i.e. ensuring that once the relatively lipophilic CDS has crossed the BBB, it is retained there. The strategy used to achieve this is to design the targetor moiety T to be susceptible to enzymatic transformation that converts it into a hydrophilic (commonly positively charged) moiety T+-D (D = drug) that is then unable to exit the BBB. (The reader will no doubt recognize the conceptually analogous trapping of phosphate esters depicted in Figure 9.4, following diffusion of their lipophilic prodrugs into cells and subsequent hydrolysis to negatively charged species). Localisation of the modified CDS in the brain allows further, predictable metabolism that eventually releases the active agent. It should be emphasised that following administration and distribution of the CDS, the same enzymatic conversion to the strongly hydrophilic species T+-D that occurs in the brain takes place elsewhere in the body, thus accelerating its peripheral elimination [24], in this way therefore contributing to brain-targeting. The most successful brain-targeting approaches have incorporated, within the CDS, redox targetors, analogous to the NAD(P)H ↔ NAD(P)+ coenzyme system [24,11]. Systems based on the 1,4-dihydropyridine ring, 400 Chapter 9 whose chemistry has been extensively studied [73], have been found to be particularly versatile. Figure 9.21 shows schematically (1) the passive diffusion across the BBB of such a CDS containing both the drug D and a targetor moiety based on the lipophilic 1,4-dihydropyridine system, and (2) oxidation of the targetor moiety to the highly hydrophilic quaternary ammonium ion, mediated by oxidases in the brain, and resulting in ‘lock-in’. Subsequent sustained, brain-specific release of the drug D is generally effected by hydrolysis mediated by appropriate esterases. The strategy above has been applied to drugs from a wide range of classes [24,63] that includes e.g. steroid hormones, anticancer agents, antiviral and antiretroviral agents. H H H H COXD COXD COXD 5 4 3 6 1 1 2 N 2 N N+ R R R BBB Fig.9.21 Targetor moiety for a drug D based on the 1,4-dihydropyridine system ( X = N, O) [24,11] Advantages of the employment of the 1,4-dihydropyridine ring as the targetor in such CDSs include the fact that it possesses a suitable degree of lipophilicity for penetrating both the BBB and other membranes, that its enzymatic oxidation to the T+-D form proceeds at a reasonable rate, and that it may be suitably functionalised to link to a given drug D. The use of phospholipid prodrugs for delivery of antiviral drugs such as AZT to the CNS was described in section 9.2.2 above. While such prodrugs may lead to improved transit into the CNS, extraction of these lipophilic compounds into other tissues may occur. This lack of selectivity may lead to serious side effects due to the potency of the antivirals. In a recent review describing the general problems associated with delivery of antiviral nucleosides to the CNS [25], the merits of chemical delivery systems based on redox trapping in the brain have also been discussed. A strategy based on the targeting methodology shown in Figure 9.21 above has been employed for delivery of drugs such as AZT to the CNS. Figure 9.22 illustrates such a CDS for AZT. As the drug molecule contains a single primary alcohol group, this is a convenient site for placement of the targetor moiety. This CDS relies on the versatile 1,4-dihydropyridine system for Strategies for drug design 401 effective targeting, and various derivatives (with R = e.g. Me, Et, Pr, i-Pr, Bz) have been investigated [74-75]. Such compounds easily penetrate the BBB and their bioactivation involves (a) conversion to the hydrophilic quaternary ammonium species by oxidoreductases, effecting ‘lock-in’, and (b) subsequent hydrolysis by esterases, releasing the AZT in a sustained manner. O O Me HN Me HN O N OCO O N OH N N 3 N3 O R O AZT-CDS AZT N O OH O OCO OH OH HO HO O O Hydrocortisone-CDS Hydrocortisone Fig.9.22 Examples of chemical delivery systems incorporating redox targetors [50,73-75] The polarity of the AZT-T+ species formed in the brain is orders of magnitude greater than that of the AZT-CDS, accounting for rapid peripheral elimination and effective ‘lock-in’ after its formation in the CNS [25]. Administration of the CDS consequently produces significantly higher AZT levels in the brain than does dosing of unmodified AZT. A systematic study of the effect of dihydronicotinate N-substitution on the brain-targeting efficacy [75] showed the N-propyl CDS to be the most efficient compound of the series examined. Brain-targeting of pharmacologically active steroids can be effected using an analogous strategy. An important example of a very promising CDS designed for the delivery of estradiol (E2) to the CNS has been described [62, 63]. This CDS was obtained by attaching the 1,4-dihydrotrigonelline targetor moiety to the 17-hydroxy function of the steroid. Intravenous administration of the E2-CDS to rats led to confirmation of the lock-in mechanism, 402 Chapter 9 sustained release of E2, and significantly elevated levels of the drug compared to those after simple E2 treatment. Potential applications of the E2- CDS include treatment of Alzheimer’s disease and menopausal hot flushes. Phase I and Phase II clinical trials were reported as being in progress in 2001 [62]. Certain chemical shortcomings of the 1,4-dihydropyridine system were, however, noted recently in connection with the development of some steroidal CDSs [73]. While 1-alkyl-1,4-dihydropyridines easily undergo oxidation to their corresponding quaternary ammonium salts, 3-substituted- 1,4-dihydropyridines are known to be susceptible to hydration at the 5, 6-double bond (see Figure 9.21 for ring-numbering), this reaction leading to products that can no longer undergo metabolism into their quaternary forms. Hydration is favoured under acidic conditions and this could have a negative impact on pharmaceutical formulation, leading to products with unacceptably short shelf-life. Thus, replacement of the hydrolytically labile 1,4-dihydropyridine ring with less reactive systems, such as those based on 1,4-dihydroquinoline and 1,2-dihydroisoquinoline, were investigated [73]. An example of a CDS for delivery of hydrocortisone (HC) based on the use of a 4-substituted-1,2-dihydroisoquinoline targetor is also shown in Figure 9.22 [73]. Its bioactivation involves formation of the hydrophilic, ‘locked-in’ species T+-HC (following diffusion of the HC-CDS across the BBB and enzymatic oxidation) and final hydrolysis, which releases HC. The testosterone analogue was also synthesised and

likewise evaluated for chemical stability studies and in vivo animal distribution studies. In the case of the HC-CDS, metabolism leading to prolonged release of HC in the brain of Sprague-Dawley rats was evident, whereas the blood levels of the CDS, its quaternary intermediate and the parent drug fell to undetectable values after a much shorter period. Interestingly, the analogous CDS for testosterone behaved differently, no testosterone being detected in the brain. This was attributed to its excessively slow release rate by hydrolysis. The general conclusion of these studies, however, was that the 4-substituted-1,2-dihydroisoquinoline targetor moiety shows promise for brain-specific CDSs owing to favourable rates of metabolic activation as well as its chemical stability [73]. In particular, the desired result, a significantly reduced tendency to undergo hydration compared with 1,4-dihydropyridine, was confirmed, as was the greater stability of the alternative targetor ring-system towards aerial oxidation, these factors being favourable for formulation. The same basic type of targetor moiety used in the above examples has been employed in considerably more elaborate CDSs designed for neuropeptide delivery to the brain, a topic that has recently been reviewed [63]. Many CNS disease states have potential for treatment with neuropeptides, but compounds in this class are notoriously difficult Strategies for drug design 403 candidates for delivery to the BBB due to their rapid degradation by peptidases. More than a decade ago, a strategy was reported for delivering peptides into the CNS via a CDS that relied on sequential metabolism [76]. The challenges that such delivery presents are considerable. In designing an appropriate peptide CDS, the strategy must ensure, at minimum, a suitable level of liphophilicity for BBB penetration, prevention of premature inactivation of the peptide, as well provision for targeting to allow controlled metabolic release of the peptide in the brain. The actual strategy employed has been described as an extension of the CDS approach to a ‘molecular packaging strategy’ [63], since it involves ‘disguising’ the peptide entity within a bulky molecule that is both lipophilic (for passive diffusion through the BBB) and that will evade recognition by peptidases. Moreover, the bioactivation of such a molecular package might involve as many as five or six metabolic steps, whose timing is crucial to successful peptide delivery. Part of the strategy therefore requires incorporation of a spacer unit between the peptide and the targetor to control the timing for targetor release. Much research using this approach has been aimed at delivery of thyrotropin-releasing hormone (TRH), or Pyr-His-Pro-NH2 and its analogues [63]. TRH is the primary neurotrophic hormone for secretion of thyroid- stimulating hormone and has beneficial effects in the treatment of e.g. memory impairment and amyotrophic lateral sclerosis. To put this into context, it should be mentioned that the simpler prodrug approach has also been employed to enhance delivery of TRH. One such study specifically focused on improving the lipophilicity of TRH and reducing its susceptibility to rapid enzymatic inactivation in the systemic circulation [77]. The prodrug strategy adopted involved N-acylation of the imidazole ring of the histidine residue with various chloroformates. N-alkoxycarbonyl prodrug derivatives were found to be resistant to enzymatic cleavage but underwent the desired facile bioreversal quantitatively to TRH via spontaneous hydrolysis or by plasma esterase-catalysed hydrolysis. These prodrugs were also significantly more lipophilic than the parent TRH. An example of the ‘molecular packaging strategy’ CDS approach to peptide delivery is illustrated in Figure 9.23 and refers to a system designed to transport a pyroglutamyl peptide amide to the CNS, namely the TRH- analogue, Pyr-Leu-Pro-NH2. This CDS incorporated a Gln-Leu-Pro-Gly progenitor sequence of the above analogue [78]. Whereas previous methodology used by the same group was applicable only to neuropeptides containing free amino and carboxylic acid terminal functions [76], the example cited thus involved an extension to peptides with N-terminal pyroglutamyl (Pyr) and C-terminal carboxamide functions. In designing the CDS, the free carboxylic acid 404 Chapter 9 function of the glycine residue was rendered lipophilic by esterification with the bulky cholesterol molecule. O H O N C O H O H2 O N N N N H O H (C O N H2)2 CONH2 CDS O NH H O 2 O N N N H O Pyr-Leu-Pro-NH2 Fig.9.23 Example of a CDS designed to deliver a peptide to the CNS [78] The targetor, a 1,4-dihydrotrigonellyl unit, was appended to the progenitor via a spacer unit, namely alanine. This elaborate CDS, comprising four units (a targetor, a peptide, a spacer unit and a lipophilic moiety) was shown to undergo a series of metabolic reactions, eventually releasing pharmacologically significant quantities of Pyr-Leu-Pro-NH2 in the CNS of mice, following intravenous injection. It should be noted that, as with the HC-CDS described above, the first step in the bioactivation of the peptide-CDS following passive transport across the BBB is oxidation (NAD ↔ NADH) in the brain. The remaining bioactivation steps include cleavage of the cholesterol moiety by an esterase or lipase, and subsequent stepwise metabolic processes that are respectively mediated by (a) peptidyl glycine alpha-amidating monooxygenase, (b) dipeptidyl peptidase and (c) glutaminyl cyclase, the latter enzyme generating the peptide Pyr-Leu-Pro-NH2. The intermediate structures in this sequence can be found in ref. 78. The application described for this particular compound thus highlighted the potential of incorporating within the CDS a Strategies for drug design 405 suitable peptide progenitor with predictable bioactivation, as an extension to the existing methodology for synthesising peptide-CDSs. The use of macromolecules in prodrug development to improve drug stability and bioavailability was described earlier. However, targeting of tissues using drugs attached to macromolecules such as neoglycoproteins and synthetic polymers has also been explored. This approach has been reviewed, with emphasis on anti-HIV therapy [79]. Many of the recently developed systems are based on a model comprising the drug molecule, solubiliser moieties (e.g. carboxylic, hydroxyl), spacer units that link drug molecules to the macromolecular carrier, and targeting moieties. The function of the spacers is to undergo chemical or enzymatic hydrolysis to release the drug. In contrast to simple prodrugs, drug-polymer conjugates can only be taken up by cells via pinocytosis. Neoglycoprotein carriers have been successfully conjugated to anti- HIV drugs to produce prodrugs [80]. An example is the drug zidovudine, in the form of its 5’-monophosphate, AZTMP. Brain-targeting of AZT has also been effected by linking the drug in the form of its succinate to e.g. the anti- transferrin receptor antibody OX-26 [81]. In vivo studies indicated that the conjugate did indeed target brain capillaries and released the drug rapidly in situ. Conjugation of AZT by means of a succinate spacer with the water- soluble synthetic polymer α,β-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) has also been achieved [82]. Release of AZT from the resulting macromolecular prodrug was tested under a range of pH conditions. Efficient bioactivation by plasmatic enzymes was evident from the fact that more than 60% of linked AZT was released from the conjugate in plasma. The intention in the above section was to highlight the range of ideas currently being employed in the creation and development of novel chemical delivery systems, with an emphasis on those that employ targeting and that take advantage of predictable metabolism for their activation. Some of the challenges encountered in these areas of research have also been mentioned. 9.3 THE ROLE OF FORMULATION All of the approaches to drug design described in the previous sections involved creation of new entities from existing, active agents in an attempt to overcome problems relating to pharmacokinetics, or to take advantage of controlled or predictable metabolism for bioactivation, or to achieve drug targeting, or a combination of these. In all cases, chemical synthesis, and hence the formation of new covalent bonds, was required to effect the necessary molecular modifications to arrive at prodrugs, hard drugs, soft drugs and chemical delivery systems. 406 Chapter 9 In concluding this chapter it is appropriate to remind the reader that several negative aspects associated with drug delivery may be satisfactorily addressed by alternative, ‘softer’ approaches. In Chapter 1, a number of methods for improving oral absorption (e.g. by increasing aqueous solubility and drug stability, by reducing first-pass metabolism) were described. To mention a few approaches, these included e.g. selection of the appropriate solid form of the compound for formulation, the use of proliposomes to effect controlled drug release, enteric-coating to reduce first-pass metabolism, achieve tissue targeting and improve drug safety profiles, bioadhesive nanoparticles to reduce pre-systemic metabolism, and cyclodextrin inclusion to promote drug stability, increase bioavailability and reduce gastrointestinal irritation caused by NSAIDs. The medicinal chemist needs to keep such alternative formulation approaches in mind, since they might well serve in overcoming specific problems presented by new drug candidates. To stress the significance of formulation issues, we highlight just two of the above aspects, which happen to fall within our own research interests, namely drug polymorphism and cyclodextrin inclusion of drugs. A very fundamental issue that is sometimes overlooked by synthetic chemists involved in drug discovery and design is the crucial importance of selecting the ‘correct’ solid form of the drug candidate intended for further development in an oral preparation (e.g. tablet, capsule). Often, this is the most stable (least soluble) solid form, but there may be good reasons for developing a metastable form of higher solubility. The recognition that every organic compound can potentially exist in multiple solid forms (polymorphs, solvates), each with a unique thermodynamic stability and solubility, and that physical and chemical factors during the manufacturing and processing stages can effect interconversion of solid forms, has alerted the pharmaceutical industry in recent years to the need to monitor the integrity of a drug substance from the point of its initial crystallization through to the finished product [83-85]. A notorious recent case involving the antiretroviral drug ritonavir illustrates the dire consequences of unexpected solid-state transformation in the pharmaceutical industry. In short, two years after successful marketing of this drug, several lots of capsules failed dissolution testing due to precipitation of the drug from semi-solid dosage forms. This was traced to conversion of the original crystalline form I of ritonavir to a thermodynamically more stable form II, with a solubility only ~25% that of form I. Rapid pervasion of form II followed and attempts to recover form I failed initially, necessitating temporary reformulation. A detailed, expensive and time-consuming investigation later revealed that the dramatic difference in solubility was due to significant differences in the hydrogen bonding arrangements in the two crystalline modifications of ritonavir. The source of Strategies for drug design 407 form II was attributed to probable heterogeneous nucleation by a degradation product. A detailed account of this unwelcome occurrence of polymorphism has been published [86] and its perusal is highly recommended. The chemical stabilities and bioavailabilities of many drugs have been improved by their encapsulation within cyclodextrins [87-88] and this is consequently one formulation technology that continues to be widely applied in the pharmaceutical industry. What may not be widely evident, however, is that the significant benefits that this technology brings may be applied not only to active drugs, but may even enhance the properties of e.g. prodrugs that have themselves been derived as carefully designed chemical drug delivery systems. In support of this statement, we cite the recent use of cyclodextrins in attempts to increase the aqueous solubility and stability of the designed soft corticosteroid loteprednol etabonate [89], whose structure, design strategy and bioactivation were described in section 9.2.3. In fact, a recent, authoritative review [90] reminds us that in the process of retrometabolic drug design of chemical delivery systems (CDSs), aimed at ‘identifying new drug candidates with improved therapeutic indices based on predictable/controlled metabolism and/or site-targeted delivery’, issues such as dosage form stability, solubility and dissolution properties are crucial for successful drug performance and pharmaceutical acceptability. Examples quoted include the use of the highly soluble 2-hydroxypropyl-β-cyclodextrin that has been successfully employed in stabilising and improving the water- solubility of a number of chemical delivery systems for parenteral administration, including, for example, the AZT-CDS and the estradiol-CDS described earlier. 9.4 CONCLUDING REMARKS In this chapter, the authors attempted to show how considerations of pharmacokinetics and metabolism guide the process of developing drugs with improved delivery characteristics and the ability to target specific organs or tissues so as to maximise therapeutic efficacy. In reviewing some of the main approaches adopted to achieve these ends, we have deliberately omitted detailed chemical methodology, which we consider as secondary

to the design concepts. Aspects of the synthetic methodology feature in many of the papers and reviews cited above. Compilation of this modest review owes everything to the eminent scientists whose original, imaginative concepts outlined above have been responsible for the creation of new generations of effective drugs. One message that is implicit in the above review is that due recognition of pharmacokinetic issues and a deeper insight 408 Chapter 9 into the nature of drug metabolism will contribute to more successful application of the principles outlined above to the design of new drug molecules. The major thrust of this monograph has in fact been to elucidate the complex nature of drug metabolism and its ramifications in medicine. 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Pharmazie 57:94-101. Index absorption 3-20 alclofenac 79 enhancers 12-13 aldehyde oxidase 98, 201 factors that influence 12-17 alicyclic amines 85 mechanisms of 17-20 aliphatic hydroxylations 57 routes of 4-11 allele 273 acetaldehyde 79 allylic positions 70 acetaminophen 63 amide hydrolysis 115 acetonitrile 71 amlodipine 99 acetyl CoA 138-141 amino azaheterocyclic compounds 87 acetylation 138 aminoacid conjugation 155 CoA-S-acetyltransferase (CoA-S-) 138 amphetamine 219, 246, 304 N-acetyltransferases 141 androgens 62, 345 reaction mechanism of, 141-144 aniline 230 polymorphism of, 284 antagonist 27 acoxyl (acetoxy-methyl) 112 antagonistic interactions 296 acyclovir 315, 379, 383 antipyrine 194, 231-232, 328 acylation 152, 403 arachidonic acid 198 additive (synergistic) interactions 296 aromatase 61-62 adenine 87 aromatic amines 130, 132, 136, 154, 212 S-adenosylmethyionine (SAM) 148 aromatic azaheterocycles 98 synthesis, biochimic role 148-149 aromatic heterocycles 201 ADEPT(antibody-directed-enzyme aromatic hydrocarbons prodrug therapy) 372 aromatic hydroxylation 52, 55, 58 ADME concept 1-2, 36 aromatic substrates 52 ADMET 350 aryl hydrocarbon hydroxylase 63, 224 adrenaline 147 autosomal dominant 273 adverse reactions 329-348 autosomal recessive 273 adverse effects 46, 215, 219, 257, 295-6al azo compounds 103, 336 effects on drug metabolism 243-244, 254-257 bacampicillin 374-375 age 15, 70, 154, 190, 217, 228, 234, benzene 53 243-244, 254-255, 257, 269, 307, carcinogenity 54 332, 345-348 hydroxylation 54 agonist 27 benzo[a]pyrene 76 alcohol 47, 59, 101, 105, 113, 135, 152, hydration of, 116 195, 202, 212, 220-221, 259-260, 270, benzoic acid 284 295-297, 315, 327-329, 337, 382, 398, bezafibrate 262 400 bilirubin 131, 134, 138,203-204, 211, inductive effect, toxicity 259-260 213 alcohol oxidation 58-59 bioavailability 2, 4-8, 12-13, 17, 25, 113, alcohol dehydrogenase 202 130, 153, 155, 162, 165, 220, 231, 257, aldehyde dehydrogenase 101 301, 306, 325, 343, 350, 372, 375, aldehyde oxidases 98, 201 387, 390, 405 aldehydes 58, 98, 105, 201 bioequivalence 12, 255 adverse reactions 327, 329-348 biophore 2 415 416 Index biotoxication 41 cyano groups 68, 71 bisphosphonates 256, 387 cyclodextrins 13, 407 budesonide 8 cyclophosphamide 218, 278, 310, 337, 344-345 caffeine 89 cyclosporine A 249 N-demethylation 85-86, 89, 311 cytochrome P450 30,49,189-193 capecitabine 378 mechanism of action, 192-193 carbamazepine 78 multiple forms of, 193-195 carbohydrates, polymorphisms of, 275-283 effects on drug metabolism 222-223 carbon oxidations 52 DAO (diamine oxidase) 95, 99 sp3-hybridised carbon atoms 59-71 dapsone 251 sp2-hybridised carbon atoms 77-79 dealkylation 49-50, 60, 82, 88-89 in aromatic rings 71-77 deamination 99-100, 246 sp-hybridised carbon atoms 79-81 debrisoquine 275-276, 298 carboxylic acid 58, 132, 141, 152, 165, dehalogenation 82, 92, 105, 195 386, 391, 403 dehydrogenases 202 carboxylesterases 107 desoxyacyclovir 379 carcinogenesis 41,54,119, 134, 224, 270, dextromethorphan 44 331,333 diazepam 89 cardiac glycosides 62 N-demethylation 89 catalyst 172 diclofenac 162-163 catechol 53 dietary factors, in enzyme cefuroxime axetil 110 induction and inhibition 220-234 chemical delivery systems 394-405 digitoxin 320 chitosan microspheres 7 digoxin 298, 301-304, 316-317, 322, 324, chloramphenicol 113 346, 348 esters of, 113 1,4-dihydropyridines 86 salts of, 114 diltiazem 195, 305, 320 chlordiazepoxide 328 diols 76, 116 chlorobenzene 146 disease, chlorpromazine 282, 299, 320 effect on drug metabolism 258-261 chlorpropamide 64, 298, 348 distribution 21 cholesterol 7, 301, 387 disulfiram 305, 318, 322 cholestyramine 301, 311, 315, 318 dopamine 149, 151, 154-155, 296, 298 chrono-pharmacogenetics 271 drug action 25-28, 41, 214, 254, 377 ciprofibrate 261 drug design 117, 166, 271, 351 cisplatin 260 strategies in, 369-417 clearance 35-36 drug-drug interactions 295-297 clofibrate 262 associated with the pharmacodynamic cocaine 195 phase, 297-300 codeine 135 pharmacokinetic interactions, 300-305 O-glucuronidation 135 during the biotransformation phase, co-enzyme A 141-142 305-308 co-factors (coenzymes) 148, 180 drug-enzyme interaction 28 condensation reactions 155 drug interactions with other entities 325 conjugation reactions 29, 41, 129, 147, drug-food interactions 325-327 152-155, 202, 229 interactions with alcohol 327-328 controlled-release preparations 7 tobacco smoking, copper-containing amine oxidases 99 effect on drug metabolism 232-234, corticosteroids 44, 211, 299, 310, 316, 328-329 319, 323 Index 417 drug metabolism 29 flampropisopropyl 93 factors affecting, 47 flavin-containing monooxigenases 96,195 differences in pregnancy 344-345 mechanism of action, 196-197 in infants and children 345-346 food in elderly 346-348 effects on drug absorption 5-6, 11, 13 phase I reactions 29, 41-42 drug-food interactions 325-327 phase II reactions 29, 41-42 free radicals 60, 117, 213, 259 the hard drug approach 385-390 functionalization (phase I) products 42 the soft drug approach 390-394 drug receptor interaction 25 GDEPT (gene-directed enzyme prodrug therapy) 372 ecogenetics 270 genetic factors 89, 234, 263 efavirenz 248 effect on drug metabolism 269-287 effectors 181 genetic polymorphism 31, 272, 274, 287 activators 183 genotype 273 inhibitors, types of, kinetics, 181-182 glucocorticoids 9-10, 147, 281,309 elimination rate 34 glucuronidation 129-138 empenthrin 252 enzymology 130-131 enalapril (enalaprilat) 165, 386 general mechanism of, 132 encainide 165 N-glucuronidation 136-137 endogenous metabolism 194 O-glucuronidation 134-136 endoplasmic reticulum 30, 42, 48-50, polymorphism of, 137 130, 159, 189-190, 202, 212 glutathione 144 environmental factors, glutathione conjugation 144-147 effects on drug metabolism 262-263 major types of, 145 enzymes 219-233 further possible metabolism, 146 general mechanisms of actions,173-174l , glutathione-S-transferases 203-204, 285 mechanism of action at molecular leve glutethimide 70 183-185 glycine conjugation 153 induction 210-214, 307 ‘green pigments’ 80 inhibition 214-219, 305 non-protein catalysts, 188 haem regulation of activities, 185-188 in cytochrome P450 biosynthesis 190 specificity of, 185 haemproteins 49, 127, 190 epoxides 52, 60, 71-72, 76-78, 116, half-life 35, 166, 211, 244, 247, 286, 300, 201, 203, 370 314 epoxide hydrolase 201-202 haloperidol 46 erythrocytes, halothane as potential carriers for drugs 7-8 oxidative dehalogenation of, 92 erythromycin esters 112 reductive metabolism of, 105 ester (hydrolytic) cleavage 108-115 hard drugs 371, 385-390 esterases 202 hepatic clearance 347 ethnopharmacology 270 hepatotoxicity 92, 134, 221, 259, 307, excretion 32, 41, 44, 62, 86, 131,147, 315, 323, 325, 327, 333,337, 339, 343 159, 162, 166, 204, 256,283, 296, 300, heterolytic cleavage 192 304, 310, 324-325, 332, 344, 348, heterozygous 273 386 hexobarbital 70 ‘extensive metabolisers’ 274 histamine 96 histamine N-methyltransferase 149 fatty acid conjugation 155 homozygous 273 felodipine 86 hormones, fenofibrate 262 effects on drug metabolism 261-262 418 Index hydration 201, 402 loteprednol etabonate 392 hydrazine 88 β-lyase 151, 339 hydralazine 88 hydrazones 336 macrolide antibiotics 69, 212, 281, 311, hydrogen peroxide 95, 98 318, 350 hydrolysis 7, 29-30, 42, 80, 82, 88, 107 magnesium 149, 188, 228, 230, 300, 303, 116-117, 129, 131, 140, 147, 165, 320, 184, 202, 251-252, 275, 375-378, 383- MDEPT (melanocyte-directed enzyme 384, 387, 391, 399-405 prodrug therapy) 382 hydrolytic cleavage 107, 136 melagatran 372-373 hydroperoxidase 97 mephenytoin 73, 278-280, 286 hydroxylation 30, 49-50, 52, 54-75, 79, mercapturic acid 144 83-84, 87-90, 93, 156, 163, 195, 217- mercury 216 218, 221, 224, 226, 250, 278, 287, metabolism 41-42 336, 349, 392, 402, 407 sequential 43-44, 399 hydroxylamines 88, 106, 136, 203, 333, parallel 44-45 335-336 reversible 45 metabolite 41 ibuprofen 280 metabonomics 271 imidazole 67, 95, 194, 422 methadone 311-312 iminium 95 methionine 148 imipramine 151 l-methionine adenosyltransferase 148 indomethacin 115, 159-160, 304, 322 methyltransferases 147, 285 insulin 11, 16, 233, 317, 321, 328 6-methylthiopurine 94 iodine 345 methylation 147-152 isoenzymes general mechanism of, 148 impact on genetic variations, 273 methyl groups 60, 67-68, 84, 136, 149 isoniazid metoprolol 69 hydrolysis of, 116 metronidazole 305 N-acetylation of, 142 microsomal-mixed function oxidase system (MMFO) 48 ketones 58, 68, 105, 391 midazolam 68 ketorolac 162 minerals, effects on drug metabolism 228-230 L-DOPA 149 molecular oxygen 48-49, 58, 95, 98-99, lead 217 180, 189-193 leukotrienes 103 monoamino oxidase system 95 lidocaine 85 MAO-A 96 lipid peroxidation 98, 201, 225-227, 259 MAO-B 96 lipids, monogenic variability 285 effects on drug metabolism 221-222 morphine, lipophilicity 42, 370, 372, 386, 397, 400, 403 N-demethylation 86 liver 5-6, 8-12, 14, 21-22, 30, 32, 42-43, glucuronidation 136 48, 64-66, 68-69, 73, 80, 83, 85-92, 98, 101,106, 108, 115, 130-131, 138, 143, NAD(P) 399 147, 149, 154-155, 162,165, 189, 192, NADH 404 195, 200, 202-203, 210-213, 216, 219- NADH-cytochrome b5- 221, 224-232, 245, 249-256, 258-261, reductase system, 102 271, 281-283, 303, 308, 310-314, 316, NADPH 49-50, 69, 96, 103, 180-181, 328, 334, 339, 343, 346, 350, 373, 378- 190, 192, 196, 223-227, 230, 278 379, 390 NADPH-cytochrome c- liver slices 249, 348 (P450)-reductase 49, 102-103 Index 419 nanoparticles 7-8, 406 pharmacodinamics 21 naphthalene 52-53, 73 pharmacogenetics 269 N-C cleavage 82, 88 consequences of, 272 N-dealkylation 60, 88-90, 245, 337 pharmaco-informatology 269 N-demethylation 85-86, 89, 311, 314 pharmacokinetics 21, 135, 254 N-oxygenation 84, 333 pharmacophore 371, 385, 388, 394 neoglycoprotein carriers 405 phenacetin 63 nephrotoxicity 48, 248, 259, 316, 324- phencyclidine 66 325, 333, 339, 342 phenelzine nicotine 13, 97, 233, 327, 329 MAO-catalysed oxidation of, 100 NIH shift 52, 56-57 phenobarbitone 79, 211, 213, 221, 345 nitrenium ions 136, 335-336 phenols 52,72, 74-75, 130, 132, 134, 138, p-nitroanisole 93 149, 153, 203, 338 nitrofurantoin 304, 344 phenotype 143, 149, 273-274, 276-280, nitrogen oxidation 82 284, 286, 350 N,N-diethylamino derivatives 84 phentermine 82 N,N-dimethylamino derivatives 84 phenylbutazone 133, 156-157, 305-306, NSAIDs 16, 135,298-299, 303-304, 310, 327, 329, 343-344 318, 321-322, 327, 342, 406 phenytoin 75 noradrenaline 147 phonophoresis 16 norbenzphetamine 333-334 phospholipid prodrugs 400 norepinephrine 150 pinazepam 89, norethindrone 81 piperazine 136 piroxicam 163-164 O-dealkylation 94 pivampicillin 109, 374 olefinic bonds 77, 79 plasma drug concentration 35 olefins 72 polychlorinated biphenyls 262 one-electron oxidation 198 polycyclic aromatic hydrocarbons 76 oral contraceptives 234, 305, 310, 328, polymorphism 14 344 ‘poor metabolisers’ 274 oxazepam 89, 232 potential toxicity 136, 210, 219, 339, 348 oxidation 30 Positive Higher Structures 7 oxidations at hetero-atoms 82 prednisone 45 oxidative deamination 95, 99, 246 pregnancy 229, 261, 329, 344 oxidoreductases 99, 401 procaine (procainamide) oxygen rebound mechanism 59 hydroxylation of, 83 oxygenases 48, 96 hydrolysis of, 108 monooxygenases 58, 73, 97, 201, 275 procarbazine 88 oxyphenbutazone 97, 156, 197 prodrugs 29, 46-47, 109-113, 251, 332, 371-385 paclitaxel 247 progesterone 256 pancreas 254 proliposomes 7 panomifene 250 propranolol 43 PAPS 154 hydroxylation 74 paracetamol 199 N-dealkylation and

deamination 91 pathological status, prostaglandins 198, 200 effect on drug metabolism, 258-261 prostaglandin-synthetase 97, 197 PEG-based prodrugs 384 co-oxidation of drugs, 198 pentobarbital 63 proteins, peroxidases 95 effects on drug metabolism 221 pesticides 263 proteomics 271 pharmacoanthropology pseudocholinesterase 107-108, 310 420 Index pulmonary toxicity 333, 343-344 terfenadine 61 purine tertiary amines 97, 132, 136 regiospecific XO metabolization of, 101 testosterone 65 pyridine (dihydro-) 379, 399, 400, 402 theophylline 90, 306, 320, 324, 328 pyrolysis products 231 thyroid hormones 232 tolazamide 67 racial differences 276, 279, 281 tolbutamide 102 reduction 6, 30, 32, 42, 82, 117, 129 tolmetin 164-165 175, 191-193, 195, 197-198, 200, toxicogenomics 271 209, 216, 223, 255, 258, 275, 282, transferases 31, 95, 130-131, 134, 141, 302, 304, 308-311, 337, 373, 395, 397 143, 147-152, 154, 171, 202-204, 283 reductive drug metabolism 102-107 trimethoprim 87 retinoic acid 227, 344 ‘retrometabolic drug design’ 394 UGTs 30, 202-203 reversible metabolism 45 polymorphism of, 283 rhein 252 ‘ultra-extensive metabolisers’ 274 riboflavin 224-225 uridine diphophoglucuronic acid ritipenem acoxyl 111 valproic acid 100, 195 S-adenosylmethionine 148 VDEPT, S-dealkylation 30, 49, 94, 338 (virus-directed prodrug therapy) 382 salycilamide 75 verapamil 22, 195, 304, 324, 327 salycilate 158-159 viral hepatitis 258 selegiline 245 vitamins, sex, effects on drug metabolism 223-228 effect on drug metabolism 253-254 volume of distribution 61 soft drugs 371, 390, 392, 394, 405 species, warfarin 297-298, 301, 303, 306-309, differences in drug metabolism 244-253 318, 344 genetic control of, 274-287 spironolactone 304, 318, 337-338 xanthine oxidase 97 steroids 10,16,42,45,80, 134, 154, 212, xanthine/xanthine oxidase system 97-98, 256, 299, 310, 316-317, 323, 392, 401 200-201 suicide substrates 216 xenobiotics 30, 41-42, 48, 77, 84, 94, sulindac 161 117, 131, 134, 138, 143-144, 147, 153, sulphanilamide (N-acetylation), 142 166, 189, 197, 200-202, 210, 212, 214, sulphation 45, 129, 134, 153-155, 162 216, 221-222, 229, 244, 254, 259, 261, sulphotransferases 203 269, 271, 277, 285, 332, 337, 342, 370 polymorphism of, 283 ximelagatran 372-373 talampicillin 374 zidovudine 378, 405 tamoxifen 195, 201, 212, 250 Ziegler’s enzyme 196 targetor moiety 399-402 zolmitriptan 246 teratogenesis 331, 333 zoxazolamine 211, 218

9th EDITION CLINICIAN’S POCKET REFERENCE EDITED BY LEONARD G. GOMELLA, MD, FACS The Bernard W. Godwin, Jr., Associate Professor Department of Urology Jefferson Medical College Thomas Jefferson University Philadelphia, Pennsylvania WITH Steven A. Haist, MD, MS, FACP Professor of Medicine Division of General Internal Medicine Department of Internal Medicine University of Kentucky Medical Center Lexington, Kentucky Based on a program originally developed at the University of Kentucky College of Medicine Lexington, Kentucky McGraw-Hill MEDICAL PUBLISHING DIVISION New York Chicago San Francisco Lisbon London Madrid Mexico City Milan New Delhi San Juan Seoul Singapore Sydney Toronto McGraw-Hill abc Copyright © 2002 by Leonard G.Gomella. All rights reserved. Manufactured in the United States of America. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written permission of the publisher. 0-07-139444-3 The material in this eBook also appears in the print version of this title: 0-8385-1552-5. All trademarks are trademarks of their respective owners. Rather than put a trademark symbol after every occurrence of a trademarked name, we use names in an editorial fash- ion only, and to the benefit of the trademark owner, with no intention of infringement of the trademark. Where such designations appear in this book, they have been printed with initial caps. McGraw-Hill eBooks are available at special quantity discounts to use as premiums and sales promotions, or for use in corporate training programs. For more information, please contact George Hoare, Special Sales, at george_hoare@mcgraw-hill.com or (212) 904- 4069. TERMS OF USE This is a copyrighted work and The McGraw-Hill Companies, Inc. (“McGraw-Hill”) and its licensors reserve all rights in and to the work. Use of this work is subject to these terms. Except as permitted under the Copyright Act of 1976 and the right to store and retrieve one copy of the work, you may not decompile, disassemble, reverse engineer, reproduce, modify, create derivative works based upon, transmit, distribute, disseminate, sell, pub- lish or sublicense the work or any part of it without McGraw-Hill’s prior consent. You may use the work for your own noncommercial and personal use; any other use of the work is strictly prohibited. Your right to use the work may be terminated if you fail to comply with these terms. THE WORK IS PROVIDED “AS IS”. McGRAW-HILL AND ITS LICENSORS MAKE NO GUARANTEES OR WARRANTIES AS TO THE ACCURACY, ADEQUACY OR COMPLETENESS OF OR RESULTS TO BE OBTAINED FROM USING THE WORK, INCLUDING ANY INFORMATION THAT CAN BE ACCESSED THROUGH THE WORK VIA HYPERLINK OR OTHERWISE, AND EXPRESSLY DISCLAIM ANY WARRANTY, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICU- LAR PURPOSE. McGraw-Hill and its licensors do not warrant or guarantee that the func- tions contained in the work will meet your requirements or that its operation will be unin- terrupted or error free. Neither McGraw-Hill nor its licensors shall be liable to you or anyone else for any inaccuracy, error or omission, regardless of cause, in the work or for any damages resulting therefrom. McGraw-Hill has no responsibility for the content of any information accessed through the work. Under no circumstances shall McGraw-Hill and/or its licensors be liable for any indirect, incidental, special, punitive, consequential or similar damages that result from the use of or inability to use the work, even if any of them has been advised of the possibility of such damages. This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise. DOI: 10.1036/0071394445 Want to learn more? We hope you enjoy this McGraw- Hill eBook! If you’d like more information about this book, its author, or related books and web- sites, please click here. To Tricia, Mom, Dad, Leonard, Patrick, Andrew, Michael and Aunt Lucy “We don’t drive the trucks, we only load them.” Nick Pavona, MD UKMC Class of 1980 This page intentionally left blank. For more information about this book, click here. CONTENTS Consulting Editors vii Contributors viii Preface xiii Abbreviations xv “So You Want to Be a Scut Monkey”: An Introduction to Clinical Medicine 1 1 History and Physical Examination 9 2 Chartwork 33 3 Differential Diagnosis: Symptoms, Signs, and Conditions 41 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 53 5 Laboratory Diagnosis: Clinical Hematology 95 6 Laboratory Diagnosis: Urine Studies 109 7 Clinical Microbiology 121 8 Blood Gases and Acid-Base Disorders 161 9 Fluids and Electrolytes 177 10 Blood Component Therapy 193 11 Diets and Clinical Nutrition 205 12 Total Parenteral Nutrition (TPN) 227 13 Bedside Procedures 239 14 Pain Management 315 15 Imaging Studies 325 16 Introduction to the Operating Room 339 17 Suturing Techniques and Wound Care 345 18 Respiratory Care 359 19 Basic ECG Reading 367 20 Critical Care 389 21 Emergencies 445 22 Commonly Used Medications 475 Appendix 639 Index 659 Emergency Medications (inside front and back covers) Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Us.e This page intentionally left blank. CONSULTING EDITORS MARIANNE BILLETER, PharmD, BCPS Associate Professor of Pharmacy Practice, Division of Distance Education, Bernard J. Dunn School of Pharmacy, Shenandoah University, Winchester, Virginia TRICIA L. GOMELLA, MD Part-Time Clinical Assistant Professor of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland IRA HOROWITZ, MD Professor of Gynecology and Obstetrics and Oncology, Director of Gynecologic Oncology, Emory University, Atlanta, Georgia ALAN T. LEFOR, MD, MPH, FACS Director, Division of Surgical Oncology, Director, Surgical Education and Academic Affairs, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California; Associate Professor of Clinical Surgery, Department of Surgery, University of California, Los Angeles, Los Angeles, California JOHN A. MORRIS, MD Director, Division of Trauma, Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use CONTRIBUTORS Aimee G. Adams, PharmD Director, Primary Care Pharmacy Practice Residency, University of Kentucky Medical Center; Assistant Professor College of Pharmacy and Department of Medicine, University of Kentucky, Lexington, Kentucky Marianne Billeter, PharmD, BCPS Associate Professor of Pharmacy Practice, Division of Distance Education, Bernard J. Dunn School of Pharmacy, Shenandoah University, Winchester, Virginia Pasquale Casale, MD Chief Resident, Department of Urology, Thomas Jefferson University, Philadelphia, Pennsylvania Murray Cohen, MD Clinical Associate Professor of Surgery, Director, Division of Trauma/Critical Care, Department of Surgery, Jefferson Medical College, Philadelphia, Pennsylvania Marisa Davis Doctor of Pharmacy Candidate, Bernard J. Dunn School of Pharmacy, Shenandoah University, Winchester, Virginia Neil M. Davis, PharmD, FASHP Professor Emeritus of Pharmacy, Temple University, Philadelphia, Pennsylvania; Director, Safe Medication Practice Consulting, Inc., Huntingdon Valley, Pennsylvania Ehab A. El Gabry, MD Fellow, Department of Urology, Thomas Jefferson University, Philadelphia, Pennsylvania Sue Fosson, MA Former Associate Dean for Student Affairs, University of Kentucky College of Medicine, Lexington, Kentucky Leonard G. Gomella, MD, FACS The Bernard W. Godwin, Jr., Associate Professor, Department of Urology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use Contributors ix Steven A. Haist, MD, MS, FACP Professor of Medicine, Division of General Internal Medicine, Department of Internal Medicine, University of Kentucky Medical Center, Lexington, Kentucky Sara Maria Haverty, MD Senior Resident, Department of Obstetrics and Gynecology, Thomas Jefferson University, Philadelphia, Pennsylvania Mohamed Ismail, MD Senior Resident, Department of Urology, Thomas Jefferson University, Philadelphia, Pennsylvania Gregory C. Kane, MD Clinical Associate Professor of Medicine, Program Director, Internal Medicine Residency, Jefferson Medical College, Philadelphia, Pennsylvania Matthew J. Killion, MD Assistant Professor of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania Alan T. Lefor, MD, MPH, FACS Director, Division of Surgical Oncology, Director, Surgical Education and Academic Affairs, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California; Associate Professor of Clinical Surgery, Department of Surgery, University of California, Los Angeles, Los Angeles, California Layla F. Makary, MD, MSC, PhD Lecturer, Department of Anesthesia, Cairo University, Clinical Fellow, Department of Anesthesia, Cleveland Clinic Foundation, Cleveland, Ohio John Moore, MD Clinical Associate Professor, Department of Surgery, Division of Plastic Surgery, Jefferson Medical College, Philadelphia, Pennsylvania Nick A. Pavona, MD Associate Professor, Department of Surgery, Division of Urology, Benjamin Franklin University Medical Center, Chadds Ford, Pennsylvania Roger J. Pomerantz, MD, FACP Professor of Medicine, Biochemistry and Molecular Pharmacology, Division of Infectious Diseases and Center for Human Virology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania Ganesh Raj, MD, PhD Senior Resident, Division of Urology, Department of Surgery, Duke University Medical Center, Durham, North Carolina x Contributors Steven Rosensweig, MD Director, Jefferson Center for Integrative Medicine, Jefferson Medical College, Philadelphia, Pennsylvania Paul J. Schenarts, MD Instructor in Surgery, Section of Surgical Sciences, Vanderbilt University, Nashville, Tennessee Francis G. Serio, DMD, MS Associate Professor and Chairman, Department of Periodontics, University of Mississippi School of Dentistry, Jackson, Mississippi Kelly Smith, PharmD Clinical Associate Professor, Division of Pharmacy Practice & Science, University of Kentucky College of Pharmacy; Director, Pharmacy Practice Residency, University of Kentucky Medical Center, Lexington, Kentucky PREFACE The Clinician’s Pocket Reference is based on a University of Kentucky house manual enti- tled So You Want to Be a Scut Monkey: Medical Student’s and House Officer’s Clinical Handbook. The Scut Monkey Program at the University of Kentucky College of Medicine began in the summer of 1978 and was developed by members of the Class of 1980 to help ease the often frustrating transition from the preclinical to the clinical years of medical school. From detailed surveys at the University of Kentucky College of Medicine and 44 other medical schools, a list of essential information and skills that third-year students should be familiar with at the start of their clinical years was developed. The Scut Monkey Program was developed around this core of material and consisted of reference manuals and a series of workshops conducted at the start of the third year. Presented originally as a pilot program for the University of Kentucky College of Medicine Class of 1981, the program has been incorporated into the third-year curriculum. It is the responsibility of each new fourth- year class to orient the new third-year students. The basis of the program’s success is the fact that it was developed and taught by students for other students. This method has al- lowed us to maintain perspective on those areas that are critical not only for learning while on the wards but also for delivering effective patient care. Information on the Scut Monkey Orientation Program is available from Todd Cheever, MD, Associate Dean for Academic Af- fairs at the University of Kentucky College of Medicine. Through the last eight editions, the book has undergone expansion and careful revisions as the practice of medicine and the educational needs of students have changed. Although the book’s original mission, providing new clinical clerks with essential patient care infor- mation in an easy-to-use format, remains unchanged, our readership has expanded. Resi- dents, practicing physicians, and allied health professionals all use the Clinician’s Pocket Reference as a “manual of manuals.” Even individuals considering careers in medicine have used the book in their decision-making process. An attempt is made to cover the most fre- quently asked basic management questions that are normally found in many different sources, such as procedure manuals, laboratory manuals, drug references, and critical care manuals, to name a few. It is not meant as a substitute for specialty-specific reference manu- als. The core of information presented is a foundation for new medical students as they move through training to more advanced medical studies. The book is designed to represent a cross section of medical practices around the coun- try. The Clinician’s Pocket Reference has been translated into six different languages with electronic media versions in development. I was honored to have been asked to grant per- mission to Warner Brothers, the producers of the TV show “ER,” to have the eighth edition of the Scut Monkey book as one of the books used on their series. I would like to express special thanks to my wife and my family for their long-term sup- port of the Scut Monkey project. Linda Davoli, our extraordinary copy editor, had an excep- tional eye for detail in helping create this final work. Janet Foltin, Harriet Lebowitz, Lester Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use xii Preface

Sheinis, and the team at McGraw-Hill were instrumental in moving the book forward and in giving the ninth edition a fresh, new two-color format. They are also responsible for helping reach our long-term goal of the new companion manual, the Clinician’s Pocket Drug Refer- ence. A special thanks to my assistant Conchita Ballard, who always kept things organized and flowing smoothly. I am indebted to all of the past contributors and readers who have helped to keep the Scut Monkey book as a useful reference for students and residents world- wide. The original coeditors of this work, G. Richard Braen, MD, and Michael J. Olding, MD, are acknowledged for their early contributions. Your comments and suggestions for improvement are always welcomed by me person- ally, since revisions to the book would not be possible if it were not for the ongoing interest of our readers. I hope this book will not only help you learn some of the basics of the art and science of medicine but also allow you to care for your patients in the best way possible. Leonard G. Gomella, MD Philadelphia, Pennsylvania Leonard.Gomella@mail.tju.edu ABBREVIATIONS The following are common abbreviations used in medical records and in this edition ÷: divided dose A.D.C. VAN DISSEL: mnemonic for ↓: decrease(d), reduce, downward Admit, Diagnosis, Condition, Vitals,
: times for multiplication sign Activity, Nursing procedures, Diet, Ins ↑: increase(d), upward (as in titrate upward) and outs, Specific drugs, Symptomatic /: per drugs, Extras, Labs ±: with or without ADH: antidiuretic hormone : with ADHD: attention-deficit hyperactivity <: less than, younger than disorder >: more than, older than ad lib: as much as needed (ad libitum) ~=: approximately equal to AEIOU TIPS: mnemonic for Alcohol, AAA: abdominal aortic aneurysm Encephalopathy, Insulin, Opiates, AaDO2: difference in partial pressures of Uremia, Trauma, Infection, Psychi- oxygen in mixed alveolar gas and atric, Syncope (diagnosis of coma) mixed arterial blood AF: afebrile, aortofemoral, atrial fibrilla- A-a gradient: alveolar-to-arterial gradient tion AAI: ankle-arm index AFB: acid-fast bacilli AAS: acute abdominal series AFP: alpha-fetoprotein AB: antibody, abortion, antibiotic A/G: albumin/globulin ratio A&B: apnea and bradycardia AHA: American Heart Association ABD: abdomen AHF: antihemophilic factor ABG: arterial blood gas AI: aortic insufficiency A/B index: ankle-brachial index AIDS: acquired immunodeficiency syn- ABMT: autologous bone marrow trans- drome plantation AJCC: American Joint Committee on ac: before eating (ante cibum), assist- Cancer controlled AKA: above-the-knee amputation ACCP: American College of Chest ALAT: alanine aminotransferase Physicians ALL: acute lymphocytic leukemia ACE: angiotensin-converting enzyme ALS: amyotrophic lateral sclerosis Ach-ase: acetylcholinesterase ALT: alanine aminotransferase ACLS: Advanced Cardiac Life Support AM: morning ACS: acute coronary syndrome, American amb: ambulate Cancer Society, American College of AMI: acute myocardial infarction Surgeons AML: acute myelocytic leukemia, acute ACTH: adrenocorticotropic hormone myelogenous leukemia A.D.C. VAAN DIML: mnemonic for AMMoL: acute monocytic leukemia Admit, Diagnosis, Condition, Vitals, amp: ampule Activity, Allergies, Nursing procedures, AMP: adenosine monophosphate Diet, Ins and outs, Medications, Labs ANA: antinuclear antibody Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use xiv Abbreviations ANC: absolute neutrophil count BEE: basal energy expenditure ANCA: antineutrophil cytoplasmic anti- bid: twice a day (bis in die) body bili: bilirubin ANLL: acute nonlymphoblastic leukemia BKA: below-the-knee amputation ANS: autonomic nervous system BM: bone marrow, bowel movement AOB: alcohol on breath BMR: basal metabolic rate AODM: adult-onset diabetes mellitus BMT: bone marrow transplantation AP: anteroposterior, abdominal-perineal BOM: bilateral otitis media APAP: acetaminophen BP: blood pressure APL: acute promyelocytic leukemia BPH: benign prostatic hypertrophy aPPT: activated partial thromboplastin bpm: beats per minute time BR: bed rest APSAC: anisoylated plasminogen strepto- BRBPR: bright red blood per rectum kinase activator complex BRP: bathroom privileges APUD: amine precursor uptake (and) bs, BS: bowel sounds, breath sounds decarboxylation BSA: body surface area Ara-C: cytarabine BS&O: bilateral salpingo-oophorectomy ARD: antibiotic removal device BUN: blood urea nitrogen ARDS: adult respiratory distress syndrome BW: body weight ARF: acute renal failure Bx: biopsy AS: aortic stenosis c: with (cum) ASA: American Society of Anesthesiolo- Ca: calcium gists CA: cancer ASAP: as soon as possible CAA: crystalline amino acid ASAT: aspartate aminotransferase CABG: coronary artery bypass graft ASCVD: atherosclerotic cardiovascular CAD: coronary artery disease disease CAF: cyclophosphamide, doxorubicin ASD: atrial septal defect (Adriamycin), 5-fluorouracil ASHD: atherosclerotic heart disease CALGB: Cancer and Leukemia Group B ASO: antistreptolysin O cAMP: cyclic adenosine monophosphate AST: aspartate aminotransferase CaO2: arterial oxygen content ATG: antithymocyte globulin caps: capsule(s) ATN: acute tubular necrosis CAT: computed axial tomography ATP: adenosine triphosphate CBC: complete blood count AUC: area under the curve CBG: capillary blood gas AV: atrioventricular CC: chief complaint A-V: arteriovenous CCI: corrected count increment (platelets) A-VO2: arteriovenous oxygen CCO: continuous cardiac output B I&II: Billroth I and II CCO2: capillary oxygen content BACOD: bleomycin, doxorubicin (Adri- CCU: clean-catch urine, cardiac care unit amycin), cyclophosphamide, vin- CCV: critical closing volume cristine (Oncovin), dexamethasone CD: continuous dose BACOP: bleomycin, doxorubicin (Adri- CDC: Centers for Disease Control and Pre- amycin), cyclophosphamide, vin- vention cristine (Oncovin), prednisone CEA: carcinoembryonic antigen BBB: bundle branch block CEP/CIEP: counterimmunoelectrophore- BC: bone conduction sis BCAA: branched-chain amino acid CF: cystic fibrosis BCG: bacille Calmette-Guérin CFU: colony-forming unit(s) BE: barium enema CGL: chronic granulocytic leukemia Abbreviations xv CH50: (total serum) hemolytic complement CVAT: costovertebral angle tenderness CHD: coronary heart disease CVH: common variable hypogammaglobu- CHF: congestive heart failure linemia CHO: carbohydrate CvO2: oxygen content of mixed venous CHOP: cyclophosphamide, doxorubicin, blood vincristine (Oncovin), prednisone CVP: central venous pressure CI: cardiac index CXR: chest x-ray CIE: counterimmunoelectrophoresis d: day CIS: carcinoma in situ D5LR: 5% dextrose in lactated Ringer’s CK: creatine phosphokinase solution CKI: cyclin-dependent kinase inhibitor D5W: 5% dextrose in water CK-MB: isoenzyme of creatine kinase DAG: diacylglycerol with muscle and brain subunits DAP: diastolic pulmonary artery pressure Cl: chlorine DAT: diet as tolerated CLL: chronic lymphocytic leukemia DAW: dispense as written cm: centimeter DC: discontinue, discharge, direct current CML: chronic myelogenous leukemia D&C: dilation and curettage CMV: cytomegalovirus ddI: dideoxyinosine CN: cranial nerve DDx: differential diagnosis CNS: central nervous system DEA: United States Drug Enforcement CO: cardiac output Administration C/O: complaining of DES: diethylstilbestrol COAD: chronic obstructive airway disease DEXA: dual-energy x-ray absorptiometer COLD: chronic obstructive lung disease DHEA: dehydroepiandrosterone COMT: catechol-O-methyltransferase DHEAS: dehydroepiandrosterone sulfate conc: concentrate DI: diabetes insipidus cont inf: continuous infusion DIC: disseminated intravascular coagulation COPD: chronic obstructive pulmonary DIP: distal interphalangeal joint disease DIT: diiodotyrosine COX-2: cyclooxygenase-2 DJD: degenerative joint disease CP: chest pain, cerebral palsy DKA: diabetic ketoacidosis CPAP: continuous positive airway pressure dL: deciliter CPK: creatinine phosphokinase DM: diabetes mellitus CPP: central precocious puberty DMSA: dimercaptosuccinic acid CPR: cardiopulmonary resuscitation DNA: deoxyribonucleic acid CR: controlled release DNP: deoxyribonucleic protein CrCl: creatine clearance DNR: do not resuscitate CREST: calcinosis cutis, Raynaud’s DOA: dead on arrival disease, esophageal dysmotility, DOCA: deoxycorticosterone acetate syndactyly, telangiectasia DOE: dyspnea on exertion CRF: chronic renal failure DOPA: dihydroxyphenylalanine CRH: corticotropin-releasing hormone DP: dorsalis pedis CRP: C-reactive protein 2,3-DPG: 2,3-diphosphoglycerate C&S: culture and sensitivity DPL: diagnostic peritoneal lavage CSF: cerebrospinal fluid, colony- DPT: diphtheria, pertussis, tetanus stimulating factor DR: delayed release C-spine: cervical spine DRG: diagnosis-related group CT: computed tomography DS: double strength CVA: cerebrovascular accident, costoverte- DSA: digital subtraction angiography bral angle DTPA: diethylenetriamine-pentaacetic acid xvi Abbreviations DTR: deep tendon reflex FiO2: fraction of inspired oxygen DVT: deep venous thrombosis FRC: functional residual capacity Dx: diagnosis FSH: follicle-stimulating hormone EAA: essential amino acid FSP: fibrin split product EBL: estimated blood loss ft: foot EBV: Epstein–Barr virus FTA-ABS: fluorescent treponemal EC: enteric-coated antibody-absorbed ECG: electrocardiogram FTT: failure to thrive ECOG: Eastern Cooperative Oncology FU: follow-up Group 5-FU: fluorouracil ECT: electroconvulsive therapy FUO: fever of unknown origin EDC: estimated date of confinement FVC: forced vital capacity EDTA: ethylenediamine tetraacetic acid Fx: fracture EDVI: end-diastolic volume index g: gram EFAD: essential fatty acid deficiency G: gravida ELISA: enzyme-linked immunosorbent GABA: gamma-aminobutyric acid assay GAD: glutamic acid decarboxylase EMD: electromechanical dissociation GC: gonorrhea (gonococcus) EMG: electromyelogram G-CSF: granulocyte colony-stimulating EMS: emergency medical system, factor eosinophilia-myalgia syndrome GDP: guanosine diphosphate EMV: eyes, motor, verbal response GERD: gastroesophageal reflux disease (Glasgow Coma Scale) GETT: general by endotracheal tube ENA: extractable nuclear antigen (anesthesia) ENT: ear, nose, and throat GFR: glomerular filtration rate eod: every other day GGT: gamma-glutamyltransferase EOM: extraocular muscle GH: growth hormone EPO: erythropoietin GHIH: growth hormone-inhibiting EPSP: excitatory postsynaptic potential hormone ER: endoplasmic reticulum, Emergency GI: gastrointestinal Room, extended release GM-CSF: granulocyte-macrophage ERCP: endoscopic retrograde cholan- colony-stimulating factor giopancreatography GNID: gram-negative intracellular ERV: expiratory reserve volume diplococci ESR: erythrocyte sedimentation rate GnRH: gonadotropin-releasing hormone ESRD: end-stage renal disease GOG: Gynecologic Oncology Group ET: endotracheal G6PD: glucose-6-phosphate ETOH: ethanol dehydrogenase ETT: endotracheal tube gr: grain EUA: examination under anesthesia GSW: gunshot wound ExU: excretory urogram gt, gtt: drop, drops (gutta) Fab: antigen-binding fragment GTP: guanosine triphosphate FANA: fluorescent antinuclear antibody GTT: glucose tolerance test FBS: fasting blood sugar GU: genitourinary Fe: iron GVHD: graft-versus-host disease FEV1: forced expiratory volume in 1 s GXT: graded exercise tolerance (cardiac FFP: fresh frozen plasma stress test) FHR: fetal heart rate HA: headache FIGO: Fédération Internationale de HAA: hepatitis B surface antigen Gynécologie et d’Obstétrique (hepatitis-associated antigen) Abbreviations xvii HAV: hepatitis A virus IgG1{k}: immunoglobulin G1 kappa HBcAg: hepatitis B core antigen IHSS: idiopathic hypertrophic subaortic HBeAg: hepatitis B e antigen stenosis HBP: high blood pressure IL: interleukin HBsAg: hepatitis B surface antigen IM: intramuscular HBV: hepatitis B virus IMV: intermittent mandatory ventilation HCG: human chorionic gonadotropin in.: inch HCL: hairy cell leukemia INF: intravenous nutritional fluid HCT: hematocrit INH: isoniazid HCTZ: hydrochlorothiazide inhal: inhalation HDL: high-density lipoprotein inj: injection HEENT: head, eyes, ears, nose, and throat INR: international normalized ratio HFV: high-frequency ventilation I&O: intake and output Hgb: hemoglobin IP3: inositol triphosphate [Hgb]: hemoglobin concentration IPPB: intermittent positive pressure H/H: hemoglobin/hematocrit, breathing Henderson–Hasselbalch equation IPSP: inhibitory postsynaptic potential HIAA: 5-hydroxyindoleacetic acid iPTH: parathyroid hormone by radioim- HIDA: hepatic 2,6-dimethyliminodiacetic munoassay acid IR: inversion recovery HIV: human immunodeficiency virus IRBBB: incomplete right bundle branch HJR: hepatojugular reflex block HLA: histocompatibility locus antigen IRDM: insulin-resistant diabetes mellitus HO: history of IRV: inspiratory reserve volume HOB: head of bed ISA: intrinsic sympathomimetic activity H&P: history and physical examination IT: intrathecal hpf: high-power field ITP: idiopathic thrombocytopenic HPI: history of the present illness purpura HPLC: high-pressure liquid IV: intravenous chromatography IVC: intravenous cholangiogram HPV: human papilloma virus IVP: intravenous pyelogram HR: heart rate JODM: juvenile-onset diabetes mellitus hs: at bedtime (hora somni) JVD: jugular venous distention HSG: hysterosalpingogram K: potassium HSM: hepatosplenomegaly katal: unit of enzyme activity HSV: herpes simplex virus kg: kilogram 5-HT3: 5-hydroxytryptamine KOR: keep open rate HTLV-III: human T-lymphotropic virus, 17-KSG: 17-ketogenic steroids type III (AIDS agent, HIV) KUB: kidneys, ureters, bladder HTN: hypertension KVO: keep vein open Hx: history L: left, liter IC: inspiratory capacity LAD: left axis deviation, left anterior ICN: Intensive Care Nursery descending ICS: intercostal space LAE: left atrial enlargement ICSH: interstitial cell-stimulating hormone LAHB: left anterior hemiblock ICU: intensive care unit LAP: left atrial pressure, leukocyte ID: identification, infectious disease alkaline phosphatase I&D: incision and drainage LBBB: left bundle branch block IDDM: insulin-dependent diabetes mellitus LDH: lactate dehydrogenase Ig: immunoglobulin LDL: low-density lipoprotein xviii Abbreviations LE: lupus erythematosus mg: milligram LH: luteinizing hormone Mg: magnesium LHRH: luteinizing hormone releasing MHA-TP: microhemagglutination- hormone Treponema pallidum LIH: left inguinal hernia MHC: major histocompatibility complex liq: liquid MI: myocardial infarction, mitral insuffi- LLL: left lower lobe ciency LLSB: left lower sternal border MIBG: metaiodobenzyl-guanidine LMP: last menstrual period MIC: minimum inhibitory concentration LNMP: last normal menstrual period min: minute, minimum LOC: loss of consciousness, level of con- MIT: monoiodotyrosine sciousness mL: milliliter LP: lumbar puncture MLE: midline episiotomy lpf: low-power field mm: millimeter LPN: licensed practical nurse MMEF: maximal midexpiratory flow LSB: left sternal border mm Hg: millimeters of mercury LSD: lysergic acid diethylamide mmol: millimole LUL: left upper lobe MMR: measles, mumps, rubella LUQ: left upper quadrant mo: month LV: left ventricle mol: mole LVD: left ventricular dysfunction MOPP: mechlorethamine, vincristine LVEDP: left ventricular end-diastolic pres- (Oncovin), procarbazine, prednisone sure 6-MP: mercaptopurine LVH: left ventricular hypertrophy MPF: M phase-promoting factor m: meter MPGN: membrane-proliferative glomeru- MAC: Mycobacterium avium complex lonephritis MACE: methotrexate, doxorubicin (Adri- MPTP: analog of meperidine (used by amycin), cyclophosphamide, drug addicts) epipodophyllotoxin MRI: magnetic resonance imaging MAG3: mercaptoacetyltriglycine mRNA: messenger ribonucleic acid MAMC: midarm muscle circumference MRS: magnetic resonance spectroscopy MAO: monoamine oxidase MRSA: methicillin-resistant Staphylococ- MAOI: monoamine oxidase inhibitor cus aureus MAP: mean arterial pressure MS: mitral stenosis, morphine sulfate, mul- MAST: military/medical

antishock trousers tiple sclerosis MAT: multifocal atrial tachycardia MSBOS: maximal surgical blood order max: maximum schedule MBC: minimum bactericidal concentration MSH: melanocyte-stimulating hormone MBT: maternal blood type MTT: monotetrazolium MCH: mean cell hemoglobin MTX: methotrexate MCHC: mean cell hemoglobin concentra- MUGA: multigated (image) acquisition tion (analysis) MCT: medium-chain triglycerides m: micrometer MCTD: mixed connective tissue disease MVA: motor vehicle accident MCV: mean cell volume MVI: multivitamin injection MEN: multiple endocrine neoplasia MVV: maximum voluntary ventilation meq: milliequivalent MyG: myasthenia gravis MESNA: 2-mercaptoethane sulfonate Na: sodium sodium NAACP: mnemonic for Neoplasm, Allergy, met-dose: metered-dose Addison’s disease, Collagen-vascular Abbreviations xix disease, Parasites (causes of PAC: premature atrial contraction eosinophilia) PAD: diastolic pulmonary artery pressure NAD: no active disease PAF: paroxysmal atrial fibrillation Na+/K+-ATPase: sodium/potassium PAL: periarterial lymphatic (sheath) adenosine triphosphate PaO2: peripheral arterial oxygen content NAPA: N-acetylated procainamide, PAO2: alveolar oxygen N-acetylparaaminophenol PAOP: pulmonary artery occlusion pres- NAS: no added sodium sure NAVEL: mnemonic for Nerve, Artery, PAP: pulmonary artery pressure, prostatic Vein, Empty space, Lymphatic acid phosphatase NCV: nerve conduction velocity PAS: systolic pulmonary artery pressure NE: norepinephrine PASG: pneumatic antishock garment neb: nebulizer PAT: paroxysmal atrial tachycardia NED: no evidence of recurrent disease PBM: pharmacy benefit manager ng: nanogram pc: after eating (post cibum) NG: nasogastric PCA: patient-controlled analgesia NIDDM: non-insulin-dependent diabetes PCI: percutaneous coronary intervention mellitus PCKD: polycystic kidney disease NK: natural killer PCN: percutaneous nephrostomy NKA: no known allergies pCO2: partial pressure of carbon dioxide NKDA: no known drug allergy PCP: Pneumocystis carinii pneumonia, nmol: nanomole phencyclidine NMR: nuclear magnetic resonance PCR: polymerase chain reaction NPC: nuclear pore complex PCWP: pulmonary capillary wedge NPO: nothing by mouth (nil per os) pressure NRM: no regular medicines PDA: patent ductus arteriosus NS: normal saline PDGF: platelet-derived growth factor NSAID: nonsteroidal antiinflammatory PDR: Physicians’ Desk Reference drug PDS: polydioxanone NSILA: nonsuppressible insulin-like PE: pulmonary embolus, physical exami- activity nation, pleural effusion NSR: normal sinus rhythm PEA: pulseless electrical activity NT: nasotracheal PEEP: positive end-expiratory pressure NTG: nitroglycerin PEG: polyethylene glycol, percutaneous OB: obstetrics gastrostomy OCD: obsessive-compulsive disorder PERRLA: pupils equal, round, reactive to OCG: oral cholecystogram light and accommodation 7-OCHS: 17-hydroxycorticosteroids PERRLADC: pupils equal, round, reactive OD: overdose, right eye (oculus dexter) to light and accommodation directly oint: ointment and consensually OM: otitis media PET: positron emission tomography OOB: out of bed PFT: pulmonary function test ophth: ophthalmic pg: picogram OPV: oral polio vaccine PGE1: prostaglandin E1 OR: operating room PI: pulmonic insufficiency (disease) OS: opening snap, left eye (oculus sinister) PICC: peripherally inserted central OTC: over-the-counter (medications) catheter OU: both eyes PID: pelvic inflammatory disease p: para PIE: pulmonary infiltrates with PA: posteroanterior, pulmonary artery eosinophilia xx Abbreviations PIH: prolactin-inhibiting hormone qid: four times a day (quater in die) PKU: phenylketonuria QNS: quantity not sufficient PMDD: premenstrual dysphoric disorder qod: every other day PMH: past medical history Qs: volume of blood (portion of cardiac PMI: point of maximal impulse output) shunted past nonventilated PMNL: polymorphonuclear leukocyte alveoli (neutrophil) Qs/Qt: shunt fraction PND: paroxysmal nocturnal dyspnea Qt: total cardiac output PNS: peripheral nervous system R: right PO: by mouth (per os) RA: rheumatoid arthritis, right atrium pO2: partial pressure of oxygen RAD: right axis deviation POD: postoperative day RAE: right atrial enlargement postop: postoperative, after surgery RAP: right atrial pressure PP: pulsus paradoxus, postprandial RBBB: right bundle branch block PPD: purified protein derivative RBC: red blood cell (erythrocyte) P&PD: percussion and postural drainage RBP: retinol-binding protein PPN: partial parenteral nutrition RCC: renal cell carcinoma PR: by rectum RDA: recommended dietary allowance PRA: plasma renin activity RDS: respiratory distress syndrome (of PRBC: packed red blood cells newborn) preop: preoperative, before surgery RDW: red cell distribution width PRG: pregnancy REF: right ventricular ejection fraction PRK: photorefractive keratectomy REM: rapid eye movement PRN: as often as needed (pro re nata) RER: rough endoplasmic reticulum PS: pulmonic stenosis, partial saturation %RH: percentage of relative humidity PSA: prostate-specific antigen RIA: radioimmunoassay PSV: pressure support ventilation RIH: right inguinal hernia PSVT: paroxysmal supraventricular RIND: reversible ischemic neurologic tachycardia deficit Pt: patient RL: Ringer’s lactate PT: prothrombin time, physical therapy, RLL: right lower lobe posterior tibial RLQ: right lower quadrant PTCA: percutaneous transluminal RME: resting metabolic expenditure coronary angioplasty RML: right middle lobe PTH: parathyroid hormone RMSF: Rocky Mountain spotted fever PTHC: percutaneous transhepatic RNA: ribonucleic acid cholangiogram RNase: ribonuclease PTT: partial thromboplastin time R/O: rule out PTU: propylthiouracil ROM: range of motion PUD: peptic ulcer disease ROS: review of systems PVC: premature ventricular contraction RPG: retrograde pyelogram PVD: peripheral vascular disease RPR: rapid plasma reagin PVR: peripheral vascular resistance rRNA: ribosomal ribonucleic acid PWP: pulmonary wedge pressure RRR: regular rate and rhythm PZI: protamine zinc insulin RSV: respiratory syncytial virus q: every (quaque) RT: rubella titer, respiratory therapy, Q: mathematical symbol for flow radiation therapy qd: every day RTA: renal tubular acidosis qh: every hour RTC: return to clinic q{_}h: every {_} hours RTOG: Radiation Therapy Oncology qhs: every hour of sleep Group Abbreviations xxi RU: resin uptake SQ: subcutaneous RUG: retrograde urethrogram SR: sustained release RUL: right upper lobe SRP: single recognition particle RUQ: right upper quadrant SRS-A: slow-reacting substance of ana- RV: residual volume phylaxis RVEDVI: right ventricular end-diastolic SSKI: saturated solution of potassium volume index iodide RVH: right ventricular hypertrophy SSRI: selective serotonin reuptake in- Rx: treatment hibitor s: without (sine), second stat: immediately (statim) SA: sinoatrial STD: sexually transmitted disease S&A: sugar and acetone supp: suppository SAA: synthetic amino acid susp: suspension SaO2: arterial oxygen saturation SVD: spontaneous vaginal delivery SBE: subacute bacterial endocarditis SvO2: mixed venous blood oxygen satura- SBFT: small bowel follow-through tion SBS: short bowel syndrome SVR: systemic vascular resistance SCr: serum creatinine SVT: supraventricular tachycardia segs: segmented cells SWOG: Southwest Oncology Group SEM: systolic ejection murmur Sx: symptoms SER: smooth endoplasmic reticulum Ṫ: one, ṪṪ: two, etc. SG: Swan–Ganz T3: triiodothyronine SGA: small for gestational age T3 RU: triiodothyronine resin uptake SGGT: serum gamma-glutamyl transpepti- T4: thyroxine dase tabs: tablet(s) SGOT: serum glutamic-oxaloacetic TAH: total abdominal hysterectomy transaminase TB: tuberculosis SGPT: serum glutamic-pyruvic transami- TBG: thyroxine-binding globulin, total nase blood gas SI: Système International (see page 55) TBLC: term birth, living child SIADH: syndrome of inappropriate antidi- T&C: type and cross-match uretic hormone TC&DB: turn, cough, and deep sig: write on label (signa) breathe SIMV: synchronous intermittent manda- TCF: triceps skin fold tory ventilation TCP: transcutaneous pacer SIRS: systemic inflammatory response Td: tetanus-diphtheria toxoid syndrome TD: transdermal SKSD: streptokinase-streptodornase TFT: thyroid function test SL: sublingual 6-TG: 6-thioguanine SLE: systemic lupus erythematosus T&H: type and hold SMA: sequential multiple analysis TIA: transient ischemic attack SMO: slips made out TIBC: total iron-binding capacity SMX: sulfamethoxazole tid: three times a day (ter in die) SOAP: mnemonic for Subjective, Objec- TIG: tetanus immune globulin tive, Assessment, Plan TKO: to keep open SOB: shortness of breath TLC: total lung capacity SOC: signed on chart TMJ: temporal mandibular joint soln: solution TMP: trimethoprim SPAG: small-particle aerosol generator TMP-SMX: trimethoprim-sulfamethoxa- SPECT: single-photon emission computed zole tomography TNF: tumor necrosis factor alpha xxii Abbreviations TNM: tumor-nodes-metastases UTI: urinary infection TNTC: too numerous to count UUN: urinary urea nitrogen TO: telephone order V: volt TOPV: trivalent oral polio vaccine VAMP: vincristine, doxorubicin TORCH: toxoplasma, rubella, cy- (Adriamycin), methylprednisolone tomegalovirus, herpes virus (O = other VC: vital capacity [syphilis]) VCUG: voiding cystourethrogram TPA: tissue plasminogen activator VDRL: Venereal Disease Research TPN: total peripheral resistance, total par- Laboratory enteral nutrition VF: ventricular fibrillation TRH: thyrotropin-releasing hormone VLDL: very low density lipoprotein TSH: thyroid-stimulating hormone VMA: vanillylmandelic acid TT: thrombin time VO: voice order TTP: thrombotic thrombocytopenic pur- VP-16: etoposide pura V̇/Q̇: ventilation-perfusion TU: tuberculin units VSS: vital signs stable TUR: transurethral resection VT: ventricular tachycardia TURBT: TUR bladder tumors W: watt TURP: TUR prostate WB: whole blood TV: tidal volume WBC: white blood cell, white blood cell TVH: total vaginal hysterectomy count Tx: treatment, transplant, transfer WD: well developed type 2 DM: noninsulin-dependent diabetes WF: white female mellitus, type 2 diabetes mellitus wk: week UA: urinalysis WM: white male UAC: uric acid WN: well nourished ud: as directed (ut dictum) wnl, WNL: within normal limits UDS: urodynamic studies WPW: Wolff-Parkinson-White UGI: upper gastrointestinal XRT: x-ray therapy UPEP: urine protein electrophoresis y: year URI: upper respiratory infection YO: years old US: ultrasonography ZE: Zollinger–Ellison USP: United States Pharmacopeia “SO YOU WANT TO BE A SCUT MONKEY”: AN INTRODUCTION TO CLINICAL MEDICINE* The transition from the preclinical years to the clinical years of medical school is often a difficult one. Understanding the new responsibilities and a set of ground rules can ease this transition. What follows is a brief introduction to clinical medicine for the new clinical clerk. THE HIERARCHY Most services can be expected to have at least one of each of the following physicians on the team. The Intern In some programs, the intern is known euphemistically as the first-year resident. This person has the day-to-day responsibilities of patient care. This duty, combined with a total lack of seniority, usually serves to keep the intern in the hospital more than the other members of the team and may limit his or her teaching of medical students. Any question concerning de- tails in the evaluation of the patient, for example, whether Mrs. Pavona gets a complete blood count this morning or this evening, is usually referred first to the intern. The Resident The resident is a member of the house staff who has completed at least 1 year of postgradu- ate medical education. The most senior resident is typically in charge of the overall conduct of the service and is the person you might ask a question such as “What might cause Mrs. Pavona’s white blood cell count to be 142,000?” You might also ask your resident for an ap- propriate reference on the subject or perhaps to arrange a brief conference on the topic for everyone on the service. A surgical service typically has a chief resident, a doctor in the last year of residency who usually runs the service. On medical services the chief resident is * Adapted, with permission, from Epstein A, Frye T (eds.): So You Want to Be a Toad. College of Medicine, Ohio State University, Columbus, OH. 1 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 2 Clinician’s Pocket Reference, 9th Edition usually an appointee of the chairman of medicine and primarily has administrative responsi- bilities with limited ward duties. The Attending Physician The attending physician is also called simply “The Attending,” and on nonsurgical services, “the attending.” This physician has completed postgraduate education and is now a member of the teaching faculty. The attending is morally and legally responsible for the care of all patients whose charts are marked with the attending’s name. All major therapeutic decisions made about the care of these patients are ultimately passed by the attending. In addition, this person is responsible for teaching and evaluating house staff and medical students. This is the member of the team you might ask, “Why are we treating Mrs. Pavona with busulfan?” The Fellow Fellows are physicians who have completed their postgraduate education and elected to do extra study in one special field, such as, nephrology, high-risk obstetrics, or surgical oncol- ogy. They may or may not be active members of the team and may not be obligated to teach medical students, but usually they are happy to answer any questions you may ask. You might ask this person to help you read Mrs. Pavona’s bone marrow smear. TEAMWORK The medical student, in addition to being a member of the medical team, must interact with members of the professional team of nurses, dietitians, pharmacists, social workers, and all others who provide direct care for the patient. Good working relations with this group of professionals can make your work go more smoothly; bad relations with them can make your rotation miserable. Nurses are generally good-tempered, but overburdened. Like most human beings, they respond very favorably to polite treatment. Leaving a mess in a patient’s room after the per- formance of a floor procedure, standing by idly while a 98-lb licensed practical nurse strug- gles to move a 350-lb patient onto the chair scale, and obviously listening to three ringing telephones while room call lights flash are acts guaranteed not to please. Do not let anyone talk you into being an acting nurse’s aide or ward secretary, but try

to help when you can. You will occasionally meet a staff member who is having a bad day, and you will be able to do little about it. Returning hostility is unwarranted at these times, and it is best to avoid confrontations except when necessary for the care of the patient. When faced with ordering a diet for your first sick patient, you will no doubt be con- fronted with the inadequacy of your education in nutrition. Fortunately for your patient, di- etitians are available. Never hesitate to call one. In matters concerning drug interactions, side effects, individualization of dosages, alter- ation of drug dosages in disease, and equivalence of different brands of the same drug, it never hurts to call the pharmacist. Most medical centers have a pharmacy resident who fol- lows every patient on a floor or service and who will gladly answer any questions you have on medications. The pharmacist or pharmacy resident can very often provide pertinent arti- cles on a requested subject. YOUR HEALTH AND A WORD ON “AGGRESSIVENESS” In your months of curing disease both day and night, it becomes easy to ignore your own right to keep yourself healthy. There are numerous bad examples of medical and surgical in- terns who sleep 3 hours a night and get most of their meals from vending machines. Do not let anyone talk you into believing that you are not entitled to decent meals and sleep. If you offer yourself as a sacrifice, it will be a rare rotation on which you will not become one. “So You Want to Be a Scut Monkey” 3 You may have the misfortune someday of reading an evaluation that says a student was not “aggressive enough.” This is an enigmatic notion to everyone. Does it mean that the student re- fused to attempt to start an intravenous line after eight previous failures? Does it mean that the student was not consistently the first to shout out the answer over the mumblings of fellow stu- dents on rounds? Whatever constitutes “aggressiveness” must be a dubious virtue at best. A more appropriate virtue might be assertiveness in obtaining your education. Ask good questions, have the house staff show you procedures and review your chartwork, read about your patient’s illness, review the surgery basics before going to the OR, participate ac- tively in your patient’s care, and take an interest in other patients on the service. This ap- proach avoids the need for victimizing your patients and comrades that the definition of aggression suggests. ROUNDS Rounds are meetings of all members of the service for discussing the care of the patient. These occur daily and are of three kinds. Morning Rounds Also known as “work rounds,” these take place anywhere from 6:30 to 9:00 AM on most ser- vices and are attended by residents, interns, and students. This is the time for discussing what happened to the patient during the night, the progress of the patient’s evaluation or therapy or both, the laboratory and radiologic tests to be ordered for the patient, and, last but not least, talking with and evaluating the patient. Know about your patient’s most recent lab- oratory reports and progress—this is a chance for you to look good. Ideally, differences of opinion and any glaring omissions in patient care are politely dis- cussed and resolved here. Writing new orders, filling out consultations, and making any nec- essary telephone calls are best done right after morning rounds. Attending Rounds These vary greatly depending on the service and on the nature of the attending physician. The same people who gathered for morning rounds will be here, with the addition of the at- tending. At this meeting, the patients are often seen again (especially on the surgical ser- vices); significant new laboratory, radiographic, and physical findings are described (often by the student caring for the patient); and new patients are formally presented to the attend- ing (again, often by the medical student). The most important priority for the student on attending rounds is to know the patient. Be prepared to concisely tell the attending what has happened to the patient. Also be ready to give a brief presentation on the patient’s illness, especially if it is unusual. The attending will probably not be interested in minor details that do not affect therapeutic decisions. Ad- ditionally, the attending will probably not wish to hear a litany of normal laboratory values, only the pertinent ones, such as, Mrs. Pavona’s platelets are still 350,000/µL in spite of her bone marrow disease. You do not have to tell everything you know on rounds, but you must be prepared to do so. Open disputes among house staff and students are bad form on attending rounds. For this reason, the unwritten rule is that any differences of opinion not previously discussed shall not be initially raised in the presence of the attending. Check-out or Evening Rounds Formal evening rounds on which the patients are seen by the entire team a second time are typically done only on surgical services and pediatrics. Other services, such as, medicine, often will have check-out with the resident on call for the service that evening (sometimes 4 Clinician’s Pocket Reference, 9th Edition called “card rounds”). Expect to convene sometime between 3:00 and 7:00 PM on most days. All new data are presented by the person who collected them (usually the student). Orders are again written, laboratory work desired for early the next day is requested, and those un- fortunates on call compile a “scut list” of work to be done that night and a list of patients who need close supervision. BEDSIDE ROUNDS Basically, these are the same as any other rounds except that tact is at a premium. The first consideration at the bedside must be for the patient. If no one else on the team says “Good morning” and asks how the patient is feeling, do it yourself; this is not a presumptuous act on your part. Keep this encounter brief and then explain that you will be talking about the patient for a while. If handled in this fashion, the patient will often feel flattered by the at- tention and will listen to you with interest. Certain points in a hallway presentation are omitted in the patient’s room. The patient’s race and sex are usually apparent to all and do not warrant inclusion in your first sentence. The patient must never be called by the name of the disease, eg, Mrs. Pavona is not “a 45-year-old CML (chronic myelogenous leukemia)” but “a 45-year-old with CML.” The patient’s general appearance need not be reiterated. Descriptions of evidence of disease must not be prefaced by words such as outstanding or beautiful. Mrs. Pavona’s massive spleen is not beautiful to her, and it should not be to the physician or student either. At the bedside, keep both feet on the floor. A foot up on a bed or chair conveys impa- tience and disinterest to the patient and other members of the team. It is poor form to carry beverages or food into the patient’s room. Although you will probably never be asked to examine a patient during bedside rounds, it is still worthwhile to know how to do so considerately. Bedside examinations are often done by the attending at the time of the initial presentation or by one member of a surgical service on postoperative rounds. First, warn the patient that you are about to examine the wound or af- fected part. Ask the patient to uncover whatever needs to be exposed rather than boldly re- moving the patient’s clothes yourself. If the patient is unable to do so alone, you may do it, but remember to explain what you are doing. Remove only as much clothing as is necessary and then promptly cover the patient again. In a ward room, remember to pull the curtain. Bedside rounds in the intensive care unit call for as much consideration as they do in any other room. That still, naked soul on the bed might not be as “out of it” as the resident (or anyone else) might believe and may be hearing every word you say. Again, exercise discretion in dis- cussing the patient’s illness, plan, prognosis, and personal character as it relates to the disease. Remember that the patient information you are entrusted with as a health care provider is confidential. There is a time and place to discuss this sensitive information and public areas such as elevators or cafeterias are not the appropriate location for these discussions. READING Time for reading is at a premium on many services, and it is therefore important to use that time effectively. Unless you can remember everything you learned in the first 20 months of medical school, you will probably want to review the basic facts about the disease that brought your patient into the hospital. These facts are most often found in the same core texts that got you through the preclinical years. Unless specifically directed to do so, avoid the temptation to sit down with MEDLINE/Index Medicus to find all the latest articles on a disease you have not read about for the last 7 months; you do not have the time. The appropriate time to head for the MEDLINE/Index Medicus is when a therapeutic dilemma arises and only the most recent literature will adequately advise the team. You may wish to obtain some direction from the attending, the fellow, or the resident before plunging into “So You Want to Be a Scut Monkey” 5 the library on your only Friday night off call this month. Ask the residents or fellow students for the pocket manuals or PDA downloads that they found most useful for a given rotation. THE WRITTEN HISTORY AND PHYSICAL Much has been written on how to obtain a useful medical history and perform a thorough physical examination, and there is little to add to it. Three things worth emphasizing are your own physical findings, your impression, and your own differential diagnosis. Trust and record your own physical findings, even if other examiners have written things different from those you found. You just may be right, and, if not, you have learned something from it. Avoid the temptation to copy another examiner’s findings as your own when you are unable to do the examination yourself. Still, it would be an unusually cruel resident who would make you give Mrs. Pavona her fourth rectal examination of the day, and in this circumstance you may write “rectal per resident.” Do not do this routinely just to avoid performing a complete physical examination. Check with the resident first. Although not always emphasized in physical diagnosis, your clinical impression is probably the most important part of your write-up. Reasoned interpretation of the medical history and physical examination is what separates physicians from the computers touted by the tabloids as their successors. Judgment is learned only by boldly stating your case, even if you are wrong more often than not. The differential diagnosis, that is, your impression, should include only those entities that you consider when evaluating your patient. Avoid including every possible cause of your pa- tient’s ailments. List only those that you are seriously considering, and include in your plan what you intend to do to exclude each one. Save the exhaustive list for the time your attending asks for all the causes of a symptom, syndrome, or abnormal laboratory value. THE PRESENTATION The object of the presentation is to briefly and concisely (usually in a few minutes) describe your patient’s reason for being in the hospital to all members of the team who do not know the patient and the story. Unlike the write-up, which contains all the data you obtained, the presentation may include only the pertinent positive and negative evidence of a disease and its course in the patient. It is hard to get a feel for what is pertinent until you have seen and done a few presentations yourself. Practice is important. Try never to read from your write-up, as this often produces dull and lengthy presentations. Most attendings will allow you to carry note cards, but this method can also lead to trouble unless content is carefully edited. Presentations are given in the same order

as a write-up: identification, chief complaint, history of the present illness, past medical history, family history, psychosocial history, review of systems, physical exam- ination, laboratory and x-ray data, clinical impression, and plan. Only pertinent positives and negatives from the review of systems should be given. These and truly relevant items from other parts of the interview often can be added to the history of the present illness. Fi- nally, the length and content of the presentation vary greatly according to the wishes of the attending and the resident, but you will learn quickly what they do and do not want. RESPONSIBILITY Your responsibilities as a student should be clearly defined on the first day of a rotation by either the attending or the resident. Ideally, this enumeration of your duties should also in- clude a list of what you might expect concerning teaching, floor skills, presentations, and all the other things you are paying many thousand dollars a year to learn. 6 Clinician’s Pocket Reference, 9th Edition On some services, you may feel like a glorified unit secretary (clinical rotations are called “clerkships” for good reason!), and you will not be far from wrong. This is not what you are going into hock for. The scut work should be divided among the house staff. You will frequently be expected to call for a certain piece of laboratory data or to go re- view an x-ray with the radiologist. You may then mutter under your breath, “Why waste my time? The report will be on the chart in a day or two!” You will feel less annoyed in this situ- ation if you consider that every piece of data ordered is vital to the care of your patient. Outpatient clinic experiences are incorporated into many rotations today. The same basic rules and skill set necessary for inpatient care can be easily transferred to the outpa- tient setting. The student’s responsibility may be summarized in three words: know your patient. The whole service relies to a great extent on a well-informed presentation by the student. The better informed you are, the more time left for education and the better your evaluation will be. A major part of becoming a physician is learning responsibility. ORDERS Orders are the physician’s instructions to the nursing and other members of the professional staff on the care of the patient. These may include the frequency of vital signs, medications, respiratory care, laboratory and x-ray studies, and nearly anything else that you can imagine. There are many formats for writing concise admission, transfer, and postoperative or- ders. Some rotations may have a precisely fixed set of routine orders, but others will leave you and the intern to your own devices. It is important in each case to avoid omitting in- structions critical to the care of the patient. Although you will be confronted with a variety of lists and mnemonics, ultimately it is helpful to devise your own system and commit it to memory. Why memorize? Because when you are an intern and it is 3:30 AM, you may over- look something if you try to think it out. One system for writing admission or transfer orders uses the mnemonic “A.D.C. Vaan Diml” and is discussed in Chapter 2. The word stat is the abbreviation for the Latin word statim, which means “immedi- ately.” When added to any order, it puts the requested study in front of all the routine work waiting to be done. Ideally, this order is reserved for the truly urgent situation, but in prac- tice it is often inappropriately used. Most of the blame for this situation rests with physi- cians who either fail to plan ahead or order stat lab results when routine studies would do. Student orders usually require a co-signature from a physician, although at some institu- tions students are allowed to order routine laboratory studies. Do not ask a nurse or pharma- cist to act on an unsigned student order; it is illegal for them to do so. The intern is usually responsible for most orders. The amount of interest shown by the resident and the attending varies greatly, but ideally you will review the orders on routinely admitted patients with the intern. Have the intern show you how to write some orders on a few patients, then take the initiative and write the orders yourself and review them with the intern. THE DAY The events of the day and the effective use of time are two of the most distressing enigmas encountered in making the transition from preclinical to clinical education. For example, there are no typical days on surgical services, as the operating room schedule prohibits mak- ing rounds at a regularly scheduled time every day. The following are suggestions that will help on any service. “So You Want to Be a Scut Monkey” 7 1. Schedule special studies early in the day. The free time after work rounds is usually ideal for this. Also, call consultants early in the morning. Often, they can see your pa- tient on the same day or at least early the next day. 2. Try to take care of all your business in the radiology department in one trip unless a given problem requires viewing a film promptly. Do not make as many separate trips as you have patients. 3. Make a point of knowing when certain services become unavailable, for example, elec- trocardiograms, contrast-study scheduling, and blood drawing. Be sure to get these pro- cedures done while it is still possible to do so. 4. Make a daily work or “scut”* list, and write down laboratory results as soon as you ob- tain them. Few people can keep all the daily data in their heads without making errors. 5. Try to arrange your travels around the hospital efficiently. If you have patients to see on four different floors, try to take care of all their needs, such as, drawing blood, remov- ing sutures, writing progress notes, and calling for consultations, in one trip. 6. Strive to work thoroughly but quickly. If you do not try to get work done early, you never will (this is not to say that you will succeed even if you do try). There is no sin in leaving at 5:00 PM or earlier if your obligations are completed and the supervising resi- dent has dismissed you. A PARTING SHOT The clinical years are when all the years of premed study in college and the first two years of medical school suddenly come together. Trying to tell you adequately about being a clini- cal clerk is similar to trying to make someone into a swimmer on dry land. The terms to describe new clinical clerks may vary at different medical centers (“scut monkey,” “scut boy,” “scut dog,” “torpedoes”). These euphemistic expressions describing the new clinical clerk acknowledge that the transition, a sort of rite of passage, into the next phase of physician training has occurred. It is hoped that this “So You Want to Be a Scut Monkey” introduction and the information contained in this book will give you a good start as you enter the “hands on” phase of becoming a successful and respected physician. * Although the origin of the word scut is obscure, it probably represents an acronym for “some common unfinished task” or “some clinically useful training.” This page intentionally left blank. 1 1 HISTORY AND PHYSICAL EXAMINATION History and Physical Examination Dental Examination Psychiatric History and Physical Dermatologic Descriptions Psychiatric Mental Status Dermatome and Cutaneous Innervation Examination Physical Symptoms and Eponyms Mini Mental Status Examination Example of a Written History and Heart Murmurs and Extra Heart Sounds Physical Examination Blood Pressure Guidelines HISTORY AND PHYSICAL EXAMINATION An example of a complete H&P write-up can be found on page 28. The details provided and length of the written H&P can vary with the particular problem and with the service to which the patient is admitted. History Identification: Name, age, sex, referring physician, and the informant (eg, patient, rela- tive, old chart) and the informant’s reliability. Chief Complaint: State, in patient’s own words, the current problem. History of the Present Illness (HPI): Defines the present illness by quality; quan- tity; setting; anatomic location and radiation; time course, including when it began; whether the complaint is progressing, regressing, or steady; of constant or intermittent frequency; and aggravating, alleviating, and associated factors. The information should be in chrono- logic order, including diagnostic tests done prior to admission. Related history, including previous treatment for the problem, risk factors, and pertinent negatives should be included. Any other significant ongoing problems should be included in the HPI in a separate section or paragraph. For instance, if a patient with poorly controlled diabetes mellitus comes to the emergency room because of chest pain, the HPI would first include information regarding the chest pain followed by a detailed history of the diabetes mellitus. If the diabetes mellitus was well controlled or diet-controlled, the history of the diabetes mellitus is placed in the past medical history. Past Medical History (PMH): Current medications, including OTC medications, vit- amins, and herbals; allergies (drugs and other—include how allergies are manifested); surg- eries; hospitalizations; blood transfusions, include when and how many units and the type of blood product; trauma; stable current and past medical problems unrelated to the HPI. Spe- cific illnesses to inquire about include diabetes mellitus, hypertension, MI, stroke, peptic ulcer disease, asthma, emphysema, thyroid and kidney disease, bleeding disorders, cancer, 9 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 10 Clinician’s Pocket Reference, 9th Edition 1 TB, hepatitis, and STDs. Also inquire about routine health maintenance. This category de- pends on the age and sex of the patient but could include last Pap smear and pelvic exam, breast exam, whether the patient does self breast examination, date of last mammogram, diphtheria/tetanus immunization, pneumococcal and flu vaccine, stool samples for hemoc- cult, sigmoidoscopy, cholesterol, HDL cholesterol, and use of seat belts. Pediatric patients: Include prenatal and birth history, feedings, food intolerance, and immunization history. Family History: Age, status (alive, dead) of blood relatives and medical problems for any blood relatives (inquiry about cancer, especially breast, colon, and prostate; TB, asthma; MI; HTN; thyroid disease; kidney disease; peptic ulcer disease; diabetes mellitus; bleeding disorders; glaucoma, and macular degeneration). Can be written out or use family tree. Psychosocial (Social) History: Stressors (financial, significant relationships, work or school, health) and support (family, friends, significant other, clergy); life-style risk factors, (alcohol, drugs, tobacco, and caffeine use; diet; and exposure to environmental agents; and sexual practices); patient profile (may include marital status and children; present and past employment; financial support and insurance; education; religion; hobbies; beliefs; living conditions); for veterans, include military service history. Pediatric patients: Include grade in school, sleep, and play habits. Review of Systems (ROS) General. Weight loss, weight gain, fatigue, weakness, appetite, fever, chills, night sweats Skin. Rashes, pruritus, bruising, dryness, skin cancer or other lesions Head. Trauma, headache, tenderness, dizziness, syncope Eyes. Vision, changes in the visual field, glasses, last prescription change, photophobia, blurring, diplopia, spots or floaters, inflammation, discharge, dry eyes, excessive tear- ing, history of cataracts or glaucoma Ears. Hearing changes, tinnitus, pain, discharge, vertigo, history of ear infections Nose. Sinus problems, epistaxis, obstruction, polyps, changes in or loss of sense of smell Throat. Bleeding gums; dental history (last checkup, etc); ulcerations or other lesions on tongue, gums, buccal mucosa Respiratory. Chest pain; dyspnea; cough; amount and color of sputum; hemoptysis; history of pneumonia, influenza, pneumococcal vaccinations, or positive PPD Cardiovascular. Chest pain, orthopnea, dyspnea on exertion, paroxysmal nocturnal dyspnea, murmurs, claudication, peripheral edema, palpitations Gastrointestinal. Dysphagia, heartburn, nausea, vomiting, hematemesis, indigestion, ab- dominal pain, diarrhea, constipation, melena (hematochezia), hemorrhoids, change in stool shape and color, jaundice, fatty food intolerance Gynecologic. Gravida/para/abortions; age at menarche; last menstrual period (frequency, duration, flow); dysmenorrhea; spotting; menopause; contraception; sexual history, in- cluding history of venereal disease, frequency of intercourse, number of partners, sexual orientation and satisfaction, and dyspareunia Genitourinary. Frequency, urgency, hesitancy; dysuria; hematuria; polyuria; nocturia; incon- tinence; venereal disease; discharge; sterility; impotence; polyuria; polydipsia; change in urinary stream; and sexual history, including frequency of intercourse, number of partners, sexual orientation and satisfaction, and history of venereal disease Endocrine. Polyuria, polydipsia, polyphagia, temperature intolerance, glycosuria, hormone therapy, changes in

hair or skin texture Musculoskeletal. Arthralgias, arthritis, trauma, joint swelling, redness, tenderness, limita- tions in ROM, back pain, musculoskeletal trauma, gout 1 History and Physical Examination 11 1 Peripheral Vascular. Varicose veins, intermittent claudication, history of thrombophlebitis Hematology. Anemia, bleeding tendency, easy bruising, lymphadenopathy Neuropsychiatric. Syncope; seizures; weakness; coordination problems; alterations in sensa- tions, memory, mood, sleep pattern; emotional disturbances; drug and alcohol problems Physical Examination General: Mood, stage of development, race, and sex. State if patient is in any distress or is assuming an unusual position, such as, sitting up leaning forward (position often seen in patients with acute exacerbation of COPD or pericarditis) Vital Signs: Temperature (note if oral, rectal, axillary), pulse, respirations, blood pressure (may include right arm, left arm, lying, sitting, standing), height, weight. Blood pres- sure and heart rate supine and after standing 1 min should always be included if volume depletion is suspected, such as in GI bleeding, diarrhea, dizziness, or syncope. Skin: Rashes, eruptions, scars, tattoos, moles, hair pattern (See page 20 for definitions of dermatologic lesions.) Lymph Nodes: Location (head and neck, supraclavicular, epitrochlear, axillary, inguinal), size, tenderness, motility, consistency Head, Eyes, Ears, Nose, and Throat (HEENT) Head. Size and shape, tenderness, trauma, bruits. Pediatric patients: Fontanels, suture lines Eyes. Conjunctiva; sclera; lids; position of eyes in orbits; pupil size, shape, reactivity; ex- traocular muscle movements; visual acuity (eg, 20/20); visual fields; fundi (disc color, size, margins, cupping, spontaneous venous pulsations, hemorrhages, exudates, A-V ratio, nicking) Ears. Test hearing, tenderness, discharge, external canal, tympanic membrane (intact, dull or shiny, bulging, motility, fluid or blood, injected) Nose. Symmetry; palpate over frontal, maxillary, and ethmoid sinuses; inspect for obstruc- tion, lesions, exudate, inflammation. Pediatric patients: Nasal flaring, grunting Throat. Lips, teeth, gums, tongue, pharynx (lesions, erythema, exudate, tonsillar size, pres- ence of crypts) Neck: ROM, tenderness, JVD, lymph nodes, thyroid examination, location of larynx, carotid bruits, HJR. JVD should be reported in relationship to the number of centime- ters above or below the sternal angle, such as “1 cm above the sternal angle,” rather than “no JVD.” Chest: Configuration and symmetry of movement with respiration; intercostal retractions; palpation for tenderness, fremitus, and chest wall expansion; percussion (include dia- phragmatic excursion); breath sounds; adventitious sounds (rales, rhonchi, wheezes, rubs). If indicated: vocal fremitus, whispered pectoriloquy, egophony (found with con- solidation) Heart: Rate, inspection, and palpation of precordium for point of maximal impulse and thrill; auscultation at the apex, LLSB, and right and left second intercostal spaces with diaphragm and apex and LLSB with bell. Heart murmurs are reviewed on pages 16 to 18. Breast: Inspection for nipple discharge, inversion, excoriations and fissures, and skin dim- pling or flattening of the contour; palpation for masses, tenderness; gynecomastia in males Abdomen: Note shape (scaphoid, flat, distended, obese); examine for scars; auscultate for bowel sounds and bruits; percussion for tympani and masses; measure liver size (span in midclavicular line); note costovertebral angle tenderness; palpate for tenderness (if present, check for rebound tenderness), note hepatomegaly, splenomegaly; guarding, in- guinal adenopathy 12 Clinician’s Pocket Reference, 9th Edition 1 Male Genitalia: Inspect for penile lesions, scrotal swelling, testicles (size, tenderness, masses, varicocele), and hernia, and observe for transillumination of testicular masses Pelvic: See Chapter 13, page 289. Rectal: Inspect and palpate for hemorrhoids, fissures, skin tags, sphincter tone, masses, prostate (size [grade from small 1+ to massively enlarged 4+], note any nodules, tender- ness); note presence or absence of stool; test stool for occult blood Musculoskeletal: Note amputations, deformities, visible joint swelling, and ROM; also pal- pate joints for swelling, tenderness, and warmth Peripheral Vascular: Note hair pattern; color change of skin; varicosities; cyanosis; club- bing; palpation of radial, ulnar, brachial, femoral, popliteal, posterior tibial, dorsalis pedis pulses; simultaneous radial pulses; calf tenderness; Homans’s sign; edema; aus- cultate for femoral bruits Neurologic Mental Status Examination. (If appropriate, see sections “Psychiatric History and Physical,” and “Psychiatric Mental Status Examination,” page 13.) Cranial Nerves. There are 12 cranial nerves, the functions of which are as follows: • I Olfactory—Smell • II Optic—Vision, visual fields, and fundi; afferent limb of pupillary response • III, IV, VI Oculomotor, trochlear, abducens—Efferent limb pupillary response, pto- sis, volitional eye movements, pursuit eye movements • V Trigeminal—Corneal reflex (afferent), facial sensation, masseter and temporalis muscle tested by biting down • VII Facial—Raise eyebrows, close eyes tight, show teeth, smile, or whistle, corneal reflex (efferent) • VIII Acoustic—Test hearing by watch tick, finger rub, Weber–Rinne test (see also page 27) to be done if hearing loss noted on history or by gross testing. (Air conduc- tion lasts longer than bone conduction in a normal person.) • IX, X Glossopharyngeal and vagus—Palate moves in midline; gag; speech • XI Spinal accessory—Shoulder shrug, push head against resistance. • XII Hypoglossal—Stick out tongue. Strength can be tested by having the patient press tongue against the buccal mucosa on each side and the examiner can press a finger against the patient’s cheek. Also look for fasciculations. Motor. Strength should be tested in upper and lower extremities proximally and distally. (Grading system: 5 active motion against full resistance; 4 active motion against some resistance; 3 active motion against gravity; 2 active motion with gravity eliminated; 1 barely detectable motion; 0 no motion or muscular contraction detected) Cerebellum. Romberg’s test (see page 27)—heel to shin (should not be with assistance from gravity), finger to nose, heel and toe walking, rapid alternating movements upper and lower extremities Sensory. Pain (sharp) or temperature distal and proximal upper and lower extremities, vibra- tion using either a 128- or 256-Hz tuning fork or position sense distally upper and lower extremities, and stereognosis or graphesthesia. Identify any deficit using the dermatome and cutaneous innervation diagrams (see Figure 1–3). Reflexes. Brachioradialis and biceps C5–6, triceps C7–8, abdominal (upper T8–10, lower T10–12), quadriceps (knee) L3–4–5, ankle S1–2, (Grading system: 4+ Hyperactive with clonus; 3+ brisker than usual; 2+ normal or average; 1+ decreased or less than normal; 0 absent). Check for pathologic reflexes: Babinski’s sign, Hoffmann’s sign, snout, others (see pages 21 to 27). Pediatric patients: Moro’s reflex (startle) and suck re- flexes 1 History and Physical Examination 13 1 Database Laboratory tests, x-rays ordered as indicated by the history and physical Problem List (See example page 31.) Should include entry date of problem, date of problem onset, prob- lem number. (With initial problem list, the more severe problems are numbered first. After the initial list is generated, problems are added chronologically.) List problem by status: ac- tive or inactive. Assessment (Impression) A discussion and evaluation of the current problems with a differential diagnosis. Plan: Additional laboratory tests, medical treatment, consults, etc. Note: The history and physical examination should be legibly signed and your title noted. Each entry should be dated and timed. PSYCHIATRIC HISTORY AND PHYSICAL The elements of the psychiatric history and physical are identical to those of the basic his- tory and physical outlined earlier. The main difference involves attention to the past psychi- atric history and more detailed mental status examination as described in the following section. Psychiatric Mental Status Examination The following factors are evaluated as part of the psychiatric status examination. • Appearance: Gestures, mannerisms, and so on • Speech: Coherence, flight of ideas, and so on • Mood and Affect: Depression, elation, anger, and so on • Thought Process: Blocking, evasion, and so on • Thought Content: Worries, hypochondriasis, lack of self-confidence, delusions, hallucinations, and so on • Motor Activity: Slow, rapid, purposeful, and so on • Cognitive Functions: Attention and concentration Memory (immediate, recent, and remote recall) Calculations Abstractions Judgment Mini Mental Status Examination A thorough mental status exam should be done on every geriatric patient, every patient with AIDS, and any patient suspected of having dementia. The mini mental status exam is a sim- ple, practical test that takes only a few minutes and can be followed over time. It may show progression, improvement, or no changes in the underlying process. The mini mental status exam developed by Folstein, Folstein, and McHugh is discussed in detail in the Journal of Psychiatric Research, 1975, Vol. 12, pages 189–198. The test is divided into two sections: one assessing orientation, memory, and attention and the other testing the patient’s ability to 14 Clinician’s Pocket Reference, 9th Edition 1 write a sentence and to copy a diagram (usually two intersecting pentagons whose intersect forms a four-sided figure. Table 1–1 is the “Mini Mental State” Examination as outlined by Folstein and associates. HEART MURMURS AND EXTRA HEART SOUNDS Table 1–2 and Figure 1–1 describe the various types of heart murmurs and extra heart sounds. BLOOD PRESSURE GUIDELINES There is a clear association between hypertension and coronary artery and cerebrovascular disease. Hypertension is defined as systolic BP >140 mm Hg or a diastolic BP >90 mm Hg in adults. Measure the BP after 5 min of rest with patient seated and arm at heart level. Use the bell of the stethoscope, the last sounds heard are the Korotkoff sounds, which are low- pitched. Take the average of two readings separated by 2 min. Elevated readings on three separate days should be obtained prior to diagnosing hypertension. Classification and fol- low-up recommendations for adults are shown in Table 1–3. In children from age 1 to 10 years, systolic blood pressure can be calculated as follows: Lower limits (5th percentile): 70 mm Hg + (child’s age in years × 2) Typical (50th percentile): 90 mm Hg + (child’s age in years × 2) DENTAL EXAMINATION The dental examination is an often overlooked part of the history and physical. Many times, the patient may have some intraoral problem that is contributing to the overall medical con- dition (ie, the inability to eat due to a toothache, abscess, or ill-fitting denture in a poorly controlled diabetic) for which a dental consult may be necessary. Loose dentures can com- promise the ability to manually maintain an open airway. In addition, in an emergency situa- tion when intubation is necessary, complications may occur if the clinician is unfamiliar with the oral structures. The patient may be able to give some dental history, including recent toothaches, ab- scesses, and loose teeth or dentures. Be sure to ask if the patient is wearing a removable par- tial denture (partial plate), which should be removed before intubation. As lost dentures are a chief dental complaint of hospitalized patients, care must be taken not to misplace the re- moved prosthesis. A brief dental examination may be performed with gloved hand, two tongue blades, and a flashlight. Look for any obvious inflammation, erythema, edema, or ulceration of the gin- giva (gums) and oral mucosa. Gently tap on any natural teeth to test for sensitivity. Place each tooth between two tongue blades and push gently to check for looseness. This is espe- cially important for the maxillary anterior teeth, which serve as the fulcrum for the laryngo- scope blade. Any abnormal dental findings should be noted and the appropriate consults obtained. Many diseases, including AIDS, STDs, pemphigus, pemphigoid, allergies, uncon- trolled diabetes, leukemia, and others, may first manifest themselves in the mouth. Hospitalized patients often have difficulty cleaning their teeth or dentures. This care should be added to the daily orders if indicated. Patients who will be receiving head and neck radiation must be examined and treated for any tooth extractions or dental infections before the initiation of the radiation therapy. Extractions after radiation to the maxilla and particularly the mandible may lead to osteoradionecrosis, a condition that may be impossi- ble to control. (text continues on page 17 ) 1 History and Physical Examination 15 1 TABLE 1–1 The Mini Mental State Examination Patient ____________________________ Examiner ___________________________ Date ______________________________ “Mini Mental State” Maximum Score Score Orientation 5 What is the (year) (season) (date) (day) (month)? 5 Where are we? (state) (county) (town) (hospital) (floor) Registration 3 Name 3 objects: 1 second to say each. Then ask the patient all 3 after you have said them. Give 1 point for each correct answer. Then repeat until he learns all 3. Count trials and record. Trials _______________________________ Attention and Calculation 5 Serial 7’s: One point for each correct. Stop after 5 an- swers. Alternatively, spell “world” backward. Recall 3 Ask for the 3 objects repeated above. Give 1 point for each correct answer. Language 9 Point to a pencil, and

watch and ask the patient to name it. (2 points) Repeat the following: “No if’s, and’s, or but’s.” (1 point) Follow a 3-stage command: “Take a paper in your right hand, fold it in half, and put it on the floor.” (3 points) Read and obey the following: Close your eyes (1 point) Write a sentence (1 point) Copy design (1 point) ________________________ Total Score Assess level of consciousness along the following continuum Alert Drowsy Stupor Coma Source: Based on data from Folstein, Folstein, and McHugh: J Psychiatr Res 1975; 12:189–198, 1975. 16 Clinician’s Pocket Reference, 9th Edition 1 TABLE 1–2 Heart Murmurs and Extra Heart Sounds* Type† Description A. Aortic stenosis (AS) Heard best at second intercostal space. Sys- tolic (medium-pitched) crescendo–de- crescendo murmur with radiation to the carotid arteries. A2 decreased, ejection click and S4 often heard at apex. Paradoxical splitting of S2. Narrow pulse pressure and delayed carotid upstroke and left ventricular hypertrophy (LVH) with lift at apex. B. Aortic insufficiency (AI) Heard best at left lower sternal border third and fourth interspace with patient sitting up, leaning forward and fully exhaled. Diastolic (high-pitched) decrescendo murmur. Often with LVH. Widened pulse pressure, bisferious pulse, Traube’s sign, Quincke’s sign, and Cor- rigan’s pulse may be seen with chronic aortic insufficiency. S3 and pulsus alternans often present with acute aortic insufficiency. C. Pulmonic stenosis (PS) Heard best at left second intercostal space. Systolic crescendo–decrescendo murmur. Louder with inspiration. Click often present. P2 delayed and soft if severe. Right ventricu- lar hypertrophy (RVH) with parasternal lift. D. Pulmonic insufficiency (PI) Heard best at left second intercostal space. Diastolic decrescendo or crescendo– decrescendo murmur. Louder with inspiration. RVH usually present. E. Mitral stenosis (MS) Localized at the apex. Diastolic (low-pitched rumbling sound) murmur heard best with the bell in the left lateral decubitus position. With increased or decreased S1. Opening snap (OS) heard best at apex with diaphragm. In- creased P2, right-sided S4, left-sided S3 often present. RVH with parasternal lift may be present. F. Mitral insufficiency (MI) Heard best at apex. Holosystolic (high- pitched) murmur with radiation to axilla. Soft S1, may be masked by murmur. S3 and LVH often present. Midsystolic click suggests mi- tral valve prolapse. G. Tricuspid insufficiency (TI) Heard best at left lower sternal border. Holosystolic (high-pitched) murmur. Increases with inspiration. Right-sided S3 often present. Large V wave in jugular venous pulsations. (continued) 1 History and Physical Examination 17 1 TABLE 1–2 (Continued) Type† Description H. Atrial septal defect (ASD) Heard best at left upper sternal border. Sys- tolic (medium-pitched) murmur. Fixed splitting of S2 and RVH, often with left- and right-sided S4. I. Ventricular septal defect Heard best at left lower sternal border. Harsh (VSD) holosystolic (high-pitched) murmur with midsystolic peak. S1 and S2 may be soft. J. Patent ductus Heard best at left first and second intercostal arteriosus (PDA) space. Continuous, machinery (medium- pitched) murmur. Increased P2 and ejection click may be present. K. Third heard sound (S3) Early diastolic sound caused by rapid ventric- ular filling. Heard best with bell. Left-sided S3 heard at apex, right-sided S3 heard at left lower sternal border. Left-sided S3 seen nor- mally in young people, also pregnancy, thy- rotoxicosis, mitral regurgitation, and congestive heart failure. L. Fourth heart sound (S4) Late diastolic sound caused by a noncompli- ant ventricle. Heard best with bell. Left-sided S4 heard at apex, right-sided S4 heard at left lower sternal border. Left-sided S4 seen with hypertension, aortic stenosis, and myocardial infarction. Right-sided S4 seen with pulmonic stenosis and pulmonary hypertension. *Refer to Figure 1–1 for graphic representations of murmurs. †Capital letters preceding type of murmur refer to graphs in Figure 1–1. Eruption of Teeth The eruption of teeth may be of great concern to new parents. Often, parents think some- thing is developmentally wrong with their child if teeth have not appeared by a certain age. The timing of tooth eruption varies tremendously. Factors contributing to this variation in- clude family history, ethnic background, vitality during fetal development, position of teeth in the arch, size and shape of the dental arch itself, and, in the case of the eruption of perma- nent teeth, when the primary tooth was lost. Radiographs of the maxilla and mandible can determine whether or not the teeth are present. Figure 1–2 serves as a guide to the chron- ology of tooth eruption. Remember that variations may be greater than 1 year in some cases. 18 Clinician’s Pocket Reference, 9th Edition 1 Systole Diastole Type S1 P2 A A S 2 4 AS S1 P B A 2 2 AI S1 A2 C P2 PS S1 P2 D A2 PI P2 OS E S1 A2 MS A2 P2 F S1 MR S1 A G 2 P2 TR S1 H A2 P2 ASD S1 A2 I P2 VSD S1 P J 2 A PDA 2 S A 1 2 P2 S K 3 S3 S1 L A S4 2 P2 S4 FIGURE 1–1 Graphic representation of common heart murmurs. See Table 1–2 for abbreviations and descriptions of murmurs. 1 History and Physical Examination 19 1 Erupt Shed (months) (years) Central incisor 8–12 6–7 Lateral incisor 9–13 7–8 Canine (cuspid) 16–22 10–12 First molar 13–19 9–11 Upper teeth Second molar 25–33 10–12 Primary Second molar 23–31 10–12 Lower teeth First molar 14–18 9–11 Canine (cuspid) 17–23 9–12 Lateral incisor 10–16 7–8 Cental incisor 6–10 6–7 Erupt (years) Central incisor 7–8 Lateral incisor 8–9 Canine (cuspid) 11–12 First premolar 10–11 (first bicuspid) Second premolar 10–12 (second bicuspid) Upper teeth First molar 6–7 Second molar 12–13 Third molar 17–21 (wisdom tooth) Permanent Third molar 17–21 (wisdom tooth) Second molar 11–13 Lower teeth First molar 6–7 Second premolar 11–12 (second bicuspid) First premolar 10–12 (first bicuspid) Canine (cuspid) 9–10 Lateral incisor 7–8 Central incisor 6–7 FIGURE 1–2 Dentition development sequences. The age when teeth are shed and erupt varies widely. (Based on data from: McDonald RE and Avery DR [eds]: Den- tistry for the Child and Adolescent, Mosby, St. Louis, 1994. Used with permission.) 20 Clinician’s Pocket Reference, 9th Edition 1 TABLE 1–3 Guidelines for Blood Pressure Management in Adults CLASSIFICATION SYSTEM Systolic Diastolic Category (mm Hg) (mm Hg) Desired <120 <80 Normal <130 <85 High normal 130–139 85–89 Hypertension Stage 1 140–159 90–99 Stage 2 160–179 100–109 Stage 3 >180 >110 FOLLOW-UP RECOMMENDATIONS INITIAL SCREENING BP (MM HG) Systolic Diastolic Action <130 <85 Recheck in 2 years 130–139 85–89 Recheck in 1 yr 140–159 90–99 Confirm within 2 months 160–179 100–109 Evaluate or refer within 1 month >180 >110 Evaluate or refer immediately or within 1 wk depending on the clinical situ- ation DERMATOLOGIC DESCRIPTIONS Atrophy: Thinning of the surface of the skin with associated loss of normal markings. Ex- amples: Aging, striae associated with obesity, scleroderma Bulla: A superficial, well-circumscribed, raised, fluid-filled lesion greater than 1 cm in diameter. Examples: Bullous pemphigoid, pemphigus, dermatitis herpetiformis Burrow: A subcutaneous linear track made by a parasite. Example: Scabies Crust: A slightly raised lesion with irregular border and variable color resulting from dried blood, serum, or other exudate. Examples: Scab resulting from an abrasion, or impetigo Ecchymoses: A flat, nonblanching, red-purple-blue lesion that results from extravasation of red blood cells into the skin. Differs from purpura in that ecchymoses are large purpura. Examples: Trauma, long-term steroid use Erosion: A depressed lesion resulting from loss of epidermis due to rupture of vesicles or bullae. Example: Rupture of herpes simplex blister 1 History and Physical Examination 21 1 Excoriation: A linear superficial lesion, which may be covered with dried blood. Early le- sions with surrounding erythema. Often self-induced. Example: Scratching associated with pruritus from any cause Fissure: A deep linear lesion into the dermis. Example: Cracks seen in athlete’s foot Keloid: Irregular, raised lesion resulting from scar tissue that is hypertrophied. Examples: Often seen with burns, and African-Americans are more prone to keloid formation. Lichenification: A thickening of the skin with an increase in skin markings resulting from chronic irritation and rubbing. Example: Atopic dermatitis Macule: A circumscribed nonpalpable discoloration of the skin less than 1 cm in diameter. Examples: Freckles, rubella, petechiae Nodule: A solid, palpable, circumscribed lesion larger than a papule and smaller than a tumor. Examples: Erythema nodosum, gouty tophi Papule: A solid elevated lesion less than 1 cm. Examples: Acne, warts, insect bites Patch: A nonpalpable discoloration of the skin with an irregular border, greater than 1 cm in diameter. Example: Vitiligo Petechiae: A flat pinhead-sized, nonblanching, red-purple lesion caused by hemorrhage into the skin. Example: Seen in DIC, ITP, SLE, meningococcemia (Neisseria meningitidis) Plaque: A solid, flat, elevated lesion greater than 1 cm in diameter. Examples: Psoriasis, discoid lupus erythematosus, actinic keratosis Purpura: A flat, nonblanching, red-purple lesion larger than petechiae caused by hemor- rhage into the skin. Examples: Henoch–Schönlein purpura, TTP. Pustule: A vesicle that is filled with purulent fluid. Examples: Acne, impetigo Scales: Partial separation of the superficial layer of skin. Examples: Psoriasis, dandruff Scar: Replacement of normal skin with fibrous tissue, often resulting from injury. Exam- ples: Surgical scar, burn Telangiectasia: Dilatation of capillaries resulting in red, irregular, clustered lines that blanch. Examples: Seen in scleroderma, Osler–Weber–Rendu disease, cirrhosis Tumor: A solid, palpable, circumscribed lesion that is greater than 2 cm in diameter. Exam- ple: Lipoma Ulcer: A depressed lesion resulting from loss of epidermis and part of the dermis. Exam- ples: Decubitus ulcers, primary lesion of syphilis, venous stasis ulcer Vesicle: A superficial, well-circumscribed, raised, fluid-filled lesion that is less than 1 cm in diameter. Examples: Herpes simplex, varicella (chickenpox) Wheal: Slightly raised, red, irregular lesions that are transient and secondary to edema of the skin. Examples: Urticaria (hives), allergic reaction to injections or insect bites DERMATOME AND CUTANEOUS INNERVATION The diagrams (Figures 1–3A and B) demonstrate dermatome levels and cutaneous innerva- tion distribution useful in the physical examination. PHYSICAL SYMPTOMS AND EPONYMS Allen’s Test: (See Chapter 13, page 246.) Apley’s Test: Determination of meniscal tear in the knee by grinding the joint manually Argyll–Robertson Pupil: Bilaterally small, irregular, unequal pupils that react to accom- modation but not to light. Seen with tertiary syphilis Austin Flint Murmur: Late diastolic mitral murmur; associated with aor1ic insufficiency with a normal mitral valve 22 Clinician’s Pocket Reference, 9th Edition 1 Peripheral nerve Nerve root Ophthalmic branch Trigeminal Maxillary branch Mandibular branch Anterior cutaneous nerve of neck C3 Supraclavicular nerves Post. Mid. Ant C4 T2 C5 Axillary nerve T3 T4 T5 T2 Medial cutaneous nerve of arm T6 Lateral cutaneous nerve of arm T7 T8 T9 T1 T10 Medial cutaneous nerve of forearm T11 C6 † T12 Lateral cutaneous nerve of forearm X L1 L1 Radial * C6 Median L2 C8 Ulnar C7 Lateral femoral cutaneous Obturator L3 Medial femoral cutaneous Anterior femoral cutaneous Lateral cutaneous nerve of calf Saphenous L4 L5 X = Iliohypogastric † = Ilioinguinal Superficial peroneal * = Genitofemoral Dorsal nerve of penis Perineal Sural S1 Lateral and medial plantar Deep peroneal A FIGURE 1–3 A: Dermatomes and cutaneous innervation patterns, anterior view. (Reprinted, with permission, from: Aminoff MJ et al [eds]: Clinical Neurology, 3rd ed, Appleton & Lange, Stamford CT, 1996.) Lateral thoracic rami Anterior thoracic rami 1 History and Physical Examination 23 1 Nerve root Peripheral nerve Great occipital C2 Lesser occipital Greater auricular C3 Posterior rami of cervical nerves C4 Supraclavicular T2 T3 Axillary T4 C5 T5 T2 T6 Medial cutaneous nerve of arm T7 T8 T9 Posterior cutaneous nerve T10 T1 of forearm T11 C6 T12 Medial cutaneous nerve L1 of forearm P X L2 lu o mste b r Lateral cutaneous nerve a io r r ra of forearm m S3 i Posterior C6 S4 sacral Radial S5 rami X = Iliohypogastric Median C7 C8 Ulnar Lateral femoral cutaneous Obturator L 3 Anterior femoral cutaneous S2 Posterior femoral cutaneous Medial femoral cutaneous Lateral cutaneous nerve of calf L L Superficial peroneal 5 4 Saphenous Sural Calcaneal S1 Lateral plantar Medial plantar B FIGURE 1–3 B: Dermatomes and cutaneous innervation patterns, posterior view. (Reprinted, with permission, from: Aminoff MJ et al [eds]: Clinical Neurology, 3rd ed, Appleton & Lange, Stamford CT, 1996.) Posterior thoracic rami Lateral thoracic rami 24 Clinician’s Pocket Reference, 9th Edition 1 Babinski’s Sign: Extension of the large toe with stimulation of the plantar surface of the foot instead of the normal flexion; indicative of upper motor neuron

disease (normal in neonates) Bainbridge’s Reflex: Increased heart rate due to increased right atrial pressure Battle’s Sign: Ecchymosis behind the ear associated with basilar skull fractures. Beau’s Lines: Transverse depressions in nails due to previous systemic disease Beck’s Triad: JVD, diminished or muffled heart sounds, and decreased blood pressure as- sociated with cardiac tamponade Bell’s Palsy: Lower motor neuron lesion of the facial nerve affecting muscles of upper and lower face. Easily distinguished from upper motor lesions, which affect predominately muscles of lower face since upper motor neurons from each side innervate muscles on both sides of the upper face Bergman’s Triad: Altered mental status, petechiae, and dyspnea associated with fat embo- lus syndrome Biot’s Breathing: Seen with brain injury; abruptly alternating apnea and equally deep breaths Bisferious Pulse: A double-peaked pulse seen in severe chronic aortic insufficiency Bitot’s Spots: Small scleral white patches suggesting vitamin A deficiency Blumberg’ Sign: Pain felt in the abdomen when steady constant pressure is quickly re- leased. Seen with peritonitis Blumer’s Shelf: Hardness palpable on rectal examination due to metastatic cancer of the rectouterine (pouch of Douglas) or rectovesical pouch Bouchard’s Nodes: Hard, nontender, painless nodules in the dorsolateral aspects of the proximal interphalangeal joints associated with osteoarthritis. Results from hypertrophy of the bone Branham’s Sign: With large AV fistulas, abrupt slowing of the heart rate with compression of the feeding artery Brudzinski’s Sign: Flexion of the neck causing flexion of the hips in meningitis Chadwick’s Sign: Bluish color of cervix and vagina, seen with pregnancy Chandelier’s Sign: Extreme pain elicited with movement of the cervix during bimanual pelvic examination. Indicates PID Charcot’s Triad: Right upper quadrant pain, fever (chills), and jaundice associated with cholangitis Cheyne–Stokes Respiration: Repeating cycle of a gradual increase in depth of breathing followed by a gradual decrease to apnea; seen with CNS disorders, uremia, some nor- mal sleep patterns Chvostek’s Sign: Tapping over the facial nerve causes facial spasm in hypocalcemia (tetany). May be normal finding in some patients Corrigan’s Pulse: A palpable hard pulse immediately followed by sudden collapse, seen in aortic regurgitation Cullen’s Sign: Ecchymosis around the umbilicus associated with severe intraperitoneal bleeding. Seen with ruptured ectopic pregnancy and hemorrhagic pancreatitis Cushing’s Triad: Hypertension, bradycardia, and irregular respiration associated with in- creased intracranial pressure Darier’s Sign: Stroking of the skin causes erythema and edema in mastocytosis Doll’s Eyes: Conjugated movement of eyes in one direction as head is briskly turned in the other direction in comatose patients. Tests oculocephalic reflex indicating intact brain stem Drawer Sign: Forward (or backward) movement of the tibia with pressure, indicating laxity or a tear in the anterior (or posterior) cruciate ligament 1 History and Physical Examination 25 1 Dupuytren’s Contracture: Proliferation of fibrosis tissue of the palmar fascia resulting in contracture of the fourth and/or fifth digits, which is often bilateral. May be hereditary or seen in patients with chronic alcoholic liver disease or seizures Duroziez’s Sign: Found in aortic regurgitation a “to and fro” murmur when stethoscope is pressed over the femoral artery Electrical Alternans: Beat to beat variation in the electrical axis, seen in large pericardial effusions, suggests impending hemodynamic compromise Ewart’s Sign: Dullness to percussion, increased fremitus and bronchial breathing beneath the angle of the left scapula found with pericardial effusion Fong Lesion/Syndrome: Autosomal-dominant anomalies of the nails and patella associated with renal abnormalities Frank’s Sign: Fissure of the ear lobe; may be associated with CAD, diabetes, and hyperten- sion Gibbus: Angular convexity of the spine due to vertebral collapse; associated with osteo- porosis or metastasis Gregg’s Triad: Cataracts, heart defects, and deafness with congenital rubella Grey Turner’s Sign: Ecchymosis in the flank associated with retroperitoneal hemorrhage Grocco’s Sign: Triangular area of paravertebral dullness, opposite side of a pleural effusion Heberden’s Nodes: Hard, nontender, painless nodules on the dorsolateral aspects of the distal interphalangeal joints associated with osteoarthritis. Results from hypertrophy of the bone Hegar’s Sign: Softening of the distal uterus. Reliable early sign of pregnancy Hellenhorst’s Plaque: A cholesterol plaque on retina seen on funduscopic examination as- sociated with amaurosis fugax Hill’s Sign: Femoral artery pressure 20 mm Hg greater than brachial pressure seen in severe aortic regurgitation Hoffmann’s Sign/Reflex: Flicking of the volar surface of the distal phalanx causing fingers to flex; associated with pyramidal tract disease Homans’ Sign: Calf pain with forcible dorsiflexion of the foot, associated with venous thrombosis Horner’s Syndrome: Unilateral miosis, ptosis, and anhidrosis (absence of sweating). From destruction of ipsilateral superior cervical ganglion often from lung carcinoma, espe- cially squamous cell carcinoma Janeway’s Lesion: Erythematous or hemorrhagic lesion seen on the palm or sole with sub- acute bacterial endocarditis Joffroy’s Reflex: Inability to wrinkle the forehead when patient asked to bend head and look up, seen in hyperthyroidism Kayser–Fleischer Ring: Brown pigment lesion due to copper deposition seen in Wilson’s disease Kehr’s Sign: Left shoulder and left upper quadrant pain associated with splenic rupture Kernig’s Sign: When the thigh is flexed at a right angle, complete extension of the leg is not possible because of inflammation of the meninges; seen with meningitis Koplik’s Spots: White papules on buccal mucosa opposite molars seen in measles Korotkoff’s Sounds: Low-pitched sounds resulting from vibration of the artery, detected when obtaining a blood pressure using the bell of the stethoscope. The last Korotkoff sound may be a more accurate estimate of the true diastolic blood pressure than the di- astolic blood pressure obtained using the diaphragm. Kussmaul’s Respiration: Deep, rapid respiratory pattern seen in coma or DKA Kussmaul’s Sign: Paradoxical rise in the jugular venous pressure on inspiration in constric- tive pericarditis or COPD 26 Clinician’s Pocket Reference, 9th Edition 1 Kyphosis: Excessive rounding of the thoracic spinal convexity, associated with aging, espe- cially in women Lasègue’s Sign/Straight-Leg-Raising Sign: The patient is extended in the supine position and raises the leg gently. Pain in the distribution of nerve root suggests sciatica. Levine’s Sign: Clenched fist over the chest while describing chest pain; associated with angina and AMI Lhermitte’s Sign: In MS, neck flexion results in a “shock sensation.” List: Lateral tilt of the spine, frequently associated with herniated disk and muscle spasm Lordosis: Accentuated normal concavity of the lumbar spine, normal in pregnancy Louvel’s Sign: Coughing or sneezing causes pain in the leg with DVT Marcus–Gunn Pupil: Dilation of pupils with swinging flashlight test. Results from unilat- eral optic nerve disease. Normal pupillary response is elicited when light is directed from the normal eye and a subnormal response when light is quickly directed from the normal eye into the abnormal eye. When light is directed into the abnormal eye, both pupils dilate rather than maintain the previous degree of miosis. McBurney’s Point/Sign: Point located one-third of the distance from the anterior superior iliac spine to the umbilicus on the right; tenderness at the site is associated with acute appendicitis. McMurray’s Test: External rotation of the foot produces a palpable or audible click on the joint line, suggesting medial meniscal injuries Möbius’ Sign: Weakness of convergence seen in thyrotoxicosis Moro’s Reflex (Startle Reflex): Abduction of hips and arms with extension of arms when infant’s head and upper body is suddenly dropped several inches while being held. Nor- mal reflex in early infancy Murphy’s Sign: Severe pain and inspiratory arrest with palpation of the right upper quad- rant during deep inspiration; associated with cholecystitis Musset’s or de Musset’s Sign: Rhythmic nodding or movement of the head with each heart beat caused by blood flow back into the heart in aortic insufficiency Obturator Sign: Flexion and internal rotation of the thigh elicits hypogastric pain in cases of inflammation of the obturator internus; positive with pelvic abscess and appendicitis Ortolani’s Test/Sign: Sign is hip click that suggests congenital hip dislocation. With the in- fant supine, point the legs toward you and flex the legs to 90 degrees at the hips and knees. Osler’s Node: Tender, red, raised lesions on the hands or feet seen with SBE. Pancoast’s Syndrome: Carcinoma involving apex of lung, resulting in arm and or shoulder pain from involvement of brachial plexus and Horner’s syndrome from involvement of the superior cervical ganglion Pastia’s Lines: Linear striations of confluent petechiae in axillary folds are antecubital fossa seen in scarlet fever Phalen’s Test: Prolonged maximum flexion of wrists while opposing dorsum of each hand against each other. A positive test results in pain and tingling in the distribution of the median nerve, indicating carpal tunnel syndrome Psoas Sign (Iliopsoas Test): Flexion against resistance or extension of the right hip, produc- ing pain; seen with inflammation of the psoas muscle; positive with appendicitis. Pulsus Alternans: Fluctuation of pulse pressure with every other beat. Seen in aortic steno- sis and CHF Queckenstedt’s Test: Tests patency of the subarachnoid space; compression of the inter- nal jugular vein during lumbar puncture; should normally immediately raise CSF pres- sure 1 History and Physical Examination 27 1 Quincke’s Sign: Alternating blushing and blanching of the fingernail bed following light compression; seen in chronic aortic regurgitation Radovici’s Sign: A frontal release sign, scratching palm causes chin contractions Raynaud’s Phenomenon/Disease: Pain and tingling in fingers after exposure to cold with characteristic color changes of white to blue and then often red. May be seen with scle- roderma, and SLE Romberg’s Test: Used to test position sense or cerebellar function. The patient stands with heels and toes together. Arms may be outstretched with palms facing upward or down or arms can be at the patient’s side. The patient may be lightly tapped by the examiner with the eyes open and then closed. A positive test is a loss of balance. A loss of balance with the eyes open indicates cerebellar dysfunction. Normal balance with eyes open and loss of balance with eyes closed indicates loss of position sense. Roth’s Spots: Oval retinal hemorrhages with a pale central area occurring in patients with bacterial endocarditis Rovsing’s Sign: Pain in the right lower quadrant with deep palpation of the left lower quad- rant. Seen in acute appendicitis Schmorl’s Node: Degeneration of the intervertebral disk resulting in herniation into the ad- jacent vertebral body Scoliosis: Lateral curvature of the spine Sentinel Loop: A single dilated loop of small or large bowel, usually occurs localized in- flammation such as pancreatitis Sister Mary Joseph’s Sign/Node: Metastatic cancer to umbilical lymph node Stellwag’s Sign: Infrequent ocular blinking Tinel’s Sign: Radiation of an electric shock sensation in the distal distribution of the median nerve elicited by percussion of the flexor surface of the wrist when fully extended. Seen in carpal tunnel syndrome Traube’s Sign: Booming or pistol shot sounds heard over the femoral arteries in chronic aortic insufficiency Trendelenburg’s Test: Observe patient from behind while patient shifts weight from one leg to the other; a pelvis tilt to opposite side suggests hip disease and weakness of the gluteus medius muscle. If normal, pelvis will not tilt. Trousseau’s Sign: Carpal spasm produced by inflating a blood pressure cuff above the sys- tolic pressure for 2–3 min, indicates hypocalcemia; also migratory thrombophlebitis as- sociated with cancer Turner’s Sign: See Grey Turner’s sign Virchow’s Node (Signal or Sentinel Node): A palpable, left supraclavicular lymph node; often first sign of a GI neoplasm, such as pancreatic or gastric carcinoma von Graefe’s Sign: Lid lag associated with thyrotoxicosis Weber–Rinne Test: For the Weber test a 512- or 1024-Hz tuning fork is placed on the mid- dle of the skull to determine if the sound lateralizes. For the Rinne test, the tuning fork is held against the mastoid process (BC) with the opposite ear covered. The patient indi- cates when the sound is gone. The tuning fork is then held next to the ear and the patient indicates whether the sound is present and when the sound (AC) disappears. Normally AC is better than BC. With sensorineural hearing loss, the Weber test lateralizes to the less affected ear and AC > BC; with conduction hearing loss, the Weber test lateralizes to the more affected ear and BC > AC. Whipple’s Triad: Hypoglycemia, CNS, and vasomotor symptoms (ie, diaphoresis, syn- cope); relief of symptoms with glucose; associated with insulinoma 28 Clinician’s Pocket Reference, 9th Edition 1 EXAMPLE OF A WRITTEN HISTORY AND PHYSICAL EXAMINATION (Adult Admitted to a Medical Service) • 7/10/01 5:30 PM Identification: Mr. Robert Jones is a 50-year-old male referred by Dr. Harry Doyle from Whitesburg,

Kentucky. The informant is the patient, who seems reliable, and a photocopy of the ER records from Whitesburg Hospital accompanies the patient. Chief Complaint: “Squeezing chest pain for 10 h, 4 d ago” HPI: Mr. Jones awoke at 6 AM 3 d ago with squeezing substernal chest pain that felt “like a ton of bricks” sitting on his chest. The chest pain was a 9 on a 10-point scale, with 10 being pain from a kidney stone. The pain was progressively worse after its onset and decreased in intensity after going to the Whitesburg ER. The pain radiated to his left neck and elbow and was associated with dyspnea and diaphoresis. He denies ex- periencing any associated nausea. He notes the pain seemed to get worse with any movement, and nothing seemed to alleviate it. He presented to the Whitesburg ER 10 h after the onset of pain and was given 3 NTG tablets SL and 2 mg morphine sulfate. ECG revealed 3 mm ST depression in leads V1 through V4. He was admitted to the ICU at Whitesburg Hospital and had an unevent- ful course. CPK increased to 850 at 24 h. He has been on aspirin 325 mg/d PO, isosor- bide dinitrate 20 mg PO q6h, and diltiazem 60 mg PO q8h. He was transferred for possible cardiac catheterization. He notes a similar chest pain that was less intense and occurred intermittently over the last 3 mo. The pain was precipitated by exercise and relieved with rest. He denies seeking medical attention in the past. He denies a history of orthopnea, paroxysmal noc- turnal dyspnea, dyspnea on exertion, or pedal edema. He has smoked two packs of cigarettes per day for 35 years, notes a 2-y history of hypertension for which he has been taking HCTZ 25 mg/d and denies a history of hy- percholesterolemia or diabetes. The patient’s father died of an MI at age 54, and his brother underwent coronary artery bypass graft surgery last year at age 48. PMH Medications. As above and ranitidine 300 mg PO qhs. Occasional ibuprofen 200 mg two to three tablets PO for back pain and acetaminophen 500 mg PO for headache Vitamins. One-a-day Herbals. None Allergies. Penicillin, rash entire body 20 years of age Surgeries. Appendectomy age 20, Dr. Smith, Whitesburg Hospitalization. See above. Trauma. Roof fall in mine accident 10 years ago, injured back. Notes occasional pain, which is relieved with ibuprofen 200 mg two or three tablets at a time Transfusions. None Illnesses. Denies asthma, emphysema, thyroid disease, kidney disease, peptic ulcer dis- ease, cancer, bleeding disorders, tuberculosis, or hepatitis. He notes a several-year history of water brash/heartburn and has been on ranitidine for 1 year 1 History and Physical Examination 29 1 Routine Health Maintenance. Last diphtheria/tetanus immunization 3 years ago. Stools for guaiac were negative times 3. Refused sigmoidoscopy. He has been seen by Dr. Doyle every 3–4 months for the last 2 years for hypertension. Family History 73 y.o. 54 y.o. MI cataracts also hypertension & gout 53 y.o. 49 y.o. 42 y.o. DM CABG A&W DM Male 17 y.o. 15 y.o. A&W asthma Female Deceased Denotes patient A&W Alive & well Psychosocial History: Mr. Jones has been married for 25 years and has three children. He and his family live in a home on 3 acres about three miles from Whitesburg. He worked in a coal mine until 10 years ago when he was injured in a “roof fall.” He is cur- rently employed in a local chair factory. He graduated from high school. He is Baptist and attends church regularly. Hobbies include woodworking and gardening. He eats breakfast and supper every day and has a soft drink and crackers for lunch. He currently works 8 h/d Monday through Friday. He notes going to bed every day by 10:00 PM and awakens at 5:30 AM. He drinks one to two cups of coffee per day and denies drinking any alcohol. He denies drug use but smokes as noted earlier. He denies exposure to environmental toxins. He denies any financial problems but is concerned about how his illness will affect his income. He has “good” health insurance. He denies any other stressor in his life. His sources of support are his wife, minister, and a sister who lives near the patient. ROS: Negative unless otherwise noted. Eyes. Has worn reading glasses since 1995; notes blurred vision for 1 year; last eye ap- pointment 1996. Denies loss of vision, double vision, or history of cataracts. Respiratory. Notes cough every morning and has produced 1 teaspoon of gray sputum for years. Denies hemoptysis or pleuritic chest pain. Last chest x-ray prior to today was 3 years ago. All other ROS negative. 30 Clinician’s Pocket Reference, 9th Edition 1 PHYSICAL EXAMINATION General: Mr. Jones is a pleasant male lying comfortably supine in bed. He appears to be the stated age. Vital Signs: Temp 98.6°F orally. Resp 16, HR 88 and regular, BP 110/70 left arm supine Skin: Tattoo left arm, otherwise no lesions Node: 1 × 1 left axillary node, nontender and mobile. No other lymphadenopathy HEENT Head. Normocephalic, atraumatic, nontender, no lesions Eyes. Visual acuity 20/40 left and right corrected. External structures normal, without lesions, PERRLA. EOM intact. Visual fields intact. Funduscopic examination disks sharp bilaterally, moderate arteriolar narrowing and A-V nicking. Ears. Hearing intact to watch tick at 3 ft bilaterally. Tympanic membranes intact with good cone of light bilaterally Nose. Symmetrical. No lesions. Sinuses nontender Mouth. Several dental fillings, otherwise normal dentition. No lesions Neck. Full ROM without tenderness. No masses or lymphadenopathy. Carotids +2/4 bi- laterally, no bruits. Internal jugular vein visible 2 cm above the sternal angle, pa- tient at 30 degrees. Chest: Symmetrical expansion. Fremitus by palpation bilaterally equal. Diaphragm moves 5.5 cm bilaterally by percussion. Lung fields clear to percussion. Breath sounds normal except end-inspiratory crackles heard at both bases that do not clear with coughing. Breast: Normal to inspection and palpation Heart: No cardiac impulse visible. Apical impulse palpable at the sixth intercostal space 2 cm lateral to the midclavicular line. Normal S1, physiologically split S2. S4 heard at apex. No murmurs, rub, or S3. Abdomen: Flat, no scars. Positive bowel sounds. No bruits. Liver 10 cm midclavicular line. No CVA tenderness. No hepatomegaly or splenomegaly by palpation. No ten- derness or guarding. No inguinal lymphadenopathy Genital: Normal circumcised male, both testes descended without masses or tenderness Rectal: Normal sphincter tone. No external lesions. Prostate smooth without tenderness or nodules. No palpable masses. Stool present, stool for occult blood negative Musculoskeletal: Lumbar spine decreased flexion to 75 degrees, extension to 5 degrees, decreased rotary and lateral movement. Otherwise full ROM of all joints, no ery- thema, tenderness, or swelling. No clubbing cyanosis or edema Peripheral Vascular: Radial, ulnar, brachial, femoral, dorsalis pedis, and posterior tibial pulses +2/4 bilaterally. Popliteal pulses nonpalpable. No femoral bruits Neurologic: Cranial nerves: I through XII intact. Motor: +5/5 upper and lower extrem- ity, proximally and distally. Sensory intact to pinprick upper and lower extremities proximally and distally. Vibratory sense intact in great toes and thumbs bilaterally. Stereognosis intact Reflexes. Biceps, triceps, brachioradialis, quadriceps, and ankles +2/4 bilaterally. Toes down going bilaterally Cerebellum. Romberg’s sign negative. Intact finger-to-nose and heel-to-shin bilaterally; gait normal—normal heel-and-heel, toe-and-toe, and heel-to-toe gaits. Rapid alter- nating movements intact upper and lower extremities bilaterally DATABASE ECG. HR 80, NSR inverted T waves V1 through V5 1 History and Physical Examination 31 1 CXR. Cardiomegaly, otherwise clear UA. Normal PT, PTT. Normal Chemistry Profile. Normal. Except elevated CPK CBC. 6700 WBC; 49 Hct; HBG 16; 40 S, 5 B, 44 L, 5 M, 6 E ASSESSMENT AND PLAN Coronary Artery Disease: Mr. Jones presented with a classic history for MI. The CPK and electrocardiogram support the diagnosis. The ST depression without evolving Q waves was consistent with a nontransmural MI. Mr. Jones is at risk for further MI since it was a nontransmural MI, and he will require further evaluation before discharge. • Continue aspirin 325 mg/d PO and diltiazem 60 mg/d PO. • Change isosorbide to tid prior to discharge. • Monitor by telemetry unit for next 24–48 h. • Stress test by modified Bruce protocol prior to discharge. • Consider cardiac catheterization especially if any further pain or if an early posi- tive stress test. • Continue cardiac rehabilitation. Hypertension: In view of the patient’s age, sex, and degree of hypertension, and the fact that there is no evidence of a secondary cause, the hypertension is most likely primary in nature. It is important that blood pressure be well controlled after this infarct. Mr. Jones’ blood pressure has been well controlled on diltiazem alone. • Continue diltiazem. • Dietary consult to instruct patient on low-sodium as well as low-fat diet prior to dis- charge. • Continue discussion of other problems as shown earlier. Signature: ______________________________ Title: __________________________________ Date Date of Date Entered Onset Problem Active Inactive Inactive 7-10-01 4-01 1 Coronary artery disease 7-10-01 7-7-01 1a Subendocardial MI—anterior 7-10-01 1998 2 Hypertension 7-10-01 1997 3 Bronchitis 7-10-01 1999 4 Heartburn/reflux esophagitis 7-10-01 1990 5 Back injury 7-10-01 7-10-01 6 Eosinophilia 7-10-01 2000 7 Blurred vision 7-10-01 1970 8 Appendicitis 1970 This page intentionally left blank. 2 CHARTWORK 2 How to Write Orders Preoperative Note Problem-Oriented Progress Note Operative Note Discharge Summary/Note Night of Surgery Note (Postop Note) On-Service Note Delivery Note Off-Service Note Outpatient Prescription Writing Bedside Procedure Note Shorthand for Laboratory Values HOW TO WRITE ORDERS The following format is useful for writing concise admission, transfer, and postoperative orders. It involves the mnemonic “A.D.C. VAAN DIML,” which stands for Admit/Attending, Diagno- sis, Condition, Vitals, Activity, Allergies, Nursing procedures, Diet, Ins and outs, Medications, and Labs. A.D.C. Vaan Diml Admit: Admitting team, room number Attending: The name of the attending physician, the person legally responsible for the pa- tient’s care. Also include the resident’s and intern’s names. Diagnosis: List admitting diagnosis or procedure if postop orders. Condition: Stable, critical, etc Vitals: Determine frequency of vital signs (temperature, pulse, blood pressure, central ve- nous pressure, pulmonary capillary wedge pressure, weight, etc) Activity: Specify bedrest, up ad lib, ambulate qid, bathroom privileges, etc Allergies: Note any drug reactions or food or environmental allergies. Nursing Procedures Bed Position. Elevate head of bed 30 degrees, etc Preps. Enemas, scrubs, showers Respiratory Care. P&PD. TC&DB, etc Dressing Changes, Wound Care. Change dressing bid, etc Notify House Officer If. Temperature >101°F, BP <90 mm Hg, etc Diet: NPO, clear liquid, regular, etc Ins and Outs: Refers to all “tubes” a patient may have. Record Daily I&O. IV Fluids. Specify type and rate. Drains. NG to low wall suction, Foley to gravity, etc 33 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 34 Clinician’s Pocket Reference, 9th Edition Endotracheal Tubes, Arterial Lines, Pulmonary-Artery Catheters. Specify care desired. 2 Medications: Write orders for specific medications (eg, diuretic, antibiotics, hormones, etc) and symptomatic drugs as needed (eg, pain medications, laxatives, “sleepers”). Include dose frequency and special instructions, ie, take with food. Labs: Indicate studies and specify times desired if applicable. This includes ECGs, x-rays, nuclear scans, consultation requests, etc. PROBLEM-ORIENTED PROGRESS NOTE (See Chapter 20 for a sample ICU progress note.) 1. List each medical, surgical, psychiatric problem separately: pneumonia, pancreatitis, congestive heart failure, etc. 2. Give each problem a call number: 1, 2, 3, . . . (as on page 31). 3. Retain the number of each problem throughout the hospitalization. 4. When the problem is resolved, mark it as such and delete it from the daily progress note. 5. Evaluate each problem by number in the following SOAP format. Or, you may do a separate assessment and plan for each problem Soap Subjective • How the patient feels, any complaints Objective • How the patient looks • Vital signs • Physical examination • Laboratory data, etc Assessment: (for each problem) • Evaluation of the data and any conclusions that can be drawn Plan: (for each problem) • Any new lab tests or medications • Changes or additions to orders • Discharge or transfer plans DISCHARGE SUMMARY/NOTE A formal discharge note is usually required for any admission that is longer than 24 h at most hospitals. This note provides a framework for the complete dictated note as well as providing a reference,

if needed, before the dictated note is transcribed and filed. The fol- lowing skeleton includes most of the information needed for a discharge note. Date of Admission: Date of Discharge: Admitting Diagnosis: Discharge Diagnosis: Attending Physician and Service Caring for Patient: Referring Physician: Provide address if available. Procedures: Include surgery and any invasive diagnostic procedures, eg, lumbar punctures, arteriograms. 2 Chartwork 35 Brief History, Pertinent Physical and Lab Data: Briefly review the main points of the history, physical, and admission lab tests. Do not repeat what is available in the admis- 2 sion note; summarize the most important points about the patient’s admission. Hospital Course: Briefly summarize the evaluation, treatment, and progress of the patient during the hospitalization. Condition at Discharge: Note if improved, unchanged, etc. Disposition: Where was the patient discharged to (eg, home, another hospital, nursing home)? Try to give specific address if transferred to another medical institution, and note who will be assuming responsibility for the patient. Discharge Medications: List medications, dosing, refills. Discharge Instructions and Follow-up: Clinic return date, diet instructions, activity re- strictions, etc Problem List: List active and past medical problems. ON-SERVICE NOTE Also known as a “pick-up note,” the on-service note is written by a new member of the team taking over the care of a patient who has been on the service for some time. The note should be brief and summarize the hospital course to date as well as demonstrate that the new team member has reviewed the patient’s care to date. The following skeleton includes most of the information needed in an on-service note. Date of Admission: Admitting Diagnosis: Procedures (with Results) to Date: Hospital Course to Date: This should be briefly summarized. Brief Physical Examination: Pertinent to the patient’s problems. Pertinent Lab Data: Problem List: Assessment: Plan: OFF-SERVICE NOTE This is written by the team member who is rotating off the service but who was primarily re- sponsible for the patient before the patient is ready for discharge. The components are iden- tical to the “On-Service” note in the previous section. BEDSIDE PROCEDURE NOTE Procedure: (eg, LP, thoracentesis, etc) Indications: (eg, R/O meningitis, symptomatic pleural effusion) Permit: Note risks and benefits explained and indicate signed and on chart Physicians: Note physicians present and responsible for procedure Description of Procedure: Indicate type of positioning, prep, anesthesia, and amount. Briefly describe technique and instruments used. Complications: List. EBL: List. Specimens/Findings Obtained: (eg, opening pressure for LP, CSF appearance, and tubes sent to lab, etc) Disposition: Describe patient’s status after procedure (eg, Patient alert and oriented with no complaints; BP stable) 36 Clinician’s Pocket Reference, 9th Edition PREOPERATIVE NOTE 2 The specific items in the preoperative note depend on institutional guidelines, the nature of the procedure, and the age and health of the patient. For example, an ECG and blood set-up may not be necessary for a 2-year-old child being treated for a hernia but essential for a 70-year-old scheduled for vascular surgery. The following list includes most of the informa- tion needed in a preoperative note. Preop Diagnosis: Such as “acute appendicitis” Procedure: The planned procedure, eg, “exploratory laparotomy” Labs: Results of CBC, electrolytes, PT, PTT, urinalysis, etc CXR: Note results. ECG: Note results. Blood: T&C 2 units PRBC, blood not needed, etc History and Physical: Should be “on chart.” Orders: Note any special preop orders, such as preop colon preps, vaginal douches, prophy- lactic antibiotics. Permit: If completed, write “signed and on chart”; if not, indicate plans for obtaining per- mit. OPERATIVE NOTE The operative note is written immediately after surgery to summarize the operation for those who were not present and is meant to complement the formal operative summary dictated by the surgeon. The following list includes most of what is needed in an operative note. Preop Diagnosis: Reason for the surgery, eg, “acute appendicitis” Postop Diagnosis: Based on the operative findings, eg, “mesenteric lymphadenitis” Procedure: Surgery performed, eg, “exploratory laparotomy” Surgeons: List the attending physicians, residents, and students who scrubbed on the case, including their titles (MD, CCIV, MSII, etc). It is often helpful to identify the dictating surgeon. Findings: Briefly note operative findings, eg, “normal appendix with marked lym- phadenopathy.” Anesthesia: Specify the type of anesthesia, eg, local, spinal, general, endotracheal, etc. Fluids: Amount and type of fluid administered during case, eg, 1500 mL NS, 1 unit PRBC, 500 mL albumin. This is usually obtained from the anesthesia records. EBL: Usually obtained from the anesthesia or nursing records. Drains: State location and type of drain, eg, “Jackson–Pratt drain in left upper quadrant,” “T-tube in midline,” etc. Specimens: State any samples sent to pathology and the results of examination of any intra- operative frozen sections. Complications: Note any complications during or after the surgery. Condition: Note where the patient is taken immediately after surgery and the patient’s con- dition. Example: “Transferred to the recovery room in stable condition.” NIGHT OF SURGERY NOTE (POSTOP NOTE) This type of progress note is written several hours after or the night of surgery. Procedure: Indicate the operation performed. Level of Consciousness: Note if the patient is alert, drowsy, etc. 2 Chartwork 37 Vital Signs: BP, pulse, respiration. I&O: Calculate amount of IV fluids, blood, urine output, and other drainage, and attempt to 2 assess fluid balance. Physical Examination: Examine and note the findings of the chest, heart, abdomen, ex- tremities, and any other part of the physical examination pertinent to the surgery; exam- ine the dressing for bleeding. Labs: Review lab results if any were obtained since surgery. Assessment: Evaluate the postop course thus far (stable, etc). Plan: Note any changes in orders. DELIVERY NOTE __ -year-old (married or single) G __ now para __, AB __, clinic (note if patient received prenatal clinic care) patient with EDC __, and a prenatal course (uncomplicated or describe any problems). Any comments concerning labor (eg, Pitocin-induced, premature rupture) and draped in the usual sterile fashion. Under controlled conditions delivered a __ lb __ oz (__ g) viable male or female infant under __ (general, spinal, pudendal, none) anesthesia. Delivery was via SVD with midline episiotomy (or forceps or cesarean section). Apgars were __ at 1 min and __ at 5 min (for Apgar scoring, see Appendix). State delivery date and time. Cord blood sent to lab and placenta expressed intact with trailing membranes. Lacera- tions of the __ degree repaired by standard method with good hemostasis and restoration of normal anatomy. • EBL: • MBT: • HCT (predelivery and postdelivery): • RT: • VDRL test: • Condition of mother: OUTPATIENT PRESCRIPTION WRITING The format for outpatient prescription writing is outlined in the following list and illustrated in Figure 2–1. Controlled substances, such as narcotics, require a DEA number on the pre- scription and in some states may require that the controlled substance be written on a special type of prescription paper (see Chapter 22 for controlled drugs indicated by a [C]). For secu- rity, the DEA number should never be preprinted on a prescription pad but written by hand at the time the prescription is written. Elements of an outpatient prescription include: Patient’s Name, Address, and Age: Print clearly where indicated. Date: State requirements vary, but most prescriptions must be filled within 6 months. Rx: Drug name, strength, and type (usually listed as the generic name); if you specifically want a brand name you must designate “no substitution.” Rx is an abbreviation from the Latin for “recipe.” List the strength of the product (usually in mg) and the form (eg, tablets, capsule, suspension, transdermal, etc). Dispense: Amount of drug (number of capsules), or time period (1 month supply, etc). Sig: Short for the Latin “signa,” which means “mark through” on patient instructions. This part can be written out or noted in shorthand. Shorthand use is generally discouraged, however, because writing out the prescription decreases the likelihood of errors. Fre- quently used abbreviations are noted here with a more complete listing provided at the front of the book. 38 Clinician’s Pocket Reference, 9th Edition 2 NICK PAVONA, MD BENJAMIN FRANKLIN UNIVERSITY MEDICAL CENTER CHADDS FORD, PA 19317 LICENSE PA MD 685-488-194 DEA NP–3612982 NAME NICK PAVONA, Sr. AGE 84 ADDRESS 34-10 75th Street DATE 10/24/2001 Wilmington, DE Rx: minoxidil (Rogaine) 2% topical solution DISP: 60 mL SIG: Apply BID to scalp Brand medically necessary REFILL X5 SUBSTITUTION PERMISSIBLE M.D. TO ENSURE BRAND NAME DISPENSING, PRESCRIBER MUST SPECIFY “DISPENSE AS WRITTEN” ON THE PRESCRIPTION.* *This can vary by state; some require that you write “Brand Medically Necessary” to specify a brand name and not a generic. FIGURE 2–1 Example of an outpatient prescription. As a safety feature DEA num- bers should never be preprinted on a prescription form. The “Dispense as Written” statement can vary by state requirements; this statement requests that the pharmacist fill the prescription as requested and not substitute a generic equivalent. ad lib = freely at pleasure PO = by mouth PR = by rectum OS = left eye OD = right eye qd = daily (this is a dangerous abbreviation and should not be used; see “Dangerous Prac- tices,” page 39) PRN = as needed Ṫ = one ˙̇T = two ˙̇̇T = three qhs = every night at bedtime bid = twice a day tid = three times a day q6h = every 6 h 2 Chartwork 39 qid = four times a day. (Note that qid and q6h are NOT the same orders: qid means that the medication is given four times a day while awake (eg, 8 AM, 12 noon, 6 PM, and 10 PM); 2 q6h means that the medication is given four times a day but by the clock (eg, 6 AM, 12 noon, 6 PM, 12 midnight). Refills: Indicate how many times this prescription can be refilled. Substitution: Can a generic drug be used instead of the one prescribed? Tips for Safe Prescription Writing Legibility 1. Take time to write legibly. 2. Print if this would be more legible than handwriting. 3. Use a typewriter or computer if necessary. In the near future, physicians will generate all prescriptions by computer to eliminate legibility problems. 4. When prescribing a new or rarely used drug, carefully print the order to avoid mis- reading. Dangerous Practices 1. NEVER use a trailing zero. Correct: 1 mg Dangerous: 1.0 mg. If the decimal is not seen, a 10-fold overdose can occur. 2. NEVER leave a decimal point “naked.” Correct: 0.5 mL Dangerous: .5 mL. If the decimal point is not seen, a 10-fold overdose can occur. 3. NEVER abbreviate a drug name because the abbreviation may be misunderstood or have multiple meanings. 4. NEVER abbreviate U for units as it can easily be read as a zero, thus “6 U regular in- sulin” can be misread as 60 units. The order should be written as “6 units regular in- sulin.” 5. NEVER use qd (abbreviation for once a day). When poorly written, the tail of the “q” can make it read qid or four times a day. SHORTHAND FOR LABORATORY VALUES (See Figure 2–2) 40 Clinician’s Pocket Reference, 9th Edition 2 CBC HgB Segs/Bands/Lymphs/Monos/Basos/Eos WBC MCV-MCH-MCHC HCT platelet count Example: 10.1 40S, 20B, 30L, 6M, 1B, 3E 11,000 80/27/32 30.5 285,000 Electrolytes Example: sodium chloride 140 98 potassium bicarbonate 4.5 24 SMA-6 (Basic Metabolic Panel) BUN sodium chloride (creatinine) potassium bicarbonate glucose Example: 10 140 98 (1.1) 4.5 24 110 FIGURE 2–2 Shorthand notation for recording laboratory values. The basic meta- bolic panel is similar to the SMA-6 except that the creatinine is also listed. 3 DIFFERENTIAL DIAGNOSIS: SYMPTOMS, SIGNS, 3 AND CONDITIONS Abdominal Distention Frequency Abdominal Pain Galactorrhea Adrenal Mass Gynecomastia Alopecia Headache Amenorrhea Heartburn (Pyrosis) Anorexia Hematemesis and Melena Anuria Hematochezia Arthritis Hematuria Ascites Hemoptysis Back Pain Hepatomegaly Breast Lump Hiccups (Singultus) Chest Pain Hirsutism Chills Impotence (Erectile Dysfunction) Clubbing Incontinence (Urinary) Coma Jaundice Constipation Lymphadenopathy and Splenomegaly Cough Melena Cyanosis Nausea and Vomiting Delirium Nystagmus Dementia Oliguria and Anuria Diarrhea Pleural Effusion Diplopia Pruritus Dizziness Seizures Dysphagia Splenomegaly Dyspnea Syncope Dysuria Tremors Earache Vaginal Bleeding Edema Vaginal Discharge Epistaxis Vertigo Failure to Thrive Vomiting Fever Weight Loss Fever of Unknown Origin (FUO) Wheezing Flatulence This chapter provides a general guide to commonly encountered symptoms and conditions and their frequent causes. Remember: “There are more uncommon presentations of com- mon diseases than common presentations of uncommon

diseases.” 41 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 42 Clinician’s Pocket Reference, 9th Edition ABDOMINAL DISTENTION Ascites, intestinal obstruction, cysts (ovarian or renal), tumors, hepatosplenomegaly, aortic aneurysm, uterine enlargement (pregnancy), bladder distention, inflammatory mass 3 ABDOMINAL PAIN Diffuse: Intestinal angina, early appendicitis, colitis, diabetic ketoacidosis, hereditary angioedema, gastroenteritis, mesenteric thrombosis, mesenteric lymphadenitis, peritonitis, porphyria, sickle cell crisis, uremia, renal colic, renal infarct, pancreatitis Right Upper Quadrant: Dissecting aneurysm, gallbladder disease (cholecystitis, cholangitis, choledocholithiasis), hepatitis, hepatomegaly, pancreatitis, peptic ulcer disease, pneumonia, PE, pyelonephritis, renal colic, renal infarct, appendicitis (retroperitoneal) Left Upper Quadrant: Dissecting aneurysm, esophagitis, hiatal hernia, esophageal rupture, gastritis, pancreatitis, peptic ulcer disease, MI, pericarditis, pneumonia, PE, pyelonephritis, renal colic, renal infarct, splenic rupture or infarction Lower Abdomen: Aortic aneurysm, colitis, diverticulitis including Meckel’s, intestinal obstruction, hernias, perforated viscus, pregnancy, ectopic pregnancy, dysmenorrhea, en- dometriosis, mittelschmerz (ovulation), ovarian cyst or tumor (especially with torsion), PID, renal colic, UTI, rectal hematoma, bladder distention. Right Lower Quadrant Specific: Appendicitis, ectopic pregnancy, ovarian cyst or tumor, salpingitis, mittelschmerz, cholecystitis, perforated duodenal ulcer, Crohn’s disease ADRENAL MASS Adrenal adenoma, adrenal hyperplasia (unilateral or bilateral), adrenal metastasis (solid tu- mors, lymphoma, leukemia), adrenocortical carcinoma, pheochromocytoma, adrenal myelolipoma, adrenal cyst, Wolman’s disease, adrenal varices, hemorrhage, congenital adrenal hyperplasia, ganglioneuroma, micronodular adrenal disease ALOPECIA Male pattern baldness (alopecia, androgenic type in both men and women), trauma and hair pulling, congenital, tinea capitis, bacterial folliculitis, telogen arrest, anagen arrest (chemotherapy/radiation therapy), alopecia areata, discoid lupus AMENORRHEA Pregnancy, menopause (physiologic or premature), severe illness, weight loss, stress, ath- letic training, “physiologically delayed puberty,” anatomic (imperforate hymen, uterine age- nesis, etc), gonadal dysgenesis (Turner’s syndrome, etc), hypothalamic and pituitary tumors, virilizing syndromes (polycystic ovaries, idiopathic hirsutism, etc). Amenorrhea is catego- rized as primary (never had menses) or secondary (cessation of menses). ANOREXIA Hepatitis, carcinoma (most types, especially advanced), anorexia nervosa, generalized debil- itating diseases, digitalis toxicity, uremia, depression, CHF, pulmonary failure, radiation ex- posure, chemotherapy 3 Differential Diagnosis: Symptoms, Signs, and Conditions 43 ANURIA See Oliguria, page 49 ARTHRITIS 3 Osteoarthritis, bursitis, tendonitis, connective tissue disease (RA, SLE, rheumatic fever, scleroderma, gout, pseudogout, rheumatoid variants [ankylosing spondylitis, psoriatic arthritis, Reiter’s syndrome]), infection (bacterial, viral, TB, fungal Lyme disease), trauma, sarcoidosis, sickle cell anemia, hemochromatosis, amyloidosis, coagulopathy ASCITES (See Chapter 13, page 296, under “Peritoneal Paracentesis” for more details.) CHF, tricuspid insufficiency, constrictive pericarditis, venous occlusion (including Budd–Chiari syndrome), cirrhosis, pancreatitis, peritonitis (ruptured viscus, TB, bile leak, spontaneous bacterial), tu- mors (most common ovarian, gastric, uterine, unknown primary, breast, lymphoma), trauma, Meigs’ syndrome (ovarian fibroma associated with hydrothorax and ascites), myxedema, anasarca (hypoalbuminemia) BACK PAIN Herniated disk, spinal stenosis, ankylosing spondylitis, metastatic tumor, multiple myeloma, mechanical back sprain, referred pain (visceral, vascular), vertebral body fracture, osteo- porosis induced fracture, infectious processes (diskitis, osteomyelitis, epidural abscess BREAST LUMP Cancer, fibroadenoma, fibrocystic breast disease, fat necrosis, gynecomastia (males, alco- holics) CHEST PAIN Deep, Dull, Poorly Localized: Angina, variant angina, unstable angina, AMI, aortic aneurysm, PE, tumor, gallbladder disease, pulmonary hypertension Sharp, Well Localized: PE, pneumothorax, epidemic pleurodynia, pericarditis, atypi- cal MI, hyperventilation, hiatal hernia, esophagitis, esophageal spasm, herpes zoster, aortic aneurysm, breast lesions, variety of bony and soft tissue abnormalities (rib fractures, costo- chondritis, muscle damage), perforated ulcer, acute cholecystitis, pancreatitis CHILLS Infection (bacterial with bacteremia, viral, TB, fungal), neoplasm (Hodgkin’s disease), drug and transfusion reactions, hypothermia, malaria CLUBBING Pulmonary causes (bronchiectasis, lung abscesses, tuberculosis, neoplasms, fibrosis), AV malformations, cardiac (congenital cyanotic heart diseases, bacterial endocarditis), GI (ul- cerative and regional enteritis, cirrhosis), hereditary, thyrotoxicosis 44 Clinician’s Pocket Reference, 9th Edition COMA Use the mnemonic AEIOU TIPS: Alcohol; Encephalitis (other CNS causes—epilepsy, he- morrhage, mass), Insulin (hypoglycemia, hyperglycemia), Opiates (drugs), Uremia (and 3 other metabolic conditions, such as hypernatremia, hyponatremia, hypercalcemia, hepatic failure, and thiamine deficiency), Trauma, Infection, Psychiatric causes, Syncope (or de- creased cardiac output such as from arrhythmias). CONSTIPATION Dehydration, lack of exercise, bedrest, medications (narcotics, anticholinergics, antidepres- sants, calcium channel blockers—verapamil, diuretics, clonidine, aluminum- or calcium- containing antacids), laxative abuse, megacolon, spastic colon, chronic suppression of the urge to defecate, fecal impaction (often with paradoxical diarrhea), neoplasm, intestinal ob- struction, vascular occlusion to the bowel, inflammatory lesions (diverticulitis, proctitis), hemorrhoids, anal fissures, neurological disorders, depression, porphyria, hypothyroidism, hypercalcemia COUGH Acute: Tracheobronchitis, pneumonia, sinusitis, pulmonary edema, foreign body, toxic inhalation, allergy, pharyngitis (viral or bacterial), asthma, GERD ACE inhibitors, impacted cerumen or foreign body in ear Chronic: Bronchitis (smoker), chronic sinusitis, emphysema, cancer (bronchogenic, head and neck, and esophageal), TB, sarcoidosis, fungal infection, bronchiectasis, mediasti- nal lymphadenopathy, thoracic aneurysm, GERD, ACE inhibitors CYANOSIS Peripheral: Arterial occlusion and insufficiency, vasospasm/Raynaud’s disease, venous stasis, venous obstruction Central: Hypoxia, congenital heart disease (right to left shunt), PE, pseudo-cyanosis (eg, polycythemia vera), methemoglobinemia DELIRIUM Metabolic: Hypoglycemia, hypoxia, sodium and calcium disorders, hypercarbia, uremia Neurologic: Stroke, subdural and epidural hematoma, subarachnoid hemorrhage, post- ictal, concussion and contusion, meningitis, encephalitis, brain tumor Drug or Toxin-Induced: Lithium intoxication, ethanol, steroids, anticholinergics, sympathomimetics, poisons (eg, mushrooms, carbon monoxide), drugs of abuse DEMENTIA Chronic CNS disease: Alzheimer’s, senile dementia, Pick’s disease, Parkinson’s, chronic demyelinating disease (MS), ALS, brain tumor, normal pressure hydrocephalus, Wilson’s disease, Huntington’s disease, lipid storage diseases (eg, Tay–Sachs) Metabolic: Usually chronic (hypoxia, hypoglycemia, hypocalcemia), hyperammonemia, dialysis, heavy-metal intoxication, pernicious anemia (B12 deficiency), niacin and thiamine 3 Differential Diagnosis: Symptoms, Signs, and Conditions 45 deficiency (usually chronic alcoholic) posthepatic coma, medications (barbiturates, phe- nothiazines, lithium, benzodiazepines, many others) Infectious: AIDS encephalopathy, brain abscess, chronic meningoencephalitis (eg, fun- gal neurosyphilis), encephalitis, Jakob–Creutzfeldt disease 3 Vascular: Vasculitis, multicerebral/cerebellar infarcts Traumatic: Contusion, hemorrhage, subdural hematoma Psychiatric: Sensory deprivation, depression (pseudodementia) DIARRHEA Acute: Infections (bacterial, viral, fungal, protozoan, parasitic), toxic (food poisoning, chemical), drugs (antibiotics, cholinergic agents, lactulose, magnesium-containing antacids, quinidine, reserpine, guanethidine, metoclopramide, bethanechol), appendicitis, diverticular disease, GI bleeding, ischemic colitis, food intolerance, fecal impaction (paradoxical diar- rhea), pseudomembranous colitis Chronic: After gastrectomy or vagotomy, ZE syndrome, regional enteritis, ulcerative coli- tis, malabsorption, diverticular disease, carcinoma, villous adenoma, gastrinomas, lym- phoma of the bowel, functional bowel disorders (irritable colon, mucous colitis), pseudomembranous colitis, endocrine disease (carcinoid, hyperthyroidism, Addison’s dis- ease), radiation enteritis, drugs, Whipple’s disease, amyloidosis, AIDS DIPLOPIA Problems with the third, fourth, or sixth cranial nerve, such as from vascular disturbances, meningitis, tumor, demyelination, orbital blow-out fracture, hyperthyroid ocular myopathy DIZZINESS Hyperventilation, depression, hypoglycemia, anemia, volume depletion, hypoxia, trauma, Ménière’s disease, benign positional vertigo, aminoglycoside toxicity, vestibular neuronitis, MS, brain stem ischemia or stroke, posterior fossa lesions, cerebellar ischemia or stroke, ar- rhythmias, aortic stenosis, carotid sinus hypersensitivity DYSPHAGIA Loss of tongue function, pharyngeal dysfunction (myasthenia gravis), Zenker’s diverticu- lum, tumors (bronchogenic, head and neck, and esophageal), stricture, esophageal web, Schatzki’s ring, lower esophageal sphincter spasm, foreign body, aortic aneurysm, achalasia, scleroderma, diabetic neuropathy, amyloidosis, infection (especially candidiasis), dermato- myositis, polymyositis, MS, brain stem infarctions DYSPNEA Laryngeal and tracheal infections and foreign bodies, tumors (both intrinsic and extrinsic), COPD, asthma, pneumonia, lung carcinoma, atelectasis, pneumothorax, pleural effusion, hemothorax, PE, pulmonary infarction, carbon monoxide poisoning, any cause of pain from respiratory movements, cardiac and noncardiac pulmonary edema, AMI, pericardial tam- ponade, anemia, abdominal distention, anxiety 46 Clinician’s Pocket Reference, 9th Edition DYSURIA Urethral stricture, stones, blood clot, tumor (bladder, prostate, urethral), prostatic enlarge- ment, infection (urethritis, cystitis, vaginitis, prostatitis), trauma, bladder spasm, dehydra- 3 tion EARACHE Otitis media and externa, mastoiditis, serous otitis, otic barotrauma, foreign body, impacted cerumen, referred pain (dental or TMJ) EDEMA CHF, constrictive pericarditis, liver disease (cirrhosis), nephrotic syndrome, nephritic syn- drome, hypoalbuminemia, malnutrition, myxedema, hemiplegia, volume overload, throm- bophlebitis, lymphatic obstruction, medications (nifedipine), venous stasis EPISTAXIS Trauma (nose picking, blunt trauma), neoplasm, polyps, foreign body, desiccation, coagu- lopathy, medications (use of cocaine, nasal sprays), infections (sinusitis), uremia, hyperten- sion (more often a result rather than a cause of epistasis) FAILURE TO THRIVE Environmental: Social deprivation, decreased food intake Organic: CNS disorder, intestinal malabsorption, CF, parasites, cleft palate, heart fail- ure, endocrine diseases, hypercalcemia, Turner’s syndrome, renal disease, chronic infection, malignancies FEVER Based on adult population studies an AM temperature above 98.8°F (37.2°C) or PM above 99.9°F (37.7°C) is generally defined as a fever. Rectal temperatures are generally 1°F (0.6°C) higher and reflect core temperature Infections (viral, bacterial, mycobacterial, fungal, parasitic), neoplasm (lymphoma, leukemia, renal and hepatic carcinoma), connective tissue disease (SLE, vasculitis, RA, adult Still’s disease, temporal arteritis), heat stroke, malignant hyperthermia, thyroid storm, adrenal insufficiency, PE, MI, atrial myxoma, inflammatory bowel disease, factitious, drugs (most common offenders: amphotericin, bleomycin, barbiturates, cephalosporins, methyl- dopa, penicillins, phenytoin, procainamide, sulfonamides, quinidine, cocaine, LSD, phency- clidine and amphetamines) FEVER OF UNKNOWN ORIGIN (FUO) Defined as a temperature of 101°F (38.3°C) or greater for at least 3 weeks and for which a diagnosis is not established after 1 week of hospitalization. In children, the minimum dura- tion is 2 weeks and the temperature is at least 101.3°F (38.5°C): TB, fungal infection, endo- carditis, abscess (especially hepatic), neoplasm (lymphoma, renal cell, hepatoma, preleukemia), atrial myxoma, connective tissue disease, drugs (see Fever, previous listing), PE, Crohn’s disease, ulcerative colitis, hypothalamic injury, factitious; in elderly, temporal arteritis 3 Differential Diagnosis: Symptoms, Signs, and Conditions 47 FLATULENCE Aerophagia, food intolerance, disturbances in bowel motility (diabetes, uremia), lactose in- tolerance, gallbladder disease, peptic ulcer fiber, cholestyramine 3 FREQUENCY Infection (bladder, prostate), excessive fluid intake, use of diuretics (also coffee, tea, or colas), diabetes mellitus, diabetes insipidus, prostatic obstruction, bladder stones, bladder tumors, pregnancy, psychogenic bladder syndrome, neurogenic bladder, interstitial cystitis GALACTORRHEA Hyperprolactinemia, prolonged breast feeding, major stress, pituitary tumors, breast lesions (benign, cancer, inflammatory), idiopathic with menses and after oral contraceptive use GYNECOMASTIA Normal (Physiologic): Newborn, adolescence, aging Pathologic: Medications or drug use(cimetidine, spironolactone, estrogens, gonado- tropins, antiandrogens, marijuana), decreased testosterone (Klinefelter’s syndrome, testicu- lar failure or absence), increased estrogen production (hermaphroditism, testicular or lung cancers, adrenal and liver diseases) HEADACHE Includes cluster, tension, and migraine (classic or simple), benign exertional, headache asso- ciated with sexual activity, benign cough headache, ice-pick (idiopathic stabbing), vascular (menstruation, hypertension), eye strain, acute glaucoma, sinusitis, dental problems, TMJ dysfunction, trauma, subarachnoid hemorrhage, intracranial mass, fever, meningitis, pseudo-tumor cerebri, trigeminal neuralgia, temporal arteritis (especially in elderly), hypo- glycemia, toxin exposure (carbon monoxide poisoning), drugs (vasodilators—nifedipine [Procardia]), vasculitis HEARTBURN (PYROSIS) GERD, esophagitis, hiatal hernia, peptic ulcer, gallbladder disease, medications, tumors, scleroderma, food intolerance. Myocardial ischemia maybe mistaken for heartburn. HEMATEMESIS AND MELENA Melena generally means that the bleeding site is in the upper GI tract (ie, proximal to the ligament of Treitz), but occasionally can be as distal as the right colon.) Swallowed blood (eg, epistaxis), esophageal varices, esophagitis, Mallory–Weiss syndrome, hiatal hernia, gastritis, peptic ulcer, duodenitis, carcinoma of the stomach, tumors (both small and large bowel), ischemic colitis, aortoenteric fistula, bleeding diathesis, anticoagulation (may un- mask GI tract pathology) HEMATOCHEZIA Massive upper GI bleeding, hemorrhoids, diverticular disease, angiodysplasia, polyps, carci- noma, inflammatory bowel disease, ischemic colitis 48 Clinician’s Pocket Reference, 9th Edition HEMATURIA (see also page 111) First rule out false-positives: myoglobinuria, hemoglobinuria, porphyria. GU neoplasms (malignant and benign), polycystic kidneys, trauma, infection (urethral, bladder, prostate, 3 etc), stones, glomerulonephritis, renal infarction, renal vein thrombosis, anticoagulation (may unmask GU tract pathology), bleeding diathesis, enterovesical fistula, sickle cell ane- mia, vigorous exercise (“runners’ hematuria”), accelerated hypertension, factitious, and vaginal and rectal bleeding HEMOPTYSIS Infection (pneumonia, bronchitis, fungal, TB), bronchiectasis, cancer (usually bron- chogenic), PE, arteriovenous malformations, Wegener’s granulomatosis, Goodpasture’s syn- drome, SLE, pulmonary hemosiderosis, foreign body, trauma, bleeding diatheses, excessive anticoagulation (may unmask respiratory tract pathology), pulmonary edema, mitral stenosis HEPATOMEGALY CHF, hepatitis (viral, alcoholic, drug-induced, autoimmune), cirrhosis (alcoholic, etc), tu- mors (primary and metastatic), amyloid, biliary obstruction, hemochromatosis, chronic granulomatous disease, infections (schistosomiasis, liver abscess). Riedel’s lobe is a normal variant, elongated right lobe of the liver with normal liver volume. HICCUPS (SINGULTUS) Uremia, electrolyte disorders, diabetes, medications (benzodiazepines, barbiturates, others), emotionally induced (excitement, fright), gastric distention, CNS disorders, psychogenic, thoracic and diaphragmatic disorders (pneumonia, MI, diaphragmatic irritation), alcohol in- gestion HIRSUTISM Idiopathic, familial, adrenal causes (Cushing’s disease, congenital adrenal hyperplasia, viril- izing adenoma or carcinoma), polycystic ovaries, medications (minoxidil, androgens) IMPOTENCE (ERECTILE DYSFUNCTION) Psychogenic, vascular, neurologic (cord injury, radical prostatectomy, rectal surgery, aortic bypass), pelvic radiation, medications (some common drugs: antihypertensives, thiazide di- uretics, beta-blockers, methyldopa; antidepressants especially the SSRIs, anticholinergics; addictive medications: alcohol, narcotics; antipsychotics; antiandrogens: histamine H2 blockers, finasteride, LHRH analogues, spironolactone, others; history of priapism, Pey- ronie’s disease, testicular failure, hyperprolactinemia INCONTINENCE (URINARY) Cystitis, dementia and delirium, stroke, prostatic hypertrophy, fecal impaction, peripheral or autonomic neuropathy, medications (diuretics, sedatives, alpha blockers), diabetes, spinal cord trauma or lesions, MS,

childbirth, surgery (prostate, rectal), aging, acute and chronic medical conditions, estrogen deficiency 3 Differential Diagnosis: Symptoms, Signs, and Conditions 49 JAUNDICE Hepatitis (alcoholic, viral, drug-induced, autoimmune), Gilbert’s disease, Crigler–Najjar syndrome, Dubin–Johnson syndrome, Wilson’s disease, drug-induced cholestasis (pheno- thiazines and estrogen), gallbladder and biliary tract disease (including inflammation, infec- 3 tion, obstruction, and tumors—primary hepatic and metastatic), hemolysis, neonatal jaundice, cholestatic jaundice of pregnancy, total parenteral nutrition LYMPHADENOPATHY AND SPLENOMEGALY Infection (bacterial, fungal, viral, parasitic), benign neoplasm (histiocytosis), malignant neo- plasm (primary lymphoma, metastatic), sarcoid, connective tissue disease, drugs (phenytoin, etc), AIDS, splenomegaly without lymphadenopathy (cirrhosis, hereditary spherocytosis, hemoglobinopathies, ITP, hairy cell leukemia, and amyloidosis) MELENA (See Hematemesis, page 47.) NAUSEA AND VOMITING Appendicitis, acute cholecystitis, chronic gallbladder disease, peptic ulcer disease, gastritis (especially alcoholic), pancreatitis, gastric distention (diabetic atony, pyloric obstruction), intestinal obstruction, peritonitis, food intolerance, intestinal infection (bacterial, viral, para- sitic), acute systemic infections (especially in children), hepatitis, toxins (food poisoning), CNS disorders ([increased intracranial pressure often cause vomiting without headache], tumor, hemorrhagic stroke, hydrocephalus, meningitis, labyrinthitis, Ménière’s disease, mi- graine headaches) AMI, CHF, endocrine disorders (DKA, adrenal crisis), hypercalcemia, hyperkalemia, hypokalemia, pyelonephritis, nephrolithiasis, uremia, hepatic failure, preg- nancy, PID, drugs (opiates, digitalis, chemotherapeutic agents, L-dopa, NSAIDs), psy- chogenic vomiting, porphyria, radiation therapy NYSTAGMUS Congenital, vision loss early in life, MS, neoplasms, infarction, toxic or metabolic en- cephalopathy, alcoholic cerebellar degeneration, medications (anticonvulsants, barbiturates, phenothiazines, lithium, others), encephalitis, vascular brainstem lesions, Arnold– Chiari malformation, nonpathologic (extreme lateral gaze), opticokinetic nystagmus (attempt to fix gaze on rapidly moving object, eg, train) OLIGURIA AND ANURIA (See also Urinary Indices, page 119.) Oliguria is <500 mL urine/24 h; anuria is <100 mL urine/24 h in adults. Prerenal: Volume depletion, shock, heart failure, fluids in the third space, renal artery compromise Renal: Glomerular disease, acute tubular necrosis, bilateral cortical necrosis, interstitial disease (acute and chronic interstitial nephritis, urate or hypercalcemic nephropathy), trans- 50 Clinician’s Pocket Reference, 9th Edition fusion reaction, myoglobulinuria, radiographic contrast media (especially in diabetics, dehy- dration, multiple myeloma and elderly), ESRD, drugs (aminoglycosides, amphotericin B, vancomycin, NSAIDs, cephalosporins, penicillins, and sulfonamides), emboli, thrombosis, 3 and DIC Postrenal: Bilateral ureteral obstruction, prostatic obstruction, neurogenic bladder PLEURAL EFFUSION (See Chapter 13, page 304, Thoracentesis, for more details.) Transudate: (Pleural to serum protein ratio <0.5, and pleural to serum LDH ratio <0.6 and pleural LDH <2⁄ 3 the upper limits of normal for serum LDH), CHF, cirrhosis, nephrotic syndrome, peritoneal dialysis Exudate: (Pleural to serum protein ratio >0.5, or pleural to serum LDH ratio >0.6, or pleural LDH >2⁄ 3 the upper limits of normal for serum LDH), bacterial or viral pneumonia, pulmonary infarction, TB, RA, SLE, malignancy (most common, breast, lung lymphoma, leukemia, ovarian, unknown primary, GI, mesothelioma, others), pancreatitis, pneumotho- rax, chest trauma, uremia Chylothorax: Traumatic or postoperative complication Empyema: Bacteria, fungi, TB, trauma, surgery Hydrothorax: Usually iatrogenic (central venous catheter complication) PRURITUS Skin lesions (papulosquamous, vesicobullous, contact dermatitis, infestations [scabies, etc], infections), dry skin (especially in winter), liver disease, uremia, diabetes, gout, Hodgkin’s disease, leukemias, polycythemia vera, intestinal parasites, drug reactions, pregnancy, psy- chosomatic, neurologic, or circulatory disturbances SEIZURES Types Generalized: Grand mal and petit mal (absence), febrile Partial Seizures: Partial motor, partial sensory, partial complex (psychomotor or tem- poral lobe, déjà vu, automatisms) Causes: Primary, CNS tumors (primary, metastatic), trauma, metabolic (hypoglycemia, hyponatremia, hypernatremia, acidosis, alkalosis, porphyria, uremia, etc), fever (especially in children), infection (meningitis, encephalitis, and abscess), anoxia (arrhythmias, stroke, carbon monoxide poisoning), drugs (alcohol or barbiturate withdrawal, cocaine, ampheta- mines), collagen-vascular disease (SLE), chronic renal failure, trauma, hypertensive en- cephalopathy, toxemia of pregnancy, psychogenic SPLENOMEGALY (See Lymphadenopathy and Splenomegaly, page 49) 3 Differential Diagnosis: Symptoms, Signs, and Conditions 51 SYNCOPE Includes vasovagal (simple faint), orthostatic (volume depletion, sympathectomy [either functional or surgical], diabetes, Shy–Drager [idiopathic], tricyclic antidepressants and di- uretics) and hysterical. Cardiac syncope (Adams–Stokes attack), paroxysmal atrial tachycar- 3 dia, atrial fibrillation, ventricular tachycardia, sinoatrial or atrioventricular block, pacemaker malfunction, aortic stenosis, IHSS, primary pulmonary hypertension, atrial myxoma, cough syncope, hypoglycemia, seizure disorder, subclavian steal syndrome, cerebrovascular acci- dent, AMI, alcohol-related TREMORS Resting (decrease with movement): Parkinson’s disease, Wilson’s disease, brain tu- mors (rare), medications (SSRI antidepressants, metoclopramide, phenothiazines [tardive dyskinesia]) Action (present with movement): Benign essential tremor (familial and senile), cere- bellar diseases, withdrawal syndromes (alcohol, benzodiazepines, opiates), normal/physio- logic (induced by anxiety, fatigue) Ataxic (worse at end of voluntary movement): MS, cerebellar diseases Others: Medication-induced (caffeines [coffee, tea], steroids, valproic acid, bronchodila- tors) febrile, hypoglycemic, hyperthyroidism, pheochromocytoma VAGINAL BLEEDING Normal menstrual period, dysfunctional uterine bleeding (premenopausal bleeding, oral contraceptives, luteal phase defect), anovulatory abnormal uterine bleeding (hypothalamic/ pituitary disorders, stress, thyroid and adrenal disease, endometriosis), pregnancy-related (ectopic pregnancy, threatened/spontaneous abortion, retained products of gestation), neoplasia (uterine fibroids; cervical polyps; and endometrial, cervical, ovarian, and vulvar carcinoma) VAGINAL DISCHARGE Vaginitis due to Candida albicans, Trichomonas vaginalis, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma, herpesvirus, chronic cervicitis, tumors, irritants, foreign bodies, estrogen deficiency VERTIGO Ménière’s disease (recurrent vertigo, deafness and tinnitus), labyrinthitis, aminoglycoside toxicity, benign positional vertigo, vestibular neuronitis, brainstem ischemia and infarction, basilar artery migraine, cerebellar infarction, acoustic neuroma motion sickness, excess of ethanol, quinine, and salicylic acid VOMITING (See Nausea and Vomiting page 49) 52 Clinician’s Pocket Reference, 9th Edition WEIGHT LOSS Normal or Increased Appetite: Diabetes, hyperthyroidism, anxiety, drugs (thyroid), carcinoid, sprue, pancreatic deficiency, parasites 3 Decreased Appetite: Depression, anorexia nervosa, GI obstruction, neoplasm, liver disease, severe infection, severe cardiopulmonary disease, uremia, adrenal insufficiency, hy- percalcemia, hypokalemia, intoxication (alcohol, lead), old age, drugs (amphetamines, digi- talis, SSRIs, such as fluoxetine [Prozac]), AIDS WHEEZING Large airway difficulty (laryngeal stridor, tracheal stenosis, foreign body), endobronchial tumor, asthma, bronchitis, emphysema, pulmonary edema, PE, anaphylactic reactions, my- ocardial ischemia 4 LABORATORY DIAGNOSIS: CHEMISTRY, IMMUNOLOGY, AND SEROLOGY 4 Acetoacetate Cortisol, Serum Acid Phosphatase Counterimmunoelectrophoresis ACTH Creatine Phosphokinase ACTH Stimulation Test Creatinine, Serum Albumin Cryoglobulins, Serum Albumin/Globulin Ratio Cytomegalovirus Antibodies Aldosterone Dehydroepiandrosterone Alkaline Phosphatase Dehydroepiandrosterone Sulfate Alpha-fetoprotein (AFP) Dexamethasone Suppression Test ALT Erythropoietin Ammonia Estradiol, Serum Amylase Estrogen/Progesterone Receptors ASO Titer Ethanol AST Fecal Fat Autoantibodies Ferritin Base Excess/Deficit Folic Acid Bicarbonate Follicle-Stimulating Hormone (FSH) Bilirubin FTA-ABS Blood Urea Nitrogen (BUN) Fungal Serologies BUN/Creatinine Ratio Gastrin, Serum C-Peptide GGT C-Reactive Protein Glucose CA 15-3 Glucose Tolerance Test, Oral CA 19-9 Glycohemoglobin CA-125 Haptoglobin Calcitonin Helicobacter pylori Antibody Titers Calcium, Serum Hepatitis Testing Captopril Test High-Density Lipoprotein Cholesterol Carbon Dioxide HLA Carboxyhemoglobin Homocysteine, Serum Carcinoembryonic Antigen (CEA) Human Chorionic Gonadotropin (HCG) Catecholamines, Fractionated Serum Human Immunodeficiency Antibody Chloride, Serum Testing (HIV) Cholesterol Immunoglobulins, Quantitative Clostridium difficile Toxin Assay, Fecal Iron Cold Agglutinins Iron-Binding Capacity, Total Complement (C3, C4, CH50) Lactate Dehydrogenase (LDH) 53 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 54 Clinician’s Pocket Reference, 9th Edition Lactic Acid Renin LAP Score Plasma LE Preparation Renal Vein Lead, Blood Retinol-Binding Protein Legionella Antibody Rheumatoid Factor 4 Lipase Rocky Mountain Spotted Fever Lipid Profile Antibodies Low-Density Lipoprotein- Semen Analysis Cholesterol SGGT Luteinizing Hormone SGOT Lyme Disease Serology SGPT Magnesium Sodium, Serum Metyrapone Test Stool for Occult Blood MHA-TP Sweat Chloride β2-Microglobulin T3 RU Monospot Testosterone Myoglobin Thyroglobulin 5
-Nucleotidase Thyroid-Stimulating Hormone Oligoclonal Banding, CSF Thyroxine Osmolality, Serum Thyroxine-Binding Globulin Oxygen Thyroxine Index, Free P-24 Antigen (HIV Antigen) TORCH Battery Parathyroid Hormone Transferrin Phosphorus Triglycerides Potassium, Serum Triiodothyronine Progesterone, Serum Troponin, Cardiac-Specific Prolactin Uric Acid Prostate-Specific Antigen (PSA) VDRL Test Protein Electrophoresis, Serum Vitamin B12 and Urine Zinc Protein, Serum This chapter outlines commonly ordered blood chemistry, immunology, and serology tests with normal values and a guide to the diagnosis of common abnormalities. Other laboratory tests can be found in the following chapters: Hematology, Chapter 5; Urine Studies, Chap- ter 6; Microbiology, Chapter 7; and Blood Gases, Chapter 8. With the institution of DRGs, it becomes imperative to understand appropriate, as well as economical, laboratory testing patterns. Laboratory testing should be guided by, but not a substitute for, an effective history, physical, and careful clinical assessment. Most laboratories offer AMA recommended “panel” tests, whereby multiple determina- tions are performed on a single sample. Although your lab may vary, some common chem- istry panels include: Basic Metabolic Panel: BUN, calcium, creatinine, electrolytes (Na, K, Cl, CO2), glucose Cardiac Enzymes: CK-MB (if total CK >150 IU/L), troponin Chem-7 Panel/SMA-7: BUN, creatinine, electrolytes (Na, K, Cl, CO2),glucose 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 55 Comprehensive Metabolic Panel: Albumin, alkaline phosphatase, ALT (SGPT), AST (SGOT), bilirubin (total), BUN, calcium, creatinine, electrolytes (Na, K, Cl, CO2), glu- cose, protein (total) Electrolytes: Sodium, potassium, chloride, CO2, (Na, K, Cl, CO2) Health Screen-12/SMA-12: Albumin, alkaline phosphatase, AST (SGOT), bilirubin (total), calcium, cholesterol, creatinine, glucose, LDH, phosphate, protein (total), uric acid Hepatic Function Panel: Albumin, alkaline phosphatase, ALT (SGPT), AST (SGOT), 4 bilirubin (total & direct), protein Lipid Panel: Cholesterol, HDL cholesterol, LDL cholesterol (calculated), triglycerides The Système International (SI) is a metric-based laboratory data-reporting system that is used internationally. The mole is the unit used most extensively in the system. The SI unit for expressing enzymatic activity is the “katal”; however, most countries have adopted units per liter (U/L) as an alternative measure of enzymatic activity. For most lab values, repre- sentative SI units have been included; however, each individual laboratory should be con- sulted for its “normal” values. If an increased or decreased value is not clinically useful, it is usually not listed. Be- cause each laboratory has its own set of normal reference values, the normals given should only be used as a guide. The range for common normal values is given in parentheses. Un- less specified, values reflect normal adult levels. This section includes the method of collec- tion since laboratories have attempted to standardize collection methods; however, be aware that some labs may have alternative collection methods. Blood specimen tubes are listed in Chapter 13, page 311. ACETOACETATE (KETONE BODIES, ACETONE) • Normal = negative • Collection: Red top tube Positive: DKA, starvation, emesis, stress, alcoholism, infantile organic acidemias, iso- propanol ingestion ACID PHOSPHATASE (PROSTATIC ACID PHOSPHATASE, PAP) • <3.0 ng/mL by RIA, or <0.8 IU/L by enzymatic • Collection: Tiger top tube Not a useful screening test for cancer; most useful as a marker of response to therapy or in confirming metastatic disease. PSA is more sensitive in diagnosis of cancer. Increased: Carcinoma of the prostate (usually outside of prostate), prostatic surgery or trauma (including prostatic massage), rarely in infiltrative bone disease (Gaucher’s disease, myeloid leukemia), prostatitis, or BPH ACTH (ADRENOCORTICOTROPIC HORMONE) • 8 AM 20–140 pg/mL (SI: 20–140 ng/L), midnight, approximately 50% of AM value • Col- lection: Tiger top tube Increased: Addison’s disease (primary adrenal hypofunction), ectopic ACTH produc- tion (small [oat] cell lung carcinoma, pancreatic islet cell tumors, thymic tumors, renal cell carcinoma, bronchial carcinoid), Cushing’s disease (pituitary adenoma), congenital adrenal hyperplasia (adrenogenital syndrome) Decreased: Adrenal adenoma or carcinoma, nodular adrenal hyperplasia, pituitary in- sufficiency, corticosteroid use 56 Clinician’s Pocket Reference, 9th Edition ACTH STIMULATION TEST (CORTROSYN STIMULATION TEST) • Collection: Tiger top tube Used to help diagnose adrenal insufficiency. Cortrosyn (an ACTH analogue) is given at a dose of 0.25 mg IM or IV in adults or 0.125 mg in children <2 years. Collect blood at time 0, 30, and 60 min for cortisol and aldosterone. 4 Normal Response: Three criteria are required: basal cortisol of at least 5 mg/dL, an in- cremental increase after cosyntropin (Cortrosyn) injection of at least 7 mg/dL, and a final serum cortisol of at least 16 mg/dL at 30 or 18 mg/dL at 60 min or cortisol increase of >10 mg/dL. Aldosterone increases >5 ng/dL over baseline. Addison’s Disease (Primary Adrenal Insufficiency): Neither cortisol nor aldos- terone increase over baseline. Secondary Adrenal Insufficiency: Caused by pituitary insufficiency or suppression by exogenous steroids, cortisol does not increase, but aldosterone does. ALBUMIN • Adult 3.5–5.0 g/dL (SI: 35–50 g/L), child 3.8–5.4 g/dL (SI: 38–54 g/L) • Collection: Tiger top tube; part of SMA-12 Decreased: Malnutrition (see page 211), overhydration, nephrotic syndrome, CF, mul- tiple myeloma, Hodgkin’s disease, leukemia, metastatic cancer, protein-losing en- teropathies, chronic glomerulonephritis, alcoholic cirrhosis, inflammatory bowel disease, collagen-vascular diseases, hyperthyroidism ALBUMIN/GLOBULIN RATIO (A/G RATIO) • Normal >1 A calculated value (Total protein minus albumin = globulins. Albumin divided by glob- ulins = A/G ratio). Serum protein electrophoresis is a more informative test (see page 85). Decreased: Cirrhosis, liver diseases, nephrotic syndrome, chronic glomerulonephritis, cachexia, burns, chronic infections and inflammatory states, myeloma ALDOSTERONE • Serum: Supine 3–10 ng/dL (SI: 0.083–0.28 nmol/L) early AM, normal sodium intake [3 g

sodium/d] • Upright 5–30 ng/dL (SI: 0.138–0.83 nmol/L); urinary 2–16 mg/24 h (SI: 5.4–44.3 nmol/d) • Collection: Green or lavender top tube Discontinue antihypertensives and diuretics 2 wk prior to test. Upright samples should be drawn after 2 h. Primarily used to screen hypertensive patients for possible Conn’s syn- drome (adrenal adenoma producing excess aldosterone). Increased: Primary hyperaldosteronism, secondary hyperaldosteronism (CHF, sodium depletion, nephrotic syndrome, cirrhosis with ascites, others), upright posture Decreased: Adrenal insufficiency, panhypopituitarism, supine posture ALKALINE PHOSPHATASE • Adult 20–70 U/L, child 20–150 U/L • Collection: Tiger top tube; part of SMA-12 A fractionated alkaline phosphatase was formerly used to differentiate the origin of the en- zyme in the bone from that in the liver. Replaced by the GGT and 5
-nucleotidase determinations 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 57 Increased: Increased calcium deposition in bone (hyperparathyroidism), Paget’s dis- ease, osteoblastic bone tumors (metastatic or osteogenic sarcoma), osteomalacia, rickets, pregnancy, childhood, healing fracture, liver disease such as biliary obstruction (masses, drug therapy), hyperthyroidism Decreased: Malnutrition, excess vitamin D ingestion 4 ALPHA-FETOPROTEIN (AFP) • (<16 ng/mL (SI: <16 mL) • third trimester of pregnancy maximum 550 ng/mL (SI: 550 mL) • Collection: Tiger top tube Increased: Hepatoma (hepatocellular carcinoma), testicular tumor (embryonal carci- noma, malignant teratoma), neural tube defects (in mother’s serum [spina bifida, anen- cephaly, myelomeningocele]), fetal death, multiple gestations, ataxia–telangiectasia, some cases of benign hepatic diseases (alcoholic cirrhosis, hepatitis, necrosis) Decreased: Trisomy 21 (Down syndrome) in maternal serum ALT (ALANINE AMINOTRANSFERASE, ALAT) OR SGPT • 0–35 U/L (SI: 0–0.58 mkat/L), higher in newborns • Collection: Tiger top tube Increased: Liver disease, liver metastasis, biliary obstruction, pancreatitis, liver conges- tion (ALT is more elevated than AST in viral hepatitis; AST elevated more than ALT in alco- holic hepatitis.) AMMONIA • Adult 10–80 mg/dL (SI: 5–50 mmol/L) • To convert mg/dL to mmol/L, multiply by 0.5872 • Collection: Green top tube, on ice, analyze immediately Increased: Liver failure, Reye’s syndrome, inborn errors of metabolism, normal neonates (normalizes within 48 h of birth) AMYLASE • 50–150 Somogyi units/dL (SI: 100–300 U/L) • Collection: Tiger top tube Increased: Acute pancreatitis, pancreatic duct obstruction (stones, stricture, tumor, sphincter spasm secondary to drugs), pancreatic pseudocyst or abscess, alcohol ingestion, mumps, parotiditis, renal disease, macroamylasemia, cholecystitis, peptic ulcers, intestinal obstruction, mesenteric thrombosis, after surgery Decreased: Pancreatic destruction (pancreatitis, cystic fibrosis), liver damage (hepatitis, cirrhosis), normal newborns in the first year of life ASO (ANTISTREPTOLYSIN O/ANTISTREPTOCOCCAL O) TITER (STREPTOZYME) • <200 IU/mL (Todd units) school-age children • <100 IU/mL preschool and adults • varies with lab • Collection: Tiger top tube Increased: Streptococcal infections (pharyngitis, scarlet fever, rheumatic fever, post- streptococcal glomerulonephritis), RA, and other collagen diseases 58 Clinician’s Pocket Reference, 9th Edition AST (ASPARTATE AMINOTRANSFERASE, ASAT) OR SGOT • 8–20 U/L (SI: 0–0.58 mkat/L) • Collection: Tiger top tube; part of SMA-12 Generally parallels changes in ALT in liver disease. Increased: AMI, liver disease, Reye’s syndrome, muscle trauma and injection, pancre- atitis, intestinal injury or surgery, factitious increase (erythromycin, opiates), burns, cardiac 4 catheterization, brain damage, renal infarction Decreased: Beriberi (vitamin B6 deficiency), severe diabetes with ketoacidosis, liver disease, chronic hemodialysis AUTOANTIBODIES • Normal = negative • Collection: Tiger top tube Antinuclear Antibody (ANA, FANA) A useful screening test in patients with symptoms suggesting collagen–vascular disease, es- pecially if titer is >1:160. Positive: SLE, drug-induced lupus-like syndromes (procainamide, hydralazine, isoni- azid, etc), scleroderma, MCTD, RA, polymyositis, juvenile RA (5–20%). Low titers are also seen in non-collagen–vascular disease. Specific Immunofluorescent ANA Patterns Homogenous. Nonspecific, from antibodies to DNP and native double-stranded DNA. Seen in SLE and a variety of other diseases. Antihistone is consistent with drug-induced lupus. Speckled. Pattern seen in many connective tissue disorders. From antibodies to ENA, includ- ing anti-RNP, anti-Sm, anti-PM-1, and anti-SS. Anti-RNP is positive in MCTD and SLE. Anti-Sm is very sensitive for SLE. Anti-SS-A and anti-SS-B are seen in Sjögren’s syndrome and subacute cutaneous lupus. The speckled pattern is also seen with sclero- derma. Peripheral Rim Pattern. From antibodies to native double-stranded DNA and DNP. Seen in SLE Nucleolar Pattern. From antibodies to nucleolar RNA. Positive in Sjögren’s syndrome and scleroderma Anticentromere: Scleroderma, Raynaud’s disease, CREST syndrome Anti-DNA (Antidouble-stranded DNA): SLE (but negative in drug-induced lupus), chronic active hepatitis, mononucleosis Antimitochondrial: Primary biliary cirrhosis, autoimmune diseases such as SLE Antineutrophil Cytoplasmic: Wegener’s granulomatosis, polyarteritis nodosa, and other vasculitides Anti-SCL 70: Scleroderma Antismooth Muscle: Low titers are seen in a variety of illnesses; high titers (>1:100) are suggestive of chronic active hepatitis. Sjögren Syndrome Antibody (SS-A): Sjögren syndrome, SLE, RA Antimicrosomal: Hashimoto’s thyroiditis 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 59 BASE EXCESS/DEFICIT • –2 to +2 • See Chapter 8, page 162 BICARBONATE (OR “TOTAL CO2”) • 23–29 mmol/L • See CARBON DIOXIDE, page 61 4 BILIRUBIN • Total, 0.3–1.0 mg/dL (SI: 3.4–17.1 mmol/L) • direct, <0.2 mg/dL (SI: <3.4 mmol/L) • indirect, <0.8 mg/dL (SI: <3.4 mmol/L) • To convert mg/dL to mmol/L, multiply by 17.10 • Collection: Tiger top tube Increased Total: Hepatic damage (hepatitis, toxins, cirrhosis), biliary obstruction (stone or tumor), hemolysis, fasting. Increased Direct (Conjugated): Note: Determination of the direct bilirubin is usually unnecessary with total bilirubin levels <1.2 mg/dL (SI: 21 mmol/L) Biliary obstruction/cholestasis (gallstone, tumor, stricture), drug-induced cholestasis, Dubin– Johnson and Rotor’s syndromes Increased Indirect (Unconjugated): Note: This is calculated as total minus direct bilirubin. So-called hemolytic jaundice caused by any type of hemolytic anemia (transfusion reaction, sickle cell, etc), Gilbert’s disease, physiologic jaundice of the newborn, Crigler–Najjar syndrome Bilirubin, Neonatal(“Baby Bilirubin”) • Normal levels dependent on prematurity and age in days • “panic levels” usually >15–20 mg/dL (SI: >257–342 mmol/L in full-term infants) • Collection: Capillary tube Increased: Erythroblastosis fetalis, physiologic jaundice (may be due to breast-feeding), resorption of hematoma or hemorrhage, obstructive jaundice, others BLOOD UREA NITROGEN (BUN) • Birth–1 year: 4–16 mg/dL (SI: 1.4–5.7 mmol/L) • 1–40 years 5–20 mg/dL (SI: 1.8–7.1 mmol/L)]] • Gradual slight increase with age • To convert mg/dL to mmol/L, multiply by 0.3570 • Collection: Tiger top tube Less useful measure of GFR than creatinine because BUN is also related to protein metabolism Increased: Renal failure (including drug-induced from aminoglycosides, NSAIDs), pre- renal azotemia (decreased renal perfusion secondary to CHF, shock, volume depletion), postrenal (obstruction), GI bleeding, stress, drugs (especially aminoglycosides) Decreased: Starvation, liver failure (hepatitis, drugs), pregnancy, infancy, nephrotic syn- drome, overhydration BUN/CREATININE RATIO (BUN/CR) • Mean 10, range 6–20 Calculated based on serum levels 60 Clinician’s Pocket Reference, 9th Edition Increased: Prerenal azotemia (renal hypoperfusion), GI bleeding, high-protein diet, ileal conduit, drugs (steroids, tetracycline) Decreased: Malnutrition, pregnancy, low-protein diet, ketoacidosis, hemodialysis, SIADH, drugs (cimetidine) 4 C-PEPTIDE, INSULIN (“CONNECTING PEPTIDE”) • Fasting, <4.0 ng/mL (SI: <4.0 mg/L) • Male >60 years, 1.5–5.0 ng/mL (SI: 1.5–5.0 mg/L) • Female 1.4–5.5 ng/mL (SI: 1.4–5.5 mg/L) • Collection: Tiger top tube Differentiates between exogenous and endogenous insulin production/administration. Liberated when proinsulin is split to insulin; levels suggest endogenous production of insulin Decreased: Diabetes (decreased endogenous insulin), insulin administration (factitious or therapeutic), hypoglycemia C-REACTIVE PROTEIN (CRP) • Normal = none detected • Collection: Tiger top tube A nonspecific screen for infectious and inflammatory diseases, correlates well with ESR. In the first 24 h, however, ESR may be normal and CRP elevated. Increased: Bacterial infections, inflammatory conditions (acute rheumatic fever, acute RA, MI, transplant rejection, embolus, inflammatory bowel disease), last half of pregnancy, oral contraceptives, some malignancies CA 15-3 Used to detect breast cancer recurrence in asymptomatic patients and monitor therapy. Lev- els related to stage of disease Increased: Progressive breast cancer, benign breast disease and liver disease Decreased: Response to therapy (25% change considered significant) CA 19-9 • <37 U/ml (SI:<37 kU/L) • Collection: Tiger top tube Primary used to determine resectability of pancreatic cancers (ie, >1000U/mL 95% unresectable) Increased: GI cancers such as pancreas, stomach, liver, colorectal, hepatobiliary, some cases of lung and prostate, pancreatitis CA-125 • <35 U/mL (SI: <35 kU/L) • Collection: Tiger top tube Not a useful screening test for ovarian cancer when used alone; best used in conjunction with ultrasound and physical examination. Rising levels after resection predictive for recur- rence Increased: Ovarian, endometrial, and colon cancer; endometriosis; inflammatory bowel disease; PID; pregnancy; breast lesions; and benign abdominal masses (teratomas) 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 61 CALCITONIN (THYROCALCITONIN) • <19 pg/mL (SI: <19 ng/L) • Collection: Tiger top tube Increased: Medullary carcinoma of the thyroid, C-cell hyperplasia (precursor of medullary carcinoma), small (oat) cell carcinoma of the lung, newborns, pregnancy, chronic renal insufficiency, Zollinger–Ellison syndrome, pernicious anemia. 4 CALCIUM, SERUM • Infants to 1 month: 7–11.5 mg/dL (SI: 1.75–2.87 mmol/L) • 1 month to 1 year: 8.6–11.2 mg/dL (SI: 2.15–2.79 mmol/L) • >1 year and adults: 8.2–10.2 mg/dL (SI: 2.05–2.54 mmol/L) • Ionized: 4.75–5.2 mg/dL (SI: 1.19–1.30 mmol/L) • To convert mg/dL to mmol/L, multiply by 0.2495 • Collection: Tiger top tube; ionized requires green or red tube When interpreting a total calcium value, albumin must be known. If it is not within nor- mal limits, a corrected calcium can be roughly calculated by the following formula. Values for ionized calcium need no special corrections. Corrected total Ca = 0.8 (Normal albumin − Measured albumin) + Reported Ca Increased: (Note: Levels >12 mg/dL [2.99 mmol/L] may lead to coma and death) Pri- mary hyperparathyroidism, PTH-secreting tumors, vitamin D excess, metastatic bone tu- mors, osteoporosis, immobilization, milk-alkali syndrome, Paget’s disease, idiopathic hypercalcemia of infants, infantile hypophosphatasia, thiazide diuretics, chronic renal fail- ure, sarcoidosis, multiple myeloma Decreased: (Note: Levels <7 mg/dL [<1.75 mmol/L] may lead to tetany and death.) Hy- poparathyroidism (surgical, idiopathic), pseudo-hypoparathyroidism, insufficient vitamin D, calcium and phosphorus ingestion (pregnancy, osteomalacia, rickets), hypomagnesemia, renal tubular acidosis, hypoalbuminemia (cachexia, nephrotic syndrome, CF), chronic renal failure (phosphate retention), acute pancreatitis, factitious decrease because of low protein and albumin CAPTOPRIL TEST • See Aldosterone, page 56, and renin (plasma renin), page 88, for normal values Used in the evaluation of renovascular hypotension, the drug is an ACE inhibitor that blocks angiotensin II. Captopril is administered (25 mg IV at 8AM). Aldosterone decreases 2 h later from baseline in normals or essential hypertension, but does not suppress in pa- tients with aldosteronism. For renovascular hypertension, the PRA increases >12 ng/mL/h and an absolute increase of 10 ng/mL/h plus a 400% increase in PRA if pretest level <3 ng/mL/h and >150% over baseline if the pretest PRA was >3 ng/mL/h. Test now also com- bined with nuclear renal scan to identify renal artery stenosis CARBON DIOXIDE (“TOTAL CO2” OR BICARBONATE) • Adult 23–29 mmol/L, child 20–28 mmol/L • (See Chapter 8 for pCO2 values • Collec- tion: Tiger top tube, do not expose sample to air Increased: Compensation for respiratory acidosis (emphysema) and metabolic alkalosis (severe vomiting, primary aldosteronism, volume contraction, Bartter’s syndrome) 62 Clinician’s Pocket Reference, 9th Edition Decreased: Compensation for respiratory alkalosis, and metabolic acidosis (starvation, diabetic ketoacidosis, lactic acidosis, alcoholic ketoacidosis, toxins [methanol, ethylene gly- col, paraldehyde], severe diarrhea, renal failure, drugs [salicylates, acetazolamide], dehydra- tion, adrenal insufficiency) CARBOXYHEMOGLOBIN (CARBON MONOXIDE) 4 • Nonsmoker <2%; smoker <9%; toxic >15%• Collection: Gray or lavender top tube; con- firm with lab Increased: Smokers, smoke inhalation, automobile exhaust inhalation, normal new- borns CARCINOEMBRYONIC ANTIGEN (CEA) • Nonsmoker <3.0 ng/mL (SI: <3.0 µg/L) • smoker <5.0 ng/mL (SI: <5.0 µg/L) • Collec- tion: Tiger top tube Not a screening test; useful for monitoring response to treatment and tumor recurrence of adenocarcinomas of the GI tract Increased: Carcinoma (colon, pancreas, lung, stomach), smokers, nonneoplastic liver disease, Crohn’s disease, and ulcerative colitis CATECHOLAMINES, FRACTIONATED SERUM • Collection: Green or lavender tube; check with lab Values vary and depend on the lab and method of assay used. Normal levels shown here are based on a HPLC technique. Patient must be supine in a nonstimulating environment with IV access to obtain sample. Catecholamine Plasma (Supine) Levels Norepinephrine 70–750 pg/mL (SI: 414–4435 pmol/L) Epinephrine 0–100 pg/mL (SI: 0–546 pmol/L) Dopamine <30 pg/mL (SI: 196 pmol/L) Increased: Pheochromocytoma, neural CREST tumors (neuroblastoma), with extra- adrenal pheochromocytoma, norepinephrine may be markedly elevated compared with epi- nephrine. CHLORIDE, SERUM • 97–107 mEq/L (SI: 97–107 mmol/L) • Collection: Tiger top tube Increased: Diarrhea, renal tubular acidosis, mineralocorticoid deficiency, hyperalimen- tation, medications (acetazolamide, ammonium chloride) Decreased: Vomiting, diabetes mellitus with ketoacidosis, mineralocorticoid excess, renal disease with sodium loss CHOLESTEROL • Total • Normal, see Table 4–1; see also LIPID PROFILE/CHOLESTEROL SCREEN- ING, page 79, and

Figure 4–4, see page 80.• To convert mg/dL to mmol/L, multiply by 0.02586 • Collection: Tiger top tube 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 63 TABLE 4–1 Normal Total Cholesterol Levels by Age Standard Units Sl Units Age (mg/dL) (mmol/L) 4 <29 <200 <5.20 30–39 <225 <5.85 40–49 <245 <6.35 Increased: Idiopathic hypercholesterolemia, biliary obstruction, nephrosis, hypothy- roidism, pancreatic disease (diabetes), pregnancy, oral contraceptives, hyperlipoproteinemia (types IIb, III, V) Decreased: Liver disease (hepatitis, etc), hyperthyroidism, malnutrition (cancer, starva- tion), chronic anemias, steroid therapy, lipoproteinemias, AMI High-Density Lipoprotein Cholesterol (HDL, HDL-C) • Fasting 30–70 mg/dL (SI: 0.8–1.80 mmol/L) • Female 30–90 mg/dL (SI: 0.80–2.35) HDL-C has the best correlation with the development of CAD; decreased HDL-C in males leads to an increased risk. Levels <45 mg/dL associated with increased risk of CAD Increased: Estrogen (females), regular exercise, small ethanol intake, medications (nicotinic acid, gemfibrozil, others) Decreased: Males, smoking, uremia, obesity, diabetes, liver disease, Tangier disease Low-Density Lipoprotein Cholesterol (LDL, LDL-C) • 50–190 mg/dL (SI: 1.30–4.90 mmol/L) Increased: Excess dietary saturated fats, MI, hyperlipoproteinemia, biliary cirrhosis, en- docrine disease (diabetes, hypothyroidism) Decreased: Malabsorption, severe liver disease, abetalipoproteinemia CLOSTRIDIUM DIFFICILE TOXIN ASSAY, FECAL • Normal negative Majority of patients with pseudomembranous colitis have positive C. difficile assay. Often positive in antibiotic associated diarrhea and colitis. Can be seen in some normals and neonates COLD AGGLUTININS • <1:32 • Collection: Lavender or blue top tube Most frequently used to screen for atypical pneumonias. 64 Clinician’s Pocket Reference, 9th Edition Increased: Atypical pneumonia (mycoplasmal pneumonia), other viral infections (espe- cially mononucleosis, measles, mumps), cirrhosis, parasitic infections, Waldenström’s macroglobulinemia, lymphomas and leukemias, multiple myeloma COMPLEMENT 4 • Collection: Tiger or lavender top tube Complement describes a series of sequentially reacting serum proteins that participate in pathogenic processes and lead to inflammatory injury. Complement C3 • 85–155 mg/dL, (SI: 800–1500 ng/L) Decreased levels suggest activation of the classical or alternative pathway, or both. Increased: RA (variable finding), rheumatic fever, various neoplasms (gastrointestinal, prostate, others), acute viral hepatitic, MI, pregnancy, amyloidosis Decreased: SLE, glomerulonephritis (poststreptococcal and membranoproliferative), sepsis, SBE, chronic active hepatitis, malnutrition, DIC, gram-negative sepsis Complement C4 • 20–50 mg/dL (SI: 200–500 ng/L) Increased: RA (variable finding), neoplasia (gastrointestinal, lung, others) Decreased: SLE, chronic active hepatitis, cirrhosis, glomerulonephritis, hereditary an- gioedema (test of choice). Complement CH50 (Total) • 33–61 mg/mL (SI: 330–610 ng/L) Tests for complement deficiency in the classical pathway. Increased: Acute-phase reactants (tissue injury, infections, etc) Decreased: Hereditary complement deficiencies CORTISOL, SERUM • 8 AM, 5.0–23.0 mg/dL (SI: 138–365 nmol/L) • 4 PM, 3.0–15.0 mg/dL (SI: 83–414 nmol/L) • Collection: Green or red top tube Increased: Adrenal adenoma, adrenal carcinoma, Cushing’s disease, nonpituitary ACTH-producing tumor, steroid therapy, oral contraceptives Decreased: Primary adrenal insufficiency (Addison’s disease), congenital adrenal hy- perplasia, Waterhouse-Friderichsen syndrome, ACTH deficiency COUNTERIMMUNOELECTROPHORESIS (CIEP, CEP) • Normal = negative An immunologic technique that allows for rapid identification of infecting organisms from fluids, including serum, urine, CSF, and other body fluids. Organisms identified in- 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 65 clude Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, and group B Streptococcus. CREATINE PHOSPHOKINASE (KINASE) (CP, CPK) • 25–145 mU/mL (SI: 25–145 U/L) • Collection: Tiger top tube Used in suspected MI or muscle diseases. Heart, skeletal muscle, and brain have high 4 levels Increased: Muscle damage (AMI, myocarditis, muscular dystrophy, muscle trauma [in- cluding injections], after surgery), brain infarction, defibrillation, cardiac catheterization and surgery, rhabdomyolysis, polymyositis, hypothyroidism CPK Isoenzymes MB: (Normal <6%, heart origin) increased in AMI (begins in 2–12 h, peaks at 12–40 h, returns to normal in 24–72 h), pericarditis with myocarditis, rhabdomyolysis, crush injury, Duchenne’s muscular dystrophy, polymyositis, malignant hyperthermia, and cardiac surgery MM: (Normal 94–100%, skeletal muscle origin) increased in crush injury, malignant hy- perthermia, seizures, IM injections BB: (Normal 0%, brain origin) brain injury (CVA, trauma), metastatic neoplasms (prostate), malignant hyperthermia, colonic infarction CREATININE, SERUM • Adult male <1.2 mg/dL (SI: 106 mmol/L) • Adult female <1.1 mg/dL (SI: 97 mmol/L) • Child 0.5–0.8 mg/dL (SI: 44–71 mmol/L) • To convert mg/dL to µmol/L, multiply by 88.40 • Collection: Tiger top tube A clinically useful estimate of GFR. As a rule of thumb, serum creatinine doubles with each 50% reduction in the GFR. Creatine clearance is discussed in Chapter 6. Increased: Renal failure (prerenal, renal, or postrenal obstruction or medication- induced [aminoglycosides, NSAIDs, others]), gigantism, acromegaly, ingestion of roasted meat, false-positive with DKA Decreased: Pregnancy, decreased muscle mass, severe liver disease CRYOGLOBULINS (CRYOCRIT) <0.4% (or negative if qualitative) {·} Collection: Tiger top tube, process immediately These abnormal proteins precipitate out of serum at low temperatures. Cryocrit, a quan- titative measure, is preferred over the qualitative method. Should be collected in nonantico- agulated tubes and transported at body temperature. Positive samples can be analyzed for immunoglobulin class, and light-chain type on request. Monoclonal: Multiple myeloma, Waldenström’s macroglobulinemia, lymphoma, CLL Mixed Polyclonal or Mixed Monoclonal: Infectious diseases (viral, bacterial, para- sitic), such as SBE or malaria; SLE; RA; essential cryoglobulinemia; lymphoproliferative diseases; sarcoidosis; chronic liver disease (cirrhosis) 66 Clinician’s Pocket Reference, 9th Edition CYTOMEGALOVIRUS (CMV) ANTIBODIES • IgM <1:8, IgG <1:16 • Collection: Tiger top tube Used in neonates (CMV is the most common intrauterine infection), posttransfusion CMV infection, and organ donors and recipients. Most of adults will have detectable titers. Increased: Serial measurements 10–14 days apart with a 4× increase in titers or a single 4 IgM >1:8 is suspicious for acute infection. Universally increased titers in AIDS. IgM most useful in neonatal infections DEHYDROEPIANDROSTERONE (DHEA) • Male 2.0–3.4 ng/mL (SI: 5.2–8.7 mmol/L) • Female, premenopausal 0.8–3.4 ng/mL (SI: 2.1–8.8 mmol/L) • Postmenopausal 0.1–0.6 ng/mL (SI: 0.3–1.6 mmol/L) • Collection: Tiger top tube Increased: Anovulation, polycystic ovaries, adrenal hyperplasia, adrenal tumors Decreased: Menopause DEHYDROEPIANDROSTERONE SULFATE (DHEAS) • Male 1.7–4.2 ng/mL (SI: 6–15 mmol/L) • Female 2.0–5.2 ng/mL (SI: 7–18 mmol/L) • Collection: Tiger top tube Increased: Hyperprolactinemia, adrenal hyperplasia, adrenal tumor, polycystic ovaries, lipoid ovarian tumors Decreased: Menopause DEXAMETHASONE SUPPRESSION TEST Used in the differential diagnosis of Cushing’s syndrome (elevated cortisol) Overnight Test: In the “rapid” version of this test, a patient takes 1 mg of dexametha- sone PO at 11 PM and a fasting 8 AM plasma cortisol is obtained. Normally the cortisol level should be <5.0 mg/dL [138 nmol/L]. A value that is >5 mg/dL [138 nmol/L] usually con- firms the diagnosis of Cushing’s syndrome; however, obesity, alcoholism, or depression may occasionally show the same result. In these patients, the best screening test is a 24-h urine for free cortisol. Low-Dose Test: After collection of baseline serum cortisol and 24-h urine-free cortisol levels, dexamethasone 0.5 mg is administered PO every 6 h for eight doses. Serum and urine cortisol are repeated on the second day. Failure to suppress to a serum cortisol of <5.0 mg/dL [138 nmol/L] and a urine-free cortisol of <30 µg/dL (82 nmol/L) confirms Cushing’s syndrome. High-Dose Test: After the low-dose test, dexamethasone, 2 mg PO every 6 h for eight doses will cause a fall in urinary-free cortisol to 50% of the baseline value in bilateral adrenal hyperplasia (Cushing’s disease) but not in adrenal tumors or ectopic ACTH pro- duction. ERYTHROPOIETIN (EPO) • 5–36 mU/L (5–36 IU/L) • Collection: Tiger top tube EPO is a renal hormone that stimulates RBC production. 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 67 Increased: Pregnancy, secondary polycythemia (high altitude, COPD, etc), tumors (renal cell carcinoma, cerebellar hemangioblastoma, hepatoma, others), PCKD, anemias with bone marrow unresponsiveness (aplastic anemia, iron deficiency, etc) Decreased: Bilateral nephrectomy, anemia of chronic disease (ie, renal failure, nephrotic syndrome), primary polycythemia (Note: The determination of EPO levels before administration of recombinant EPO for renal failure is not usually necessary.) 4 ESTRADIOL, SERUM • Collection: Tiger top tube Serial measurements useful in assessing fetal well-being, especially in high-risk preg- nancy. Also useful in evaluation of amenorrhea and gynecomastia in males. Female Normal Values Follicular phase 25–75 pg/mL Midcycle peak 200–600 pg/mL Luteal phase 100–300 pg/mL Pregnancy 1st trimester 1–5 ng/mL 2nd trimester 5–15 ng/mL 3rd trimester 10–40 ng/mL Postmenopause 5–25 pg/mL Oral contraceptives <50 pg/mL Male Prepubertal 2–8 pg/mL Adult 10–60 pg/mL ESTROGEN/PROGESTERONE RECEPTORS These are typically determined on fresh surgical (breast cancer) specimens. The presence of the receptors is associated with a longer disease-free interval, survival from breast cancer, and increased likelihood of responding to endocrine therapy. Fifty to seventy-five percent of breast cancers are estrogen-receptor-positive. ETHANOL (BLOOD ALCOHOL) • 0 mg/dL (0 mmol/L) • Collection: Tiger top tube; do not use alcohol to clean venipunc- ture site, use povidone-iodine Physiologic changes can vary with degree of alcohol tolerance of an individual. • <50 mg/dL [<10.85 mmol/L]: Limited muscular incoordination • 50–100 [10.85–21.71]: Pronounced incoordination • 100–150 [21.71–32.57]: Mood and personality changes; legally intoxicated in most states • 150–400 [32.57–87]: Nausea, vomiting, marked ataxia, amnesia, dysarthria • ≥400: Coma, respiratory insufficiency and death FECAL FAT • 2–6 g/d on an 80–100 g/d fat diet • 72-h collection time • Sudan III stain, random <60 droplets fat/hpf Increased: CF, pancreatic insufficiency, Crohn’s disease, chronic pancreatitis, sprue 68 Clinician’s Pocket Reference, 9th Edition FERRITIN • Male 15–200 ng/mL (SI: 15–200 mg/L) • Female 12–150 ng/mL (SI: 12–150 mg/L) • Collection: Tiger top tube Increased: Hemochromatosis, hemosiderosis, sideroblastic anemia 4 Decreased: Iron deficiency (earliest and most sensitive test before red cells show any morphologic change), severe liver disease FOLIC ACID Serum Folate • >2.0 ng/mL (SI: >5 nmol/L) RBC • 125–600 ng/mL (283–1360 nmol/L) • Collection: Lavender top tube Serum folate can fluctuate with diet. RBC levels are more indicative of tissue stores. Vi- tamin B12 deficiency can result in the RBC unable to take up folate in spite of normal serum folate levels. Increased: Folic acid administration Decreased: Malnutrition/malabsorption (folic acid deficiency), massive cellular growth (cancer) or cell turnover, ongoing hemolysis, medications (trimethoprim, some anticonvul- sants, oral contraceptives), vitamin B12 deficiency (low RBC levels), pregnancy FOLLICLE-STIMULATING HORMONE (FSH) • Males: <22 IU/L • Females: nonmidcycle <20 IU/L, midcycle surge <40 IU/L (Midcycle peak should be two times basal level • Postmenopausal 40–160 IU/L • Collection: Tiger top tube Used in the workup of impotence, infertility in men, and amenorrhea in women Increased: (Hypergonadotropic >40 IU/L) postmenopausal, surgical castration, gonadal failure, gonadotropin-secreting pituitary adenoma Decreased: (Hypogonadotropic <5 IU/L) prepubertal, hypothalamic and pituitary dys- function, pregnancy FTA-ABS (FLUORESCENT TREPONEMAL ANTIBODY ABSORBED) • Normal = nonreactive • Collection: Tiger top tube FTA-ABS may be negative in early primary syphilis and remain positive in spite of ade- quate treatment. Positive: Syphilis (test of choice to confirm diagnosis after a reactive VDRL test), other treponemal infections can cause false-positive (Lyme disease, leprosy, malaria) FUNGAL SEROLOGIES • Negative <1:8 • Collection: Tiger top tube This is a screening technique for complement-fixed fungal antibodies, which usually detects antibodies to Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neo- formans, Aspergillus species, Candida species, and Coccidioides immitis. 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 69 GASTRIN, SERUM • Fasting <100 pg/mL (SI: 47.7 pmol/L) • Postprandial 95–140 pg/mL (SI: 45.3–66.7 pmol/L) • Collection: Tiger top tube, freeze immediately Make sure patient is not on H2 blockers or antacids. Increased: Zollinger–Ellison syndrome, medications (antacids, cimetidine, others) py- 4 loric stenosis, pernicious anemia, atrophic gastritis, ulcerative colitis, renal insufficiency, and steroid and calcium administration Decreased: Vagotomy and antrectomy GGT (SERUM GAMMA-GLUTAMYL TRANSPEPTIDASE, SGGT) • Male 9–50 U/L • Female 8–40 U/L • Collection: Tiger top tube Generally parallels changes in serum alkaline phosphatase and 5
-nucleotidase in liver disease. Sensitive indicator of alcoholic liver disease Increased: Liver disease (hepatitis, cirrhosis, obstructive jaundice), pancreatitis. GLUCOSE • Fasting, 70–105 mg/dL (SI: 3.89–5.83 nmol/L) • 2 h postprandial <140 mg/dL (SI: <7.8 nmol/L) • To convert mg/dL to nmol/L, multiply by 0.05551 • Collection: Tiger top tube American Diabetes Association Diagnostic Criterion for Diabetes: normal fasting <110, Impaired fasting 110–126, diabetes >126 or any random level >200 when associated with other symptoms. Confirm with repeat testing. Increased: Diabetes mellitus, Cushing’s syndrome, acromegaly, increased epinephrine (injection, pheochromocytoma, stress, burns, etc), acute pancreatitis, ACTH administration, spurious increase caused by drawing blood from a site above an IV line containing dextrose, elderly patients, pancreatic glucagonoma, drugs (glucocorticoids, some diuretics) Decreased: Pancreatic disorders (pancreatitis, islet cell tumors), extrapancreatic tumors (carcinoma of the adrenals, stomach), hepatic disease (hepatitis, cirrhosis, tumors), en- docrine disorders (early diabetes, hypothyroidism, hypopituitarism), functional disorders (after gastrectomy), pediatric problems (prematurity, infant of a diabetic mother, ketotic hy- poglycemia, enzyme diseases), exogenous

insulin, oral hypoglycemic agents, malnutrition, sepsis GLUCOSE TOLERANCE TEST (GTT), ORAL (OGTT) A fasting plasma glucose level >126 mg/dl (7.0 mmol/L) or a casual plasma glucose –200 mg/dL (11.1 mmol/L) meets the threshold for the diagnosis of diabetes, if confirmed on a subsequent day, and precludes the need for any glucose challenge. GTT is usually unneces- sary to diagnose asymptomatic diabetes mellitus; it may be useful in gestational diabetes. The GTT is unreliable in the presence of severe infection, prolonged fasting, or after the in- jection of insulin. After an overnight fast, a fasting blood glucose is drawn, and the patient is given a 75-g oral glucose load (100 g for gestational diabetes screening, 1.75 mg/kg ideal body weight in children up to a dose of 75 g). Plasma glucose is then drawn at 30, 60, 120, and 180 min. 70 Clinician’s Pocket Reference, 9th Edition Interpretation of GTT Adult-Onset Diabetes: Any fasting blood sugar >126, or >200 at both 120 min and one other time interval measured Gestational Diabetes: Any fasting blood sugar >126, 60 min >180, 120 min >155, 180 min >140 4 GLYCOHEMOGLOBIN (GHB, GLYCATED HEMOGLOBIN, GLYCOHEMOGLOBIN, HBA1C, HBA1 HEMOGLOBIN A1C, GLYCOSYLATED HEMOGLOBIN) • 4.6–7.1% or new standard: Nondiabetic <6, near normal 6–7 • Excellent glucose control 7–8 • Good control 8–9 • Fair control 9–10 • Poor control >10 • Collection: Lavender top tube Useful in long-term monitoring control of blood sugar in diabetics; reflects levels over preceding 3–4 months. Glycated serum protein (GSP) under study and may reflect serum glucose over the preceding 1–2 weeks Increased: Diabetes mellitus (uncontrolled), lead intoxication Decreased: Chronic renal failure, hemolytic anemia, pregnancy, chronic blood loss HAPTOGLOBIN • 40–180 mg/dL (SI: 0.4–1.8 g/L) • Collection: Tiger top tube Increased: Obstructive liver disease, any cause of increased ESR (inflammation, collagen-vascular diseases) Decreased: Any type of hemolysis (transfusion reaction, etc), liver disease, anemia, oral contraceptives, children and infants HELICOBACTER PYLORI ANTIBODY TITERS • IgG <0.17 = negative Most patients with gastritis and ulcer disease (gastric or duodenal) have chronic H. py- lori infection that should be treated. Positive in 35–50% asymptomatic patients (increases with age). Use in dyspepsia controversial. Four diagnostic methods are available to test for H. pylori, the organism associated with gastritis and ulcers. These include noninvasive (serology and a 13C breath test) and invasive (gastric mucosal biopsy and the Campylobacter- like organism test). The IgG subclass is found in all patient populations; occasionally only IgA antibodies can be detected. Serology is most useful in the evaluation of newly diag- nosed H. pylori infection or in monitoring response to therapy. IgG levels decrease slowly after treatment, but can remain elevated after clearing infection. Positive: Active or recent H. pylori infection, some asymptomatic carriers HEPATITIS TESTING Recommended hepatitis panel tests based on clinical settings is shown in Table 4–2. Inter- pretation of testing patterns is shown in Table 4–3. Profile patterns of hepatitis A and B are shown in Figures 4–1 and 4–2, respectively. Hepatitis Tests (Collection: Tiger top tube) 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 71 TABLE 4–2 Hepatitis Panel Testing to Guide the Ordering of Hepatitis Profiles for Given Clinical Settings Clinical Setting Test Purpose SCREENING TESTS 4 Pregnancy HBsAg* All expectant mothers should be screened during third trimester High-risk patients on HBsAg To screen for chronic or admission (homosexuals, active infection dialysis patients) Percutaneous inoculation Donor HBsAg To test patient’s blood (esp. Anti-HBc IgM dialysis and HIV patients) for Anti-Hep C infectivity with hepatitis B and C if a health care worker is exposed Victim HBsAg To test exposed health care Anti-HBc worker for immunity or Anti-Hep C chronic infection Pre-HBV vaccine Anti-HBc To determine if an individual Anti-HBs is infected or has antibod- ies to HBV Screening blood donors HBsAg Used by blood banks to Anti-HBc screen donors for hepatitis Anti-Hep C B and C DIAGNOSTIC TESTS Differential diagnosis of HBsAg To differentiate between HBV, acute jaundice, hepatitis, Anti-HBc IgM HAV, and hepatitis C in an or fulminant liver failure Anti-HAV IgM acutely jaundiced patient Anti-Hep C with hepatitis or fulminant liver failure Chronic hepatitis HBsAg To diagnose HBV infection: HBeAg if positive for HBsAg to Anti-HBe determine infectivity Anti-HDV If HBsAg patient worsens or (total + IgM) is very ill, to diagnose concomitant infection with hepatitis delta virus MONITORING Infant follow-up HBsAg To monitor the success of Anti-HBc vaccination and passive (continued) 72 Clinician’s Pocket Reference, 9th Edition TABLE 4–2 (Continued) Clinical Setting Test Purpose 4 Anti-HBs immunization for perinatal transmission of HBV 12–15 mo after birth Postvaccination screening Anti-HBs To ensure immunity has been achieved after vaccination (CDC recommends “titer” determination, but usually qualitative assay is ade- quate) Sexual contact HBsAg To monitor sexual partners of Anti-HBc a patient with chronic HBV Anti-Hep C or hepatitis C *See text for abbreviations. TABLE 4–3 Interpretation of Viral Hepatitis Serologic Testing Patterns Anti-HAV Anti-HBc Anti-HBc Anti-C (IgM) HBsAg (IgM) (Total) (ELISA) Interpretation + − − − − Acute hepatitis A + + − + − Acute hepatitis A in hepatitis B carrier − + − + − Chronic hepatitis B* − − + + − Acute hepatitis B − + + + − Acute hepatitis B − − − + − Past hepatitis B infection − − − − + Hepatitis C† − − − − − Early hepatitis C or other cause (other virus, toxin) *Patients with chronic hepatitis B (either active hepatitis or carrier state) should have HBeAg and anti-HBe checked to determine activity of infection and relative infectivity. Anti-HBs is used to determine response to hepatitis B vaccination. †Anti-C often takes 3–6 mo before being positive. PCR may allow earlier detection. 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 73 Early Incubation Acute Acute Recovery 28–45 Days 0–14 Days 3–6 Months Years 4 Onset of Jaundice Total Anti-HAV HAAg Anti-HAV IgM Time After Exposure to HAV FIGURE 4–1 Hepatitis A diagnostic profile. (Courtesy of Abbott Laboratories, Di- agnostic Division, North Chicago, Illinois.) Early nvalescent Acute Acute Co Window Recovery Time 4–12 1–2 2 weeks–3 months 3–6 months Years wks wks Symptoms Anti-HBc IgM HBsAg Anti- HBc Anti-HBs HBeAg Anti-HBe Time FIGURE 4–2 Hepatitis B diagnostic profile. (Courtesy of Abbott Laboratories, Di- agnostic Division, North Chicago, Illinois.) Relative Concentration Incubation Late Incubation Early Acute Relative Concentration Acute/Sero- Conversion in Progress Early Recovery 74 Clinician’s Pocket Reference, 9th Edition Hepatitis A Anti-HAV Ab: Total antibody to hepatitis A virus; confirms previous exposure to hep- atitis A virus, elevated for life. Anti-HAV IgM: IgM antibody to hepatitis A virus; indicative of recent infection with 4 hepatitis A virus; declines typically 1–6 months after symptoms Hepatitis B HBsAg: Hepatitis B surface antigen. Earliest marker of HBV infection. Indicates either chronic or acute infection with hepatitis B virus. Used by blood banks to screen donors; vac- cination does not affect this test Anti-HBc-Total: IgG and IgM antibody to hepatitis B core antigen; confirms either previous exposure to hepatitis B virus (HBV) or ongoing infection. Used by blood banks to screen donors Anti-HBc IgM: IgM antibody to hepatitis B core antigen. Early and best indicator of acute infection with hepatitis B HBeAg: Hepatitis Be antigen; when present, indicates high degree of infectivity. Order only when evaluating for chronic HBV infection HBV-DNA: Most sensitive and specific for early evaluation of hepatitis B and may be detected when all other markers are negative Anti-HBe: Antibody to hepatitis Be antigen; associated with resolution of active inflam- mation Anti-HBs: Antibody to hepatitis B surface antigen; when present, typically indicates immunity associated with clinical recovery from HBV infection or previous immunization with hepatitis B vaccine. Order only to assess effectiveness of vaccine and request titer levels Anti-HDV: Total antibody to delta hepatitis; confirms previous exposure. Order only in patients with known acute or chronic HBV infection. Anti-HDV IgM: IgM antibody to delta hepatitis; indicates recent infection. Order only in cases of known acute or chronic HBV infection Hepatitis C Anti-HCV: Antibody against hepatitis C. Indicative of active viral replication and infec- tivity. Used by blood banks to screen donors. Many false-positives HCV-RNA: Nucleic acid probe detection of current HCV infection HIGH-DENSITY LIPOPROTEIN CHOLESTEROL • See CHOLESTEROL, page 62. HLA (HUMAN LEUKOCYTE ANTIGENS; HLA TYPING) • Collection: Green top tube 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 75 This test identified a group of antigens on the cell surface that are the primary determi- nants of histocompatibility and useful in assessing transplantation compatibility. Some are associated with specific diseases but are not diagnostic of these diseases. HLA-B27: Ankylosing spondylitis, psoriatic arthritis, Reiter’s syndrome, juvenile RA HLA-DR4/HLA DR2: Chronic Lyme disease arthritis 4 HLA-DRw2: MS HLA-B8: Addison’s disease, juvenile-onset diabetes, Grave’s disease, gluten-sensitive enteropathy HOMOCYSTEINE, SERUM • Normal fasting 5 and 15 µmol/L • Fasting target <10 µmol/L Under investigation as a risk factor for CAD and atherosclerosis. Moderate, intermedi- ate, and severe hyperhomocystinemia refer to concentrations between 16 and 30, between 31 and 100, and >100 µmol/L, respectively. May be useful to screen high-risk patients and recommend strategies to obtain target of <10 (ie, dietary, lifestyle changes, vitamin supple- mentation) Increased: Vitamin B12, B6 and folate deficiency, kidney and renal failure, medications (nicotinic acid, theophylline, methotrexate, L-dopa, anticonvulsants) advanced age, hypothy- roidism, impaired kidney function, SLE, and certain medications HUMAN CHORIONIC GONADOTROPIN, SERUM (HCG, BETA SUBUNIT) • Normal, <3.0 mIU/mL • 10 days after conception, >3 mIU/mL • 30 days, 100–5000 mIU/mL • 10 weeks, 50,000–140,000 mIU/mL • >16 weeks, 10,000–50,000 mIU/mL • Thereafter, levels slowly decline (SI units IU/L equivalent to mIU/mL) • Collection: Tiger top tube Increased: Pregnancy, some testicular tumors (nonseminomatous germ cell tumors, but not seminoma), trophoblastic disease (hydatidiform mole, choriocarcinoma levels usually >100,000 mIU/mL) HUMAN IMMUNODEFICIENCY VIRUS (HIV) TESTING See Figure 4–3 CDC guidelines. Any HIV-positive person over 13 years of age with a CD4+ T-cell level <200/mL or an HIV-positive patient with a series of CDC-defined indicator con- ditions (eg, pulmonary candidiasis, disseminated histoplasmosis, HIV wasting, Kaposi’s sarcoma, TB, various lymphomas, PCP, and others) is considered to have AIDS. HIV Antibody • Normal = negative • Collection: Tiger top tube Assay kits recognize both HIV-1 and HIV-2 antibodies. Used in the diagnosis of AIDS and to screen blood for use in transfusion. Antibodies appear in blood 1–4 mo after infection in most cases. HIV Antibody, ELISA • Normal = negative 76 Clinician’s Pocket Reference, 9th Edition ELISA antibody test 4 + – Repeat ELISA on separate sample + – Retest if result is unexpected Western blot confirmation + Indeterminate – HIV infection Repeat Western blot diagnosis within 3 months FIGURE 4–3 Diagnostic algorithm for HIV infection. (Courtesy of Burroughs- Wellcome Company, Research Triangle Park, North Carolina.) Initial screen to detect HIV antibody; a positive test is often repeated or confirmed by Western blot. Positive: AIDS, asymptomatic HIV infection False-Positive: Flu vaccine within 3 months, hemophilia, rheumatoid factor, alcoholic hepatitis, dialysis patients HIV Western Blot • Normal = negative The technique is used as the reference procedure for confirming the presence or absence of HIV antibody, usually after a positive HIV Antibody by ELISA Determination Positive: AIDS, asymptomatic HIV infection (if indeterminate, repeat in 1 mo or per- form PCR for HIV-1 DNA or RNA) False-Positive: Autoimmune or connective tissue diseases, hyperbilirubinemia, HLA antibodies, others 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 77 HIV DNA PCR • Normal = negative Performed on peripheral blood mononuclear cells. Preferred test to diagnose HIV infec- tion in children <18 months of age HIV RNA PCR 4 • Normal = <400 copies/mL Used to quantify plasma “viral load.” Establishes the diagnosis before antibody produc- tion begins or when HIV antibody test is indeterminate. Obtained at baseline diagnosis, serves as an important parameter to initiate or modify HIV therapy (see the following details of viral load). Not recommended for routine testing of children <18 months HIV VIRAL LOAD • Normal <50 copies/mL Single best predictor of progression to AIDS and death among HIV-infected individu- als. Also used as a baseline and for initiation and modification of HIV therapy, but not for diagnosis. For example, antiretroviral therapy is uniformly initiated when the viral load is >20,000 copies/mL RNA or RT PCR. HIV Antigen (P-24 antigen) • Normal = negative Detects early HIV infection before antibody conversion, used along with PCR testing IMMUNOGLOBULINS, QUANTITATIVE • IgG: 65–1500 mg/dL or 6.5–15 g/L • IgM: 40–345 mg/dL or

0.4–3.45 mg/L • IgA: 76–390 mg/dL or 0.76–3.90 g/L • IgE: 0–380 IU/mL or KIU/L • IgD: 0–8 mg/dL or 0–80 mg/L • Collection: Tiger top tube Levels are determined in the evaluation of immunodeficiency diseases, during replace- ment therapy, and to evaluate humoral immunity. Increased: Multiple myeloma (myeloma immunoglobulin increased, other im- munoglobulins decreased); Waldenström’s macroglobulinemia (IgM increased, others de- creased); lymphoma; carcinoma; bacterial infection; liver disease; sarcoidosis; amyloidosis; myeloproliferative disorders Decreased: Hereditary immunodeficiency, leukemia, lymphoma, nephrotic syndrome, protein-losing enteropathy, malnutrition IRON • Males 65–175 mg/dL (SI: 11.64–31.33 mmol/L) • Females 50–170 mg/dL (SI: 8.95–30.43 mmol/L) • To convert mg/dL to mmol/L, multiply by 0.1791 • Collection: Tiger top tube Increased: Hemochromatosis, hemosiderosis caused by excessive iron intake, excess destruction or decreased production of erythrocytes, liver necrosis Decreased: Iron deficiency anemia, nephrosis (loss of iron-binding proteins), nor- mochromic anemia of chronic diseases and infections 78 Clinician’s Pocket Reference, 9th Edition IRON-BINDING CAPACITY, TOTAL (TIBC) • 250–450 mg/dL (SI: 44.75–80.55 mmol/L) • Collection: Tiger top tube The normal iron/TIBC ratio is 20–50%. Decreased ratio (<10%) is almost diagnostic of iron deficiency anemia. Increased ratio is seen with hemochromatosis. Increased: Acute and chronic blood loss, iron deficiency anemia, hepatitis, oral contra- 4 ceptives Decreased: Anemia of chronic diseases, cirrhosis, nephrosis/uremia, hemochromatosis, iron therapy overload, hemolytic anemia, aplastic anemia, thalassemia, megaloblastic ane- mia LACTATE DEHYDROGENASE (LD, LDH) • Adults <230 U/L, (<3.82 mkat/L) • Higher levels in childhood • Collection: Tiger top tube; carefully avoid hemolysis because this can increase LDH levels Increased: AMI, cardiac surgery, prosthetic valve, hepatitis, pernicious anemia, malig- nant tumors, pulmonary embolus, hemolysis (anemias or factitious), renal infarction, muscle injury. megaloblastic anemia, liver disease LDH Isoenzymes (LDH 1 to LDH 5) Normally, the ratio LDH 1/LDH 2 is <0.6–0.7. If the ratio becomes >1 (also termed “flipped”), suspect a recent MI (change in ratio can also be seen in pernicious or hemolytic anemia). With an AMI, the LDH will begin to rise at 12–48 h, peak at 3–6 days, and return to normal at 8–14 days. LDH 5 is >LDH 4 in liver diseases. (Largely replaced by troponin.) LACTIC ACID (LACTATE) • 4.5–19.8 mg/dL (SI: 0.5–2.2 mmol/L) • Collection: Gray top tube on ice Suspect lactic acidosis with elevated anion gap in the absence of other causes (renal failure, ethanol or methanol ingestion) Increased: Lactic acidosis due to hypoxia, hemorrhage, shock, sepsis, cirrhosis, exer- cise, ethanol, DKA, regional ischemia (extremity, bowel) spurious (prolonged use of a tourniquet) LAP SCORE (LEUKOCYTE ALKALINE PHOSPHATASE SCORE/STAIN) • 50–150 • Collection: Finger stick blood sample directly on slide; air dry Used to differentiate among various hematologic conditions Increased: Leukemoid reaction, acute inflammation, Hodgkin’s disease, pregnancy, liver disease Decreased: Chronic myelogenous leukemia, nephrotic syndrome LE (LUPUS ERYTHEMATOSUS) PREPARATION • Normal = no cells seen Positive: SLE, scleroderma, RA, drug-induced lupus (procainamide, others) 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 79 LEAD, BLOOD • Adult <40 mg/dL (1.93 mmol/L) • Child <25 mg/dL (1.21 mmol/L) • Collection: Lavender, navy, or green top tube; lab-specific Neurologic findings can be detected at 15 mg/dL in children and 30 mg/dL in adults; se- vere symptoms (lethargy, ataxia, coma) are present >60 mg/dL. Increased: Lead poisoning, occupational exposure 4 LEGIONELLA ANTIBODY • <1:32 titers Obtain two sera, acute (within 2 wk of onset) and convalescent (at least 3 wk after onset of fever). A fourfold rise in titers or a single titer of 1:256 is diagnostic. Increased: Legionella infection; false-positives with Bacteroides fragilis, Francisella tularensis, Mycoplasma pneumoniae. LIPASE • 0–1.5 U/mL (SI: 10–150 U/L) by turbidimetric method • Collection: Tiger top tube Increased: Acute or chronic pancreatitis, pseudo-cyst, pancreatic duct obstruction (stone, stricture, tumor, drug-induced spasm), fat embolus syndrome, renal failure, dialysis (usually normal in mumps) gastric malignancy, intestinal perforation, diabetes (usually in DKA only) LIPID PROFILE/LIPOPROTEIN PROFILE/LIPOPROTEIN ANALYSIS • See also CHOLESTEROL, page 62, and TRIGLYCERIDES, page 91. Usually includes cholesterol, HDL cholesterol, LDL cholesterol (calculated), trigly- cerides. Useful in the evaluation of CAD and allows classification of dyslipoproteinemias to direct treatment. Initial screening for cardiac risk includes total cholesterol and HDL as out- lined in Figure 4–4 (page 80). The main lipids in the blood are cholesterol and triglycerides. These lipids are carried by lipoproteins. Lipoproteins are further classified by density (least dense to most dense): • Chylomicrons (least dense, rise to surface of unspun serum) and are normally found only after a fatty meal is eaten (a “lipemic specimen” on a lab report usually refers to these chylomicrons). • VLDL consist mainly of triglycerides. • LDL in the fasting state; the LDL carry most cholesterol. • HDL are the densest and consist of mostly apoproteins and cholesterol. Table 4–4 (see page 81) indicates the dyslipoproteinemias based on the lipid profile. LOW-DENSITY LIPOPROTEIN-CHOLESTEROL (LDL, LDL-C) • See CHOLESTEROL, page 62. LUTEINIZING HORMONE, SERUM (LH) • Male 7–24 IU/L • Female 6–30 IU/L, midcycle peak increase two- to threefold over baseline, postmenopausal >35 IU/L • Collection: Tiger top tube 80 Clinician’s Pocket Reference, 9th Edition Nonfasting cholesterol and HDL Cholesterol <200 Cholesterol 200–239 Cholesterol >240 4 HDL HDL HDL <35 or HDL >35 or <35 ≥35 ≥2 risk factors <2 risk factors Repeat every Patient education 5 years Repeat in 1–2 years Lipoprotein analysis (LA) •Fasting 9–12 hours •Average 2 values 1–8 weeks apart •Average 3 values if LDL varies more than 30 No evidence of CHD Definitive evidence of CHD or other atherosclerotic disease LDL LDL LDL Optimal High risk <130 130–159 ≥160 LDL < 100 LDL > 100 Dietary consult <2 RF ≥2 RF Repeat LA yearly Patient education Evaluate and Evaluate and consider risk factors Repeat cholesterol consider Educate on Step II diet and LDL in 5 years risk factors Repeat LA in 6–12 weeks Keep LDL <100 Patient education Keep LDL <130 Keep LDL <160 or <130 Step I diet Step I diet for 3 mo if >2 risk factors Keep LDL <160 Step II diet for 3 mo Step I diet for 3 mo Repeat LA annually Recheck LA after 6 mo and Recheck LA after 6 mo and if LDL >130 begin meds if LDL not <160 or <130 If risk factors cannot be with 2 RF start meds altered consider meds Risk Factors (RF) Step I Diet Step II Diet •Age: male ≥45, •Saturated fat 8–10% •Saturated fat <7% female ≥55 •<300 mg cholesterol daily •<200 mg cholesterol daily •Family history •HDL <35 Adapted from the Second Report of the Expert Panel on Detection, •Tobacco use Evaluation and Treatment of High Blood Cholesterol in Adults from •Diabetes the National Cholesterol Education Program (NCEP), NIH Publication No. 93-3096, September 1993. FIGURE 4–4 Cholesterol and lipoprotein screening. (Reprinted, with permission, from: Gordon JD [ed]: Obstetrics, Gynecology, and Infertility, 4th ed. Scub Hill Press, Menlo Park CA, 1995.) 4 81 TABLE 4–4 Lipoproteins Fredrickson Classification Type I Type IIa Type IIb Type III Type IV Type V System (Rare) (Common) (Common) (Uncommon) (Uncommon) (Uncommon) Cholesterol N or slightly ⇑ Very ⇑ Very ⇑ Very ⇑ N or slightly ⇑ ⇑ LDL N ⇑ ⇑ ⇑ N N HDL N or ⇓ N or ⇓ N or ⇓ N or ⇓ N or ⇓ N or ⇓ Triglycerides Very ⇑ N I Very ⇑ Very ⇑ ⇑ Increased Chylomicrons LDL LDL, VLDL IDL VLDL VLDL and lipoproteins chylomicrons Atherogenesis No increase Very ⇑ ⇑ ⇑ No increase No increase risk 82 Clinician’s Pocket Reference, 9th Edition Increased: (Hypergonadotropic >40 IU/L) postmenopausal, surgical or radiation castra- tion, ovarian or testicular failure, polycystic ovaries Decreased: (Hypogonadotropic <40 IU/L prepubertal) hypothalamic, and pituitary dys- function, Kallmann’s syndrome, LHRH analogue therapy 4 LYME DISEASE SEROLOGY • Normal varies with assay, ELISA <1:8 • Western blot nonreactive Most useful when comparing acute and convalescent serum levels for relative titers. Normal values differ among labs. IgM antibody becomes detectable 2–4 weeks after onset of rash; IgG rises in 4–6 weeks and peaks up to 6 mo after infection and may stay elevated for months to years. Positive: Infection with Borrelia burgdorferi, syphilis, and other rickettsial diseases Negative: After antibiotic therapy or during first few weeks of disease MAGNESIUM • 1.6–2.6 mg/dL (SI: 0.80–1.20 mmol/L) • Collection: Tiger top tube Increased: Renal failure, hypothyroidism, magnesium-containing antacids, Addison’s disease, diabetic coma, severe dehydration, lithium intoxication Decreased: Malabsorption, steatorrhea, alcoholism and cirrhosis, hyperthyroidism, al- dosteronism, diuretics, acute pancreatitis, hyperparathyroidism, hyperalimentation, NG suc- tioning, chronic dialysis, renal tubular acidosis, drugs (cisplatin, amphotericin B, aminoglycosides), hungry bone syndrome, hypophosphatemia, intracellular shifts with res- piratory or metabolic acidosis METYRAPONE TEST • See Chapter 22, page 570 MHA-TP (MICROHEMAGGLUTINATION, TREPONEMA PALLIDUM) • Normal <1:160 • Collection: Tiger top tube Confirmatory test for syphilis, similar to FTA-ABS. Once positive, remains so, there- fore cannot be used to judge effect of treatment. False-positives with other treponemal infec- tions (pinta, yaws, etc), mononucleosis, and SLE Β2-MICROGLOBULIN • 0.1–0.26 mg/dL )1–2.6 mg/L) • Collection: Tiger top tube A portion of the class I MHC antigen. A useful marker to follow the progression of HIV infections Increased: HIV infection, especially during periods of exacerbation, lymphoid malig- nancies, renal diseases (diabetic nephropathy, pyelonephritis, ATN, nephrotoxicity from medications), transplant rejection, inflammatory conditions Decreased: Treatment of HIV with AZT (zidovudine) 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 83 MONOSPOT • Normal = negative • Collection: Tiger top tube Positive: Mononucleosis, rarely in leukemia, serum sickness, Burkitt’s lymphoma, viral hepatitis, RA MYOGLOBIN 4 • 30–90 ng/mL • Collection: Tiger top tube Increased: Skeletal muscle injury (crush, injection, surgical procedures), delirium tremens, rhabdomyolysis (burns, seizures, sepsis, hypokalemia, others) 5
-NUCLEOTIDASE • 2–15 U/L Used in the workup of increased alkaline phosphatase and biliary obstruction Increased: Obstructive or cholestatic liver disease, liver metastasis, biliary cirrhosis OLIGOCLONAL BANDING, CSF • Normal = negative • Collection: Serum tiger top tube and simultaneous CSF sample col- lected in a plain tube by LP This is performed simultaneously on CSF and serum samples when MS is clinically suspected. Agarose gel electrophoresis will reveal multiple bands in the IgG region not seen in the serum. Oligoclonal banding is present in up to 90% of patients with MS. Occasionally seen in other CNS inflammatory conditions and CNS syphilis OSMOLALITY, SERUM • 278–298 mOsm/kg (SI: 278–298 mmol/kg) • Collection: Tiger top tube A rough estimation of osmolality is [2(Na) + BUN/2.8 + glucose/18]. Measured value is usually less than calculated value. If measured value is 15 mOsm/kg less than calculated, consider methanol, ethanol, or ethylene glycol ingestion. Increased: Hyperglycemia; ethanol, methanol, mannitol, or ethylene glycol ingestion; increased sodium because of water loss (diabetes, hypercalcemia, diuresis) Decreased: Low serum sodium, diuretics, Addison’s disease, SIADH (seen in bron- chogenic carcinoma, hypothyroidism), iatrogenic causes (poor fluid balance) OXYGEN • See Chapter 8, Table 8–1, page 162 P-24 ANTIGEN (HIV CORE ANTIGEN) • Normal = negative • Collection: Tiger top tube • See also Human Immunodeficiency Virus Testing, page 75 Used to diagnose recent acute HIV infection; becomes positive earlier than HIV anti- bodies. Decreases “window” period. Can be positive as early as 2–4 weeks but becomes un- detectable during antibody seroconversion (periods of latency). With progression of disease, P-24 usually becomes evident again. Used to screen blood donors 84 Clinician’s Pocket Reference, 9th Edition PARATHYROID HORMONE (PTH) • Normal based on relationship to serum calcium, usually provided on the lab report • Also, reference values vary depending on the laboratory and whether the N-terminal, C-terminal or midmolecule is measured. • PTH midmolecule: 0.29– –0.85 ng/mL (SI: 29–85 pmol/L) • With calcium: 8.4–10.2 mg/dL (SI: 2.1–2.55 mmol/L) • Collection: 4 Tiger top tube Increased: Primary hyperparathyroidism, secondary hyperparathyroidism (hypocal- cemic states, such as chronic renal failure, others) Decreased: Hypercalcemia not due to hyperparathyroidism, hypoparathyroidism PHOSPHORUS • Adult 2.5–4.5 mg/dL (SI: 0.81–1.45 mmol/L) • Child 4.0–6.0 mg/dL (SI: 1.29–1.95 mmol/L) • To convert mg/dL to mmol/L, multiply by 0.3229 • Collection: Tiger top tube Increased: Hypoparathyroidism (surgical, pseudo-hypoparathyroidism), excess vitamin D, secondary hyperparathyroidism, renal failure, bone disease (healing fractures), Addison’s disease, childhood, factitious increase (hemolysis of specimen) Decreased: Hyperparathyroidism, alcoholism, diabetes, hyperalimentation, acidosis, al- kalosis, gout, salicylate poisoning, IV steroid, glucose or insulin administration, hy- pokalemia, hypomagnesemia, diuretics, vitamin D deficiency, phosphate-binding antacids POTASSIUM, SERUM • 3.5–5 mEq/L (SI: 3.5–5 mmol/L) • Collection: Tiger top tube Increased: Factitious increase (hemolysis of specimen, thrombocytosis), renal failure, Addison’s disease, acidosis, spironolactone, triamterene, ACE inhibitors, dehydration, he- molysis, massive tissue damage, excess

intake (oral or IV), potassium-containing medica- tions, acidosis Decreased: Diuretics, decreased intake, vomiting, nasogastric suctioning, villous ade- noma, diarrhea, Zollinger–Ellison syndrome, chronic pyelonephritis, renal tubular acidosis, metabolic alkalosis (primary aldosteronism, Cushing’s syndrome) PREALBUMIN • See Chapter 11, page 211 PROGESTERONE • Collection: Tiger top tube Used to confirm ovulation and corpus luteum function Sample Collection Normal Values (female) Follicular phase <1 ng/mL Luteal phase 5–20 ng/mL Pregnancy 1st trimester 10–30 ng/mL 2nd trimester 50–100 ng/mL 3rd trimester 100–400 ng/mL Postmenopause –1 ng/mL 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 85 PROLACTIN • Males 1–20 ng/mL (SI: 1–20 mg/L) • Females 1–25 ng/mL (SI: 1–25 mg/L) • Collec- tion: Tiger top tube Used in the workup of infertility, impotence, hirsutism, amenorrhea, and pituitary neo- plasm Increased: Pregnancy, nursing after pregnancy, prolactinoma, hypothalamic tumors, sar- 4 coidosis or granulomatous disease of the hypothalamus, hypothyroidism, renal failure, Ad- dison’s disease, phenothiazines, haloperidol PROSTATE-SPECIFIC ANTIGEN (PSA) • <4 ng/dL by monoclonal, eg, Hybritech assay Most useful as a measure of response to therapy of prostate cancer; approved for screening for prostate cancer. Although any elevation increases suspicion of prostate cancer, levels >10.0 ng/dL are frequently associated with carcinoma. Age corrected levels gaining popularity (40–50 y 2.5 ng/dL; 50–60 y 3.5 ng/dL; 60–70 years 4.5 ng/dL; >70 years 6.5 ng/dL.) Increased: Prostate cancer, acute prostatitis, some cases of BPH, prostatic infarction, prostate surgery (biopsy, resection), vigorous prostatic massage (routine rectal exam does not elevate levels), rarely postejaculation Decreased: Radical prostatectomy, response to therapy of prostatic carcinoma (radiation or hormonal therapy) PSA Velocity A rate of rise in PSA of 0.75 ng/mL or greater per year is suspicious for prostate cancer based on at least three separate assays 6 mo apart. PSA Free and Total Patients with prostate cancer tend to have lower free PSA levels in proportion to total PSA. Measurement of the free/total PSA can improve the specificity of PSA in the range of total PSA from 2.0–10.0 ng/mL. Some recommend prostate biopsy only if the free PSA percent- age is low. Threshold for biopsy is controversial, ranging from a ratio of less than 15% to less than 25%, with a higher threshold having improved sensitivity and lower threshold hav- ing improved specificity. PROTEIN ELECTROPHORESIS, SERUM AND URINE (SERUM PROTEIN ELECTROPHORESIS, SPEP) (URINE PROTEIN ELECTROPHORESIS, UPEP) Qualitative analysis of the serum proteins is often used in the workup of hypoglobuline- mia, macroglobulinemia, α1-antitrypsin deficiency, collagen disease, liver disease, myeloma, and occasionally in nutritional assessment. Serum electrophoresis yields five dif- ferent bands (Figure 4–5 and Table 4–5, pages 86 and 87). If a monoclonal gammopathy or a low globulin fraction is detected, quantitative immunoglobulins should be ordered. Urine protein electrophoresis can be used to evaluate proteinuria and can detect Bence Jones protein (light chain) that is associated with myeloma, Waldenström’s macroglobuline- mia, and Fanconi’s syndrome. 86 Clinician’s Pocket Reference, 9th Edition Normal serum Hypoalbuminemia 4 ALB α1 α2 β γ ALB α1 α2 β γ Nephrotic syndrome Multiple myeloma (Hypoalbuminemia, Hyperlipidemina) ALB α1 α2 β γ ALB α1 α2 β γ α1–Antitrypsin deficiency Polyclonal gammopathy ALB α1 α2 β γ ALB α1 α2 β γ A Normal urine Nephrotic syndrome ALB α β γ ALB α β γ Bence-Jones protein B ALB α β γ FIGURE 4–5 Examples of (A) serum and (B) urine protein electrophoresis patterns. See also Table 4–5. (Courtesy of Dr. Steven Haist.) 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 87 TABLE 4–5 Normal Serum Protein Components and Fractions as Determined by Electrophoresis, Along with Associated Conditions* Percentage 4 Protein of Total Fraction Protein Constituents Increased Decreased Albumin 52–68 Albumin Dehydration Nephrosis, (only known malnutri- cause) tion, chronic liver disease Alpha-1 (α1) 2.4–4.4 Thyroxine- Inflammation, Nephrosis, globulin binding neoplasia α1-anti- globulin, trypsin antitrypsin, deficiency lipoproteins, (emphy- glycoprotein, sema transcortin related) Alpha-2 (α2) 6.1–10.1 Haptoglobin, Inflammation, Severe liver globulin glycoprotein, infection, disease, macroglobulin, neoplasia, acute ceruloplasmin cirrhosis hemolytic anemia Beta (β) 8.5–14.5 Transferrin, Cirrhosis, Nephrosis globulin glycoprotein, obstructive lipoprotein jaundice Gamma (γ) 10–21 IgA, IgG, IgM, Infections, Agammaglob- globulins IgD, IgE collagen ulinemia, (immuno- vascular hypo- globulins) diseases, gamma- leukemia, globuline- myeloma mia,neph- rosis *(See also Figure 4–5). PROTEIN, SERUM • 6.0–8.0 g/dL • See also Serum Protein Electrophoresis, page 85. • Collection: Tiger top tube Increased: Multiple myeloma, Waldenström’s macroglobulinemia, benign monoclonal gammopathy, lymphoma, chronic inflammatory disease, sarcoidosis, viral illnesses 88 Clinician’s Pocket Reference, 9th Edition Decreased: Malnutrition, inflammatory bowel disease, Hodgkin’s disease, leukemias, any cause of decreased albumin RENIN Plasma (Plasma Renin Activity [PRA]) 4 • Adults, Normal sodium diet, upright 1–6 ng/mL/h (SI: 0.77–4.6 nmol/L/h) • Renal vein renin: L & R should be equal) Useful in the diagnosis of hypertension associated with hypokalemia. Values highly de- pendent on salt intake and position. Stop diuretics, estrogens for 2–4 wk before testing. Increased: Medications (ACE inhibitors, diuretics, oral contraceptives, estrogens), preg- nancy, dehydration, renal artery stenosis, adrenal insufficiency, chronic hypokalemia, up- right posture, salt-restricted diet, edematous conditions (CHF, nephrotic syndrome), secondary hyperaldosteronism Decreased: Primary aldosteronism (renin will not increase with relative volume deple- tion, upright posture) Renal Vein • Normal L & R should be equal A ratio of >1.5 (affected/nonaffected) suggestive of renovascular hypertension RETINOL-BINDING PROTEIN (RBP) • Adults 3–6 mg/dL • Children 1.5–3.0 mg/dL • Collection: Tiger top tube Decreased: Malnutrition, vitamin A deficiency, intestinal malabsorption of fats, chronic liver disease RHEUMATOID FACTOR (RA LATEX TEST) • <15 IU by Microscan kit or <1:40 • Collection: Tiger top tube Increased: Collagen-vascular diseases (RA, SLE, scleroderma, polyarteritis nodosa, others), infections (TB, syphilis, viral hepatitis), chronic inflammation, SBE, some lung dis- eases, MI ROCKY MOUNTAIN SPOTTED FEVER ANTIBODIES (RMSF) • Normal: <4(times) increase in paired acute and convalescent sera • IgG <1:64 • IgM <1:8 • Collection: Tiger top tube acute and convalescent The diagnosis of RMSF is made by acute and convalescent titers that demonstrate a 4× rise or a single convalescent titer >1:64 in the clinical setting of RMSF. Occasional false- positives in late pregnancy SEMEN ANALYSIS • Volume 2–5 mL • Sperm count >20-40 × 106/mL • Motility >60% • Forward migra- tion • Morphology >60% normal Specimen must be collected after 48–72 h abstinence and analyzed within 1–2 h. Test may not be valid after a recent illness or high fever. Verify abnormal analysis by serial tests. 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 89 Decreased: After vasectomy (should be 0 sperm after 3 mo), varicocele, primary testic- ular failure (ie, Klinefelter’s syndrome), secondary testicular failure (chemotherapy, radia- tion, infections),varicocele, after recent illness, congenital obstruction of the vas, retrograde ejaculation, endocrine causes (hyperprolactinemia, low testosterone, others) SGGT (SERUM GAMMA-GLUTAMYL TRANSPEPTIDASE) 4 • See GGT, page 69. SGOT (SERUM GLUTAMIC-OXALOACETIC TRANSAMINASE) • See AST, page 58. SGPT SERUM (GLUTAMIC-PYRUVIC TRANSAMINASE) • See ALT, page 57. SODIUM, SERUM • 136–145 mmol/L • Collection: Tiger top tube In factitious hyponatremia due to hyperglycemia, for every 100 mmol/L blood glucose above normal, serum sodium decreases 1.6. For example, a blood glucose of 800 and a sodium of 129 would factitiously lower the sodium value by about 7 × 1.6, or 11.6. Cor- rected serum sodium would therefore be 129 + 11 = 140. Increased: Associated with low total body sodium (glycosuria, mannitol, or lactulose use urea, excess sweating), normal total body sodium (diabetes insipidus [central and nephrogenic], respiratory losses, and sweating), and increased total body sodium (adminis- tration of hypertonic sodium bicarbonate, Cushing’s syndrome, hyperaldosteronism) Decreased: Associated with excess total body sodium and water (nephrotic syndrome, CHF, cirrhosis, renal failure), excess body water (SIADH, hypothyroidism, adrenal insuffi- ciency), decreased total body water and sodium (diuretic use, renal tubular acidosis, use of mannitol or urea, mineralocorticoid deficiency, vomiting, diarrhea, pancreatitis), and pseudo-hyponatremia (hyperlipidemia, hyperglycemia, and multiple myeloma) STOOL FOR OCCULT BLOOD (HEMOCCULT TEST) Normal-Negative: Apply small amount of stool to test site on Hemoccult card and close. Open test panel on other side of card and apply 2–3 drops developer to the test and the positive control panels; read in 30 s. Blue color is positive. Detects >5 mg hemoglobin/g feces. Repeat three times for maximum yield. (A positive test more informative than a nega- tive test) Positive: Any GI tract ulcerated lesion (ulcer, carcinoma, polyp, diverticulosis, inflam- matory bowel disease), hemorrhoids, telangiectasias, drugs that cause GI irritation (eg, NSAIDs) swallowed blood, ingestion of rare red meat, certain foods (horseradish, turnips) (vitamin C [>500 mg/d], antacids may result in false-negative test) SWEAT CHLORIDE • 5–40 mEq/L (SI: 5–40 mmol/L) • Collection: 100–200 mg sweat on filter paper after electrical stimulation of sweating by pilocarpine iontophoresis on an extremity 90 Clinician’s Pocket Reference, 9th Edition Increased: CF (not valid on children <3 wk); Addison’s disease, meconium ileus, and renal failure can occasionally raise levels. T3 RU (RESIN UPTAKE; THYROXINE-BINDING GLOBULIN RATIO) 4 • 30–40% This test is used in conjunction with a T4 to yield the Free T4 Index [FTI]), an estimate of the free T4. Increased: Hyperthyroidism, medications (phenytoin [Dilantin], steroids, heparin, as- pirin, others), nephrotic syndrome Decreased: Hypothyroidism, medications (iodine, propylthiouracil, others), any cause of increased TBG, such as oral estrogen or pregnancy TESTOSTERONE • Male free: 9–30 ng/dL, total 300–1200 ng/dL • Female, see following table Sample Collection Normal Values (female) Follicular phase 20–80 ng/dL Midcycle peak 20–80 ng/dL Luteal phase 20–80 ng/dL Postmenopause 10–40 ng/dL Increased: Adrenogenital syndrome, ovarian stromal hyperthecosis, polycystic ovaries, menopause, ovarian tumors. Decreased: Some cases of impotence, hypogonadism, hypopituitarism, Klinefelter’s syndrome THYROGLOBULIN • 1–20 ng/mL (mg/L) • Collection: Tiger top tube Useful for following patients with nonmedullary thyroid carcinomas Increased: Differentiated thyroid carcinomas (papillary, follicular), Graves’ disease, nontoxic goiter Decreased: Hypothyroidism, testosterone, steroids, phenytoin THYROID-STIMULATING HORMONE (TSH) • 0.7–5.3 mU/mL • Collection: Tiger top tube Excellent screening test for hyperthyroidism as well as hypothyroidism. Differentiates between a low normal and a decreased TSH Increased: Hypothyroidism Decreased: Hyperthyroidism. Less than 1% of hypothyroidism is from pituitary or hy- pothalamic disease resulting in a decreased TSH. THYROXINE (T4 TOTAL) • 5–12 mg/dL (SI: 65–155 nmol/L) • Males: >60 years, 5–10 mg/dL (SI: 65–129 nmol) • Females: 5.5–10.5 µg/dL (SI: 71–135 nmol/L) • Collection: Tiger top tube 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 91 Good screening test for hyperthyroidism. Measures both bound and free T4, therefore, can be affected by TBG levels. Increased: Hyperthyroidism, exogenous thyroid hormone, estrogens, pregnancy, severe illness, euthyroid sick syndrome Decreased: Hypothyroidism, euthyroid sick syndrome, any cause of decreased TBG 4 THYROXINE-BINDING GLOBULIN (TBG) • 21–52 mg/dL (270–669 nmol/L) • Collection: Tiger top tube Increased: Hypothyroidism, pregnancy, oral contraceptives, estrogens, hepatic disease, acute porphyria Decreased: Hyperthyroidism, androgens, anabolic steroids, prednisone, nephrotic syn- drome, severe illness, surgical stress, phenytoin, hepatic disease THYROXINE INDEX, FREE (FTI) • 6.5–1.25 Practically speaking, the FTI is equivalent to the free thyroxine. Useful in patients with clinically suspected hyper- or hypothyroidism. Determined as follows: Thyroxine (Total T4) × T3 RU Increased: Hyperthyroidism, high-dose beta-blockers, psychiatric illnesses Decreased: Hypothyroidism, phenytoin (Dilantin) TORCH BATTERY • Normal = negative • Collection: Tiger top tube Serial determinations best (acute and convalescent titers). Test is based on serologic evidence of exposure to toxoplasmosis, rubella, cyto- megalovirus, and herpesviruses. TRANSFERRIN • 220–400 mg/dL (SI: 2.20–4.0 g/L) • Collection: Tiger top tube, avoid hemolysis Used in the workup of anemias; transferrin levels can also be assessed by the total iron- binding capacity. Increased: Acute and chronic blood loss, iron deficiency, hemolysis, oral contracep- tives, pregnancy, viral hepatitis Decreased: Anemia of chronic disease, cirrhosis, nephrosis, hemochromatosis, malig- nancy TRIGLYCERIDES • Recommended values: • Males: 40–160 mg/dL (SI: 0.45–1.81 mmol/L) • Females: 35–135 mg/dL (SI: 0.40–1.53 mmol/L) • Can vary with age. • Collection: Tiger top tube • Fasting preferred • See also LIPID PROFILE page 79 Increased: Nonfasting specimen, hyperlipoproteinemias (types I, IIb, III, IV, V), hy- pothyroidism, liver diseases, poorly controlled diabetes mellitus, alcoholism, pancreatitis, 92 Clinician’s Pocket Reference, 9th Edition AMI, nephrotic syndrome, familial, medications (oral contraceptives, estrogens, beta- blockers, cholestyramine) Decreased: Malnutrition, malabsorption, hyperthyroidism, Tangier disease, medications (nicotinic acid, clofibrate, gemfibrozil) congenital abetalipoproteinemia 4 TRIIODOTHYRONINE (T3 RIA) • 120–195 ng/dL (SI: 1.85–3.00 nmol/L) • Collection: Tiger top tube Useful when hyperthyroidism is suspected, but T4 is normal; not useful in the diagnosis of hypothyroidism Increased: Hyperthyroidism, T3 thyrotoxicosis, pregnancy, exogenous T4, any cause of increased TBG, such as oral estrogen or pregnancy Decreased: Hypothyroidism and euthyroid sick state, any cause of decreased TBG TROPONIN, CARDIAC-SPECIFIC • Troponin 1 (cTn1) <0.35 ng/mL •

Troponin T cTnT <0.2 µg/L Used to diagnose AMI; increases rapidly 3–12 h, peak at 24 h and may stay elevated for several days (cTn1 5–7 days, cTnT up to 14 days). More cardiac-specific than CK-MB Positive: Myocardial damage, including MI, myocarditis (false-positive: renal failure) URIC ACID (URATE) • Males: 3.4–7 mg/dL (SI: 202–416 mmol/L) • Females: 2.4–6 mg/dL (SI: 143–357 mmol/L) • To convert mg/dL to mmol/L, multiply by 59.48 • Collection: Tiger top tube Increased uric acid is associated with increased catabolism, nucleoprotein synthesis, or decreased renal clearing of uric acid (ie, thiazide diuretics or renal failure). Increased: Gout, renal failure, destruction of massive amounts of nucleoproteins (leukemia, anemia, chemotherapy, toxemia of pregnancy), drugs (especially diuretics), lactic acidosis, hypothyroidism, PCKD, parathyroid diseases Decreased: Uricosuric drugs (salicylates, probenecid, allopurinol), Wilson’s disease, Fanconi’s syndrome VDRL TEST (VENEREAL DISEASE RESEARCH LABORATORY) OR RAPID PLASMA REAGIN (RPR) • Normal = nonreactive • Collection: Tiger top tube Good screening for syphilis. Almost always positive in secondary syphilis, but fre- quently becomes negative in late syphilis. Also, in some patients with HIV infection, the VDRL can be negative in primary and secondary syphilis. Positive (Reactive): Syphilis, SLE, pregnancy and drug addiction. If reactive, confirm with FTA-ABS (false-positives with bacterial or viral illnesses). VITAMIN B12 (EXTRINSIC FACTOR, CYANOCOBALAMIN) • >100–700 pg/mL (SI: 74–516 pmol/L) • Collection: Tiger top tube Increased: Excessive intake, myeloproliferative disorders 4 Laboratory Diagnosis: Chemistry, Immunology, and Serology 93 Decreased: Inadequate intake (especially strict vegetarians), malabsorption, hyperthy- roidism, pregnancy ZINC • 60–130 mg/dL (SI: 9–20 mmol/L) • Collection: Check with lab; special collection to limit contamination 4 Increased: Atherosclerosis, CAD Decreased: Inadequate dietary intake (parenteral nutrition, alcoholism); malabsorption; increased needs, such as pregnancy or wound healing; acrodermatitis enteropathica; dwarfism This page intentionally left blank. 5 LABORATORY DIAGNOSIS: CLINICAL HEMATOLOGY Blood Collection CBC Differential Diagnosis Blood Smears: Wright’s Stain Lymphocyte Subsets Normal CBC Values RBC Morphology Differential Diagnosis 5 Normal CBC Variations WBC Morphology Differential Hematocrit Diagnosis Three-Cell Differential Count Coagulation and Other Hematologic The “Left Shift” Tests Reticulocyte Count BLOOD COLLECTION Venipuncture is discussed in detail in Chapter 13, page 39. The best CBC sample is venous blood drawn with at least a 22-gauge or larger needle. For a routine CBC, venous blood needs to be placed in a special hematology lab tube, usually a purple top tube, that has an anticoagulant (EDTA) and that is mixed gently. Blood for a CBC should be fresh, less than 3 h old. Most coagulation studies are submitted in a blue top (citrate) tube. (See page 311 for detailed description of blood collection tubes.) If a capillary fingerstick or heelstick (see page 274) is used, the hematocrit may be falsely low. If the finger needs to be “milked,” sludging of the RBCs can create a falsely high hematocrit. In practice, you can draw the blood up in a capillary tube, seal an end with clay, and spin a tube on the hematocrit centrifuge for 2–3 min and rapidly determine a hematocrit. Wright’s staining can also be done and viewed as outlined in the next section. BLOOD SMEARS: WRIGHT’S STAIN Making the Blood Smear In some clinical situations a quick interpretation of a smear can be useful. 1. Place a small drop of blood from the anticoagulated lab sample tube (usually purple top) in the center of a clean glass slide, about 1–2 cm from the end. 2. Place the spreading slide (a glass slide with a perfectly smooth edge) at a 45-degree angle on the slide with the blood sample and slowly move it back to make contact with the drop. The drop should spread out quickly along the line of contact between the two slides. The moment this occurs, spread the film by a rapid, smooth forward movement of the spreader (Figure 5–1). 3. The drop of blood should result in a film about 3 cm long. The faster a film is spread, the more even it is and the better the slide it produces. The ideal thickness shows some overlap by the RBCs throughout much of the film’s length with separation and lack of distortion toward the feathered edge of the film. Leukocytes should be easily recogniz- able throughout the length of the film. 95 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 96 Clinician’s Pocket Reference, 9th Edition Count 1 or 2 strips Blood 5 Lymphs + Polys ++ Tail Head Film too thick Ideal thickness Film too thin FIGURE 5–1 The technique of preparing a blood smear for staining and the distri- bution of white blood cells on the standard smear. Staining the Blood Smear (Wright’s stain) Make sure that all reagents are fresh, or the slide may not turn out properly. 1. Let the slide air dry, and mark the patient’s name and date in pencil on the blood film it- self. It will not be removed by staining. An alternative method is to bring the slide to the hematology lab where instruments can automatically stain the slides. 2. Fix the slide in methanol for 1 min. 3. Shake off excess methanol from the slide, but do not rinse or dry it. 4. Flood the slide with Wright’s stain, and allow the slide to stand for 3–5 min. (This time can vary with the batch of stain.) 5. Flood the slide with Wright’s buffer (pH 6.4) until about 50% of the Wright’s stain is washed off. Blow air gently over the top of the slide to mix the fluids, and look for a greenish copper sheen that appears on the surface. Let the slide stand for about 8 min. 6. Rinse the slide with tap water, wipe the back of the slide with methanol, and air dry it. Viewing the Film: The Differential WBC 1. The film should not be so thick that the leukocytes in the body of the film shrink. Ex- amine the smear in an area where the red cells approximate but do not overlap. 2. If the film is too thin or if a rough-edged spreader is used, up to 50% of the WBCs may accumulate in the edges and tail (See Fig. 5–1). 3. WBCs are NOT randomly dispersed even in a well-made smear. Polys and monos pre- dominate at the margins and tail, and lymphs are prevalent in the middle of the film. To overcome this problem, use the “high dry” or oil immersion objective, and count cells in a strip running the whole length of the film. Avoid the lateral edges of the film. 5 Laboratory Diagnosis: Clinical Hematology 97 TABLE 5–1 Estimated WBC Based on Cells Counted in a Blood Smear WBC/hpf Estimated WBC (high dry or 40×) (per mm3) 2–4 4000–7000 4–6 7000–10,000 6–10 10,000–13,000 5 10–20 13,000–18,000 Abbreviations: WBC = white blood cell; hpf = high-power field. 4. If fewer than 200 cells are counted in a strip, count another strip until at least 200 are seen. The special white cell counter found in most labs is ideal for this purpose. In pa- tients receiving chemotherapy, the total count may be so small that only a 25–50 cell differential is possible. 5. In smears of blood from patients with very high white counts, such as those with leukemia, count the cells in any well-spread area where the different cell types are easy to identify. Table 5–1 shows the correlation between the number of cells in a smear and the estimated white cell count. A platelet count can be estimated by averaging the num- ber of platelets seen in 10 hpf (oil immersion) and multiplying by 20,000. NORMAL CBC VALUES A CBC panel generally includes WBC count, RBC count, hemoglobin, hematocrit, MCH, MCHC, MCV, and the RDW and platelets. The differential is usually ordered separately. Normal CBC, differential, and platelet values are outlined in Tables 5–2 and 5–3. NORMAL CBC VARIATIONS Hemoglobin and hematocrit are highest at birth (20 g/100 mL and 60%, respectively). The values fall steeply to a minimum at 3 mo (9.5 g/100 mL and 32%). Then they slowly rise to near adult levels at puberty, and thereafter both values are higher in males. A normal de- crease occurs in pregnancy. The number of WBCs is highest at birth (mean of 25,000/mm3) and slowly falls to adult levels by puberty. Lymphs predominate (up to 60% from the second week of life until age 5–7 y when polys begin to predominate. HEMATOCRIT The hematocrit is a simple screening test and can be performed on the medical floor as de- scribed previously (page 95). Always remember that because an equal amount of plasma and red cells are lost in acute blood loss, the hematocrit will not reflect the loss until some- time later (sometimes 2–3 h). If an anemia is suspected, the red cell indices and reticulocyte count should be checked. THREE-CELL DIFFERENTIAL COUNT Instead of a manual differential count of WBCs, many labs now rely on a three-cell differ- ential count that is automatically performed by newer instruments. White cells are sepa- rated on the basis of three sizes: small cells (mostly normal lymphocytes), middle cells 5 98 TABLE 5–2 Normal CBC for Selected Age Ranges WBC Count RBC Count Hemoglobin MCH MCHC MCV (cells/mm3) (106/µL) (g/dL) Hematocrit (pg) (g/dL) (µm3) Age [SI: 109/L] [SI: 1012/L] [SI: g/L] (%) [SI: pg] [SI: g/L]* [SI: fL] RDW Adult ? 4500–11,000 4.73–5.49 14.40–16.60 42.9–49.1 27–31 33–37 76–100 11.5–14.5 [4.5–11.0] [4.73–5.49] [144–166] Adult / As above 4.15–4.87 12.2–14.7 37.9–43.9 As As As As above above above above [4.15–5.49] [122–147] 11–15 years 4500–13,500 4.8 13.4 39 28 34 82 6–10 years 5000–14,500 4.7 12.9 37.5 27 34 80 4–6 years 5500–15,500 4.6 12.6 37.0 27 34 80 2–4 years 6000–17,000 4.5 12.5 35.5 25 32 77 4 mo–2 y 6000–17,500 4.6 11.2 35.0 25 33 77 1 wk–4 mo 5500–18,000 4.7±0.9 14.0±3.3 42.0±7.0 30 33 90 24 hr–1 wk 5000–21,000 5.1 18.3±4.0 52.5 36 35 103 First day 9400–34,000 5.1±1.0 19.5±5.0 54.0±10.0 38 36 106 *To convert standard reference value to SI units, multiply by 10. Abbreviations: WBC = white blood cell; MCH = mean cell hemoglobin; MCHC = mean cell hemoglobin concentration; MCV = mean cell volume; RDW = red cell distribution width. 5 99 TABLE 5–3 Normal CBC for Selected Age Ranges Platelet Lymphocytes, Neutrophils, Neutrophils, Count Total Band Segmented Eosinophils Basophils Monocytes (103/µL) (% WBC (% WBC (% WBC (% WBC (% WBC (% WBC Age [SI: 109/L] count) count) count) count) count) count) Adult ? 238±49 34 3.0 56 2.7 0.5 4.0 Adult / 270±58 As above As above As above As above As above As above 11–15 years 282±63 38 3.0 51 2.4 0.5 4.3 6–10 years 351±85 39 3.0 50 2.4 0.6 4.2 4–6 years 357±70 42 3.0 39 2.8 0.6 5.0 2–4 years 357±70 59 3.0 30 2.6 0.5 5.0 4 mo–2 y As above 61 3.1 28 2.6 0.4 4.8 1 wk–4 mo As above 56 4.5 30 2.8 0.5 6.5 24 hr–1 wk 240–380 24–41 6.8–9.2 39–52 2.4–4.1 0.5 5.8–9.1 First day As above 24 10.2 58 2.0 0.6 5.8 Abbreviations: CBC = complete blood count; WBC = white blood cell. 100 Clinician’s Pocket Reference, 9th Edition (monocytes, eosinophils, large lymphocyte variants), and large cells (neutrophils [stabs and band cells]). Each lab sets its own reference ranges based on “normal” populations. If one of the three cell populations falls outside the reference range, the sample is made into a slide, and a microscopic differential count is performed. With the anticipated shortage of health care workers and the expense of manual counting, these types of determinations will be- come more widely used. As an example of the three-cell count, a patient with sepsis may have a large-cell count of 95% and a small-cell count of 5% with no middle cells. On manual examination of the 5 slide, there may be 70% segmented neutrophils and 25% stabs, for a total of 95%. THE “LEFT SHIFT” The degree of nuclear lobulation of PMNs is thought to give some indication of cell age. A predominance of immature cells with only one or two nuclear lobes separated by a thick chromatin band is called a “shift to the left.” Conversely, a predominance of cells with four nuclear lobes is called a “shift to the right.” (For historical information, left and right des- ignations

come from the formerly used manual lab counters, in which the keys for entering the stabs were located on the left of the keyboard.) As a general rule, 40–50% of PMNs have three lobes, approximately 5% have two lobes, and 15–25% have four lobes. More than 20 five-lobed cells/100 WBCs suggest incip- ient megaloblastic anemia, and a six-lobed or seven-lobed poly is virtually diagnostic. “Bands” or “stabs,” the more immature forms of PMNs (the more mature are called “segs”), are identified by the fact that the connections between ends or lobes of a nucleus are greater than one-half the width of the hypothetical round nucleus. In bands or stabs, the connection between the lobes of the nucleus is by a thick band; in segs, by a thin filament. A band is defined as a connecting strip wide enough to reveal two distinct margins with nu- clear material in between. A filament is so narrow that no intervening nuclear material is present. When in doubt if a cell is a band or seg, call it a seg. For practical purposes, a left shift is present in the CBC when more than 10–12% bands are seen or when the total PMN count (segs plus bands) is greater than 80. Left Shift: Bacterial infection, toxemia, hemorrhage Right Shift: Liver disease, megaloblastic anemia, iron deficiency anemia RETICULOCYTE COUNT • Collection: Lavender top tube The reticulocyte count is not a part of the routine CBC. The count is used in the initial workup of anemia (especially unexplained) and in monitoring the effect of hematinic or ery- thropoietin therapy, monitoring the recovery from myelosuppression or monitoring engraft- ment following bone marrow transplant. Reticulocytes are juvenile RBCs with remnants of cytoplasmic basophilic RNA. These are suggested by basophilia of the RBC cytoplasm on Wright’s stain; however, confirmation requires a special reticulocyte stain. The result is re- ported as a percentage, and you should calculate the corrected reticulocyte count for inter- pretation of the results Reported count × Patient' s HCT Corrected reticulocyte count = Normal HCT 5 Laboratory Diagnosis: Clinical Hematology 101 This corrected count is an excellent indicator of erythropoietic activity. The normal corrected reticulocyte count is <1.5. Normal bone marrow responds to a decrease in erythrocytes (shown by a decreased hematocrit) with an increase in the production of reticulocytes. Lack of increase in a reticu- locyte count with an anemia suggests a chronic disease, a deficiency disease, marrow re- placement, or marrow failure. CBC DIFFERENTIAL DIAGNOSIS • See Tables 5–2 and 5–3 for normal age and sex-specific ranges. 5 Basophils • 0–1% Increased: Chronic myeloid leukemia, after splenectomy, polycythemia, Hodgkin’s dis- ease, and, rarely, in recovery from infection and from hypothyroidism Decreased: Acute rheumatic fever, pregnancy, after radiation, steroid therapy, thyrotoxi- cosis, stress Eosinophils • 1–3% Increased: Allergy, parasites, skin diseases, malignancy, drugs, asthma, Addison’s dis- ease, collagen–vascular diseases (handy mnemonic NAACP: Neoplasm, Allergy, Addison’s disease, Collagen–vascular diseases, Parasites), pulmonary diseases including Löffler’s syn- drome and PIE Decreased: Steroids, ACTH, after stress (infection, trauma, burns), Cushing’s syndrome Hematocrit (Male 40–54%; Female 37–47%) Decreased: Megaloblastic anemia (folate or B12 deficiency); iron deficiency anemia; sickle cell anemia; acute or chronic blood loss; hemolysis; anemia due to chronic disease, dilution, alcohol, or drugs Increased: Primary polycythemia (polycythemia vera), secondary polycythemia (re- duced fluid intake or excess fluid loss, congenital and acquired heart disease, lung disease, high altitudes, heavy smoking, tumors [renal cell carcinoma, hepatoma], renal cysts) Lymphocytes • 24–44% • See also Lymphocyte Subsets, page 103 Increased: Virtually any viral infection (AIDS, measles, rubella, mumps, whooping cough, smallpox, chickenpox, influenza, hepatitis, infectious mononucleosis), acute infec- tious lymphocytosis in children, acute and chronic lymphocytic leukemias Decreased: (Normal finding in 22% of population) Stress, burns, trauma, uremia, some viral infections, AIDS, AIDS-related complex, bone marrow suppression after chemother- apy, steroids, MS 102 Clinician’s Pocket Reference, 9th Edition Atypical Lymphocytes >20%: Infectious mononucleosis, CMV infection, infectious hepatitis, toxoplasmosis <20%: Viral infections (mumps, rubeola, varicella), rickettsial infections, TB MCH (Mean Cellular [Corpuscular] Hemoglobin) • 27–31 pg (SI: pg) 5 The weight of hemoglobin of the average red cell. Calculated by Hemoglobin (g / L) MCH = RBC (106 / µL) Increased: Macrocytosis (megaloblastic anemias, high reticulocyte counts) Decreased: Microcytosis (iron deficiency, sideroblastic anemia, thalassemia) MCHC (Mean Cellular [Corpuscular] Hemoglobin Concentration) • 33–37 g/dL (SI:330–370 g/L) The average concentration of hemoglobin in a given volume of red cells. Calculated by the formula Hemoglobin (g / dL) MCHC = Hematocrit Increased: Very severe, prolonged dehydration; spherocytosis Decreased: Iron deficiency anemia, overhydration, thalassemia, sideroblastic anemia MCV (Mean Cell [Corpuscular] Volume) • 76–100 cu µm (SI: fL) The average volume of red blood cells. Calculated by the formula Hematocrit × 1000 MCV = RBC (106 / µL) Increased/Macrocytosis: Megaloblastic anemia (B12, folate deficiency), macrocytic (normoblastic) anemia, reticulocytosis, myelodysplasias, Down syndrome, chronic liver dis- ease, treatment of AIDS with AZT, chronic alcoholism, cytotoxic chemotherapy, radiation therapy, Dilantin use, hypothyroidism, newborns Decreased/Microcytosis: Iron deficiency, thalassemia, some cases of lead poisoning or polycythemia Monocytes • 3–7% Increased: Bacterial infection (TB, SBE, brucellosis, typhoid, recovery from an acute in- fection), protozoal infections, infectious mononucleosis, leukemia, Hodgkin’s disease, ul- cerative colitis, regional enteritis 5 Laboratory Diagnosis: Clinical Hematology 103 Decreased: Lymphocytic leukemia, aplastic anemia, steroid use Platelets • 150–450,000 µL Platelet counts may be normal in number, but abnormal in function as occurs in aspirin therapy. Abnormalities of platelet function are assessed by bleeding time. Increased: Sudden exercise, after trauma, bone fracture, after asphyxia, after surgery (espe- cially splenectomy), acute hemorrhage, polycythemia vera, primary thrombocytosis, 5 leukemias, after childbirth, carcinoma, cirrhosis, myeloproliferative disorders, iron deficiency Decreased: DIC, ITP, TTP, congenital disease, marrow suppressants (chemotherapy, alco- hol, radiation), burns, snake and insect bites, leukemias, aplastic anemias, hypersplenism, infectious mononucleosis, viral infections, cirrhosis, massive transfusions, eclampsia and preeclampsia, prosthetic heart valve, more than 30 different drugs (NSAIDs, cimetidine, as- pirins, thiazides, others) PMNs (Polymorphonuclear Neutrophils) (Neutrophils) • 40–76% • See also the “Left Shift” page 100. Increased Physiologic (Normal). Severe exercise, last months of pregnancy, labor, surgery, new- borns, steroid therapy Pathologic. Bacterial infections, noninfective tissue damage (MI, pulmonary infarction, pancreatitis, crush injury, burn injury), metabolic disorders (eclampsia, DKA, uremia, acute gout), leukemias Decreased: Pancytopenia, aplastic anemia, PMN depression (a mild decrease is referred to as neutropenia, severe is called agranulocytosis), marrow damage (x-rays, poisoning with benzene or antitumor drugs), severe overwhelming infections (disseminated TB, sep- ticemia), acute malaria, severe osteomyelitis, infectious mononucleosis, atypical pneumo- nias, some viral infections, marrow obliteration (osteosclerosis, myelofibrosis, malignant infiltrate), drugs (more than 70, including chloramphenicol, phenylbutazone, chlorpro- mazine, quinine), B12 and folate deficiencies, hypoadrenalism, hypopituitarism, dialysis, fa- milial decrease, idiopathic causes RDW (Red Cell Distribution Width) • 11.5–14.5 RDW is a measure of the degree of anisocytosis (variation in RBC size) and measured by the automated hematology counters. Increased: Many anemias (iron deficiency, pernicious, folate deficiency, thalassemias), liver disease LYMPHOCYTE SUBSETS Specific monoclonal antibodies are used to identify specific T and B cells. Lymphocyte sub- sets (also called lymphocyte marker assays, or T- and B-cell assay) are useful in the diagno- sis of AIDS and various leukemias and lymphomas. The designation CD (“clusters of differentiation”) has largely replaced the older antibody designations (eg, Leu 3a or OKT3). Results are most reliable when reported as an absolute number of cells/µL rather 104 Clinician’s Pocket Reference, 9th Edition than a percentage of cells. A CD4/CD8 ratio < 1 is seen in patients with AIDS. Absolute CD4 count is used to initiate therapy with antiretrovirals or prophylaxis for PCP (see page 75). The CDC includes in the category of AIDS any patient with a CD4 count < 200 who is HIV-positive. Normal Lymphocyte Subsets • Total lymphocytes 0.66–4.60 thousand/µL • T cell 644–2201 µL (60–88%) 5 • B cell 82–392 µL (3–20%) • T helper/inducer cell (CD4, Leu 3a, OKT4) 493–1191 µL (34–67%) • Suppressor/cytotoxic T cell (CD8, Leu 2, OKT8) 182–785 µL (10–42%) • CD4/CD8 ratio > 1 RBC MORPHOLOGY DIFFERENTIAL DIAGNOSIS The following lists some erythrocyte abnormalities and the associated conditions. General terms include poikilocytosis (irregular RBC shape such as sickle or burr) and anisocytosis (irregular RBC size such as microcytes and macrocytes). Basophilic Stippling: Lead or heavy-metal poisoning, thalassemia, severe anemia Burr Cells (Acanthocytes): Severe liver disease; high levels of bile, fatty acids, or toxins Helmet Cells (Schistocytes): Microangiopathic hemolysis, hemolytic transfusion re- action, transplant rejection, other severe anemias, TTP Howell–Jolly Bodies: After splenectomy, some severe hemolytic anemias, pernicious anemia, leukemia, thalassemia Nucleated RBCs: Severe bone marrow stress (hemorrhage, hemolysis, etc), marrow re- placement by tumor, extramedullary hematopoiesis Polychromasia (Basophilia): The appearance of a bluish gray red cell on routine Wright’s stain suggests reticulocytes. Sickling: Sickle cell disease and trait Spherocytes: Hereditary spherocytosis, immune or microangiopathic hemolysis, severe burns, ABO transfusion reactions Target Cells (Leptocytes): Thalassemia, hemoglobinopathies, obstructive jaundice, any hypochromic anemia, after splenectomy WBC MORPHOLOGY DIFFERENTIAL DIAGNOSIS The following gives conditions associated with certain changes in the normal morphology of WBCs. Auer Rods: AML Döhle’s Inclusion Bodies: Severe infection, burns, malignancy, pregnancy Hypersegmentation: Megaloblastic anemias Toxic Granulation: Severe illness (sepsis, burn, high temperature) 5 Laboratory Diagnosis: Clinical Hematology 105 COAGULATION AND OTHER HEMATOLOGIC TESTS The coagulation cascade is shown in Figure 5–2. A variety of coagulation-related and other blood tests follow. Activated Clotting Time (ACT) • 114–186 s • Collection: Black top tube from instrument manufacturer This is a bedside test used in the operating room, dialysis unit, or other facility to docu- ment neutralization of heparin (ie, after coronary artery bypass, heparin is reversed.) 5 Increased: Heparin, some platelet disorders, severe clotting factor deficiency Antithrombin-III (AT-III) • 17–30 mg/dL or 80–120% of control • Collection: Blue top tube, patient must be off heparin for 6 h Used in the evaluation of thrombosis. Heparin must interact with AT-III to produce anti- coagulation effect. Decreased: Autosomal-dominant familial AT-III deficiency, PE, severe liver disease, late pregnancy, oral contraceptives, nephrotic syndrome, heparin therapy (>3 days) Increased: Coumadin, after MI Bleeding Time • Duke, Ivy <6 min; Template <10 min • Collection: Specialized bedside test performed by technicians. A small incision is made, and the wound is wicked with filter paper every 30 s until the fluid is clear. In vivo test of hemostasis that tests platelet function, local tissue factors, and clotting factors. Nonsteroidal medications should be stopped 5–7 d before the test because these agents can affect platelet function. Increased: Thrombocytopenia (DIC, TTP, ITP), von Willebrand’s disease, defective platelet function (NSAIDs such as aspirin) Coombs’ Test, Direct (Direct Antiglobulin Test) • Normal = negative • Collection: Purple top tube Uses patient’s erythrocytes; tests for the presence of antibody on the patient’s cells and used in the screening for autoimmune hemolytic anemia. Positive: Autoimmune hemolytic anemia (leukemia, lymphoma, collagen–vascular dis- eases), hemolytic transfusion reaction, some drug sensitizations (methyldopa, levodopa, cephalosporins, penicillin, quinidine), hemolytic disease of the newborn (erythroblastosis fetalis) Coombs’ Test, Indirect (Indirect Antiglobulin Test/Autoantibody Test) • Normal = negative • Collection: Purple top tube Uses serum that contains antibody, usually from the patient. Used to check cross-match prior to blood transfusion in the blood bank. 106 Clinician’s Pocket Reference, 9th Edition Intrinsic Extrinsic (Prekallikrein + Surface contact) Factor XII Factor XIIa [Tissue damage] High-molecular- 5 weight kininogen Factor XI Factor XIa Tissue factor * [thromboplastin] †Factor IX Factor IXa + Ca2+ †Factor VII * Factor VIII Ca2+ Ca2+ Platelets (phospholipid) †Factor X Factor Xa Ca2+ Platelets Factor V (phospholipid) †Prothrombin (II) Thrombin (IIa) Factor XIII Ca2+ Factor XIIIa Fibrin Fibrinogen (I) Fibrin Fibrin split monomer Ca2+ products Plasminogen proactivator Fibrinolysis XIIa Activator Plasminogen Plasmin (profibrinolysin) (fibrinolysin) FIGURE 5–2 Blood coagulation cascade. Nearly all of the coagulation factors ap- parently exist as inactive proenzymes (Roman numeral) that, when activated (Roman numeral + a), serve to activate the next proenzyme in the sequence. Symbol key: * = Heparin acts to inhibit. Ü = Plasma content decreased by Coumadin. (Reprinted, with permission, from: Krupp MA [ed]: The Physician’s Handbook. Lange Medical Publi- cations, Los Angeles CA, 1985.) 5 Laboratory Diagnosis: Clinical Hematology 107 Positive: Isoimmunization from previous transfusion, incompatible blood due to im- proper cross-matching or medications such as methyldopa. Fibrin D-Dimers • Negative or <0.5 µg/mL • Collection: Blue, green, or purple top tube Fibrin broken into various D-dimer fragments by plasmin. Increased: DIC, thromboembolic diseases (PE, arterial or venous thrombosis) 5 Fibrin Degradation Products (FDP), Fibrin Split Products (FSP) • <10 µg/mL • Collection: Blue top tube

Generally replaced by the fibrin D-dimer as a screen for DIC Increased: DIC (usually >40 µg/mL), any thromboembolic condition (DVT, MI, PE), hepatic dysfunction Fibrinogen • 200–400 mg/dL (SI:2.0–4.0 g/L) • Collection: Blue top tube Most useful in the diagnosis of DIC and congenital hypofibrinogenemia. Fibrinogen is cleaved by thrombin to form insoluble fragments that polymerize to form a stable clot. Increased: Inflammatory reactions, oral contraceptives, pregnancy, cancer (kidney, stomach, breast) Decreased: DIC (sepsis, amniotic fluid embolism, abruptio placentae), surgery (prostate, open heart), neoplastic and hematological conditions, acute severe bleeding, burns, venomous snake bite, congenital Lee-White Clotting Time • 5–15 min • Collection: Draw into plain plastic syringe; clotting time measured in sepa- rate tube Increased: Heparin therapy, plasma–clotting factor deficiency (except Factors VII and XIII). (Note: This is not a sensitive test and so is therefore not considered a good screening test.) Partial Thromboplastin Time (Activated Partial Thromboplastin Time, PTT, APTT) • 27–38 s • Collection: Blue top tube Evaluates the intrinsic coagulation system (See Figure 5–2). Most commonly used to monitor heparin therapy Increased: Heparin and any defect in the intrinsic coagulation system (includes Fac- tors I, II, V, VIII, IX, X, XI, and XII), prolonged use of a tourniquet before drawing a blood sample, hemophilia A and B Prothrombin Time (PT) • 11.5–13.5 s • Figure 5–2, page 106 • Collection: Blue top tube 108 Clinician’s Pocket Reference, 9th Edition PT evaluates the extrinsic coagulation system that includes Factors I, II, V, VII, and X. The use of INR instead of the Patient/Control ratio to guide anticoagulant (Coumadin) ther- apy is becoming standard. INR provides a more universal and standardized result be- cause it measures the control against a WHO standard reference reagent. Therapeutic INR levels are 2–3 for DVT, PE, TIAs, and atrial fibrillation. Recurrent DVT on adequate treatment requires an INR of 3–4.5. Mechanical heart valves require an INR of 3–4.5 (See also Chapter 22, Table 22–10 [page 637].) Increased: Drugs (sodium warfarin [Coumadin]), vitamin K deficiency, fat malabsorp- 5 tion, liver disease, prolonged use of a tourniquet before drawing a blood sample, DIC Sedimentation Rate (Erythrocyte Sedimentation Rate, ESR) • Collection: Lavender top tube The ESR is a very nonspecific test with a high sensitivity and a low specificity. Most useful in serial measurement to follow the course of disease (eg, polymyalgia rheumatica or temporal arteritis). ZETA rate is not affected by anemia. ESR correlates well with C-reactive protein levels. Wintrobe Scale: Males, 0–9 mm/h, females, 0–20 mm/h ZETA Scale: 40–54% normal, 55–59% mildly elevated, 60–64% moderately elevated, >65% markedly elevated Westergren Scale: Males <50 years 15 mm/h, >50 years 20 mm/h; female <50 years 20 mm/h, >50 years 30 mm/h Increased: Any type of infection, inflammation, rheumatic fever, endocarditis, neo- plasm, AMI Thrombin Time • 10–14 s • Collection: Blue top tube A measure of the rate of conversion of fibrinogen to fibrin and fibrin polymerization. Used to detect the presence of heparin and hypofibrinogenemia and as an aid in the evalua- tion of prolonged PTT Increased: Systemic heparin, DIC, fibrinogen deficiency, congenitally abnormal fib- rinogen molecules 6 LABORATORY DIAGNOSIS: URINE STUDIES Urinalysis Procedure 24-Hour Urine Studies Urinalysis, Normal Values Other Urine Studies Differential Diagnosis for Routine Urinary Indices in Renal Failure Urinalysis Urine Output Urine Sediment Urine Protein Electrophoresis 6 Spot or Random Urine Studies Creatinine and Creatinine Clearance URINALYSIS PROCEDURE For a routine screening urinalysis, a fresh (less than 1-h old), clean-catch urine is accept- able. If it cannot be interpreted immediately, it should be refrigerated (urine standing at room temperature for long periods causes lysis of casts and red cells and becomes alkalin- ized.) See Chapter 13 under Urinary Tract Procedures, page 306, for the different ways to collect the sample. 1. Pour about 5–10 mL of well-mixed urine into a centrifuge tube. Check the specific gravity with a urinometer or optic refractory urinometer (refractometer) on the remain- ing sample. 2. Check for appearance (color, turbidity, odor). 3. Spin the capped sample at 3000 rpm (450 g) for 3 min. 4. While the sample is in the centrifuge and using the dipstick (Chemstrip, etc) supplied by your lab, perform the dipstick evaluation on the remaining portion of the sample. Read the results according to the color chart and instructions on the bottle. Make sure to allow the time noted before reading the test because reading before the time (up to 60 s) may yield false results. Record glucose, ketones, blood, protein, pH, nitrite, and leukocyte esterase if available. Be sure to recap the bottle tightly after use. Agents that color the urine (phenazopyridine [Pyridium]) may interfere with the results of the dipstick. 5. Decant and discard the supernatant. Mix the remaining sediment by flicking it with your finger and pour or pipette one or two drops on a microscope slide. Cover with a coverslip. If a urine sample looks very grossly cloudy, it is sometimes advisable to ex- amine an unspun sample. If an unspun sample is used, note this on the report. In gen- eral, for routine urinalysis, a spun sample is more desirable. 6. Examine 10 lpf (10× objective) for epithelial cells, casts, crystals, and mucus. Casts are usually reported per low-power field. Casts tend to collect around the periphery of the coverslip. 7. Examine several high-power fields (40× objective) for epithelial cells, crystals, RBCs, WBCs, bacteria, and parasites (trichomonads). RBCs, WBCs, and bacteria are usually reported per high-power field. Two reporting systems are commonly used: 109 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 110 Clinician’s Pocket Reference, 9th Edition System One System Two Rare = <2 per field Trace = <¹₄ of field Occasional = 3–5 per field 1+ = ¹₄ of field Frequent = 5–9 per field 2+ = ¹₂ of field Many = “large number” per field 3+ = ³₄ of field TNTC = too numerous to count 4+ = field is full URINALYSIS, NORMAL VALUES 1. Appearance: “Yellow, clear,” or “straw-colored, clear” 2. Specific Gravity 6 a. Neonate: 1.012 b. Infant: 1.002–1.006 c. Child and Adult: 1.001–1.035 (with normal fluid intake 1.016–1.022) 3. pH a. Newborn/Neonate: 5–7 b. Child and Adult: 4.6–8.0 4. Negative for: Bilirubin, blood, acetone, glucose, protein, nitrite, leukocyte esterase, re- ducing substances 5. Trace: Urobilinogen 6. RBC: Male 0–3/hpf, female 0–5/hpf 7. WBC: 0–4/hpf 8. Epithelial Cells: Occasional 9. Hyaline Casts: Occasional 10. Bacteria: None 11. Crystals: Some limited crystals based on urine pH (see below) DIFFERENTIAL DIAGNOSIS FOR ROUTINE URINALYSIS Appearance Colorless: Diabetes insipidus, diuretics, excess fluid intake Dark: Acute intermittent porphyria, malignant melanoma Cloudy: UTI (pyuria), amorphous phosphate salts (normal in alkaline urine), blood, mucus, bilirubin Pink/Red: Heme-positive. Blood, hemoglobin, sepsis, dialysis, myoglobin Heme-negative. Food coloring, beets, sulfa drugs, nitrofurantoin, salicylates Orange/Yellow: Dehydration, phenazopyridine (Pyridium), rifampin, bile pigments Brown/Black: Myoglobin, bile pigments, melanin, cascara, iron, nitrofurantoin, alkap- tonuria Green: Urinary bile pigments, indigo carmine, methylene blue Foamy: Proteinuria, bile salts pH Acidic: High-protein (meat) diet, ammonium chloride, mandelic acid and other medica- tions, acidosis, (due to ketoacidosis [starvation, diabetic], COPD) 6 Laboratory Diagnosis: Urine Studies 111 Basic: UTI, renal tubular acidosis, diet (high-vegetable, milk, immediately after meals), sodium bicarbonate therapy, vomiting, metabolic alkalosis Specific Gravity Usually corresponds with osmolarity except with osmotic diuresis. Value >1.023 indicates normal renal concentrating ability. Random value 1.003–1.030 Increased: Volume depletion; CHF; adrenal insufficiency; diabetes mellitus; SIADH; increased proteins (nephrosis); if markedly increased (1.040–1.050), suspect artifact or ex- cretion of radiographic contrast media Decreased: Diabetes insipidus, pyelonephritis, glomerulonephritis, water load with nor- 6 mal renal function Bilirubin Positive: Obstructive jaundice (intrahepatic and extrahepatic), hepatitis. (Note: False- positives occur with stool contamination.) Blood Note: If the dipstick is positive for blood, but no red cells are seen, free hemoglobin from trauma may be present; a transfusion reaction may have occurred, from lysis of RBCs (RBCs will lyse if the pH is <5 or >8); or myoglobin may be present because of a crush in- jury, burn, or tissue ischemia. Positive: Stones, trauma, tumors (benign and malignant, anywhere in the urinary tract), urethral strictures, coagulopathy, infection, menses (contamination), polycystic kidneys, in- terstitial nephritis, hemolytic anemia, transfusion reaction, instrumentation (Foley catheter, etc) Glucose Positive: Diabetes mellitus, pancreatitis, pancreatic carcinoma, pheochromocytoma, Cushing’s disease, shock, burns, pain, steroids, hyperthyroidism, renal tubular disease, iatro- genic causes. (Note: Glucose oxidase technique in many kits is specific for glucose and will not react with lactose, fructose, or galactose.) Ketones Detects primarily acetone and acetoacetic acid and not β-hydroxybutyric acid. Positive: Starvation, high-fat diet, DKA, vomiting, diarrhea, hyperthyroidism, preg- nancy, febrile states (especially in children) Nitrite Many bacteria will convert nitrates to nitrite. (See also the section on Leukocyte Esterase, page 112.) Positive: Infection (A negative test does not rule out infection because some organisms, such as Streptococcus faecalis and other gram-positive cocci, do not produce nitrite, and the urine must also be retained in the bladder for several hours to allow the reaction to take place.) 112 Clinician’s Pocket Reference, 9th Edition Protein Indication by dipstick of persistent proteinuria should be quantified by 24-h urine studies. Positive: Pyelonephritis, glomerulonephritis, Kimmelstiel–Wilson syndrome (diabetes), nephrotic syndrome, myeloma, postural causes, preeclampsia, inflammation and malignan- cies of the lower tract, functional causes (fever, stress, heavy exercise), malignant hyperten- sion, CHF Leukocyte Esterase Test detects ≥5 WBC/hpf or lysed WBCs. When combined with the nitrite test, it has a pre- 6 dictive value of 74% for UTI if both tests are positive and a value of >97% if both tests are negative. Positive: UTI (false-positive with vaginal contamination) Reducing Substances Positive: Glucose, fructose, galactose, false-positives (vitamin C, salicylates, antibiotics, etc) Urobilinogen Positive: Cirrhosis, CHF with hepatic congestion, hepatitis, hyperthyroidism, suppres- sion of gut flora with antibiotics URINE SEDIMENT Many labs no longer do microscopic examinations unless specifically requested or if evi- dence exists for an abnormal finding on dipstick test (such as positive leukocyte esterase). Figure 6–1 is a pictorial representation of materials found in urine sediments. Red Blood Cells (RBCs): Trauma, pyelonephritis, genitourinary TB, cystitis, prostati- tis, stones, tumors (malignant and benign), coagulopathy, and any cause of blood on dipstick test (See previous section on blood pH, page 111.) White Blood Cells (WBCs): Infection anywhere in the urinary tract, TB, renal tumors, acute glomerulonephritis, radiation, interstitial nephritis (analgesic abuse) Epithelial Cells: ATN, necrotizing papillitis. (Most epithelial cells are from an other- wise unremarkable urethra.) Parasites: Trichomonas vaginalis, Schistosoma haematobium infection Yeast: Candida albicans infection (especially in diabetics, immunosuppressed patients, or if a vaginal yeast infection is present) Spermatozoa: Normal in males immediately after intercourse or nocturnal emission Crystals Abnormal. Cystine, sulfonamide, leucine, tyrosine, cholesterol Normal. Acid urine: Oxalate (small square crystals with a central cross), uric acid. Alka- line urine: Calcium carbonate, triple phosphate (resemble coffin lids) Contaminants: Cotton threads, hair, wood fibers, amorphous substances (all usually unimportant) 6 Laboratory Diagnosis: Urine Studies 113 Urine Sediment Acid Urine Alkaline Urine Amorphous phosphates Calcium Uric acid crystals oxalate 6 crystals RBC's Calcium Mucus carbonate threads Renal crystals tubular Leucine cells spheres Cylindroids RBC cast Epithelial cells Ammonium Sodium urate Hyaline Granular urate crystals crystals cast cast Waxy cast Convoluted hyaline Spermatozoa cast Tyrosine Bacteria WBC's Calcium needles phosphate Yeast WBC cast crystals Triple phosphate Cystine Amorphous crystals crystals urates FIGURE 6–1 Urine sediment as seen under the microscope. (Reprinted, with per- mission, from: Greene MG [ed]: The Harriet Lane Handbook: A Manual for Pediatric House Officers, 12th ed,. Yearbook Medical Publishers, Chicago IL, 1991.) 114 Clinician’s Pocket Reference, 9th Edition Mucus: Large amounts suggest urethral disease (normal from ileal conduit or other forms of urinary diversion) Glitter Cells: WBCs lysed in hypotonic solution Casts: The presence of casts in a urine localizes some or all of the disease process to the kidney itself. Hyaline Casts. (Acceptable unless they are “numerous”), benign hypertension, nephro- tic syndrome, after exercise RBC Casts. Acute glomerulonephritis, lupus nephritis, SBE, Goodpasture’s disease, after a streptococcal infection, vasculitis, malignant hypertension 6 WBC Casts. Pyelonephritis Epithelial (Tubular) Casts. Tubular damage, nephrotoxin, virus Granular Casts. Breakdown of cellular casts, leads to waxy casts; “dirty brown granular casts” typical for ATN Waxy Casts. (End stage of granular cast). Severe chronic renal disease, amyloidosis Fatty Casts. Nephrotic syndrome, diabetes mellitus, damaged renal tubular epithelial cells Broad Casts. Chronic renal disease SPOT OR RANDOM URINE STUDIES The so-called spot urine, which is often ordered to aid in diagnosing various conditions, re-

lies on only a small sample (10–20 mL) of urine. Spot Urine for β2-microglobulin • <0.3 mg/L A marker for renal tubular injury Increased: Diseases of the proximal tubule (ATN, interstitial nephritis, pyelonephritis), drug-induced nephropathy (aminoglycosides), diabetes, trauma, sepsis, HIV, lymphoprolif- erative and lymphodestructive diseases Spot Urine for Electrolytes The usefulness of this assay is limited because of large variations in daily fluid and salt in- take, and the results are usually indeterminate if a diuretic has been given. 1. Sodium <10 mEq/L (mmol/L): Volume depletion, hyponatremic states, prerenal azotemia (CHF, shock, etc), hepatorenal syndrome, glucocorticoid excess 2. Sodium >20 mEq/L (mmol/L): SIADH, ATN (usually >40 mEq/L), postobstructive diuresis, high salt intake, Addison’s disease, hypothyroidism, interstitial nephritis 3. Chloride <10 mEq/L (mmol/L): Chloride-sensitive metabolic alkalosis (vomiting, ex- cessive diuretic use), volume depletion 4. Potassium <10 mEq/L (mmol/L): Hypokalemia, potassium depletion, extrarenal loss Spot Urine for Erythrocyte Morphology The morphology of red blood cells in a sample of urine that tests positive for blood may give some indication of the nature of the hematuria. Eumorphic red cells are typically seen in cases of postrenal, nonglomerular bleeding. Dysmorphic red cells are more likely associated with glomerular causes of bleeding. Each reference lab has standards, but as a general rule, the 6 Laboratory Diagnosis: Urine Studies 115 presence of >90% dysmorphic erythrocytes in patients with asymptomatic hematuria indicates a renal glomerular source of bleeding, especially if associated with proteinuria and or casts (ie, IgA nephropathy, poststreptococcal glomerular, sickle cell disease or trait, etc). If ≥90% eu- morphic erythrocytes or even “mixed” results (10–90% eumorphic erythrocytes) indicates a postrenal cause of hematuria requiring a complete urologic evaluation (ie, hypercalciuria, urolithiasis, cystitis, trauma, tumors, hemangioma, exercise induced, BPH, etc). Spot Urine for Microalbumin • Normal <30 µg albumin/mg creatinine Used to determine which patients with diabetes are at risk for nephropathy. Clinical al- buminuria occurs at >300 µg albumin/mg creatinine. Base test on two or three separate de- 6 terminations over 6 mo. Diabetic patients with levels between 30–300 µg have microalbuminuria and are usually initiated on ACE inhibitor or angiotensin receptor blocker. Spot Urine for Myoglobin • Qualitative negative Positive: Skeletal muscle conditions (crush injury, electrical burns, carbon monoxide poisoning, delirium tremens, surgical procedures, malignant hyperthermia), polymyositis. Spot Urine for Osmolality • 75–300 mOsm/kg (mmol/kg) • Varies with water intake Patients with normal renal function should concentrate >800 mOsm/kg (mmol/kg) after a 14-h fluid restriction; <400 mOsm/kg (mmol/kg) is a sign of renal impairment. Increased: Dehydration, SIADH, adrenal insufficiency, glycosuria, high-protein diet Decreased: Excessive fluid intake, diabetes insipidus, acute renal failure, medications (acetohexamide, glyburide, lithium) Spot Urine for Protein • Normal <10 mg/dL (0.1 g/L) or <20 mg/dL (0.2 g/L) for a sample taken in the early AM See page 112 for the differential diagnosis of protein in the urine. CREATININE AND CREATININE CLEARANCE Normal Adult Male. Total creatinine 1–2 g/24 h (8.8–17.7 mmol/d); clearance 85–125 mL/min/1.73 m2 Adult Female. Total creatinine 0.8–1.8 g/24 h (7.1–15.9 mmol/d); clearance 75–115 mL/min 1.73 m2 (1.25–1.92 mL/s/1.73 m2) Child. Total creatinine (>3 years) 12–30 mg/kg/24 h; clearance 70–140 mL/min/1.73 m2(1.17–2.33 mL/s/1.73 m2) Decreased: A decreased creatinine clearance results in an increase in serum creatinine usually secondary to renal insufficiency. See Chapter 4, page 65, for differential diagnosis of increased serum creatinine. Increased: Early diabetes mellitus, pregnancy 116 Clinician’s Pocket Reference, 9th Edition Creatinine Clearance Determination Creatinine clearance is one of the most sensitive indicators of early renal insufficiency. Clearances are ordered for patients with suspected renal disease and are useful for following patients who are taking nephrotoxic medications, (eg, gentamicin). Clearance normally de- creases with age. A creatinine clearance of 10–20 mL/min indicates severe renal failure, and a clearance of <10 mL/min usually indicates the need for dialysis. To determine a creatinine clearance, order a concurrent serum creatinine and a 24-h urine creatinine. A shorter time interval can be used, for example, 12 h, but remember that the formula must be corrected for this change and that a 24-h sample is less prone to collec- tion error. 6 Example: (A quick formula is also found under “Aminoglycoside Dosing,” page 620.) The following are calculations of (a) the creatinine clearance from a 24-h urine sample with a volume of 1000 mL, (b) a urine creatinine of 108 mg/100 mL, and (c) a serum creatinine of 1 mg/100 mL (1 mg/dL). Urine creatinine × Total urine volume Clearance = Plasma creatinine × Time where time = 1440 min if 24-h collection. (108 mg / 100 mL) (1000 mL) Clearance = = 75 mL / min (1 mg / 100 mL) (1440 min) To see if the urine sample is valid, some clinicians advocate a preliminary evaluation by determining first if the sample contains at least 18–25 mg/kg/24 h of creatinine for adult males or 12–20 mg/kg/24 h for adult females. This preliminary test is not a requirement, but can confirm if a 24–h sample was collected or if some of the sample was lost. If the patient is an adult (150 lb = body surface area of 1.73 m2), adjustment of the clearance for body size is not routinely done. Adjustment for pediatric patients is a neces- sity. If the values in the previous example were for a 10-year-old boy who weighed 70 lb (1.1 m2), the clearance would be: 1.73 m2 75 mL / min × 2 = 118 mL / min 1.1 m 24-HOUR URINE STUDIES A wide variety of diseases, most of them endocrine, can be diagnosed by assays of 24-h urine samples. The following information gives the normal values for certain agents and the conditions associated with changes in these values. Calcium, Urine Normal: On a calcium-free diet <150 mg/24 h (3.7 mmol/d), average calcium diet (600–800 mg/24 h) 100–250 mg/24 h (2.5–6.2 mmol/d) Increased: Hyperparathyroidism, hyperthyroidism, hypervitaminosis D, distal renal tubular acidosis (type I), sarcoidosis, immobilization, osteolytic lesions (bony metastasis, multiple myeloma), Paget’s disease, glucocorticoid excess, immobilization, furosemide Decreased: Medications (thiazide diuretics, estrogens, oral contraceptives), hypothy- roidism, renal failure, steatorrhea, rickets, osteomalacia 6 Laboratory Diagnosis: Urine Studies 117 Catecholamines, Fractionated Used to evaluate neuroendocrine tumors, including pheochromocytoma and neuroblastoma. Avoid caffeine and methyldopa (Aldomet) prior to test Normal: Values are variable and depend on the assay method used. Norepinephrine 15–80 mg/24 h [SI: 89–473 nmol/24 h], epinephrine 0–20 mg/24 h [0–118 nmol/24 h], dopamine 65–400 mg/24 h [SI: 384–2364 nmol/24 h]. Increased: Pheochromocytoma, neuroblastoma, epinephrine administration, presence of drugs (methyldopa, tetracyclines cause false increases) Cortisol, Free 6 Used to evaluate adrenal cortical hyperfunction, screening test of choice for Cushing’s syn- drome Normal: 10–110 mg/24 h [SI: 30–300 nmol] Increased: Cushing’s syndrome (adrenal hyperfunction), stress during collection, oral contraceptives, pregnancy Creatinine • See pages 65 and 115 Cysteine Used to detect cystinuria, homocystinuria, monitor response to therapy Normal: 40–60 mg/g creatinine Increased: Heterozygotes < 300 mg/g creatinine/day; homozygotes > 250 mg/g creati- nine 5-HIAA (5-Hydroxyindoleacetic Acid) 5–HIAA is a serotonin metabolite useful in diagnosing carcinoid syndrome. Normal: (2–8 mg [SI: 10.4–41.6] mmol/24–h urine collection) Increased: Carcinoid tumors (except rectal), certain foods (banana, pineapple, tomato, walnuts, avocado), phenothiazine derivatives Metanephrines Detects metabolic products of epinephrine and norepinephrine, a primary screening test for pheochromocytoma Normal: <1.3 mg/24 h (7.1 mmol/L) for adults, but variable in children Increased: Pheochromocytoma, neuroblastoma (neural crest tumors), false-positive with drugs (phenobarbital, guanethidine, hydrocortisone, MAO inhibitors) Protein • See also Urine Protein Electrophoresis, pages 85 and 112. 118 Clinician’s Pocket Reference, 9th Edition Normal: <150 mg/24 h (<0.15 g/d) Increased: Nephrotic syndrome usually associated with >4 g/24 h 17-Ketogenic Steroids (17-KGS, Corticosteroids) Overall adrenal function test, largely replaced by serum or urine cortisol levels Normal: Males 5–24 mg/24 h (17–83 mmol/24 h); females 4–15 mg/24 h (14–52 mmol/24 h) Increased: Adrenal hyperplasia (Cushing’s syndrome), adrenogenital syndrome 6 Decreased: Panhypopituitarism, Addison’s disease, acute steroid withdrawal 17-Ketosteroids, Total (17-KS) Measures DHEA, androstenedione (adrenal androgens); largely replaced by assay of indi- vidual elements Normal: Adult males 8–20 mg/24 h (28–69 mmol/L); adult female 6–15 mg/dL (21–52 mmol/L). Note: Low values in prepubertal children Increased: Adrenal cortex abnormalities (hyperplasia [Cushing’s disease], adenoma, carcinoma, adrenogenital syndrome), severe stress, ACTH or pituitary tumor, testicular in- terstitial tumor and arrhenoblastoma (both produce testosterone) Decreased: Panhypopituitarism, Addison’s disease, castration in men Vanillylmandelic Acid VMA is the urinary product of both epinephrine and norepinephrine; good screening test for pheochromocytoma, also used to diagnose and follow up neuroblastoma and ganglioneu- roma Normal: <7–9 mg/24 h (35–45 mmol/L) Increased: Pheochromocytoma, other neural crest tumors (ganglioneuroma, neuroblas- toma), factitious (chocolate, coffee, tea, methyldopa) OTHER URINE STUDIES Drug Abuse Screen • Normal = negative Tests urine for common drugs of abuse, often used for employment screening for criti- cal jobs. Assay will vary by facility and may include tests for amphetamines, barbiturates, benzodiazepines, marijuana (cannabinoid metabolites), cocaine metabolites, opiates, phen- cyclidine. Xylose Tolerance Test (D-Xylose Absorption Test) • 5 g xylose in 5-h urine specimen after 25 g oral dose of xylose or 1.2 g after 5-g oral dose • Collection: Patient is NPO after midnight except for water. • After voiding at 8 AM, 25 g of D-xylose (or 5 g if GI irritation is a concern) is dissolved in 250 mL water. • An addi- tional 750 mL water is drunk and the urine collected for the next 5 h. 6 Laboratory Diagnosis: Urine Studies 119 TABLE 6–1 Urinary Indices Useful in the Differential Diagnosis of Oliguria Index Prerenal Renal (ATN)* Urine osmolality >500 <350 Urinary sodium <20 >40 Urine/serum creatinine >40 <20 Urine/serum osmolarity >1.2 <1.2 Fractional excreted sodium† <1 >1 Renal failure index (RFI)‡ <1 >1 6 *Acute tubular necrosis (intrinsic renal failure). †Fractional excreted sodium = Urine / Serum sodium ×100 Urine / Serum creatinine ‡ Urine sodium × Serum creatinine Renal failure index = Urine creatinine Used to assess proximal bowel function; differentiates between malabsorption due to pancreatic insufficiency or intestinal problems. Decreased: Celiac disease (nontropical sprue, gluten-sensitive enteropathy), false de- creas0e with renal disease URINARY INDICES IN RENAL FAILURE Use Table 6–1 to help differentiate the causes (renal or prerenal) of oliguria. (See also Oli- guria and Anuria, page 49.) URINE OUTPUT Although clinical situations vary greatly, the usual, minimal acceptable urine output for an adult is 0.5–1.0 mL/kg/h (daily volume normally 1000–1600 mL/d). URINE PROTEIN ELECTROPHORESIS See Protein Electrophoresis, Serum and Urine, page 85, and Figure 4–5, page 86. This page intentionally left blank. 7 CLINICAL MICROBIOLOGY Staining Techniques Nasopharyngeal Cultures Acid-Fast Stain Blood Cultures Darkfield Examination Sputum Cultures Giemsa Stain Stool Cultures Gonorrhea Smear Throat Cultures Gram Stain Urine Cultures Gram Stain Characteristics Viral Cultures and Serology of Common Pathogens Scotch Tape Test 7 India Ink Preparation Molecular Microbiology KOH Preparation Susceptibility Testing (MIC, MBC, Stool Leukocyte Stain Schlichter Test) Tzanck Smear Differential Diagnosis of Common Vaginal Wet Preparation Infections and Empiric Therapy Wayson Stain SBE Prophylaxis Gonorrhea (GC) Cultures and Smear Isolation Protocols STAINING TECHNIQUES Acid-Fast Stain (AFB Smear, Kinyoun Stain) Clinical microbiology labs can also perform a “modified” acid-fast stain for organisms that are weakly acid-fast-staining (eg, Nocardia species). Procedure 1. Spread the smear on a slide, allow it to air dry, and then gently heat fix it. 2. Stain the smear for 3–5 min with terpinol in carbol-fuchsin red solution. 3. Rinse the slide with tap water. 4. Decolorize with acid–alcohol solution for no longer than 30 s. 5. Rinse with tap water. 6. Counterstain with methylene blue for 1 min. 7. Rinse the slide with tap water and allow it to air dry. 8. Examine the smear with high dry and oil immersion lenses; search for the acid-fast bacilli that stain red to bright pink against the light blue background (Mycobacterium tuberculosis [TB], M. scrofulaceum, M. avium-intracellulare, others). These organisms have a beaded rod appearance under oil immersion. 9. These organisms must be cultured on specialized media. Rapid-growing AFB include M. abscessus, M. chelonae, M. fortuitum and can usually be cultured in fewer than 7 days. Most other AFB (M. tuberculosis, M. avium complex, M. kansasii, M. mari- num) require at least 7–10 d to grow. M. gordonae is thought to be nonpathogenic. 121 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 122 Clinician’s Pocket Reference, 9th Edition Darkfield Examination Darkfield examination is used to identify Treponema pallidum, the organism responsible for syphilis. Rectal and oral lesions cannot be examined by this technique due to the presence

of nonpathogenic spirochetes. Procedure 1. The chancre is cleansed with a saline-moistened swab and a slide is touched on the le- sion and examined under darkfield illumination within 15 min of applying the speci- men to the slide. 2. The organisms resemble tight corkscrews and are 1–1¹₂ times the diameter of an RBC in length. 7 Giemsa Stain Used to identify intracellular organisms such as chlamydiae, Plasmodium spp. (malaria), and other parasites. Gonorrhea Smear (See the following section on Gonorrhea [GC] Cultures) Gram Stain The Gram stain is used to determine whether an organism can be decolorized with alcohol after being stained with crystal violet. This determination is based on the organism’s cell wall characteristics. Gram staining is performed on bacteria from a variety of body fluids, including exudates, abscesses, sputum, and others as clinically indicated. Procedure 1. Smear the specimen (sputum, peritoneal fluid, etc) on a glass slide in a fairly thin coat. If time permits, allow the specimen to air dry. The smear may also be fixed under very low heat (excessive heat can cause artifacts). If a Bunsen burner is not available, other possible methods for heating the sample include using a hot light bulb or setting an al- cohol swab on fire. Heat the slide until it is warm, but not hot, when touched to the back of the hand. 2. Timing for the stain is not critical, but allow at least 10 s for each set of reagents. 3. Apply the crystal violet (Gram stain), rinse the slide with tap water, apply iodine so- lution, and rinse with water. 4. Decolorize the slide carefully with the acetone–alcohol solution until the blue color is barely visible in the runoff. (Be careful; this is the step where most Gram stains are ruined.) 5. Counterstain with a few drops of safranin, rinse the slide with water, and blot it dry with lint-free bibulous or filter paper. 6. Use the high dry (100×) and oil immersion lenses on the microscope to examine the slide. If the Gram stain is satisfactory, any polys on the slide should be pink with light blue nuclei. On a Gram stain of sputum, an excessive number of epithelial cells (>25/hpf) means the sample contained more saliva than sputum. Gram-positive organ- isms stain dark blue to purple; gram-negative ones stain red. Gram Stain Characteristics of Common Pathogens: Initial lab reports identify the Gram stain characteristics of the organisms. Complete identi- fication usually requires culturing the organism. The lab algorithm for gram-positive and 7 Clinical Microbiology 123 Gram stain Gram stain + Cocci Rods (bacilli) Catalase + Catalase – (clusters) (chains) Clostridium (anaerobe) Staphylococcus Streptococcus Corynebacterium Listeria 7 Bacillus Coagulase + Coagulase – S. aureus S. epidermidis S. saprophyticus Hemolysis Green (partial) Clear No hemolysis hemolysis hemolysis α β γ S. pneumoniae* Viridans streptococci Peptostreptococcus Capsule ( + Quellung) (S. mutans) and Optochin sensitive No capsule Enterococcus Optochin resistant Group A Group B Group D S. pyogenes (S. agalactiae) (Enterococcus) Bacitracin sensitive *Important pathogens are in bold type. Note: Enterococcus is Group D but it is not β-hemolytic; it is α- or γ-hemolytic. FIGURE 7–1 Lab algorithm for the identification of gram-positive organisms. (Reprinted, with permission, from: Bhushan V [ed]: First Aid for the USMLE, Step 1, Appleton & Lange, Norwalk, CT, 1999.) 124 Clinician’s Pocket Reference, 9th Edition Gram stain Gram – “Coccoid” rods Haemophilus influenzae (requires factors V and X) Pasteurella–animal bites Brucella–brucellosis 7 Bordetella pertussis Cocci Rods Neisseria meningitidis* N. gonorrhoeae Lactose Lactose Lactose fermenter nonfermenter Fast fermenter Slow fermenter Escherichia coli Citrobacter Oxidase Enterobacter Serratia Klebsiella Others Oxidase – Oxidase + Shigella Pseudomonas Salmonella Proteus *Important pathogens are in bold type. FIGURE 7–2 Lab algorithm for the identification of gram-negative organisms. (Reprinted, with permission, from: Bhushan V [ed]: First Aid for the USMLE, Step 1, Appleton & Lange, Norwalk, CT, 1999.) 7 Clinical Microbiology 125 TABLE 7–1 Gram Stain Characteristics and Key Features of Common Organisms* Gram Staining Pattern and Organisms Identifying Key Features GRAM-POSITIVE COCCI Enterococcus spp. (E. faecalis) Pairs, chains; catalase-negative (Note: These are equivalent group D Streptococcus) Peptostreptococcus spp. Anaerobic 7 Staphylococcus spp. Clusters; catalase-positive Staphylococcus aureus Clusters; catalase-positive; coagulase- negative; beta-hemolytic; yellow pigment Staphylococcus epidermidis Clusters; catalase-positive; coagulase- positive; skin flora Staphylococcus saprophyticus Clusters; catalase-positive; coagulase- positive Streptococcus spp. Pairs, chains; catalase-negative Streptococcus agalactiae (group B) Pairs, chains; catalase-negative; vaginal flora Streptococcus bovis (group D Pairs, chains; catalase-negative Enterococcus) Streptococcus faecalis (group D Pairs, chains; catalase-negative Enterococcus) Streptococcus pneumoniae Pairs, lancet-shaped; alpha-hemolytic; (Pneumococcus, group B) Optochin-sensitive Streptococcus pyogenes (group A) Beta-hemolytic Streptococcus viridans Pairs, chains; catalase-negative; alpha- hemolytic, Optochin-resistant GRAM-NEGATIVE COCCI Acinetobacter spp. Filamentous, branching pattern Moraxella (Branhamella) Diplococci in pairs catarrhalis Neisseria gonorrhoeae Diplococci in pairs, often intracellular; (gonococcus) ferments glucose but not maltose Neisseria meningitidis Diplococci in pairs;ferments glucose (meningococcus) and maltose Veillonella spp. Anaerobic GRAM-POSITIVE BACILLI Actinomyces Branching, beaded, rods; anaerobic Bacilli anthracis (anthrax) Spore forming rod (continued ) 126 Clinician’s Pocket Reference, 9th Edition TABLE 7–1 (Continued) Gram Staining Pattern and Organisms Identifying Key Features GRAM-POSITIVE BACILLI Clostridium spp. (C. difficile, Large, with spores; anaerobic C. botulinum, C. tetani) Corynebacterium spp. Small, pleomorphic diphtheroid; skin (C. diphtheriae) flora 7 Eubacterium spp. Anaerobic Lactobacillus spp. Common vaginal bacterium; anaerobic Listeria monocytogenes Beta-hemolytic Mycobacterium spp. (limited Only rapidly growing species gram staining) stain (M. abscessus, M. chelonae, M. fortuitum) Nocardia Beaded, branched rods; partially acid-fast-staining Propionibacterium acne Small, pleomorphic diphtheroid; anaerobic GRAM-NEGATIVE BACILLI Acinetobacter spp. Lactose-negative, oxidase-negative Aeromonas hydrophilia Lactose-negative (usually), oxidase- positive Bacteroides fragilis Anaerobic Bordetella pertussis Coccoid rod Brucella (brucellosis) Coccoid rod Citrobacter spp. Lactose-positive (usually) Enterobacter spp. Lactose-positive (usually) Escherichia coli Lactose-positive Fusobacterium spp. Long, pointed shape; anaerobic Haemophilus ducreyi (chancroid) Gram-negative bacilli Haemophilus influenzae Coccoid rod, requires chocolate agar to support growth Klebsiella spp. Lactose-positive Legionella pneumophila Stains poorly, use silver stain and special media Morganella morganii Lactose-negative, oxidase-negative Proteus mirabilis Lactose-negative, oxidase-negative, indole-negative Proteus vulgaris Lactose-negative, oxidase-negative, indole-positive (continued ) 7 Clinical Microbiology 127 TABLE 7–1 (Continued) Gram Staining Pattern and Organisms Identifying Key Features GRAM-NEGATIVE BACILLI Providencia spp. Lactose-negative, oxidase-negative Pseudomonas aeruginosa Lactose-negative, oxidase-positive blue-green pigment Salmonella spp. Lactose-negative, oxidase-negative Serratia spp. Lactose-negative, oxidase-negative 7 Serratia marcescens Lactose-negative, oxidative-negative, red pigment Shigella spp. Lactose-negative, oxidase-negative Stenotrophomonas (Xanthomonas) Lactose-negative, oxidase-negative maltophilia Vibrio cholerae (cholera) Gram-negative bacilli Yersinia enterocolitica Gram-negative bacilli Yersinia pestis (bubonic plague) Gram-negative bacilli *Organisms are aerobic unless otherwise specified. gram-negative organisms is shown in Figures 7–1 and 7–2. Gram stain characteristics of clinically important bacteria are shown in Table 7–1. India Ink Preparation India ink is used primarily on CSF to identify fungal organisms (especially cryptococci). KOH Preparation KOH (potassium hydroxide) preps are used to diagnose fungal infections. Vaginal KOH preps are discussed in detail in Chapter 13, page 291. Procedure 1. Apply the specimen (vaginal secretion, sputum, hair, skin scrapings) to a slide. Skin scrapings of a lesion are usually obtained by gentle scraping with a #15 scalpel blade (see page 242 for description). 2. Add 1–2 drops of 10% KOH solution and mix. Gentle heating (optional) may acceler- ate dissolution of the keratin. A fishy odor from a vaginal prep suggests the presence of Gardnerella vaginalis (see page 291) 3. Put a coverslip over the specimen, and examine the slide for the branching hyphae and blastospores that indicate the presence of a fungus. KOH should destroy most elements other than fungus. If dense keratin and debris are present, allow the slide to sit for sev- eral hours and then repeat the microscopic examination. Lowering the substage con- denser provides better contrast between organisms and the background. 128 Clinician’s Pocket Reference, 9th Edition Stool Leukocyte Stain (Fecal Leukocytes, Löeffler Methylene Blue Stain) Used to differentiate treatable diarrhea (ie, bacterial) from other causes. This method detects causes from Crohn’s disease, ulcerative colitis, TB, and amebic infection as well, but it should be remembered that many causes of severe diarrhea are viral. The positive predictive value of the bacterial pathogen as a cause for the diarrhea is 70%. Procedure 1. Mix a small amount of stool or mucus on a slide with 2 drops of Löeffler (methylene blue) stain. Mucus is preferred; if no mucus is present, use a small amount of stool from the outside of a formed stool. 2. Examine the smear after 2–3 min to allow the white cells to take up the stain; then place a coverslip. The presence of many leukocytes suggests a bacterial cause. Increased 7 white cells (usually polys) are seen in Shigella, Salmonella, Campylobacter, Clostridium difficile, and enteropathogenic Escherichia coli infections, as well as ulcerative colitis and pseudo-membranous colitis-related diarrhea. White cells are absent or normal in cholera and in Giardia and viral (rotavirus, Norwalk virus, etc) infections. Tzanck Smear This technique (named after Arnault Tzanck) is used in the diagnosis of herpesvirus infec- tions (ie, herpes zoster or simplex). Procedure 1. Clean a vesicle (not a pustule or crusted lesion) with alcohol, allow it to air dry, and gently unroof it with a #15 scalpel blade. Scrape the base with the blade, and place the material on a glass slide. 2. Allow the sample to air dry, and stain with Wright’s stain as used for peripheral blood. Giemsa stain can also be used, however, the sample must be fixed for 10 min with methyl alcohol before the Giemsa is applied. 3. Scan the slide under low power, and identify cellular areas. Then use high-power oil immersion to identify multinucleated giant cells (epithelial cells infected with herpes viruses). This strongly suggests viral infection; culture is necessary to identify the spe- cific virus. Vaginal Wet Preparation • See Chapter 13, page 291 Wayson Stain Wayson stain is a good quick scout stain that colors most bacteria. Procedure 1. Spread the smear on a slide, and air or heat dry it. 2. Pour freshly filtered Wayson stain onto the slide, and allow it to stand for 10–20 s (tim- ing is not critical). 3. Rinse the slide gently with tap water, and dry it with filter paper. 4. Use the high dry and oil immersion lenses to examine the slide. 7 Clinical Microbiology 129 GONORRHEA (GC) CULTURES AND SMEAR Neisseria gonorrhea can be cultured from many different sites, including female genital tract (endocervix is the preferred site), male urethra, urine, anorectum, throat, and synovial fluid, and the specimen is plated on selective (Thayer–Martin or Transgrow) media. Due to the high incidence of coinfection with Chlamydia and T. pallidum (syphilis), Chlamydia cultures and syphilis serology should also be performed, especially in females with genital infections with GC. Anorectal stains may contain nonpathogenic Neisseria species; avoid fecal contact; apply swab to anal crypts. In males with a urethral discharge, insert a calcium alginate swab (Calgiswab) into the urethra to collect the specimen and then plate. The GC smear (see Chapter 13, page 291) has a low sensitivity (<50% in female endo- cervical smear, but is fairly reliable (>95%) in males with urethral discharge. A rapid en- zyme immunoassay (gonococcal antigen assay [Gonozyme]) is available to diagnose cervical or urethral GC (not throat or anus) infections in less than 1 h. DNA probe testing is 7 becoming widespread for rapid diagnosis. NASOPHARYNGEAL CULTURES Ideally, the specimen for culture should be obtained from deep in the nasopharynx and not the anterior nares, and the swab should not touch the skin. Cultures of nasopharyngeal spec- imens are useful in identifying Staphylococcus aureus and N. meningitidis infections. Normal nasal flora include Staphylococcus epidermidis and S. aureus, Streptococcus pneu- moniae, Haemophilus influenzae, and several others. BLOOD CULTURES Blood cultures are not usually indicated for the routine workup of fever. The best use is for 1. Fever of unknown origin, especially in adults with white blood counts of > 15,000/mm3 and no localizing signs or symptoms to suggest the source. 2. Clinical situations in which the diagnosis is established by a positive blood culture (eg, acute and SBE). 3. Febrile elderly, neutropenic, or immunocompromised patients. Chills and fever usually ensue from ¹₂–2 h after sudden entry of bacteria into the circu- lation (bacteremia). If bacteremia is suspected, several sets of cultures are usually needed to improve the chances of culturing the offending organism. Ideally, more than one set of cul- tures should be done at least 1 h apart; drawing more than three sets of specimens a day does not usually increase the yield. Obtain the blood through venipuncture,

and avoid sampling through venous lines. Each “set” of specimens for blood culture consists of both an aerobic and anaerobic culture bottle. If possible, culture the specimens before antibiotics are initi- ated; if the patient is already on antibiotics, use ARD culture bottles, which absorb the an- tibiotic that may otherwise destroy any bacteria. Legionella, Mycobacterium, Bordetella, and Histoplasma may require special blood collection devices. Procedure 1. Review the section on the technique of venipuncture (Chapter 13 page 309). Apply a tourniquet above the chosen vein. 2. Paint the venipuncture site with a povidone–iodine solution. Repeat this procedure three times with a different pad. Then wipe the area around the vein with alcohol and allow the alcohol to dry. 3. Use an 18–22-gauge needle (or smaller if needed) and a 10–20-mL syringe. Enter the skin over the prepped vein, and aspirate a sufficient volume of blood (10–20 mL in adults, 130 Clinician’s Pocket Reference, 9th Edition 1–5 mL in children); adequate volume will increase the detection rate. Be careful not to touch the needle or the prepped skin site. Draw about 10 mL of blood. Remove the tourniquet, and compress the venipuncture site and apply an adhesive bandage. 4. Discard the needle used in the puncture and replace it with a new, sterile 20–22- gauge needle. Place the blood in each of the bottles by allowing the vacuum to draw in the appropriate volume, usually specified on the collection device. Submit the samples to the lab promptly with the appropriate lab slips completed including current antibiotics being given. Interpretation Preliminary results are usually available in 12–48 h; cultures should not be formally re- ported as negative before 4 d. A single blood culture that is positive for one of the following organisms usually suggests contamination; however, on rare occasions these agents are the causative pathogen: Staphylococcus epidermidis, Bacillus sp., Corynebacterium diphtheriae 7 (and other diphtheroids), Streptococcus viridans. Negative results do not rule out bac- teremia, and false-positives can result for the contaminants noted. Gram-negative organ- isms, fungi, and anaerobes are considered to be pathogenic until proven otherwise. SPUTUM CULTURES Cultures of sputum remain controversial. Many clinicians do not even order them and treat only based on the Gram stain and clinical findings. One problem is that “sputum” samples often contain only saliva. If you do a Gram stain on the specimen and see only a few squa- mous cells, with many polys and histiocytes, the sample is good, and the culture will proba- bly be reliable. Excessive numbers of squamous cells (see previous section on Gram stain) suggests that the sample is more saliva than sputum. An early morning sample is most likely to be from deep within the bronchial tree. Steps to improve the quality of the sputum collection 1. Careful instructions to the patient. 2. If the patient cannot mobilize the secretions, P&PD along with nebulizer treatments may help. 3 Careful nasotracheal suctioning using a specimen trap. In general most labs will not accept anaerobic sputum cultures (critical in the diagnosis of aspiration pneumonia and lung abscesses) unless obtained by transtracheal aspirate or en- dobronchial endoscopic collection and submitted in special anaerobic transport media. Viral, Legionella, Mycoplasma, and TB cultures require special culture materials available at most labs. PCP can be diagnosed by sputum culture only about 10% of the time; there- fore open-lung biopsy, endobronchial lavage, or other invasive techniques must be used to demonstrate the organisms. Specialized staining techniques for identifying Pneumocystis carinii include the methenamine silver, Giemsa, and toluidine blue stains. STOOL CULTURES Stool cultures are most often done to diagnose the cause of diarrhea or to identify disease carriers. A fresh sample is essential to isolate the organisms. Most common pathogens (Sal- monella, Shigella, enteropathogenic E. coli, etc) can be grown on standard media. Yersinia and Campylobacter, however, usually require a special culture medium, and a special lab re- quest is usually necessary. A quick bedside test for bacterial causes of diarrhea is to check the stool for white cells (fecal leukocyte smear) see page 128. 7 Clinical Microbiology 131 Clostridium difficile Assay Clostridium difficile is usually best diagnosed by determining the presence of C. difficile en- terotoxin on the stool and not by culture. A positive C. difficile assay is found in the follow- ing cases: >90% of pseudo-membranous colitis; 30–40% antibiotic associated colitis, and 6–10% cases of antibiotic-associated diarrhea. Stool for Ova and Parasites With toxic diarrhea, the possibility of parasitic disease must be considered and stool for “ova and parasites” should be ordered. Protozoa (ameba [Entamoeba histolytica, others], Blastocystis, Giardia) cannot be cultured and are identified by seeing mature, mobile organ- isms or cysts on microscopic examination of freshly passed feces. Immunosuppressed (eg,. HIV-positive) individuals may demonstrate Cryptosporidium, Microsporidia, and Strongy- loides. The ova are most frequently identified in the stool of parasites such as nematodes 7 (Ascaris, Strongyloides), cestodes (Taenia, Hymenolepsis), and trematodes (Schistosoma). THROAT CULTURES Used to differentiate viral from bacterial (usually group A beta-hemolytic streptococci, eg, Streptococcus pyogenes) pharyngitis. Procedure 1. The best culture is obtained with the help of a tongue blade and a good light source. 2. If epiglottitis (croup) is suspected (stridor, drooling), a culture should not be at- tempted. 3. The goal is to use the culture swab and try not to touch the oral mucosa or tongue, but only the involved area. In the uncooperative patient, an arch-like swath touching both the tonsillar areas and posterior pharynx should be attempted. Many labs perform a specific “strep screen” to rapidly identify group A beta-hemolytic streptococci. Normal flora on routine culture can include alpha-hemolytic strep, non- hemolytic staph, saprophytic Neisseria species, Haemophilus, Klebsiella, Candida, and diphtheroids. Other pathogens can cause pharyngitis. If Neisseria gonorrhoeae is suspected, use the Thayer–Martin medium. Diphtheria (C. diphtheriae) with its characteristic pseudo-mem- brane, should be cultured on special media and the lab notified. URINE CULTURES As is true for sputum cultures, culturing for urinary tract pathogens is often controversial. Some clinicians base their decision to treat only when the culture is positive, whereas others rely on the presence of white blood cells or bacteria in the urinalysis, using cultures only for sensitivities in refractory infections. The introduction of urine dipsticks to detect leukocytes (by the detection of leukocyte esterase) aids in the decision making when cultures are not obtained or are confusing. Routine cultures fail to diagnose other urinary tract pathogens such as N. gonorrhea or Chlamydia. A clean-catch urine (see Chapter 13, page 306) is about 85% accurate in women and uncircumcised males. In general, a positive culture is a colony count of >100,000 bacte- ria/mL of urine or a count from 10,000–100,000 bacteria/mL of urine in the presence of pyuria. If the culture is critical for diagnosis, obtain an in-and-out catheterized urine (page 132 Clinician’s Pocket Reference, 9th Edition 308) or suprapubic aspiration in children (page 309). Any growth of bacteria on an in-and- out catheterized or suprapubic specimen is considered to represent a true infection. If a urine specimen cannot be taken to the lab within 60 min, refrigerate it. The lab as- sumes that more than three organisms growing on a culture represents a contaminant and the specimen collection should be repeated. The exception occurs in patients with a chronic in- dwelling Foley catheter that may be colonized with multiple bacterial or fungal organisms; the lab should be told to “culture all organisms” in such cases. VIRAL CULTURES AND SEROLOGY The laboratory provides the proper collection container for the specific virus. Common pathogenic viruses cultured include herpes simplex (from genital vesicles, throat), CMV (from urine or throat), varicella-zoster (from skin vesicles in children with chickenpox and 7 adults with shingles), and enterovirus (rectal swab, throat). For serologic testing, obtain an acute specimen (titer) as early as possible in the course of the illness, and take a convalescent specimen (titer) 2–4 wk later. A fourfold or greater rise in the convalescent titer compared with the acute titer indicates an active infection (see Chapter 4 for selected viral antibody titers). With the development of PCR techniques, biopsies performed on older lesions may yield useful information when cultures might be negative. SCOTCH TAPE TEST Also known as a “pinworm preparation,” this method is used to identify infestation with En- terobius vermicularis. A 3-in. piece of CLEAR Scotch tape is attached around a glass slide (sticky side out). The slide is applied to the perianal skin in four quadrants and examined under the microscope for pinworm eggs. The best sample is collected either in the early morning prior to bathing or several hours after retiring. MOLECULAR MICROBIOLOGY Molecular techniques can now identify many bacterial and viral organisms without cultur- ing. Many tests rely on DNA probes to identify the pathogens. The following includes some microbes commonly identified from clinical specimens (ie, swab, serum, tissue). Availabil- ity varies with each clinical facility. Common Microorganisms Identifiable by PCR/DNA Probe • Chlamydia trachomatis • Borrelia burgdorferi (Lyme disease) • HIV • Mycoplasma pneumoniae • Mycobacterium tuberculosis • Neisseria gonorrhoeae • Hepatitis B • HPV • Many others under development 7 Clinical Microbiology 133 SUSCEPTIBILITY TESTING To more effectively treat a specific infection by choosing the right antibiotic, many labs rou- tinely provide the MIC or MBC. For more complex infections (endocarditis), Schlichter testing is sometimes used. MIC (Minimum Inhibitory Concentration) This is the lowest concentration of antibiotic that prevents an in vitro growth of bacteria. The organism is tested against a battery of antimicrobials in concentrations normally achieved in vivo and reported as Susceptible (S): The organism is inhibited by the agent in the usual dose and route, and the drug should be effective. Intermediate (I): Sometimes also reported as “indeterminate,” this implies that high 7 doses of the drug, such as those achieved with parenteral therapy (IM, IV), most likely in- hibit the organism. Resistant (R): The organism is resistant to the usual levels achieved by the drug. MBC (Minimum Bactericidal Concentration) Similar to the MIC, but indicates the lowest antibiotic concentration that will kill 99.9% of the organisms. The MBC results in killing the organisms, and the MIC prevents growth but may not kill the organism. Schlichter Test (Serum Bacteriocidal Level) Used to determine the antibacterial level of the serum or CSF of patients who are receiving antibiotic therapy. The test uses eight serial dilutions of the patient’s serum (1:1 through 1:128) to determine what dilution is bactericidal to the infecting organism. The test is usu- ally coordinated by the departments of infectious disease and microbiology. One set of blood or CSF cultures must be negative for the infecting organism before the test is per- formed. Opinion varies greatly as to interpretation of the results. Optimal killing of the or- ganism occurs at dilutions of blood (and CSF) ranging anywhere from a trough of 1:4 to a peak of 1:8. That is, a result such as “S. aureus bactericidal level = 1:8” means the infecting organism was killed at a serum dilution of 1:8. Some data suggest higher titers (1:32) are needed to treat bacterial endocarditis. For the test to be performed, the organisms responsi- ble for the infection must be isolated from a patient specimen. DIFFERENTIAL DIAGNOSIS OF COMMON INFECTIONS AND EMPIRIC THERAPY The pathogens causing common infectious diseases are outlined in Table 7–2 along with some empiric therapeutic recommendations. The antimicrobial drug of choice for the treat- ment of infection is usually the most active drug against the pathogenic organism or the least toxic alternative among several effective agents. The choice of drugs is modified by the site of infection, clinical status (allergy, renal disease, pregnancy, etc), and susceptibility testing. Tables 7–3 through 7–7 provide empiric treatment guidelines for some common infec- tious diseases, including bacterial, fungal, viral, HIV, parasitic, and tick-borne diseases. 7 134 TABLE 7–2 Organisms Responsible for Common Infectious Diseases with Recommended Empiric Therapy* Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) BONES AND JOINTS Osteomyelitis Staphylococcus aureus Oxacillin, nafcillin Enterobacteriaceae If nail puncture: Pseudomonas spp. Joint, septic arthritis S. aureus Oxacillin; ceftriaxone if gonococci Group A strep Enterobacteriaceae Gonococci Joint, prosthetic S. aureus, S. epididymis, Vancomycin plus ciprofloxacin Streptococcus spp. BREAST Mastitis, postpartum S. aureus Cefazolin, nafcillin, oxacillin BRONCHITIS In adolescent/young patient: Treatment controversial because most infections Mycoplasma pneumoniae are viral; treat if febrile,

or associated with Respiratory viruses sinusitis, positive sputum culture in patients In chronic adult infection: with COPD or if duration >7 days; doxy- Streptococcus pneumoniae cycline, erythromycin, azithromycin, Haemophilus influenzae clarithromycin M. catarrhalis Chlamydia pneumoniae (continued ) 7 135 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) CERVICITIS Chlamydia, M. hominis, Azithromycin single dose, doxycycline (nongonococcal) Ureaplasma, others (evaluate and treat partner) CHANCHROID Haemophilus ducreyi Ceftriaxone or azithromycin as single dose CHLAMYDIA Urethritis, cervicitis, conjunctivitis, Chlamydia trachomatis Azithromycin, doxycycline (amoxicillin if proctitis pregnant) Neonatal ophthalmia, pneumonia Erythromycin Lymphogranuloma C. trachomatis (specific serotypes, Doxycycline venereum L1, L2, L3) DIVERTICULITIS Enterobacteriaceae, enterococci, TMP–SMX, ciprofloxacin plus metronidazole (no perforation or peritonitis) bacteroids EAR Acute mastoiditis S. pneumoniae Amoxicillin, ampicillin/clavulanic acid, Group A strep cefuroxime S. aureus Chronic mastoiditis Polymicrobial: Anaerobes Ticarcillin/clavulanic acid, imipenem Enterobacteriaceae Rarely: M. tuberculosis Otitis externa (swimmer’s ear) Pseudomonas spp. Topical agents such as Cortisporin otic, Enterobacteriaceae TobraDex (continued ) 7 136 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) EAR Otitis externa (continued) In diabetic or malignant otitis: Malignant otitis externa: acutely aminoglycoside, Pseudomonas spp. plus ceftazidime, imipenem or piperacillin Otitis media S. pneumoniae, H. influenzae, Amoxicillin, ampicillin/clavulanic acid, M. catarrhalis, viral causes cefuroxime S. aureus, group A strep In nasal intubation: Enterobacteriaceae, Pseudomonas spp. EMPYEMA S. pneumoniae, S. aureus Cefotaxime, ceftriaxone ENDOCARDITIS Native valve S. viridans Parenteral: penicillin or ampicillin plus oxacillin S. pneumoniae or nafcillin plus gentamicin; vancomycin Enterococci plus gentamicin S. bovis IV drug user S. aureus Nafcillin plus gentamicin Pseudomonas spp. Prosthetic valve If early (<6 mo after implant) Vancomycin plus rifampin plus gentamicin S. epidermidis S. aureus Enterobacteriaceae (continued ) 7 137 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) Prosthetic valve (continued) If late (>6 mo after implant) S. viridans Enterococci S. epidermidis S. aureus EPIGLOTTITIS H. influenzae Chloramphenicol plus ceftriaxone, cefotaxime S. pneumoniae or ampicillin S. aureus Group A strep GALL BLADDER Cholecystitis Acute: E. Coli, Klebsiella, Ampicillin plus gentamicin w/wo metronidazole, Enterococcus imipenem Chronic obstruction: anaerobes, coliforms, Clostridium Cholangitis E. coli, Klebsiella, Enterococcus GASTROENTERITIS Afebrile, no gross blood or no Virus, mild bacterial infection Supportive care only WBC in stool Febrile, gross blood, and WBC Enteropathogenic E. coli Empiric treatment pending cultures: in stool Shigella ciprofloxacin, norfloxacin Salmonella (continued ) 7 138 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factor such as Gram stain) Febrile gastroenteritis (continued) Campylobacter Vibrio C. difficile L. monocytogenes GRANULOMA INGUINALE Calymmatobacterium granulomatis Doxycycline, trimethoprim/sulfamethoxazole GONORRHEA N. gonorrhea Cefixime, ciprofloxacin, ofloxacin, (urethra, cervix, rectal, ceftriaxone all as single dose; (treat also pharyngeal) for Chlamydia) MENINGITIS (Empiric therapy before cultures) Neonate Group B strep, E. coli, Listeria Ampicillin plus cefotaxime monocytogenes Infant 1–3 mo S. pneumoniae N. meningitidis Child/adult, community acquired S. pneumoniae Vancomycin plus ceftriaxone N. meningitidis, H. influenzae Postoperative or traumatic S. epidermitis, S. aureus, Vancomycin plus ceftazidime S. pneumoniae, Pseudomonas Immunosuppressed (ie steroids) Gram-negative bacilli, L. monocytogenes Ampicillin plus ceftazidime History of alcohol abuse S. pneumoniae Ampicillin plus ceftriaxone or cefotaxime N. meningitidis, gram-negative bacilli (continued ) 139 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clini7c factors such as Gram stain) Meningitis (continued) Pseudomonas spp. H. influenzae HIV infection Cryptococcus Amphotericin B (acutely), fluconazole NOCARDIOSIS Nocardia asteroides Sulfisoxazole, TMP–SMX PELVIC INFLAMMATORY DISEASE Gonococci Ofloxacin and metronidazole or ceftriaxone Enterobacteriaceae (single dose) plus doxycycline; parenteral Bacteroides spp. cefotetan or cefoxitin plus doxycycline Chlamydia Enterococci M. hominis PERITONITIS Primary (spontaneous) S. pneumoniae Cefotaxime or ceftriaxone Enterobacteriaceae Secondary to (bowel Enterobacteriaceae, Bacteroides spp. Suspect small bowel: piperacillin, mezlocillin, perforation, etc) Enterococci meropenem, cefoxitin Pseudomonas spp. Suspect large bowel: clindamycin plus aminoglycoside Peritoneal dialysis-related S. epidermidis Based on culture S. aureus Enterobacteriaceae Candida (continued ) 7 140 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) PHARYNGITIS Respiratory virus Exudative (group A strep): benzathine penicilllin Group A strep G, erythromycin, loracarbef, azithromycin Gonococci C. diphtheria Epstein–Barr virus (infectious mono); spirochetes, anaerobes PNEUMONIA Neonate Viral (CMV, herpes), bacterial Ampicillin or nafcillin plus gentamicin (group B strep, L. monocytogenes, coliforms, S. aureus, Chlamydia) Infant (1–24 mo) Most viral such as RSV; S. pneumonia, Cefuroxime; if critically ill, cefotaxime, Chlamydia, Mycoplasma ceftriaxone plus cloxacillin Child (3 mo– 5 y) As above Erythromycin, clarithromycin; if critically ill, cefuroxime plus erythromycin Child (5–18 y) Mycoplasma, respiratory viruses, Clarithromycin, azithromycin; erythromycin S. pneumoniae, C. pneumoniae Adult community-acquired M. pneumoniae, C. pneumoniae, Clarithromycin, azithromycin S. pneumoniae If hospitalized, third-generation cephalosporin Smokers: As above plus plus erythromycin or azithromycin M. catarrhalis, H. influenzae (continued ) 7 141 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) Adult, community-acquired S. pneumoniae oral flora, including Clindamycin aspiration anaerobes (eg, Fusobacterium, Bacteroides sp.) Enterobacteriaceae Adult hospital-acquired or S. pneumonia, coliforms, Imipenem, meropenem ventilator-associated Pseudomonas, Legionella HIV-associated Pneumocystis Pneumocystis: TMP–SMX; may require steroids Others as above TB, fungi SINUSITIS S. pneumoniae Acute: TMP-SMX ampicillin, amoxicillin/ H. influenzae clavulanic acid, ciprofloxacin, clarithromycin M. catarrhalis Anaerobes In nosocomial, nasal intubations, etc: S. aureus Pseudomonas spp. Enterobacteriaceae SKIN/SOFT TISSUE Acne Propionibacterium acne Tetracycline, minocycline, topical clindamycin Acne rosacea Possible skin mite Topical: metronidazole, doxycycline Burns S. aureus, Enterobacteriaceae, Topical: silver sulfadiazine (continued ) 7 142 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) Burns (continued) Pseudomonas, Proteus Sepsis: Aztreonam or tobramycin plus Herpes simplex virus, Providencia, cefoperazone, ceftazidime or piperacillin Serratia, Candida Bite (human and animal) Anaerobes Ampicillin/sulbactam IV or amoxicillin/ P. multiloculada clavulanic acid PO Cellulitis Streptococcus spp. (group, A. B. C, G) Diabetic: nafcillin, oxacillin with or S. aureus without penicillin; if anaerobic, high-dose Anaerobic penicillin G, cefoxitin, cefotetan Decubitus Group A strep (S. pyogenes) If acutely ill: imipenem, meropenem, Anaerobes, S. aureus, ticarcillin/clavulanic acid Enterobacteria Polymicrobial anaerobic Erysipelas Group A strep (S. pyogenes) Nafcillin, oxacillin, dicloxacillin, cefazolin Impetigo Group A strep Penicillin, erythromycin; oxacillin or S. arueus nafcillin if S. aureus Tinea capitis (scalp) Fungus: Trichophyton spp., Terbinafine, itraconazole, fluconazole, “ringworm” Microsporum spp. Tinea corporis (body) Fungus: Trichophyton spp., Topical: ciclopirox, clotrimazole, econazole, Epidermophyton ketoconazole, miconazole, terconazole, others Tinea unguium Various fungi Itraconazole, fluconazole, terbinafine (continued ) 7 143 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) SYPHILIS Treponema pallidum Benzathine penicillin G one dose; (less than 1 y duration) doxycycline, tetracycline, ceftriaxone TUBERCULOSIS Mycobacterium tuberculosis Pulmonary, HIV (−) INH, rifampin ethambutol plus pyrazinamide at least 6 mo (+/− pyridoxine) TB exposure, PPD (−) Children <5 INH X3 mo (+/− pyridoxine), others observe Prophylaxis in high-risk INH 6–12 mo (+/− pyridoxine) patients (diabetics, IV drug users, immuno- suppressed, etc) PPD + conversion INH 6–12 mo (+/− pyridoxine) URINARY TRACT INFECTIONS Cystitis Enterobacteriaceae Quinolone, TMP–SMX (E. coli most common) S. saprophyticus (young female) Candida Candida: fluconazole or amphotericin B bladder irrigation Urethritis Gonococci, C. trachomatis, Ceftriaxone, cefixime, ciprofloxacin, Trichomonas ofloxacin (all one dose) plus (continued ) 7 144 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) Urethritis (continued) Herpesvirus azithromycin (single dose) or doxycycline Ureaplasma urealyticum (treat partner) Prostatitis, acute <35 y C. trachomatis Ofloxacin Gonococci Coliforms Cryptococcus (AIDS) Prostatitis, acute >35 y Coliforms Quinolone, TMP–SMX; if acutely ill gentamicin/ampicillin IV Prostatitis, chronic Coliforms, enterococci, Long-term ciprofloxacin or ofloxacin bacterial Pseudomonas Pyelonephritis Enterobacteriaceae If acutely ill, gentamicin/ampicillin IV; (E. coli) quinolone, TMP–SMX Enterococci Pseudomonas spp. ULCER DISEASE Helicobacter pylori Omeprazole plus amoxicillin plus (duodenal or gastric, clarithromycin not NSAID related) VAGINA Candidiasis C. albicans Fluconazole, itraconazole C. glabrata, C. tropicalis (continued ) 7 145 TABLE 7–2 (Continued) Site/Condition Common Common Empiric Therapy Uncommon but Important (Modify based on clinic factors such as Gram stain) Trichomonas Trichomonas vaginalis Metronidazole (treat partner) Vaginosis, bacterial Polymicrobial (Gardnerella Metronidazole (PO or vaginal gel); vaginalis, Bacteroides, M. hominis clindamycin, PO or intravaginally *All antimichrobial therapy should be based on complete clinical data, including results of Gram’s stains and cultures. See also Tables 7–3 (Viral), 7–4 (HIV), 7–5 (Fungal), and 7–6 (Parasitic) 7–7 (Tick-Borne). Note: These guidelines are based on agents commonly involved in adult infections. Actual microbial treatment should be guided by microbiologic studies inter- preted in the clinical setting. Abbreviations: AIDS = acquired immunodeficiency syndrome; COPD = chronic obstructive pulmonary disease; HIV = human immunodeficiency virus; INH = isoniazid; IV = intravenous; NSAID = nonsteroidal antiinflammatory drug; PO = by mouth; PPD = purified protein derivative; TB = tuberculosis; TMP–SMX = trimethoprim–sulfamethoxazole. 7 146 TABLE 7–3 Pathogens and Drugs of Choice for Treating Common Viral Infections* Viral Infection Drug of Choice Adult Dosage CMV Retinitis, colitis, esophagitis Ganciclovir (Cytovene)† 5 mg/kg IV q12h × 14–21d, 5 mg/kg/d IV or 6 mg/kg IV 5×/wk or 1 g PO tid (Vitrasert) implants 4.5 mg intraocularly q 5–8 mo or Foscarnet (Foscavir) 60 mg/kg IV q8h or 90 mg/kg IV q1–2 h x 14–21 d followed by 90–120 mg/kg/d IV or Cidofovir (Vistide) 5 mg/kg/wk IV × 2 wk, then 5 mg/kg IV q2 wk or Fomivirsen (Vitravene) 330 µg intravitreally q2 wk × 2 then 1/mo EBV Infectious mononucleosis None HAV None, but gamma globulin 0.2 mL/kg IM × 1 within 2 wk of exposure may limit infection HBV Chronic hepatitis Lamivudine (Epivir HBV) 100 mg PO 1×/d × 1–3 y Interferon alfa-2b (Intron A) 5 million units/d or 10 million units 3×/wk SC or IM × 4 mo HCV Chronic hepatitis Interferon alfa-2b plus 3 million units 3×/wk SC plus ribavirin Ribavirin (Rebetron) 1000–1200 mg/d PO × 12 mo Interferon alfa-2b (Intron A) 3 million units SC or IM 3x/wk × 12–24 mo Interferon alfa-2a (Roferon-A) 3 million units SC or IM 3x/wk × 12–24 mo (continued ) 7 147 TABLE 7–3 (Continued) Viral Infection Drug of Choice Adult Dosage Chronic hepatitis (continued) Interferon alfacon-1 (Infergen) 9 µg 3×/wk × 6 mo HSV Orolabial herpes in the Penciclovir (Denavir) 1% cream applied q2h while awake × 4 d immunocompetent with multiple recurrences Genital herpes first episode Acyclovir (Zovirax) 400 mg PO tid or 200 mg PO 5×/d × 7–10 d or Famciclovir (Famvir) 250 mg PO tid × 5–10 d or Valacyclovir (Valtrex) 1 g PO bid × 7–10 d recurrence Acyclovir (Zovirax) 400 mg PO tid × 5 d or Famciclovir (Famvir) 125 mg PO bid × 5 d 17 or Valacyclovir (Valtrex) 500 mg PO bid × 5 d chronic suppression Acyclovir (Zovirax) 400 mg PO bid or Valacyclovir (Valtrex) 500–1000 mg PO 1×/d or Famciclovir (Famvir) 250 mg PO bid Mucocutaneous in the Acyclovir (Zovirax) 5 mg/kg IV q8h × 7–14 d immunocompromised or Acyclovir (Zovirax) 400 mg PO 5x/d × 7–14 d Encephalitis Acyclovir (Zovirax) 10–15 mg/kg IV q8h × 14–21 d Neonatal Acyclovir (Zovirax) 20 mg/kg IV q8h × 14–21 d Acyclovir-resistant Foscarnet (Foscavir) 40 mg/kg IV q8h × 14–21 d Keratoconjunctivitis Trifluridine (Viroptic) 1 drop 1% solution topically, q2h, up to 9 gtt/d × 10 d HIV (See Table 7–4) INFLUENZA A AND B VIRUS Zanamivir (Relenza) 10 mg bid × 5d by inhaler Oseltamivir (Tamiflu) 75 mg PO bid × 5 d (continued ) 7 148 TABLE 7–3 (Continued) Viral Infection Drug of Choice Adult Dosage INFLUENZA A VIRUS Rimantadine (Flumadine) 200 mg PO 1×/d or 100 mg PO bid × 5 d Amantadine (Symmetrel) 100 mg PO bid × 5 d MEASLES Children None (immunize, See Table 22–9) Adults None or ribavirin 20–35 mg/kg/d × 7 d PAPILLOMA VIRUS (HPV) Anogenital warts Podofilox or podophyllin Topical application (see Chapter 22) Interferon alfa-2b (Intron A) 1 million units intralesional 3×/wk × 3 wk Imiquimod, 5% cream (Aldara) Apply 3/wk hs, remove 6–10 h later up to 16 wk RSV (bronchiolitis) Ribavirin (Virazole) Aerosol treatment 12–18 h/d × 3–7 d VZV Exposure prophylaxis in the VZIG, Varicella Zoster Immune See package insert immunocompromised Globulin (HIV, steroids, etc) Varicella (>12 y old) Acyclovir (Zovirax) 20 mg/kg (800 mg max) PO qid × 5 d Herpes zoster Valacyclovir (Valtrex) 1 g PO tid × 7 d or Famciclovir (Famvir) 500 mg PO tid × 7 d or Acyclovir (Zovirax) 800 mg PO

5x/d × 7–10 d (continued ) 7 149 TABLE 7–3 (Continued) Viral Infection Drug of Choice Adult Dosage Varicella or zoster in the Acyclovir (Zovirax) 10 mg/kg IV q8h × 7 d immunocompromised Acyclovir-resistant Foscarnet (Foscavir) 40 mg/kg IV q8h × 10 d *Based on Guidelines from the CDC published in MMWR and the Medical Letter Vol. 41 December 3, 1999. †The generic drug name appears in regular type; the trade name appears in parentheses afterward in italics. Abbreviations: CMV = cytomegalovirus; EBV = Epstein–Barr virus; HAV = hepatitis A virus; HBV = hepatitis B virus; HCV = hepatitis C virus; HIV = human im- munodeficiency virus; HPV = human papilloma virus; HSV = herpes simplex virus; RSV = respiratory syncytial virus; VZV = varicella zoster virus. 150 Clinicians Pocket Reference, 9th Edition TABLE 7–4 Drugs of Choice for Treating HIV Infection in Adults DRUGS OF CHOICE 2 nucleosides1 + 1 protease inhibitor2 2 nucleosides1 + 1 nonnucleoside3 2 nucleosides1 + ritonavir4 + another protease inhibitor5 ALTERNATIVES 1 protease inhibitor2 + 1 nucleoside + 1 nonnucleoside3 2 protease inhibitors (each in low dose)5 + 1 nucleoside + 7 1 nonnucleoside3 abacavir + 2 other nucleosides1 2 protease inhibitors (each full dose) 1. One of the following: zidovudine + lamivudine; zidovudine + didanosine; stavudine + lamivudine; stavudine + didanosine; zidovudine + zalcitabine. 2. Nelfinavir, indinavir, saquinavir soft gel capsules, amprenavir or ritonavir. Ritonavir is used less frequently because of troublesome adverse effects. The Invirase formulation of saquinavir generally should not be used. 3. Efavirenz is often preferred. Nevirapine causes more adverse effects. Nevirapine and delavirdine require more doses, and have had shorter follow-up in reported studies. Combinations of Efavirenz and nevirapine with protease inhibitors require increasing the dosage of the protease inhibitor. 4. Ritonavir is usually given in dosage of 100–400 mg bid when used with another pro- tease inhibitor. 5. Protease inhibitors that have been combined with ritonavir 100–400 mg bid include indinavir 400–800 mg bid, amprenavir 600–800 mg bid, saquinavir 400–600 mg bid and nelfinavir 500–750 mg bid. Source: Reproduced, with permission, from The Medical Letter Vol 42, Issue 1089, Janu- ary 10, 2000. 7 151 TABLE 7–5 Systemic Drugs for Treating Fungal Infections Infection Drug of Choice Alternatives ASPERGILLOSIS Amphotericin B or itraconazole Amphotericin B lipid complex, amphotericin cholesteryl complex liposomal amphotericin B BLASTOMYCOSIS Itraconazole or amphotericin B Fluconazole CANDIDIASIS Oral (thrush) Fluconazole or itraconazole Nystatin lozenge or swish and swallow Stomatitis, eosphagitis, Fluconazole or itraconazole Parenteral or oral amphotericin B vaginitis in AIDS Systemic Amphotericin B or fluconazole Cystitis/vaginitis See Table 7–2 COCCIDIOIDOMYCOSIS Pulmonary (normal individual) No drug usually recommended Pulmonary (high risk) Itraconazole or fluconazole Amphotericin B CRYPTOCOCCOSIS In non-AIDS patient Amphotericin B or fluconazole Amphotericin B fluconazole Meningitis (HIV/AIDS) Amphotericin B plus 5-flucytosine; then Amphotericin B lipid complex long-term suppression with fluconazole HISTOPLASMOSIS Pulmonary, disseminated Normal individual Moderate disease: itraconazole Severe: amphotericin B HIV/AIDS Amphotericin B, followed by Itraconazole itraconazole suppression (continued ) 7 152 TABLE 7–5 (Continued) Infection Drug of Choice Alternatives MUCORMYCOSIS Amphotericin B No dependable alternative PARACOCCIDIOIDOMYCOSIS Itraconazole Amphotericin B SPOROTRICHOSIS Cutaneous Itraconazole Potassium iodide 1–5 mL tid Systemic Itraconazole Amphotericin B Abbreviations: AIDS = acquired immunodeficiency syndrome; HIV = human immunodeficiency virus. TABLE 7–6 Drugs for Treating Selected Parasitic Infections Infection Drug Amebiasis (Entamoeba histolytica) Asymptomatic Iodoquinol or paramomycin Mild to moderate intestinal disease Metronidazole or tinidazole Severe intestinal disease, hepatic Metronidazole or tinidazole abscess Ascariasis (Ascaris lumbricoides, Albendazole, mebendazole or roundworm) pyrantel pamoate Cryptosporidiosis (Cryptosporidium) Paromomycin Cutaneous larva migrans (creeping Albendazole, thiabendazole or eruption, dog and cat hookworm ivermectin 7 Cyclospora infection Trimethoprim–sulfamethoxazole Enterobius vermicularis (pinworm) Pyrantel pamoate, mebendazole or albendazole Filariasis (Wuchereria bancrofti, Diethylcarbamazine Brugia malayi, Loa loa) Giardiasis (Giardia lamblia) Metronidazole Hookworm infection (Ancylostoma Albendazole, mebendazole, or duodenale, Necator americanus) pyrantel pamoate Isosporiasis (Isospora belli) Trimethoprim–sulfamethoxazole Lice (Pediculus humanus, P. capitis, 1% permethrin (topical) or 0.5% Phthirus pubis) malathion Malaria (Plasmodium falciparum, P. ovale, P. vivax, and P. malariae) Chloroquine-resistant P. falciparum Quinine sulfate plus doxycycline, tetracycline, clindamycin or pyrimethamine–sulfadoxine (oral) Chloroquine-resistant P. vivax Quinine sulfate plus doxycycline, or pyrimethamine–sulfadoxine (oral) All Plasmodium except chloroquine- Chloroquine phosphate (oral) resistant P. falciparum All Plasmodium (parenteral) Quinine gluconate or quinine dihydrochloride Prevention of relapses: P. vivax, Primaquine phosphate and P. ovale only Malaria, prevention Chloroquine-sensitive areas Chloroquine phosphate Chloroquine-resistant areas Mefloquine or doxycycline Mites, see Scabies Pinworm, see Enterobius Pneumocystis carinii pneumonia Trimethoprim–sulfamethoxazole Alternative: pentamidine Primary and secondary Trimethoprim–sulfamethoxazole prophylaxis (continued ) 153 154 Clinician’s Pocket Reference, 9th Edition TABLE 7–6 (Continued) Infection Drug Roundworm, see Ascariasis Scabies (Sarcoptes scabiei) 5% Permethrin topically Alternatives: ivermectin, 10% crotamiton Strongyloidiasis (Strongyloides Ivermectin stercoralis) Tapeworm infection 7 —Adult (intestinal stage) Diphyllobothrium latum (fish), Praziquantel Taenia saginata (beef), Taenia solium (pork), Dipylidium caninum (dog), Hymenolepis nana (dwarf tapeworm) —Larval (tissue stage) Echinococcus granulosus (hydatid Albendazole cyst) Cysticercus cellulosae (cysticercosis) Albendazole or praziquantel Toxoplasmosis (Toxoplasma gondii) Pyrimethamine plus sulfadiazine Trichinosis (Trichinella spiralis) Steroids for severe symptoms plus mebendazole Trichomoniasis (Trichomonas Metronidazole or tinidazole vaginalis) Hairworm infection (Trichostrongylus Pyrantel pamoate colubriformis) Trypanosomiasis (Trypanosoma cruzi, Benznidazole Chagas’ disease) Trichuriasis (Trichuris trichiuria, Mebendazole or albendazole whipworm) Visceral larva migrans, toxocariasis Albendazole or mebendazole (Toxocara canis) Source: Based on data from The Medical Letter March 2000 www.medletter.com. 7 155 TABLE 7–7 Guide to Common Tick-borne Diseases Causative Disease Agent Season Vector Habits Rocky Mountain spotted Rickettsia rickettsii Mostly spring, summer American Dog Tick fever (bacterium) Found in high grass and low shrubs, fields Lone Star Tick Found in woodlands, forest edge, and old fields Human granulocytic Ehrlichia spp. (bacterium) Under study Deer (black-legged) ehrlichiosis Tick found in woodlands, old fields, landscaping with significant ground cover vegetation Lyme disease Borrelia burgdorferi Mostly spring, but year- Same as for the deer (bacterium) around tick Babesiosis Babesia microti (protozoan) Mostly spring/summer Same as for the deer tick (continued ) 7 156 TABLE 7–7 (Continued) Classic Clinical Incubation Presentation Period Diagnosis Treatment Sudden moderate to 2–14 d Clinical serology Adults—doxycycline high fever, severe Children/pregnant headache, maculopapular women—chloram- rash (with planer/palmer phenicol presentation) Fever, headache, constitutional 1–30 d Clinical serology Adults—tetracyclines symptoms Children/pregnant women— consult specialist EM rash, constitutional symptoms, 3–30 d Clinical serology, culture Doxycycline, amoxicillin, arthritis, cardiovascular- cefuroxime for 14–21 d and nervous system in- volvement Fever, hemolytic anemia, con- 1–52 wk Thick and thin blood smears Clindamycin/quinine stitutional symptoms Abbreviation: EM = erythema multiforme. 7 Clinical Microbiology 157 Secretion/Discharge Precautions: (Handwashing and gloves with direct patient contact) Conjunctivitis, minor skin wounds, decubiti, colonization (but not infection that requires Wound and Skin Precautions) with MRSA, herpes, mucocutaneous candidiasis, ulcerative STDs, coccidioidomycosis, others Pregnancy Precautions: (Handwashing) CMV, rubella, parvovirus SBE PROPHYLAXIS The following recommendations are based on guidelines published by the American Heart Association. (JAMA 1997;277:1794–1801). The guidelines now specify which patients are at high, moderate, or low risk of bacteremia and provide general guidelines for procedures that are more likely to be associated with bacterial endocarditis. SBE prophylaxis is recom- mended only for patients who are at high or moderate risk. See Tables 7–8 and 7–9 for regi- mens. 7 High-risk: Prosthetic cardiac valves, history of bacterial endocarditis, complex cyanotic congenital heart disease, surgically constructed systemic pulmonary shunts Moderate-risk: Most other congenital cardiac malformations (other than those in the pre- vious or following lists), acquired valvular disease (eg, rheumatic heart disease), hyper- trophic cardiomyopathy, mitral valve prolapse with regurgitation or thickened leaflets Low-risk: Isolated ASD secundum; repair of atrial/ventricular septal defect, or PDA; prior CABG; mitral valve prolapse without regurgitation; innocent heart murmurs; previous Kawasaki disease or rheumatic fever without valve dysfunction; pacemakers or implanted defibrillator ISOLATION PROTOCOLS To prevent the spread of infectious diseases from patient to patient, visitors, and hospital personnel, isolation procedures are recommended for various pathogens and clinical settings by various agencies such as the CDC in Atlanta, Georgia. Local hospital procedures may vary slightly from these recommendations. Strict Isolation: (Single room, controlled airflow, handwashing, gown, gloves, mask) Varicella, herpes (localized, disseminated, neonatal), wound or burns infected with S. aureus or group A Streptococcus, S. aureus or group A Streptococcus pneumoniae, congenital rubella, rabies, smallpox, others Contact Isolation: (Single room, controlled airflow, handwashing, gown, gloves, mask) All acute respiratory infections in infants and children (cough, cold, pneumonia, croup, pharyngitis, etc), extensive impetigo, gonococcal conjunctivitis in the newborn, others Respiratory Isolation: (Single room, controlled airflow, handwashing, mask) TB (known or suspected), measles, mumps, rubella, pertussis, meningitis (suspected N. meningitidis or H. influenzae infection), pneumonia due to H. influenzae, epiglottitis, others Wound and Skin Precautions: (Single room; handwashing; for direct contact with pa- tient secretions: gown, gloves, mask) Major wound and skin infections, group A streptococ- cal endometritis, gas gangrene. Scabies and lice require only 24 h after effective therapy. 158 Clinician’s Pocket Reference, 9th Edition Enteric Precautions:(Single room; handwashing; for direct contact with patient secre- tions: gown, gloves) Known or suspected infectious gastroenteritis, including from ro- tavirus, enterovirus, Salmonella, Shigella, E. coli, Giardia, and C. difficile enterocolitis, acute hepatitis (all types) Blood and Body Fluid Precautions:(Handwashing; for direct contact with patient se- cretions: gown, gloves) Known or suspected HIV infection, hepatitis (in acute and chronic carriers), syphilis, malaria, Lyme disease, all rickettsial infections, others TABLE 7–8 SBE Prophylaxis for Oral, Respiratory 7 or Esophageal Procedures* Prophylaxis Agent Regimen† Standard prophylaxis Amoxicillin Adults: 2.0 g; children: 50 mg/kg PO 1 h before procedure Unable to take oral Ampicillin Adults: 2.0 g IM or IV; children: medications 50 mg/kg or IV 30 min before procedure Allergic to penicillin Clindamycin Adults: 600 mg; children: 20 mg/kg or PO 1 h before procedure Cephalexin Adults: 2.0 g; children; 50 mg/kg or cefa- PO 1 h before procedure droxil Azithromycin Adults: 500 mg; children: 15 mg/kg or clarith- PO 1 h before procedure romycin Adults: 600 mg; children: 20 mg/kg IV 30 min before procedure Penicillin allergic and Clindamycin Adults: 1.0 g; children: 25 mg/kg IM unable to take or cefa- or IV 30 min before procedure oral medications zolin *See text page 157 for recommended risk groups. †Total children’s dose should not exceed adult dose. 7 Clinical Microbiology 159 TABLE 7–9 SBE Prophylaxis for GU/GI (Excluding Esophageal) Procedures* Patient Agents Regimen High-risk Ampicillin + Adults: ampicillin 2.0 g IM/IV + gentamicin gentamicin 1.5 mg/kg (max 120 mg) within 30 min of procedure; 6 h later, ampicillin 1 g IM/IV or amoxicillin 1 g PO Children: ampicillin 50 mg/kg IM or IV (2.0 g max) + gentamicin 1.5 7 mg/kg within 30 min of procedure; 6 h later, ampicillin 25 mg/kg IM/IV or amoxicillin 25 mg/kg PO High-risk allergic Vancomycin + Adults: vancomycin 1.0 g IV over to ampicillin/ gentamicin 1–2 h + gentamicin 1.5 mg/kg amoxicillin IV/IM (120 mg max); dose within 30 min of starting procedure Children: vancomycin 20 mg/kg IV over 1–2 h + gentamicin 1.5 mg/kg IV/IM; complete dose within 30 min of starting procedure Moderate-risk Amoxicillin or Adults: amoxicillin 2.0 g PO 1 h ampicillin before procedure, or ampicillin 2.0 g IM/IV within 30 min of starting procedure Children: amoxicillin 50 mg/kg PO 1 h before procedure, or ampi- cillin 50 mg/kg IM/IV within 30 min of starting procedure Moderate-risk Vancomycin Adults: vancomycin 1.0 g IV over allergic to 1–2 h complete infusion within ampicillin/ 30 min of starting procedure amoxicillin Children: vancomycin 20 mg/kg IV over 1–2 h; complete infusion within 30 min of starting procedure *See text page 157 for recommended risk groups. Total children’s dose should not exceed adult dose. This page intentionally left blank. 8 BLOOD GASES AND ACID–BASE DISORDERS Normal Blood Gas Values Metabolic Alkalosis: Diagnosis Venous Blood Gases and Treatment Capillary Blood Gases Respiratory Acidosis: Diagnosis General Principles of Blood Gas and Treatment Determinations Respiratory Alkalosis: Diagnosis Acid–Base Disorders: Definition and Treatment Mixed Acid–Base Disorders Hypoxia Interpretation of Blood Gases Sample Acid–Base Problems Metabolic Acidosis: Diagnosis and Treatment 8 NORMAL BLOOD GAS VALUES The results of testing ABG are usually given as pH, pO − 2, pCO2, [HCO3 ], base excess/deficit (difference), and oxygen saturation. This test gives information on acid–base homeostasis (pH, pCO2, [HCO − 3 ], and base difference) and on blood oxygenation (pO2, O2 saturation). Less frequently, venous blood gases and mixed venous blood gases are measured. Normal values for blood gas analysis are given in Table 8–1, page 162, and capillary blood gases are discussed in a following section. Note that the HCO − 3 from the blood gas is a calculated value and should not be

used in the interpretation of the blood gas levels, instead the HCO − 3 from a chemistry panel should be used. The ABG and the chemistry panel [HCO − 3 ] should be obtained at the same time. VENOUS BLOOD GASES There is little difference between arterial and venous pH and bicarbonate (except in cases of CHF and shock); therefore, the venous blood gas level may occasionally be used to assess acid–base status. Venous oxygen levels, however, are significantly less than arterial levels (see Table 8–1). CAPILLARY BLOOD GASES A CBG is obtained from a highly vascularized capillary bed. (The heel is the most com- monly used site.) The CBG is often used for pediatric patients because it is easier to obtain than the ABG and is less traumatic (no risk of arterial thrombosis, hemorrhage). The proce- dure is fully described in Chapter 13, page 274, under Heelstick. When interpreting a CBG, apply the following rules: • pH: Same as arterial or slightly lower (Normal = 7.35–7.40) • pCO2: Same as arterial or slightly higher (Normal = 40–45) • pO2: Lower than arterial (Normal = 45–60) • O2 Saturation: >70% is acceptable. Saturation is probably more useful than the pO2 itself when interpreting a CBG. 161 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 162 Clinician’s Pocket Reference, 9th Edition TABLE 8–1 Normal Blood Gas Values Arterial Mixed Measurement Blood Venous* Venous pH 7.40 7.36 7.36 (range) (7.37–7.44) (7.31–7.41) (7.31–7.41) pO2 (mm Hg) 80–100 35–40 30–50 (decreases with age) pCO2 (mm Hg) 36–44 41–51 40–52 O2 saturation >95 60–80 60–85 (decreases with age) HCO − 3 (mEq/L) 22–26 22–26 22–28 [SI: mmol/L] 8 Base difference −2 to +2 −2 to +2 −2 to +2 (deficit/excess) *Obtained from the right atrium, usually through a pulmonary artery catheter. GENERAL PRINCIPLES OF BLOOD GAS DETERMINATIONS (Oxygen values are discussed on page 171.) 1. The blood gas machines in most labs actually measure the pH and the pCO2 (as well as the pO2). The [HCO − 3 ] and the base difference are calculated values using the Hender- son–Hasselbalch equation: log[ΗCO− 3 ] in mEq / L pH = pKa + 0.03 × pCO2 in mmHg or the Henderson equation: 2 × [Η + 4 pCO2 in mmHg ] in mEq / L = [HCO− 3 ] in mEq / L 2. For a rough estimate of [H+], [H+] = (7.80 – pH) × 100. This is accurate from a pH 7.25 – 7.48; 40 mEq/L = [H+] at the normal pH of 7.40. Also pH is a log scale, and for every change of 0.3 in pH from 7.40 the [H+] doubles or halves. For pH 7.10 the [H+] = 2 × 40, or 80 nmol/L, and for pH 7.70 the [H+] = ¹₂ × 40, or 20 nmol/L. 3. The calculated [HCO − 3 ] should be within 2 mEq/L of the bicarbonate concentration from a venous chemistry determination (eg, BMP) drawn at the same time. If not, an error has been made in the collection or the determination of the values, and the blood gas and serum bicarbonate should be recollected. 4. Two additional relationships that are derived from the Henderson–Hasselbalch equa- tion should be committed to memory. These two rules are helpful in interpreting blood gas results, particularly in defining a simple versus a mixed blood gas disorder: 8 Blood Gases and Acid–Base Disorders 163 Rule I: A change in pCO2 up or down 10 mm Hg is associated with an increase or de- crease in pH of 0.08 units. As the pCO2 decreases, the pH increases; as the pCO2 increases, the pH decreases. Rule II: A pH change of 0.15 is equivalent to a base change of 10 mEq/L. A decrease in base (ie, [HCO − 3 ]) is termed a base deficit, and an increase in base is termed a base excess. ACID–BASE DISORDERS: DEFINITION 1. Acid–base disorders are very common clinical problems. Acidemia is a pH <7.37, and alkalemia is a pH >7.44. Acidosis and alkalosis are used to describe how the pH changes. The primary causes of acid–base disturbances are abnormalities in the respira- tory system and in the metabolic or renal system. As from the Henderson–Hasselbalch equation, a respiratory disturbance leading to an abnormal pCO2 alters the pH, and sim- ilarly a metabolic disturbance altering the [HCO − 3 ]changes the pH. 2. Any primary disturbance in acid–base homeostasis invokes a normal compensatory response. A primary metabolic disorder leads to respiratory compensation, and a pri- 8 mary respiratory disorder leads to an acute metabolic response due to the buffering ca- pacity of body fluids, and a more chronic compensation (1–2 days) due to alterations in renal function. 3. The degree of compensation is well known and can be expressed in terms of the degree of the primary acid–base disturbance. Table 8–2, page 164, lists the major categories of primary acid–base disorders, the primary abnormality, the secondary compensatory re- sponse, and the expected degree of compensation in terms of the magnitude of the pri- mary abnormality. These changes are defined graphically in Figure 8–1, page 165. The types of simple acid–base disorders are discussed in the following sections. MIXED ACID–BASE DISORDERS 1. Most acid–base disorders result from a single primary disturbance with the normal physiologic compensatory response and are called simple acid–base disorders. In cer- tain cases, however, particularly in seriously ill patients, two or more different primary disorders may occur simultaneously, resulting in a mixed acid–base disorder. The net effect of mixed disorders may be additive (eg, metabolic acidosis and respiratory acido- sis) and result in extreme alteration of pH; or they may be opposite (eg, metabolic acidosis and respiratory alkalosis) and nullify each other’s effects on the pH. 2. To determine a mixed acid–base disorder from a blood gas value, follow the six steps in the Interpretation of Blood Gases (in the following section). Alterations in either [HCO − 3 ] or pCO2 that differ from expected compensation levels indicate a second process. Two of the examples given in the following section illustrate the strategies em- ployed in identifying a mixed acid–base disorder. INTERPRETATION OF BLOOD GASES Use a uniform, stepwise approach to the interpretation of blood gases. (See also Fig- ure 8–1.) Step 1: Determine if the numbers fit. Η + 24 × pCO [ ] = 2 [HCO− 3 ] 8 164 TABLE 8–2 Simple Acid–Base Disturbances Expected Primary Expected Degree of Acid–Base Disorder Abnormality Compensation Compensation Metabolic acidosis ↓↓↓[HCO − 3 ] ↓↓pCO2 pCO2 = (1.5 × [HCO3])+8 Metabolic alkalosis ↑↑↑[HCO − 3 ] ↑↑pCO2 ↑ in pCO2 = ∆ [HCO − 3 ] × 0.6 Acute respiratory ↑↑↑pCO − 2 ↑[HCO3 ] ↑ in [HCO − 3 ] = ∆pCO2/10 acidosis Chronic respiratory ↑↑↑pCO2 ↑↑[HCO − 3 ] ↑ in [HCO − 3 ] = 4 × ∆pCO2/10 acidosis Acute respiratory ↓↓↓pCO2 ↓[HCO − 3 ] ↓in [HCO − 3 ] = 2 × ∆pCO2/10 alkalosis Chronic respiratory ↓↓↓pCO2 ↓↓[HCO − 3 ] ↓in [HCO − 3 ] = 5 × ∆pCO2/10 alkalosis 8 Blood Gases and Acid–Base Disorders 165 Arterial blood [H +] (nmol/L) 100 90 80 70 60 50 40 35 30 25 20 60 120 100 90 80 70 60 50 40 56 52 Meta- 35 48 bolic alkalosis 44 30 Chronic 40 respiratory acidosis 36 Acute 25 32 respiratory acidosis 8 28 Acute 20 respiratory 24 Normal alkalosis 15 20 16 Metabolic 10 12 acidosis Chronic 8 respiratory alkalosis PCO (mm Hg) 2 4 0 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 Arterial blood pH FIGURE 8–1 Nomogram for acid–base disorders. (Reprinted, with permission, from: Cogan MG: Fluid and Electrolytes, Appleton & Lange, Norwalk CT, 1991.) The right side of the equation should be within about 10% of the left side. If the num- bers do not fit, you need to obtain another ABG and chemistry panel for HCO − 3 . Example. pH 7.25, pCO2 48, HCO − 3 29 mmol/L 48 56 = 24 × 29 56 ≠ 40 The blood gas is uninterpretable, and the ABG and HCO − 3 need to be recollected. The most common reason for the numbers not fitting is that the ABG and the chemistry panel [HCO − 3 ] were obtained at different times. Step 2: Next, determine if an acidemia (pH <7.37) or an alkalemia (pH >7.44) is present. Arterial plasma [HCO –3 ] (meq/L) 166 Clinician’s Pocket Reference, 9th Edition Step 3: Identify the primary disturbance as metabolic or respiratory. For example, if acidemia is present, is the pCO2 >44 mm Hg (respiratory acidosis), or is the [HCO − 3 ] <22 mmol/L (metabolic acidosis). In other words, identify which component, respiratory or metabolic, is altered in the same direction as the pH abnormality. If both components act in the same direction (eg, both respiratory [pCO2 > 44 mm Hg] and metabolic [HCO − 3 <22 mmol/L] acidosis are present), then this is a mixed acid–base problem, discussed later in this section. The primary disturbance will be the one that varies from normal the greatest, that is, with a [HCO − 3 ] = 6 mmol/L and pCO2 = 50 mm Hg, the primary disturbance would be a metabolic acidosis, the [HCO − 3 ] is about one-quarter normal, whereas the increase in pCO2 is only 25%. Step 4: After identifying the primary disturbance, use the equations in Table 8–2, page 164, to calculate the expected compensatory response. If the difference between the actual value and the calculated value is significant, then a mixed acid–base disturbance is present. Step 5: Calculate the anion gap. Anion gap = Na+ – (Cl− + HCO − 3 ). Normal anion gap is 8 8–12 mmol. If the anion gap is increased, proceed to step 6. Step 6: If the anion gap is elevated, then compare the changes from normal between the anion gap and [HCO − 3 ]. If the change in the anion gap is greater than the change in the [HCO − 3 ] from normal, then a metabolic alkalosis is present in addition to a gap metabolic acidosis. If the change in the anion gap is less than the change in the [HCO − 3 ] from normal, then a nongap metabolic acidosis is present in addition to a gap metabolic acidosis. See Examples 5, 6, and 7, page 174. Finally, be sure the interpretation of the blood gas is consistent with the clinical setting. METABOLIC ACIDOSIS: DIAGNOSIS AND TREATMENT Metabolic acidosis represents an increase in acid in body fluids reflected by a decrease in [HCO − 3 ] and a compensatory decrease in pCO2. Differential Diagnosis The diagnosis of metabolic acidosis (Figure 8–2) can be classified as an anion gap or a nonanion gap acidosis. The anion gap (Normal range, 8–12 mmol/L) is calculated as: Anion gap = [Na+ ] − ([Cl- − ] + [HCO3 ]) Anion Gap Acidosis: Anion gap >12 mmol/L; caused by a decrease in [HCO − 3 ] bal- anced by an increase in an unmeasured acid ion from either endogenous production or ex- ogenous ingestion (normochloremic acidosis). Nonanion Gap Acidosis: Anion gap = 8–12 mmol/L; caused by a decrease in [HCO − 3 ] balanced by an increase in chloride (hyperchloremic acidosis). Renal tubular acidosis is a type of nongap acidosis that can be associated with a variety of pathologic conditions (Table 8–3 page 168). The anion gap is helpful in identifying metabolic gap acidosis, nongap acidosis, mixed metabolic gap and nongap acidosis. If an elevated anion gap is present, a closer look at the anion gap and the bicarbonate helps differentiate among (a) a pure meta- bolic gap acidosis, (b) a metabolic nongap acidosis, (c) mixed metabolic gap and nongap acidosis, and (d) a metabolic gap acidosis and metabolic alkalosis. 8 Blood Gases and Acid–Base Disorders 167 Metabolic acidosis Anion gap Normal Increased Gut Renal Exogenous Endogenous 1. Diarrhea 1. Renal 1. Salicylates 1. Lactic acidosis 2. Fistulae tubular 2. Methanol 2. Ketoacidosis 3. Ileal loop acidosis 3. Paraldehyde a. Diabetic 2. Carbonic 4. Ethylene b. Starvation anhydrase glycol 3. Uremia 8 inhibitor 5. Hyperalimentation 3. Post 6. Ketoacidosis hypocapnia ETOH FIGURE 8–2 Differential diagnosis of metabolic acidosis. Treatment of Metabolic Acidosis 1. Correct any underlying disorder (control diarrhea,

etc). 2. Treatment with bicarbonate should be reserved for severe metabolic gap acidosis. If the pH <7.20, correct with sodium bicarbonate. The total replacement dose of [HCO − 3 ] can be calculated as follows: − Base deficit (mEq) × Patient' s weight (kg) [ΗCO3 ] needed in mEq = 4 3. Replace with one-half the total amount of bicarbonate over 8–12 h and reevaluate. Be aware of sodium and volume overload during replacement. Normal or isotonic bicarbonate drip is made with 3 ampules NaHCO3 (50 mmol NaHCO3/ampule) in 1 L D5W. METABOLIC ALKALOSIS: DIAGNOSIS AND TREATMENT Metabolic alkalosis represents an increase in [HCO − 3 ] with a compensatory rise in pCO2. Differential Diagnosis In two basic categories of diseases the kidneys retain [HCO − 3 ] (Figure 8–3). They can be differentiated in terms of response to treatment with sodium chloride and also by the level of urinary [Cl−] as determined by ordering a “spot,” or “random” urinalysis for chloride (UCl). Chloride-Sensitive (Responsive) Metabolic Alkalosis: The initial problem is a sustained loss of chloride out of proportion to the loss of sodium (either by renal or GI 8 168 TABLE 8–3 Renal Tubular Acidosis: Diagnosis and Management Serum Serum Minimal Clinical Renal [HCO − 3 ] [K+] Urine Condition Defect GFR (meq/L) (mEq/L) pH Associated Disease States Treatment Normal None N 24–28 3.5–5 4.8–5.2 None N/A Proximal RTA Proximal H+ N 15–18 ↓ <5.5 Drugs, Fanconi’s syndrome, various NaHCO3 or (type II RTA) secretion genetic disorders, dysproteinemic KHCO3 states, secondary hyperparathy- (10–15 roidism, toxins (heavy metals), tub- mEq/kg/d), ulointerstitial diseases, nephrotic thiazides syndrome, paroxysmal nocturnal hemoglobinuria Classic distal Distal H+ N 20–30 ↓ >5.5 Various genetic disorders, autoim- NaHCO3 RTA (type I secretion mune diseases, nephrocalcinosis, (1–3 meq/kg/d) RTA) drugs, toxins, tubulointerstitial dis- eases, hepatic cirrhosis, empty sella syndrome Buffer deficiency Distal NH3 ↓ 15–18 N <5.5 Chronic renal insufficiency, renal NaHCO3 (type III RTA) delivery osteodystrophy, severe hypophos- (1–3 mEq/kg/d) phatemia Generalized Distal Na+ ↓ 24–28 ↑ <5.5 Primary mineralocorticoid deficiency Fludrocortisone distal RTA reabsorption, (eg, Addison’s Disease), hyporenin- (0.1–0.5 (type IV RTA) K+ secretion, emic hypoaldosteronism, diabetes mg/d) dietary and H+ mellitus, tubulointerstitial diseases, K+ restriction, secretion nephrosclerosis, drugs), salt-wasting NaHCO3 mineralocorticoid-resistant hyper- (1–3 meq/kg/d) kalemia furosemide (40–160 mg/d) 8 Blood Gases and Acid–Base Disorders 169 Urine chloride (UCl) <10 mEq/L >10 mEq/L (chloride responsive) (chloride resistant) Renal loss of GI loss of H+, Cl– Excess mineralocorticoid chloride 1. NG suctioning 1. Adrenal 1. Diuretics 2. Vomiting a. Cushing's syndrome 2. Miscellaneous 3. Chloride-wasting b. Hyperaldosteronism a. Cystic fibrosis diarrhea (Conn's syndrome) 3. Posthypercapnia a. Congenital in 2. Exogenous steroid 8 children administration b. Villous adenoma 3. Bartter's syndrome FIGURE 8–3 Differential diagnosis of metabolic alkalosis. losses). This chloride depletion results in renal sodium conservation leading to a corre- sponding reabsorption of [HCO − 3 ] by the kidney. In this category of metabolic alkalosis, the urinary [Cl−] is <10 mEq/L, and the disorders respond to treatment with intravenous NaCl. Chloride-Insensitive (Resistant) Metabolic Alkalosis: The pathogenesis in this category is direct stimulation of the kidneys to retain bicarbonate irrespective of electrolyte intake and losses. The urinary [Cl−] >10 mEq/L, and these disorders do not respond to NaCl administration. Treatment of Metabolic Alkalosis Correct the underlying disorder. 1. Chloride-responsive a. Replace volume with NaCl if depleted. b. Correct hypokalemia if present. c. NH4Cl and HCl should be reserved for extreme cases. 2. Chloride-resistant a. Treat underlying problem, such as stopping exogenous steroids. RESPIRATORY ACIDOSIS: DIAGNOSIS AND TREATMENT Respiratory acidosis is a primary rise in pCO2 with a compensatory rise in plasma [HCO − 3 ]. Increased pCO2 occurs in clinical situations in which decreased alveolar ventilation occurs. Differential Diagnosis 1. Neuromuscular Abnormalities with Ventilatory Failure a. Muscular dystrophy, myasthenia gravis, Guillain–Barré syndrome, hypophos- phatemia 170 Clinician’s Pocket Reference, 9th Edition 2. Central Nervous System a. Drugs: Sedatives, analgesics, tranquilizers, ethanol b. CVA c. Central sleep apnea d. Spinal cord injury (cervical) 3. Airway Obstruction a. Chronic (COPD) b. Acute (asthma) c. Upper airway obstruction d. Obstructive sleep apnea 4. Thoracic–Pulmonary Disorders a. Bony thoracic cage: Flail chest, kyphoscoliosis b. Parenchymal lesions: Pneumothorax, severe pulmonary edema, severe pneumonia c. Large pleural effusions d. Scleroderma 8 e. Marked obesity (Pickwickian syndrome) Treatment of Respiratory Acidosis Improve Ventilation: Intubate patient and place on ventilator, increase ventilator rate, reverse narcotic sedation with naloxone (Narcan), etc RESPIRATORY ALKALOSIS: DIAGNOSIS AND TREATMENT Respiratory alkalosis is a primary fall in pCO2 with a compensatory decrease in plasma [HCO − 3 ]. Respiratory alkalosis occurs with increased alveolar ventilation. Differential Diagnosis 1. Central stimulation a. Anxiety, hyperventilation syndrome, pain b. Head trauma or CVA with central neurogenic hyperventilation c. Tumors d. Salicylate overdose e. Fever, early sepsis 2. Peripheral stimulation a. PE b. CHF (mild) c. Interstitial lung disease d. Pneumonia e. Altitude f. Hypoxemia: Any cause (See the section on Hypoxia, page 171.) 3. Miscellaneous a. Hepatic insufficiency b. Pregnancy c. Progesterone d. Hyperthyroidism e. Iatrogenic mechanical overventilation Treatment of Respiratory Alkalosis Correct the underlying disorder. 8 Blood Gases and Acid–Base Disorders 171 Hyperventilation Syndrome: Best treated by having the patient rebreathe into a paper bag to increase pCO2, decrease ventilator rate, increase amount of dead space with ventila- tor, or treat underlying cause. HYPOXIA 1. The second type of information gained from a blood gas level, in addition to acid–base results, pertains to the level of oxygenation. Usually, results are given as pO2 and oxy- gen saturation (See Table 8–1 for normal values in page 162). These two parameters are related to each other. 2. Oxygen saturation at any given pO2 is influenced by temperature, pH, and the level of 2,3-DPG as shown in Figure 8–4. Differential Diagnosis 1. V/Q abnormalities a. COPD: Emphysema, chronic bronchitis 8 b. Asthma c. Atelectasis d. Pneumonia e. PE f. ARDS g. Pneumothorax O2 dissociation curve of blood at 37°C 100 90 80 70 60 O2 affinity O2 affinity 50 (shift to right) (shift to left) 40 acidosis alkalosis hypoxemia hypothermia 30 fever banked blood increased decreased 20 2,3 DGP 2,3 DGP 10 0 0 10 20 30 40 50 60 70 80 90 100 110 120 Blood oxygen tension (pO2) FIGURE 8–4 Oxyhemoglobin dissociation curve. % Hemoglobin saturation p 7 H 7 .5 . 7 4 0 . 0 30 172 Clinician’s Pocket Reference, 9th Edition h. Pneumoconiosis i. CF j. Obstructed airway 2. Alveolar hypoventilation a. Skeletal abnormalities b. Neuromuscular disorders c. Pickwickian syndrome d. Sleep apnea 3. Decreased pulmonary diffusing capacity a. Pneumoconiosis b. Pulmonary edema c. Drug-induced pulmonary fibrosis (Bleomycin) d. Collagen–vascular diseases 4. Right-to-left shunt a. Congenital heart disease: Tetralogy of Fallot, transposition, etc 8 SAMPLE ACID–BASE PROBLEMS In each of the following examples, use the technique for blood gas interpretation on page 163 in this chapter to identify the acid–base disorder. Example 1 A patient with COPD has a blood gas of pH 7.34, pCO − 2 55, and [HCO3 ] of 29. Step 1: 55 46 = 24 × 29 46 ≈ 45 The numbers fit because the difference between the calculated and observed is <10%. Step 2: pH < 7.37, the problem is an acidemia. Step 3: pCO2 > 44 and [HCO − 3 ] is not < 22, so it represents a respiratory acidosis. Step 4: Normal compensation for chronic (COPD) respiratory acidosis (from Table 8–2). ∆[HCO − ∆ 15 3 ] = 4 × (pCO2 / 10) = 4 × = 6 10 Expected [HCO − 3 ] is 24 mEq/L + 6 = 30, which is reasonably close to the measured [HCO − 3 ] of 29, therefore this is a simple respiratory acidosis. This patient has a chronic res- piratory acidosis due to hypoventilation (simple acid–base disorder). Example 2 Immediately after a cardiac arrest a patient has a pH 7.25, pCO2 28, and [HCO − 3 ] 12. Step 1: 28 56 = 24 × 12 56 = 56 The numbers fit. 8 Blood Gases and Acid–Base Disorders 173 Step 2: pH < 7.37, so the problem is an acidemia. Step 3: [HCO − 3 ] is < 22 mEq/L and pCO2 is not > 44, so this is a metabolic acidosis. Step 4: (See Table 8–2, page 164) pCO2 = (1.5 × [ΗCO3 ] + 8) = (1.5 × 12) + 8 = 26 The expected pCO2 of 26 mm Hg is very similar to the actual measured value of 28 mm HG, so this is a simple metabolic acidosis. This patient has a lactic acidosis following a car- diopulmonary arrest (simple acid–base disorder). Example 3 A young man with a fever of 103.2°F and a fruity odor on his breath has a blood gas with pH = 7.36, pCO − 2 = 9, and [HCO3 ] = 5. 8 Step 1: = 24 45 × 9 5 43 ≈ 45 The numbers fit. Step 2: The pH < 7.37 indicates an acidemia. Step 3: [HCO − 3 ] < 22 and pCO2 is not >44, thus a metabolic acidosis is present. Step 4: The expected compensation in pCO2 can be calculated as follows (formula from Table 8–2): pCO2 = (1.5 × [HCO− 3 ]) + 8 = (1.5 × 9) + 8 = 21.5 The expected pCO2 is 15.5, but the actual result is 9 mm Hg, indicating a second process, which is a respiratory alkalosis. This patient had a metabolic acidosis due to dia- betic ketoacidosis and a concomitant respiratory alkalosis due to early sepsis and fever (mixed acid–base disorder). Example 4 A 30-y-old 30-wk pregnant female presents with nausea and vomiting. Blood gas reveals a pH 7.55, pCO2 = 25 and [HCO − 3 ] = 22. Step 1: 25 28 = 24 × 22 28 ≈ 27 The numbers fit. Step 2: pH < 7.44 indicates alkalemia. Step 3: pCO2 < 36 and the [HCO − 3 ] is not >26, thus a respiratory alkalosis is present. 174 Clinician’s Pocket Reference, 9th Edition Step 4: The expected compensation for a chronic (pregnancy) respiratory alkalosis is cal- culated from Table 8–2, page 164: ∆[HCO − 3 ] = 5 × ∆pCO2 / 10 = × 15 5 = 7.5 10 The calculated [HCO − 3 ] is then 24 – 7.5, or 16–17 mmol, but the actual bicarbonate level is 22, indicating a relative secondary metabolic alkalosis ([HCO − 3 ] is higher than ex- pected). This patient has a respiratory acidosis due to pregnancy and a relative secondary meta- bolic alkalosis due to vomiting. Example 5 A 19-y-old diabetic has an anion gap of 29 and a [HCO − 3 ] of 6. 8 Step 1: 29 mmol/L actual gap −10 mmol/L normal gap 19 mmol/L expected change in [HCO – 3 ] Step 2: 24 mmol/L normal [HCO– 3] −19 mmol/L expected change in [HCO – 3 ] 5 mmol/L expected change in [HCO – 3 ] Actual bicarbonate is 6 mmol/L, which is very close to the expected of 5 mmol/L. Thus, a pure metabolic gap acidosis is present from DKA. Example 6 A 21-y-old diabetic presents with nausea, vomiting, and abdominal pain. The anion gap was 23, and the [HCO − 3 ] was 18. Step 1: 23 mmol/L actual gap −10 mmol/L normal gap 13 mmol/L expected change in [HCO – 3 ] from normal Step 2: 24 mmol/L normal [HCO – 3 ] −13 mmol/L expected change in [HCO – 3 ] 11 mmol/L expected change in [HCO – 3 ] Actual bicarbonate is 18 mmol and not the 11 mmol/L expected from a pure metabolic gap acidosis. Because the actual bicarbonate was higher than expected, this must be a mixed metabolic gap acidosis and metabolic alkalosis. The patient has a metabolic gap acidosis from DKA and a metabolic alkalosis from the vomiting. 8 Blood Gases and Acid–Base Disorders 175 Example 7 A 55-y-old alcoholic with a 2-wk history of diarrhea. The anion gap was 17, and [HCO − 3 ] was 10. Step 1: 17 mmol/L actual gap −10 mmol/L normal gap 7 mmol/L expected change in [HCO – 3 ] from normal Step 2: 24 mmol/L normal [HCO– 3] −7 mmol/L expected change in [HCO – 3 ] 17 mmol/L expected change in [HCO – 3 ] Actual bicarbonate is 10 mmol/L and not the expected 17 mmol/L if there was

a pure metabolic gap acidosis. Since the actual bicarbonate is lower than expected, there must be a 8 mixed metabolic gap acidosis and metabolic nongap acidosis. The patient has a metabolic nongap acidosis from diarrhea and a metabolic gap acidosis from the alcoholic ketoacidosis. This page intentionally left blank. 9 FLUIDS AND ELECTROLYTES Principles of Fluids and Electrolytes Electrolyte Abnormalities: Diagnosis Composition of Parenteral Fluids and Treatment Composition of Body Fluids Ordering IV Fluids Determining an IV Rate PRINCIPLES OF FLUIDS AND ELECTROLYTES Fluid Compartments 9 • Example: 70-kg male Total Body Water: 42,000 mL (60% of BW) • Intracellular: 28,000 mL (40% of BW) • Extracellular: 14,000 mL (20% of BW) • Plasma: 3500 mL (5% of BW) • Interstitial: 10,500 mL (15% of BW) Total Blood Volume Total blood volume = 5600 mL (8% of BW) Red Blood Cell Mass Male, 20–36 mL/kg (1.15–1.21 L/m2); female, 19–31 mL/kg (0.95–1.0 L/m2) Water Balance • 70-kg male The minimum obligate water requirement to maintain homeostasis (assuming normal temperature and renal concentrating ability and minimal solute [urea, salt] excretion) is about 800 mL/d, which would yield 500 mL of urine. “Normal” Intake: 2500 mL/d (about 35 mL/kg/d baseline) • Oral liquids: 1500 mL • Oral solids: 700 mL • Metabolic (endogenous): 300 mL “Normal” Output: 1400–2300 mL/d • Urine: 800–1500 mL • Stool: 250 mL 177 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 178 Clinician’s Pocket Reference, 9th Edition • Insensible loss: 600–900 mL (lungs and skin). (With fever, each degree above 98.6°F adds 2.5 mL/kg/d to insensible loss; insensible losses are decreased if a pa- tient is on a ventilator; free water gain may occur from humidified ventilation.) Baseline Fluid Requirement Afebrile 70-kg Adult: 35 mL/kg/24 h If not a 70-kg Adult: Calculate the water requirement according to the following “kg Method”: • For the first 10 kg of body weight: 100 mL/kg/d plus • For the second 10 kg of body weight: 50 mL/kg/d plus • For the weight above 20 kg: 20 mL/kg/d Electrolyte Requirements • 70-kg adult, unless otherwise specified 9 Sodium (as NaCl): 80–120 mEq (mmol)/d (Pediatric patients, 3–4 mEq/kg/ 24 h [mmol/kg/24 h]) Chloride: 80–120 mEq (mmol)/d, as NaCl Potassium: 50–100 mEq/d (mmol/d) (Pediatric patients, 2–3 mEq/kg/24 h [mmol/kg/24 h]). In the absence of hypokalemia and with normal renal function, most of this is excreted in the urine. Of the total amount of potassium, 98% is intracellular, and 2% is extracellular. Thus, assuming the serum potassium level is normal, about 4.5 mEq/L (mmol/L), the total extracellular pool of K+ = 4.5 × 14 L = 63 mEq (mmol). Potassium is easily interchanged between intracellular and extracellular stores under conditions such as acidosis. Potassium demands increase with diuresis and building of new body tissues (anabolic states). Calcium: 1–3 gm/d, most of which is secreted by the GI tract. Routine administration is not needed in the absence of specific indications. Magnesium: 20 mEq/d (mmol/d). Routine administration is not needed in the absence of specific indications, such as parenteral hyperalimentation, massive diuresis, ethanol abuse (frequently needed) or preeclampsia. Glucose Requirements 100–200 g/d (65–75 g/d/m2). During starvation, caloric needs are supplied by body fat and protein; the majority of protein comes from the skeletal muscles. Every gram of nitrogen in the urine represents 6.25 g of protein broken down. The protein-sparing effect is one of the goals of basic IV therapy. The administration of at least 100 g of glucose/d reduces protein loss by more than one-half. Virtually all IV fluid solutions supply glucose as dextrose (pure dextrorotatory glucose). Pediatric patients require about 100–200 mg/kg/h. COMPOSITION OF PARENTERAL FLUIDS Parenteral fluids are generally classified based on molecular weight and oncotic pressure. Colloids have a molecular weight of >8000 and have high oncotic pressure; crystalloids have a molecular weight of <8000 and have low oncotic pressure. 9 Fluids and Electrolytes 179 Colloids • Albumin (see page 200) • Blood products (RBCs, single-donor plasma, etc) (Chapter 10, page 197) • Plasma protein fraction (Plasmanate) (See Chapter 22) • Synthetic colloids (hetastarch [Hespan], dextran) (Chapter 22) Crystalloids Table 9–1 describes common crystalloid parenteral fluids. COMPOSITION OF BODY FLUIDS Table 9–2 gives the average daily production and the amount of some major electrolytes present in various body fluids. ORDERING IV FLUIDS One of the most difficult tasks to master is choosing appropriate IV therapy for a patient. The patient’s underlying illness, vital signs, serum electrolytes, and a host of other variables 9 all must be considered. The following are general guidelines for IV therapy. Specific re- quirements for each patient can vary tremendously from these guidelines. Maintenance Fluids These amounts provide the minimum requirements for routine daily needs: 1. 70-kg Male: Five% dextrose in one-quarter concentration normal saline (D5¹₄NS) with 20 mEq KCl/L (20 mmol/L) at 125 mL/h. (This will deliver about 3 L of free water/day.) 2. Other Adult Patients: Also use D5¹₄ NS with 20 mEq KCl/L. Determine their 24-h water requirement by the “kg method” (page 178) and divide by 24 h to determine the hourly rate. 3. Pediatric Patients: Use the same solution, but determine the daily fluid requirements by either of the following methods: a. kg Method: (page 181) b. Meter Squared Method: Maintenance fluids are 1500 mL/m2/d. Divide by 24 to get the flow rate per hour. To calculate the surface area, use Table 9–3, page 181 “rule of sixes nomogram.” Formal body surface area charts are in the Appendix. Specific Replacement Fluids These fluids are used to replace excessive, nonphysiologic losses. Gastric Loss (Nasogastric Tube, Emesis): D5¹₂ NS with 20 mEq/L (mmol/L) potassium chloride (KCl) Diarrhea: D5LR with 15 mEq/L (mmol/L) KCl. Use body weight as a replacement guide (about 1 L for each 1 kg, or 2.2 lb, lost) Bile Loss: D5LR with 25 mEq/L (¹₂ ampule) of sodium bicarbonate mL for mL Pancreatic Loss: D5LR with 50 mEq/liter (1 amp) HCO3 mL for mL. Burn Patients: Use the Parkland or “Rule of Nines” Formulas: 9 180 TABLE 9–1 Composition of Commonly Used Crystalloids Electrolytes (mEq/L) Glucose Fluid (g/L) Na+ Cl− K+ Ca2+ HCO −* Mg2+ − 3 HPO 2 4 kcal/L D5W (5% dextrose 50 — — — — — — — 170 in water) D10W (10% dextrose 100 — — — — — — — 340 in water) D20W (20% dextrose 200 — — — — — — — 680 in water) D50W (50% dextrose 500 — — — — — — — 1700 in water) ¹₂ NS (0.45% NaCl) — 77 77 — — — — — — 3% NS — 513 513 — — — — — — NS (0.9% NaCl) — 154 154 — — — — — — D5¹₄NS 50 38 38 — — — — — 170 D5¹₄NS (0.45% NaCl) 50 77 77 — — — — — 170 D5¹₂NS (0.9% NaCl) 50 154 154 — — — — — 170 D5LR (5% dextrose 50 130 110 4 3 27 — — 180 in lactated Ringer’s) Lactated Ringer’s — 130 110 4 3 27 — — <10 Ionosol MB 50 25 22 20 — 23 3 3 170 Normosol M 50 40 40 13 — 16 3 — 170 *HCO3 is administered in these solutions as lactate that is converted to bicarbonate. 9 Fluids and Electrolytes 181 TABLE 9–2 Composition and Daily Production of Body Fluids Electrolytes (mEq/L) Average Daily Fluid Na+ Cl− K+ HCO − 3 Production* (mL) Sweat 50 40 5 0 Varies Saliva 60 15 26 50 1500 Gastric juice 60–100 100 10 0 1500–2500 Duodenum 130 90 5 0–10 300–2000 Bile 145 100 5 15 100–800 Pancreatic juice 140 75 5 115 100–800 Ileum 140 100 2–8 30 100–9000 Diarrhea 120 90 25 45 — *In adults. 9 TABLE 9–3 “Rule of Sixes” Nomogram for Calculating Fluids in Children* Weight Body Surface Area (lb) (m2) 3 0.1 6 0.2 12 0.3 18 0.4 24 0.5 30 0.6 36 0.7 42 0.8 48 0.9 60† 1.0 *Over 100 lb, treat as an adult. †After 60 lb, add 0.1 for each additional 10 lb. 182 Clinician’s Pocket Reference, 9th Edition A A A 1 1 13 13 2 2 2 2 1.5 1.5 1.5 1.5 2.5 2.5 1.25 1 1.25 1.25 1.25 9 B B B B C C C C 1.75 1.75 1.75 1.75 Relative Percentages of Areas Affected by Growth Age Area 10 15 Adult A = half of head 5.5 4.5 3.5 B = half of one thigh 4.25 4.5 4.75 C = half of one leg 3 3.25 3.5 FIGURE 9–1 Tables for estimating the extent of burns in adults and children. In adults, a reasonable system for calculating the percentage of the body surface burned is the “rule of nines”: Each arm equals 9%, the head equals 9%, the anterior and posterior each equal 18%, and the perineum equals 1%. (Reprinted, with per- mission, from: Way LW [ed]: Current Surgical Diagnosis and Treatment, 10th ed,. Appleton & Lange, Norwalk CT, 1994.) 9 Fluids and Electrolytes 183 B A A 1 1 2 2 2 2 13 13 1 1 1 1 1.25 1. 2 2 1 25 1.25 1.25 B B B B C C C C 1 1 1.75 1.75 9 Relative Percentages of Areas Affected by Growth Age Area 0 1 5 A = half of head 9.5 8.5 6.5 B = half of one thigh 2.75 3.25 4 C = half of one leg 2.5 2.5 2.75 FIGURE 9–1 Continued. Parkland Formula. Total fluid required during the first 24 h = (% body burn) × (body weight in kg) × 4 mL Replace with lactated Ringer’s solution over 24 h. Use • One-half the total over first 8 h (from time of burn) • One-quarter of the total over second 8 h. One-quarter of the total over third 8 h • Rule of Nines. Used for estimating percentage of body burned in adults. See Figure 9–1 for the exact calculation for the body burn in adults and children. This is also useful for determining ongoing fluid losses from a burn until it is healed or grafted. Fluid losses can be estimated as Loss in mL = (25 × % Body burn) × m2 Body surface area DETERMINING AN IV RATE Most IV infusions are regulated by infusion pumps. If a mechanical infusion device is not available, use the following formulas to determine the infusion rate. 184 Clinician’s Pocket Reference, 9th Edition For a MAXI Drip Chamber: Use 10 drops/mL; thus • 10 drops/min = 60 mL/h or • 16 drops/min = 100 mL/h For a MINI Drip Chamber: Use 60 drops/mL; thus • 60 drops/min = 60 mL/h or • 100 drops/min = 100 mL/h ELECTROLYTE ABNORMALITIES: DIAGNOSIS AND TREATMENT In all of the following situations, the primary goal should be to correct the underlying condi- tion. Unless specified, all dosages are for adults. The complete differential diagnosis of lab- oratory findings can be found in Chapter 4. Hypernatremia (Na+ >144 mEq/L [mmol/L]) Mechanisms: Most frequently, a deficit of total body water. 9 • Combined Sodium and Water Losses (“hypovolemic hypernatremia”). Water loss in excess of sodium loss results in low total body sodium. Due to renal (diuret- ics, osmotic diuresis due to glycosuria, mannitol, etc) or extrarenal (sweating, GI, respiratory) losses • Excess Water Loss (“isovolemic hypernatremia”). Total body sodium remains normal, but total body water is decreased. Caused by diabetes insipidus (central and nephrogenic), excess skin losses, respiratory loss, others. • Excess Sodium (“hypervolemic hypernatremia”). Total body sodium increased, caused by iatrogenic sodium administration (ie, hypertonic dialysis, sodium-contain- ing medications) or adrenal hyperfunction (Cushing’s syndrome, hyperaldoster- onism). Symptoms: Depend on how rapidly the sodium level has changed • Confusion, lethargy, stupor, coma • Muscle tremors, seizures Signs: Hyperreflexia, mental status changes Treatment: Check the serum sodium levels frequently while attempting to correct hy- pernatremia. • Hypovolemic Hypernatremia. Determine if the patient volume is depleted by de- termining if orthostatic hypotension (see page 286) is present; if volume is depleted, rehydrate with NS until hemodynamically stable, then administer hypotonic saline (¹₂ NS). • Euvolemic/Isovolemic. (No orthostatic hypotension) calculate the volume of free water needed to correct the Na+ to normal as follows: Body water deficit = Normal TBW − Current TBW where Normal TBW = 0.6 × Body weight in

kg and 9 Fluids and Electrolytes 185 Normal serum sodium × TBW Current TBW = Measured serum sodium • Give free water as D5W, one-half the volume in the first 24 h and the full volume in 48 h. (Caution: The rapid correction of the sodium level using free water (D5W) can cause cerebral edema and seizures.) • Hypervolemic Hypernatremia. Avoid medications that contain excessive sodium (carbenicillin, etc).Use furosemide along with D5W. Hyponatremia (Na+ <136 mEq/L [mmol/L]) Mechanisms: Most often due to excess body water as opposed to decreased body sodium. To define the cause, determine serum osmolality. • Isotonic Hyponatremia. Normal osmolality • Pseudo-Hyponatremia. An artifact caused by hyperlipidemia or hyperproteinemia. • Hypertonic Hyponatremia. High osmolality. Water shifts from intracellular to ex- tracellular in response to high concentrations of such solutes as glucose or mannitol. The shift in water lowers the serum sodium; however, the total body sodium remains 9 the same. • Hypotonic Hyponatremia. Low osmolality. Further classified based on clinical as- sessment of extracellular volume status • Isovolemic. No evidence of edema, normal BP. Caused by water intoxication (uri- nary osmolality <80 mOsm), SIADH, hypothyroidism, hypoadrenalism, thiazide di- uretics, beer potomania • Hypovolemic. Evidence of decreased skin turgor and an increase in heart rate and de- crease in BP after going from lying to standing. Due to renal loss (urinary sodium >20 mEq/L) from diuretics, postobstructive diuresis, mineralocorticoid deficiency (Addison’s disease, hypoaldosteronism) or extrarenal losses (urinary sodium <10 mEq/L) from sweating, vomiting, diarrhea, third spacing fluids (burns, pancreatitis, peritonitis, bowel obstruction, muscle trauma) • Hypervolemic. Evidence of edema.(urinary sodium <10 mEq/L). Seen with CHF, nephrosis, renal failure, and liver disease • Excess Water Intake. Primary (psychogenic water drinker) or secondary (large vol- ume of sterile water used in procedures, eg, transurethral resection of the prostate or multiple tap water enemas) Symptoms: Usually with Na+ <125 mEq/L (mmol/L); severity of symptoms correlates with the rate of decrease in Na+. • Lethargy, confusion, coma • Muscle twitches and irritability, seizures • Nausea, vomiting Signs: Hyporeflexia, mental status changes Treatment: Based on determination of volume status. Evaluate volume status by physi- cal examination HR and BP lying and standing after 1 min, skin turgor, edema and by deter- mination of the plasma osmolality. Do not need to treat hyponatremia from pseudo-hyponatremia (increased protein or lipids) or hypertonic hyponatremia (hyper- glycemia), treat underlying disorder (see above). 186 Clinician’s Pocket Reference, 9th Edition • Life-Threatening. (Seizures, coma) 3–5% NS can be given in the ICU setting. At- tempt to raise the sodium to about 125 mEq/L with 3–5% NS. • Isovolemic Hyponatremia. (SIADH) Restrict fluids (1000–1500 mL/d). Demeclocycline can be used in chronic SIADH. • Hypervolemic Hyponatremia Restrict sodium and fluids (1000–1500 mL/d). Treat underlying disorder. CHF may respond to a combination of ACE inhibitor and furosemide. • Hypovolemic Hyponatremia Give D5NS or NS. Hyperkalemia • (K+ >5.2 mEq/L (mmol/L) 9 Mechanisms: Most often due to iatrogenic or inadequate renal excretion of potassium. • Pseudo-Hyperkalemia. Due to leukocytosis, thrombocytosis, hemolysis, poor veni- puncture technique (prolonged tourniquet time) • Inadequate Excretion. Renal failure, volume depletion, medications that block potassium excretion (spironolactone, triamterene, others), hypoaldosteronism (in- cluding adrenal disorders and hyporeninemic states [such as Type IV renal tubular acidosis], NSAIDs, ACE inhibitors), long-standing use of heparin, digitalis toxicity, sickle cell disease, renal transplant • Redistribution. Tissue damage, acidosis (a 0.1 decrease in pH increases serum K+ approximately 0.5–1.0 mEq/L due to extracellular shift of K+), beta-blockers, de- creased insulin, succinylcholine • Excess Administration. Potassium-containing salt substitutes, oral replacement, potassium in IV fluids Symptoms: Weakness, flaccid paralysis, confusion. Signs: • Hyperactive deep tendon reflexes, decreased motor strength • ECG changes, such as, peaked T waves, wide QRS, loss of P wave, sine wave, asystole • K+ = 7–8 mEq/L (mmol/L) yields ventricular fibrillation in 5% of cases • K+ = 10 mEq/L (mmol/L) yields ventricular fibrillation in 90% of cases Treatment • Monitor patient on ECG if symptomatic or if K+ >6.5 mEq/L; discontinue all potas- sium intake, including IV fluids; order a repeat stat potassium to confirm. • Pseudo-hyperkalemia should be ruled out. If doubt exists, obtain a plasma potassium in a heparinized tube; the plasma potassium will be normal if pseudo-hyperkalemia is present. • Rapid Correction. These steps only protect the heart from potassium shifts, and total body potassium must be reduced by one of the treatments shown under Slow Correction. 9 Fluids and Electrolytes 187 Calcium chloride, 500 mg, slow IV push (only protects heart from effect of hyperkalemia) Alkalinize with 50 mEq (1 ampule) sodium bicarbonate (causes intracellular potassium shift) 50 mL D50, IV push, with 10–15 units regular insulin, IV push (causes intracellular potas- sium shift) • Slow Correction Sodium polystyrene sulfonate (Kayexalate) 20–60 g given orally with 100–200 mL of sor- bitol or 40 g Kayexalate with 40 g sorbitol in 100 mL water given as an enema. Repeat doses qid as needed. Dialysis (hemodialysis or peritoneal) • Correct Underlying Cause. Such as stopping potassium-sparing diuretics, ACE in- hibitors, mineralocorticoid replacement for hypokalemia Hypokalemia • K+ <3.6 mEq/L (mmol/L) Mechanisms: Due to inadequate intake, loss, or intracellular shifts 9 • Inadequate Intake. Oral or IV • GI Tract Loss. (Urinary chloride usually <10 mEq/d; “chloride-responsive alkalo- sis”) vomiting, diarrhea, excess sweating, villous adenoma, fistula • Renal Loss. Diuretics and other medications (amphotericin, high-dose penicillins, aminoglycosides, cisplatin), diuresis other than diuretics (osmotic, eg, hyper- glycemia or ethanol-induced), vomiting (from metabolic alkalosis from volume de- pletion), renal tubular disease (renal tubular acidosis type II [distal], and [proximal]), Bartter’s syndrome (due to increased renin and aldosterone levels), hy- pomagnesemia, natural licorice ingestion, mineralocorticoid excess (primary and secondary hyperaldosteronism, Cushing’s syndrome, steroid use), and ureterosig- moidostomy • Redistribution (Intracellular Shifts). Metabolic alkalosis (each 0.1 increase in pH lowers serum K+ approximately 0.5–1.0 mEq/L, due to intracellular shift of K+), in- sulin administration, beta-adrenergic agents, familial periodic paralysis, treatment of megaloblastic anemia Symptoms • Muscle weakness, cramps, tetany • Polyuria, polydipsia Signs • Decreased motor strength, orthostatic hypotension, ileus • ECG changes, such as flattening of T waves, “U” wave becomes obvious (U wave is the upward deflection after the T wave.) Treatment: The therapy depends on the cause. • A history of hypertension, GI symptoms, or use of certain medications may suggest the diagnosis. • A 24-h urine for potassium may be helpful if the diagnosis is unclear. Levels <20 mEq/d suggest extrarenal/redistribution, >20 mEq/d suggest renal losses. 188 Clinician’s Pocket Reference, 9th Edition • A serum potassium level of 2 mEq/L (mmol/L) probably represents a deficit of at least 200 mEq (mmol) in a 70-kg adult; to change potassium from 3 mEq/L (mmol/L) to 4 mEq/L (mmol/L) takes about 100 mEq (mmol) of potassium in a 70- kg adult. • Treat underlying cause. • Hypokalemia potentiates the cardiac toxicity of digitalis. In the setting of digoxin use, hypokalemia should be aggressively treated. • Treat hypomagnesemia if present. It will be difficult to correct hypokalemia in the presence of hypomagnesemia. • Rapid Correction. Give KCl IV. Monitor heart with replacement >20 mEq/h. IV potassium can be painful and damaging to veins. Patient <40 kg: 0.25 mEq/kg/h × 2 h Patient >40 kg: 10–20 mEq/h × 2 h Severe [<2 mEq/L (mmol/L)]: Maximum 40 mEq/h IV in adults In all cases check a stat potassium following each 2–4 h of replacement. • Slow Correction. Give KCl orally (see also Table 22–8, page 626) for potassium supplements). 9 Adult: 20–40 mEq two to three times a day (bid or tid) Pediatric patients: 1–2 mEq/kg/d in divided doses Hypercalcemia • Ca2+ > 10.2 mg/dL (2.55 mmol/L) Mechanisms • Parathyroid-Related. Hyperparathyroidism with secondary bone resorption • Malignancy-Related. Solid tumors with metastases (breast, ovary, lung, kidney), or paraneoplastic syndromes, (squamous cell, renal cell, transitional cell carcinomas, lymphomas, and myeloma) • Vitamin-D-Related. Vitamin D intoxication, sarcoidosis, other granulomatous dis- ease • High Bone Turnover. Hyperthyroidism, Paget’s disease, immobilization, vitamin A intoxication • Renal Failure. Secondary hyperparathyroidism, aluminum intoxication • Other. Thiazide diuretics, milk–alkali syndrome, exogenous intake Symptoms • Stones (renal colic) bones (osteitis fibrosa), moans (constipation), and groans (neu- ropsychiatric symptoms—confusion), as well as polyuria, polydipsia, fatigue, anorexia, nausea, vomiting Signs • Hypertension, hyporeflexia, mental status changes • Shortening of the QT interval on the ECG. Treatment: Usually emergency treatment if patient is symptomatic and Ca+2 >13 mEq/L (3.24 mmol/L) • Use saline diuresis: D5NS at 250–500 mL/h. 9 Fluids and Electrolytes 189 • Give furosemide (Lasix) 20–80 mg or more IV (saline and Lasix will treat most cases). • Euvolemia or hypervolemia must be maintained. Hypovolemia results in calcium re- absorption. • Other Second-Line Therapies: Calcitonin 2–8 IU/kg IV or SQ q6–12h if diuresis has not worked after 2–3 h Pamidronate 60 mg IV over 24 h (one dose only) Gallium nitrate 200 mg/m2 IV infusion over 24 h for 5 d Plicamycin 25 µg/kg IV over 2–3 h (use as last resort—very potent) Corticosteroids. Hydrocortisone 50–75 mg IV every 6 h. Consider hemodialysis. • Chronic Therapy: Treat underlying condition, discontinue contributing medications (ie, thiazides). Oral medications (prednisone 30 mg PO bid or phosphorus/potassium/sodium supplement [Neutra-Phos] 250–500 mg PO qid) can be effective in chronic therapy for such dis- eases as breast cancer or sarcoidosis. 9 Hypocalcemia • Ca2+ < 8.4 mg/dL (2.1 mmol/L) Mechanisms: Decreased albumin can result in decreased calcium (see discussion on page 61). • PTH. Responsible for the immediate regulation of calcium levels • Critical Illness. Sepsis and other ICU-related conditions can cause decreased cal- cium because of the fall in albumin often seen in critically ill patients, ionized cal- cium may be normal. • PTH Deficiency. Acquired (surgical excision or injury, infiltrative diseases such as amyloidosis or hemachromatosis and irradiation) hereditary hypoparathyroidism (pseudo-hypoparathyroidism), hypomagnesemia • Vitamin D deficiency. Chronic renal failure, liver disease, use of phenytoin or phe- nobarbital, malnutrition, malabsorption (chronic pancreatitis, postgastrectomy) • Other. Hyperphosphatemia, acute pancreatitis, osteoblastic metastases, medullary carcinoma of the thyroid, massive transfusion Symptoms • Hypertension, peripheral and perioral paresthesia, abdominal pain and cramps, lethargy, irritability (in infants) Signs • Hyperactive DTRs, carpopedal spasm (Trousseau’s sign, see page 27). • Positive Chvostek’s sign (facial nerve twitch, can be present in up to 25% of normal adults). • Generalized seizures, tetany, laryngospasm • Prolonged QT interval on ECG 190 Clinician’s Pocket Reference, 9th Edition Treatment • Acute Symptomatic 100–200 mg of elemental calcium IV over 10 min in 50–100 mL of D5W followed by an in- fusion containing 1–2 mg/kg/h over 6–12 h 10% calcium gluconate contains 93 mg of elemental calcium. 10% calcium chloride contains 272 mg of elemental calcium. Check magnesium levels and replace if low. • Chronic For renal insufficiency, use vitamin D along with oral calcium supplements (see the follow- ing lists) and phosphate-binding antacids (Phospho gel, ALTernaGEL). Calcium supplements Calcium carbonate (Os-Cal) 650 mg PO qid (28% calcium) Calcium citrate (Critical) 950-mg tablets (21% calcium) 9 Calcium gluconate 500- or 1000-mg tablets (9% calcium) Calcium glubionate (Neo-Calglucon) syrup 115 mg/5 mL (6.4% calcium) Calcium lactate 325- or 650-mg tablets (13% calcium) Hypermagnesemia • Mg2+ > 2.1 mEq/L (mmol/L) Mechanisms • Excess Administration. Treatment of preeclampsia with magnesium sulfate • Renal Insufficiency. Exacerbated by ingestion of magnesium-containing antacids • Others. Rhabdomyolysis, adrenal insufficiency Symptoms and Signs • 3–5 mEq/L(mmol/L): Nausea, vomiting, hypotension • 7–10 mEq/L (mmol/L): Hyperreflexia, weakness, drowsiness • >12 mEq/L (mmol/L): Coma, bradycardia, respiratory failure Treatment: Clinical hypermagnesemia requiring therapy is infrequently encountered in the patient with normal renal function. • Calcium gluconate: 10 mL of 10% solution (93 mg elemental calcium) over 10–20 min in 50–100 mL of D5W given IV to reverse symptoms (useful in patients being treated for eclampsia). • Stop magnesium-containing medications (hypermagnesemia is most often encoun- tered in patients in renal failure on magnesium-containing antacids). • Insulin and glucose as for hyperkalemia (page 186). Furosemide and saline diuresis • Dialysis Hypomagnesemia • Mg2+ <1.5 mEq/L (mmol/L) 9 Fluids and Electrolytes 191 Mechanisms • Decreased Intake or Absorption. Malabsorption, chronic GI losses, deficient in- take (alcoholics), TPN without adequate supplementation • Increased Loss. Diuretics, other medications (gentamicin, cisplatin, amphotericin B, others), RTA, diabetes mellitus (especially DKA), alcoholism, hyperaldostero- nism, excessive lactation • Other. Acute pancreatitis, hypoalbuminemia, vitamin D therapy. Symptoms • Weakness, muscle twitches, asterixis • Vertigo • Symptoms of hypocalcemia (hypomagnesemia may cause hypocalcemia and hy- pokalemia) Signs • Tachycardia, tremor, hyperactive reflexes, tetany, seizures •

ECG may show prolongation of the PR, QT, and QRS intervals as well as ventricular ectopy, sinus tachycardia 9 Treatment • Severe: Tetany or Seizures Monitor patient with ECG in ICU setting. 2 g magnesium sulfate in D5W infused over 10–20 min. Follow with magnesium sulfate: 1 g/h for 3–4 h follow DTR and levels. Repeat replacement if necessary. These patients are often hypokalemic and hypophosphatemic as well and should be supple- mented. Hypocalcemia may also result from hypomagnesemia. • Moderate Mg2+ <1.0 mg/dL but asymptomatic Magnesium sulfate: 1 g/h for 3–4 h, follow TR and levels and repeat replacement if necessary. • Mild Magnesium oxide: 1 g/d PO (available over the counter in 140-mg capsules, and in 400- and 420-mg tablets). May cause diarrhea. Hyperphosphatemia • PO –3 4 > 4.5 mg/dL (1.45 mmol/L) Mechanisms • Increased Intake/Absorption. Iatrogenic, abuse of laxatives or enemas containing phosphorus, vitamin D, granulomatous disease • Decreased Excretion (Most Common Cause). Renal failure, hypoparathyroidism, adrenal insufficiency, hyperthyroidism, acromegaly, sickle cell anemia • Redistribution/Cellular Release. Rhabdomyolysis, acidosis, chemotherapy-induced tumor lysis, hemolysis, plasma cell dyscrasias 192 Clinician’s Pocket Reference, 9th Edition Symptoms and Signs: Mostly related to tetany as a result of hypocalcemia (see page 189) caused by the hyperphosphatemia or metastatic calcification (deposition of calcium phosphate in various soft tissues) Treatment • Low-phosphate diet • Phosphate binders like aluminum hydroxide gel (Amphojel) or aluminum carbonate gel (Basaljel) orally • Acute, severe cases: Acetazolamide 15 mg/kg q4h or insulin and glucose infusion, dialysis as last resort Hypophosphatemia • PO –3 4 < 2.5 mg/dL (0.8 mmol/L) Mechanisms • Decreased Dietary Intake. Starvation, alcoholism, iatrogenic (hyperalimentation without adequate supplementation), malabsorption, vitamin D deficiency, phos- 9 phate-binding antacids (ie, ALTernaGEL) • Redistribution. Conditions associated with respiratory or metabolic alkalosis (alco- hol withdrawal, salicylate poisoning, etc), endocrine (insulin, catecholamine, etc), anabolic steroids, hyper- or hypothermia, leukemias and lymphomas, hypercalcemia, hypomagnesemia • Renal Losses. RTA, diuretic phase of ATN, hyperparathyroidism, hyperthyroidism, hypokalemia, diuretics, hypomagnesemia, alcohol abuse, diabetes mellitus (poorly controlled) • Other. Refeeding in the setting of severe protein-calorie malnutrition, severe burns, treatment of DKA Symptoms and Signs: < 1 mg/dL (0.32 mmol/L): Weakness, muscle pain and tender- ness, paresthesia, cardiac and respiratory failure, CNS dysfunction (confusion and seizures). rhabdomyolysis, hemolysis, impaired leukocyte and platelet function Treatment: IV therapy is reserved for severe potentially life-threatening hypophos- phatemia (<1.0–1.5 mg/dL) because too rapid correction can lead to severe hypocalcemia. With mild to moderate hypophosphatemia (1.5–2.5 mg/dL), oral replacement is preferred. • Severe. (<1.0–1.5 mg/dL) Potassium or sodium phosphate. 2 mg/kg (0.08 mM/kg) given IV over 6 h. (Caution: Rapid replacement can lead to hypocalcemic tetany.) • Mild to Moderate. (levels > 1.5 mg/dL) Sodium–potassium phosphate (Neutra-Phos) or potassium phosphate (K-Phos): 1–2 tablets (250–500 mg PO4 or 8 mM/tablet) PO bid or tid Sodium phosphate (Fleet’s Phospho-soda). 5 mL PO, bid or tid (128 mg PO4 or 4 mM/mL) 10 BLOOD COMPONENT THERAPY Blood Banking Procedures Blood Groups Routine Blood Donation Basic Principles of Blood Component Autologous Blood Donation Therapy Donor-Directed Blood Products Blood Bank Products Irradiated Blood Components Transfusion Procedures Apheresis Transfusion Reactions Preoperative Blood Set-Up Transfusion-Associated Infectious Emergency Transfusions Disease Risks BLOOD BANKING PROCEDURES 10 T&S or T&H: The blood bank types the patient’s blood (ABO and Rh) and screens for antibodies. If a rare antibody is found, the physician will usually be notified, and if it is likely that blood will be needed, the type and screen order may be changed to a type and cross. This usually takes less than 1 h. T&C: The blood bank types and screens the patient’s blood as described in the previous section and matches specific donor units for the patient. The cross-match involves testing the recipient’s serum against the donor blood cells. STAT Requests: The bank sets up blood immediately and usually holds it for 12 h. For routine requests, the blood is set up at a date and time that you specify and usually held for 36 h. ROUTINE BLOOD DONATION Voluntary blood donation is the mainstay of the blood system in the United States. Donors must usually be >18 y old, in good health, afebrile. and weigh >110 lb. Donors are usually limited to 1 unit every 8 wk and 6 donations/y. Patients with a history of hepatitis, HBsAg positivity, insulin-dependent diabetes, IV drug abuse, heart disease, anemia, and homosex- ual activity are excluded from routine donation. Patients are counseled about high-risk be- haviors that may risk others if they have transmissible diseases and donate blood. Donor blood is tested for ABO, Rh, antibody screen, HBsAg, antihepatitis B core antigen, hepatitis C antibody, anti-HIV-1 and 2, and anti-HTLV-1 and 2. AUTOLOGOUS BLOOD DONATION Preadmission autologous blood banking (predeposit phlebotomy) is popular for some pa- tients anticipating elective surgery in which blood may be needed. General guidelines for autologous banking include good overall health status, a hematocrit greater than 34%, and 193 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 194 Clinician’s Pocket Reference, 9th Edition arm veins that can accommodate a 16-gauge needle. Patients can usually donate up to 1 unit every 3–7 days, until 3–7 days prior to surgery (individual blood banks have their own spec- ifications), depending on the needs of the planned surgery. Iron supplements (eg, ferrous gluconate 325 mg PO tid) are usually given prior to and several months after the donation. The use of erythropoietin is being investigated in this preoperative setting. Units of whole blood can be held for up to 35 days. DONOR-DIRECTED BLOOD PRODUCTS This method of donation involves a relative or friend donating blood for a specific patient. This technique cannot be used in the emergency setting because it takes up to 48 h to process the blood for use. This system has some drawbacks: Relatives may be unduly pressured to give blood, risk factors that would normally exclude the use of the blood (hepatitis or HIV positivity) be- come problematic, and ultimately the routine donation of blood for emergency transfusion may be adversely affected. These units are usually stored as packed red cells and released into the general transfusion pool 8 h after surgery unless otherwise requested. IRRADIATED BLOOD COMPONENTS Transfusion-associated GVHD, a frequently fatal condition, can be minimized through the 10 highly selected irradiation of blood components. Patients who are at risk for GVHD include recipients of donor-directed units or HLA-matched platelets, fetal intrauterine transfusions, and selected immunocompromised and bone marrow recipients. APHERESIS Apheresis procedures are used to collect single-donor platelets (plateletpheresis) or white blood cells (leukapheresis); the remaining components are returned to the donor. Thera- peutic apheresis is the separation and removal of a particular component to achieve a thera- peutic effect (eg, erythrocytapheresis to treat polycythemia). PREOPERATIVE BLOOD SET-UP Most institutions have established parameters (MSBOS) for setting up blood before proce- dures. Some typical guidelines are given in Table 10–1 for the number of units of packed red cells or if only a T&S is requested. EMERGENCY TRANSFUSIONS Non-cross-matched blood is rarely transfused because most blood banks can do a complete cross-match within 1 h. In cases of massive, exsanguinating hemorrhage, type-specific blood (ABO- and Rh-matched only), usually available in 10 min, can be used. If even this delay is too long, type O, Rh-negative, packed red blood cells can be used as a last resort. When possible, it is generally preferable to support blood pressure with colloid or crystal- loid until properly cross-matched blood is available. BLOOD GROUPS Table 10–2 gives information on the major blood groups and their relative occurrences. O– is the “universal donor” and AB+ is the “universal recipient.” 10 Blood Component Therapy 195 TABLE 10–1 Guidelines for Blood Required for Surgical Procedures Number of Units Procedure Needed Amputation (lower extremity) 2 Cardiac procedure (CABG, valve) 4 Cholecystectomy (open and laparoscopic) T&S Colon resection 2 Colostomy T&S Cystectomy, radical with diversion 4 Esophageal resection 2 Exploratory laparotomy 2 Gastrectomy 2 Gastrostomy T&S Hemorrhoidectomy T&S Hernia T&S Hysterectomy 2 Liver resection 6 Live transplant 6 10 Mastectomy T&S Nephrectomy 2 Pancreatectomy 4 Parathyroidectomy T&S Pulmonary resection 2 Radical neck dissection 2 Radical prostatectomy 3—4 Renal transplant 2 Small bowel resection 2 Splenectomy 2 Thyroidectomy T&S Tracheostomy 2 Total hip replacement 2 TURP 2 VASCULAR PROCEDURES Abdominal aortic aneurysm 6 Aortofemoral bypass 4 Aortoiliac bypass 4 Carotid endarterectomy T&S Femoral popliteal bypass 4 Iliofemoral bypass 4 Portacaval shunt 6 Splenorenal shunt 6 Vein stripping T&S Abbreviations: CABG = coronary artery bypass graft; T&S = type and screen; TURP = transurethral resection of the prostate. 196 Clinician’s Pocket Reference, 9th Edition TABLE 10–2 Blood Groups and Guidelines for Transfusion Type Can Usually Receive* (ABO/Rh) Occurrences Blood From O+ 1 in 3 O (+/−) O− 1 in 15 O (−) A+ 1 in 3 A (+/−) or O (+/−) A− 1 in 16 A (−) or O (−) B+ 1 in 12 B (+/−) or O (+/−) B− 1 in 67 B (−) or O (−) AB+ 1 in 29 AB, A, B, or O (all + or −) AB− 1 in 167 AB, A, B, or O (all −) *First choice is always the identical blood type, other acceptable combinations are shown. An attempt is also made to match Rh status of donor and recipient; Rh negative can usually be given to an RH+ recipient safely 10 BASIC PRINCIPLES OF BLOOD COMPONENT THERAPY Table 10–3 provides some common indications and uses for transfusion products. The fol- lowing are the basic transfusion principles for adults. Red Cell Transfusions Acute Blood Loss: Normal, healthy individuals can usually tolerate up to 30% blood loss without need for transfusion; patients may manifest tachycardia, mild hypotension without evidence of hypovolemic shock. Replace loss with volume (IV fluids, etc) replace- ment. • Hgb >10 g/dL, rarely needs transfusion. • Hgb 6–10 g/dL, transfuse based on clinical symptoms, unless patient has severe medical problems (ie, CAD, respiratory conditions). • Hgb <6 g/dL usually requires transfusion. “Allowable Blood Loss”: Often used to guide acute transfusion in the operating room setting. Losses less than allowable are usually managed with IV fluid replacement. Weight in kg x 0.08 = Total blood volume Total volume x 0.3 = Allowable blood loss (assumes normal hemoglobin) Example: A 70-kg adult Estimated allowable blood loss = 70 x 0.08 = 5600 mL x 0.3 = 1680 mL Chronic Anemia: Common in certain chronic conditions such as renal failure, rarely managed with blood transfusion; typically managed with pharmacologic therapy (eg, eryth- ropoietin). However, transfusion is generally indicated if Hgb < 6 g/dL or in the face of symptoms due to low hemoglobin. 10 197 TABLE 10–3 Blood Bank Products Product Description Common Indications Whole blood No elements removed Not for routine use (see also page 196) 1 unit = 450 mL ± 45 mL (HCT ≈ 40%) Acute, massive bleeding Contains RBC, WBC, plasma and Open heart surgery platelets (WBC & platelets may be Neonatal total exchange nonfunctional) Deficient in factors V & VII Packed Red Cells (PRBC) Most plasma, WBC, platelets removed; unit = Replacement in chronic and acute (see also page 196) 250–300 mL. (HCT ≈ 75%) blood loss, GI bleeding, trauma 1 unit should raise HCT 3% Universal Pedi-Packs 250–300 mL divided into 3 bags Transfusion of infants Contains red cells, some white cells, some plasma and platelets Leukocyte-Poor Most WBC removed by filtration to make Potential renal transplant patients (Leukocyte-reduced) it less antigenic Previous febrile transfusion reactions Red Cells <5 × 106 WBC, few platelets, Patients requiring multiple transfusions minimal plasma (leukemia, etc.) 1 unit = 200–250 mL Washed RBCs Like leukocyte-poor red cells, but WBC As for leukocyte-poor red cells, but very almost completely removed expensive and much more purified <5 × 108 WBC, no plasma 1 unit = 300 mL (continued ) 10 198 TABLE 10–3 (Continued) Product Description Common Indications Granulocytes 1 unit = ≈220 mL See page 194 (pheresis) Some RBC, >1 × 1010 PMN/unit, Lymphocytes, platelets Platelets (see also page 201) 1 “pack” should raise count by 5000–8000 Decreased production or destruction (ie, “6-pack” means a pool of platelets from aplastic anemia, acute leukemia, 6 units of blood postchemo, etc) Counts <5000–10,000 (risk of spontaneous hemorrhage) must transfuse 1 pack = about 50 mL Counts 10,000–30,000 if risk of bleeding >5 × 1010 platelets unit, contains RBC, WBC (headache, GI losses, contiguous petechiae) or active bleeding

Counts <50,000 if life-threatening bleed Prophylactic transfusion >20,000 for minor surgery or >50,000 for major surgery Usually not indicated in ITP or TTP unless life-threatening bleeding or preoperatively Platelets, pheresis >3 × 1010 platelets/unit See Platelets, may be HLA matched 1 unit = 300 mL Platelets, leukocyte-reduced As above, but <5 × 106 WBC/unit See Platelets, may decrease febrile reactions and CMV transmission, alloimmunization to HLA antigens Cryoprecipitated Contains factor VIII, factor XIII, Hemophilia A (factor VII deficiency), when Antihemophilic von Willebrand’s factor, and fibrinogen safer factor VIII concentrate not available; Factor (“Cryo”) 1 unit = 10 mL von Willebrand’s disease, fibrinogen deficiency, fibrin surgical glue (continued ) 10 199 TABLE 10–3 (Continued) Product Description Common Indications Fresh-Frozen Contains factors II, VII, IX, X, XI, XII, XIII Emergency reversal of Coumadin Plasma (FFP) and heat-labile V and VII Massive transfusion (>5 L in adults) About 1 h to thaw Hypoglobulinemia (IV immune globulin 150–250 mL (400–600 mL if single-donor preferred) pheresis) Suspected or documented coagulopathy (congenital or acquired) with active bleeding or before surgery) Clotting factor replacement when concentrate unavailable Not recommended for volume replacement If PT <22 s or PTT <70 s, 1 unit is usually sufficient Single Donor Like FFP, but lacks factors V and VIII No longer routinely used for plasma Plasma About 1 h to thaw; 150–200 ml replacement Stable clotting factor replacement Coumadin reversal, hemophilia B (Christmas disease) Rho Gam Antibody against Rh factor Rh-mother with Rh+ baby, within 72 h (Rho D immune globulin) (volume = 1 mL) of delivery, to prevent hemolytic disease of newborn; autoimmune thrombocytopenia (continued ) 10 200 TABLE 10–3 (Continued) Product Description Common Indications ALL OF THE AFOREMENTIONED ITEMS USUALLY REQUIRE A “CLOT TUBE” TO BE SENT FOR TYPING. THE FOLLOWING PRODUCTS ARE USUALLY DISPENSED BY MOST H OSPITAL PHARMACIES AND ARE USUALLY ORDERED AS A MEDICATION. Factor VII (purified From pooled plasma, pure Factor VIII Routine for hemophilia A (factor VII deficiency) antihemophilic factor) Increased hepatitis risk Factor IX concentrate Increased hepatitis risk Active bleeding in Christmas disease (prothrombin complex) Factors II, VII, IX, and X (Hemophilia B or factor IX deficiency) Equivalent to 2 units of plasma Immune serum globulin Precipitate from plasma “gamma globulin” Immune globulin deficiency Disease prophylaxis (hepatitis A, measles, etc.) 5% Albumin or 5% plasma Precipitate from plasma (see Drugs, Plasma volume expanders in acute blood loss protein fraction Chapter 22) 25% Albumin Precipitate from plasma Hypoalbuminemia, volume expander, burns Draws extravascular fluid into circulation Abbreviations: RBC = red blood cells; WBC = white blood cells; HCT = hematocrit; GI = gastrointestinal; ITP = idiopathic thrombocy- topenic purpura; TTP = thrombotic thrombocytopenic purpura; HLA = histocompatibility locus antigen; PT = prothrombin time; PTT = par- tial thromboplastin time. 10 Blood Component Therapy 201 RBC Transfusion Formula: As a guide, one unit of packed RBCs raises the HCT by 3% (Hgb 1 g/dL) in the average adult. To roughly determine the volume of whole blood or packed red cells needed to raise a hematocrit to a known amount, use the following formula: Total blood volume of patient × (Desired HCT − Actual HCT) Volume of cells = HCT of transfusion product where total blood volume is 70 mL/kg in adults, 80 mL/kg in children; the HCT of packed cells is approximately 70, and that of whole blood is approximately 40. White Cell Transfusions • The use of white cell transfusions is rarely indicated today due to the use of geneti- cally engineered myeloid growth factors such as GM-CSF (see Chapter 22) • Indicated for patients being treated for overwhelming sepsis and severe neutropenia (<500 PMN/µL) Platelet Transfusions For indications, see Table 10–3 Platelet Transfusion Formula: Platelets are often transfused at a dose of 1 unit/10 kg of body weight. After administration of 1 unit of multiple-donor platelets, the count should 10 rise 5000–8000/mm3 within 1 h of transfusion and 4500 mm3 within 24 h. Normally, stored platelets that are transfused survive in vivo 6–8 d after infusion. Clinical factors (DIC, al- loimmunization) can significantly shorten these intervals. To standardize the corrected platelet count to an individual patient, use the CCI. Measure the platelet count immediately before and 1 h after the platelet infusion. If the correction is less than expected, do a workup to determine the possible cause (antibodies, splenomegaly, etc). Many institutions are now using platelet pheresis units. One platelet pheresis unit has enough platelets to raise the count by 6000–8000/mm3. Using a single unit has the advantage of exposing the patient to only one donor versus possibly six to eight. This limits HLA exposures and reduces the risks of infection transmission. Posttransfusion platelet count − Pretransfusion count × Body surface area (m2 ) CCI = Platelets given × 1011 BLOOD BANK PRODUCTS Table10–3 describes products used in blood component therapy and gives recommendations for use of these products. TRANSFUSION PROCEDURES 1. Draw a clot tube (red top), and sign the lab slips to verify that the sample came from the correct patient. The patient should be identified by referring to the ID bracelet and asking the patient, if able, to state his or her name. Place the patient’s name, hospital number, date, and your signature on the tube label. Prestamped labels are not ac- cepted by most blood banks. 2. Obtain the patient’s informed consent by discussing the reasons for the transfusion and the potential risks and benefits from it. Follow hospital procedure regarding the need 202 Clinician’s Pocket Reference, 9th Edition for the patient to sign a specific consent form. At most hospitals, chart documentation is usually all that is necessary. 3. When the blood products become available, ensure good venous access for the transfu- sion (18-gauge needle or larger is preferred in an adult). 4. Verify the information on the request slip and blood bag with another person, such as a nurse, and with the patient’s ID bracelet. Many hospitals have defined protocols for this procedure; check your institutional guidelines. 5. Mix blood products to be transfused with isotonic (0.9%) NS only. Using hypotonic products such as D5W may result in hemolysis of the blood in the tubing. Lactated Ringer’s should NOT be used because the calcium could chelate the anticoagulant cit- rate. 6. Red cells are infused through a special filter. Specific leukocyte reduction filters are available and may be used in very specific circumstances (febrile transfusion reactions, to reduce potential CMV transmission, to reduce risk of alloimmunization to WBC antigens). 7. When transfusing large volumes of packed red cells (>10 units), monitor coagulation, Mg2+, Ca2+, and lactate levels. It is usually necessary to also transfuse platelets and FFP. Also, a calcium replacement is sometimes needed because the preservative used in the blood is a calcium binder and hypocalcemia can result after large amounts of blood are transfused. Also, for massive transfusions (usually >50 mL/min in adults and 15 10 mL/min in children), the blood should be warmed to prevent hypothermia and cardiac arrhythmias. TRANSFUSION REACTIONS Several types of transfusion reactions are possible: 1. Acute intravascular hemolysis. Over 85% of adverse hemolytic reactions involving the transfusion of RBCs result from clerical error. Usually caused by ABO incompati- ble transfusion. Can result in renal failure (<1/250,000 units transfused). 2. Nonhemolytic febrile reaction. Usually mild, fever, chills, rigors, mild dyspnea. Due to a reaction to donor white cells (HLA) and more common in patients who have had multiple transfusions or delivered several children. (≅2–3:100 units transfused) 3. Mild allergic reaction. Urticaria or pruritus can be caused by sensitization to plasma proteins in transfusion product. (≅1/100 units transfused) 4. Anaphylactic reaction. Acute hypotension, hives, abdominal pain and respiratory dis- tress; seen mostly in IgA-deficient recipients. (<1/1000 units transfused) 5. Sepsis. Usually caused by transfusion of a bacterially infected transfusion product, with platelets becoming an increasing risk. E. coli, Pseudomonas, Serratia, Salmonella, and Yersinia some of the more commonly implicated bacteria. (<1/500,000 RBC units transfused, 1/12,000 platelet units transfused) 6. Acute lung injury. Fever, chills, and life-threatening respiratory failure; probably in- duced by antibodies from donor against recipient white cells. (<1/5000 units trans- fused) 7. Volume overload. Usually due to excess volume infusion; can exacerbate CHF. Detection of a Transfusion Reaction 1. Spin an HCT to look for a pink plasma layer (indicates hemolysis). 2. Order serum for free hemoglobin and serum haptoglobin assays (haptoglobin decreases with a reaction) and urine for hemosiderin levels. Obtain a stat CBC to determine the presence of schistocytes, which can be present with a reaction. 10 Blood Component Therapy 203 3. If you suspect acute hemolysis, request a DIC screen (PT, PTT, fibrinogen, and fibrin degradation products). Treatment of Transfusion Reactions 1. Stop the blood product immediately, and notify the blood bank. 2. Keep the IV line open with NS, and monitor the patient’s vital signs and urine output carefully. 3. Save the blood bag, and have the lab verify the type and cross-match. Verify that the proper patient received the proper transfusion. Redraw blood samples for the blood bank. 4. Make specific recommendations, using the following guidelines; modifications should be based on clinical judgment. • Nonhemolytic febrile reaction: Antipyretics can be used and the transfusion con- tinued with monitoring. Use leukocyte-washed transfusion products in future. • Mild allergic reaction: Administer Benadryl (25–50 mg IM/PO/IV). Resume the transfusion carefully only if the patient improves promptly. • Anaphylactic reaction. Terminate transfusion, monitor closely, give antihistamines (Benadryl 25–50 mg IM/PO/IV), corticosteroids (Solu-Medrol 125 mg IV, 2 mg/kg Peds IV), epinephrine (1:1000 0.3–0.5 mL SQ adults, 0.1 mL/kg Peds), and pressors as needed. Premedicate (antihistamines, steroids) for future transfusions; use only 10 leukocyte-washed red cells. • Acute lung injury. Give ventilatory support as needed; use only leukocyte-washed red cells for future transfusions. • Sepsis: Culture the transfusion product and specimens from the patient; treat sepsis empirically by monitoring and administering pressors and antibiotics (third/fourth- generation cephalosporin or piperacillin/tazobactam along with an aminoglycoside) until cultures return. • Volume overload. Employ a slow rate of infusion with selective use of diuretics. • Acute intravascular hemolysis. Prevent acute renal failure. Place a Foley catheter, monitor the urine output closely, and maintain a brisk diuresis with plain D5W, man- nitol (1–2 g/kg IV), furosemide (20–40 mg IV), and/or dopamine (2–10 µg/kg/min IV) as needed. Consider alkalinization of the urine with bicarbonate (see Chapter 22). Beware of DIC. A renal and hematology consult are usually indicated with a se- vere hemolytic reaction. Support pressure as needed (fluids, vasopressors such as dopamine). TRANSFUSION-ASSOCIATED INFECTIOUS DISEASE RISKS Hepatitis Incidence of posttransfusion hepatitis for Hep B is 1:63,000 units transfused and for Hep C is 1:103,000 units transfused. Anicteric hepatitis is much more common than hepatitis with jaundice. Screening of donors for HBsAg and hepatitis C has greatly reduced these forms of hepatitis. Historically, the greatest risk is with pooled factor products (concentrates of Factor VIII). Use of albumin and globulins involves no risk of hepatitis. HIV Incidence is <1:600,000 units transfused. Antibody testing is routinely performed on the donor’s blood. A positive antibody test means that the donor may be infected with the HIV virus; a confirmatory Western blot is necessary. Do a follow-up test on any donor found to 204 Clinician’s Pocket Reference, 9th Edition be HIV-positive because false-positives can occur. With screening, AIDS transmission has decreased. Because there is a delay of 22 d between HIV exposure and the development of the HIV antibody, a potential risk of HIV transmission exists even with blood from a donor who is HIV-negative. Newer molecular detection methods should decrease this to approxi- mately 11 d. CMV Incidence in donors is very high (approaches 100% in many series), but clinically represents a major risk mostly for immunocompromised recipients and neonates. Leukocyte filters can reduce the risk of transmission if procedures are strictly followed. HTLV-I, II Very rare (<<1/641,000 units transfused). Use of leukocyte filters can decrease risk of trans- mission of HTLV. Bacteria and Parasites Sepsis due to bacteria is discussed on page 414. Parasites are very rarely transmitted, but careful donor screening is necessary, especially in endemic regions (eg, Chagas’ disease in Central America). 10 11 DIETS AND CLINICAL NUTRITION Hospital Diets Principles of Enteral Tube Feeding Nutritional Assessment Postoperative Nutritional Support Nutritional Requirements Infant Formulas and Feeding Determining the Route of Nutritional Support HOSPITAL DIETS The most commonly ordered standard hospital diets and their indications

are listed in Table 11–1, page 206. The vast majority of patients admitted to the hospital can be given one of these hospital diets without any specific supplementation or modification. Most hos- 11 pitals have diet manuals available for reference, and registered dietitians are usually on staff for nutritional consultation. A physician order for diet instruction by a clinical dietitian is recommended for all patients being discharged with a therapeutic or modified diet. NUTRITIONAL ASSESSMENT Nutritional screening should be incorporated into the history and physical evaluation of all patients. Identifying patients at nutrition risk is crucial because malnutrition is prevalent among hospitalized patients and has been associated with adverse clinical outcomes. Situa- tions that predispose a patient to malnutrition include recent and continuing nausea, vomit- ing, diarrhea, inability to feed oneself, inadequate food intake (cancer-related, others), decreased nutrient absorption or utilization, and increased nutrient losses and nutritional re- quirements. If needed, detailed nutritional assessment may be needed for some patients and is discussed in the following section. Although many patients are admitted to the hospital in a nutritionally depleted state, some patients become malnourished during their hospital stay. According to guidelines from the American Society for Parenteral and Enteral Nutrition, “patients should be considered malnourished or at risk of developing malnutrition if they have inadequate nutrient intake for 7 days or more or if they have a weight loss of 10% or more of their preillness body weight.” Formal evaluation is often necessary to identify patients at nutritional risk and to provide a baseline to assess whether therapeutic goals are being achieved with specialized nutritional support. The patient’s history is useful in evaluating weight loss; dietary intolerance, including that for glucose or lactose; and disease states that may influence nutritional tolerance. Anthropometric evaluations include comparisons of actual body weight to ideal and usual body weight. Other anthropometric measurements, such as MAMC and TCF, have much 205 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 11 206 TABLE 11–1 Hospital Diets Diet Guidelines Indications House/regular Adequate in all essential nutrients No diet restrictions or modifications All foods are permitted Can be modified according to patient’s food preferences Mechanical soft Includes soft-textured or ground foods that are Decreased ability to chew or swallow easily masticated and swallowed Presence of oral mucositis or esophagitis May be appropriate for some patients with dysphagia Pureed Includes liquids as well as strained and pureed Inability to chew or swallow solid foods foods Presence of oral mucositis or esophagitis May be appropriate for some patients with dysphagia Full liquid Includes foods that are liquid at body temperature May be appropriate for patients with severely impaired Includes milk/milk products chewing ability Can provide approximately: Not appropriate for a lactase-deficient patient unless 2500–3000 mL fluid commercially available lactase enzyme tablets are 1500–2000 Cal provided 60–80 g high-quality protein <10 g dietary fiber 60–80 g fat per day Clear liquid Includes foods that are liquid at body temperature Ordered as initial diet in the transition from NPO Foods are very low in fiber to solids lactose-free Used for bowel preparation before certain medical or virtually fat-free surgical procedures Can provide approximately: For management of acute medical conditions warranting 2000 mL fluid minimized biliary contraction or pancreatic exocrine 400–600 Cal secretion (continued) 207 TABLE 11–1 (Continued) Diet Guidelines Indications Clear liquid <7 g low-quality protein (continued) <1 g dietary fiber <1 g fat/day This diet is inadequate in all nutrients and should not be used >3 d without supplementation Low-fiber Foods that are low in indigestible carbohydrates Management of acute radiation enteritis and inflammatory Decreases stool volume, transit time, and bowel disease when narrowing or stenosis of the gut frequency lumen is present Carbohydrate Calorie level should be adequate to maintain or Diabetes mellitus controlled achieve desirable body weight diet (ADA) Total carbohydrates are limited to 50–60% of total calories Ideally fat should be limited to ≈30% of total calories Acute renal failure Protein (g/kg DBW) 0.6 For patients in renal failure who are not undergoing dialysis Calories 35–50 Sodium (g/day) 1–3 Potassium (g/day) Variable Fluid (mL/day) Urine output + 500 Renal failure/ Protein (g/kg DBW) 1.0–1.2 For patients in renal failure on hemodialysis Hemodialysis Calories (per kilogram DBW) 30–35 Sodium (g/d) 1–2 Potassium (g/d) 1.5–3 Fluid (mL/d) Urine output + 500 (continued) 11 208 TABLE 11–1 (Continued) Diet Guidelines Indications Peritoneal dialysis Protein (g/kg DBW) 1.2–1.6 For patients in renal failure on peritoneal dialysis Calories (per kilogram DBW) 25–35 Sodium (g/d) 3—4 Potassium (g/d) 3–4 Fluid (mL/d) Urine output + 500 Liver failure In the absence of encephalopathy do not restrict Management of chronic liver disorders protein In the presence of encephalopathy initially restricted protein to 40–60 g/d then liberalize in increments of 10 g/d as tolerated Sodium and fluid restriction should be specified based on severity of ascites and edema Low lactose/ Limits or restricts mild products Lactase deficiency Lactose-free Commercially available lactase enzyme tablets are available on the market Low-fat <50 g total fat per day Pancreatitis Fat malabsorption Fat/cholesterol Total fat >30% total calories Hypercholesterolemia restricted Saturated fat limited to 10% of calories <300 mg cholesterol <50% calories from complex carbohydrates Low-sodium Sodium allowance should be as liberal as possible Indicated for patients with hyptertension, ascites, and to maximize nutritional intake yet control symptoms edema associated with the underlying disease “No-added salt” is 4 g/d; no added salt or highly salted food; 2 g/d avoids processed foods (ie, meats) <1 g/d is unpalatable and thus compromises adequate intake 11 Diets and Clinical Nutrition 209 interobserver variability and are generally not useful unless performed by an experienced eval- uator. Absolute lymphocyte count is sometimes used as a marker of visceral proteins and im- munocompetence. Visceral protein markers, such as prealbumin and transferrin, may be helpful in evaluating nutritional insult as well as catabolic stress. Although the most com- monly quoted laboratory parameter of nutritional status is albumin, the albumin concentration often reflects hydration status and metabolic response to injury (ie, the acute phase response) more than the nutritional state of the patient, especially in patients with intravascular volume deficits. Due to its long half-life, albumin may be normal in the malnourished patient. Preal- bumin is superior as an indicator of malnutrition only because of its shorter half-life. Use of these serum proteins as indicators of malnutrition is subject to the same limitation, however, because they are all affected by catabolic stress. Table 11–2, page 210, lists the parameters for identifying potentially malnourished patients; however, no single criterion should be used to assess a patient’s nutritional status. Patients can generally be classified as mildly, moderately, or severely nutritionally depleted based on these parameters. NUTRITIONAL REQUIREMENTS Determining the patient’s nutritional requirements is one of the first steps in prescribing a modified diet order or supplementation for a patient. The following list provides guidelines for estimating nutritional needs. Monitoring the patient’s progress and adjusting nutritional goals on the basis of clinical judgment is important for ensuring that the patient’s specific needs are being met. Caloric needs can be determined by one of two means: the Harris–Benedict BEE and the “rule of thumb” method. Caloric Needs 11 A patient’s caloric needs can be calculated by the following methods: Harris–Benedict BEE For men: BEE = 66.47 + 13.75 (w) + 5.00 (h) − 6.76 (a) For women: BEE = 655.10 + 9.56 (w) + 1.85 (h) − 4.689 (a) where w = weight in kilograms; h = height in centimeters; and a = age in years. After the BEE has been determined from the Harris–Benedict equation, the patient’s total daily maintenance energy requirements are estimated by multiplying the BEE by an ac- tivity factor and a stress factor. Total energy requirements = BEE × Activity factor × Stress factor Use the following correction factors: Activity Level Correction Factor Bedridden 1.2 Ambulatory 1.3 Level of Physiologic Stress Correction Factor Minor operation 1.2 Skeletal trauma 1.35 Major sepsis 1.60 Severe burn 2.10 11 210 TABLE 11–2 Parameters Used to Identify the Malnourished Patient Parameters Measurement/Interpretation Usefulness/Limitations ANTHROPOMETRIC MEASUREMENT Actual body weight (ABW) compared “Rule-of-thumb” method to determine IBW with ideal body weight (IBW) Step 1 For men: IBW (lb) = 106 lb for 5 ft of height, plus 6 lb for each inch of height over 5 ft For women: IBW (lb) = 100 lb for first 5 ft of height plus an additional 5 lb for each inch over 5 ft Step 2 % IBW = ABW × 100 IBW % of IBW 90–110 Normal nutritional status 80–90 Mild malnutrition 70–80 Moderate malnutrition <70 Severe malnutrition Actual body weight compared with % UBW = ABW × 100 usual body weight (UBW) UBW % of UBW 85–95% Mild malnutrition 75–84% Moderate malnutrition <75% Severe malnutrition (continued) 11 211 TABLE 11–2 (Continued) Parameters Measurement/Interpretation Usefulness/Limitations BIOCHEMICAL PARAMETERS Serum albumin 3.5–5.2 g/dL Normal Routinely available 2.8–3.4 g/dL Mild depletion Valuable prognostic indicator: depressed levels 2.1–2.7 g/dL Moderate depletion predict increased mortality and morbidity <2 g/dL Severe depletion Inexpensive Large body stores and relatively long half-life (approximately 20 d) limit usefulness in evaluating short-term changes in nutritional status Transferrin (TFN) 200–300 mg/dL Normal Frequently available 150–200 mg/dL Mild visceral depletion Depressed levels predict increased mortality 100–150 mg/dL Moderate depletion and morbidity <100 mg/dL Severe depletion Smaller body pool and shorter half-life (8–10 days) than serum albumin TFN can be calculated from the total iron- If TFN is calculated from TIBC, levels will be binding capacity (TIBC) as follows: increased with the presence of iron defi- TFN = (0.8 × TIBC) − 43 ciency or chronic blood loss Levels are increased during pregnancy Levels are decreased if iron stores are increased as a result of hemosiderosis, hemochromatosis, thalassemia (continued) 11 212 TABLE 11–2 (Continued) Parameters Measurement/Interpretation Usefulness/Limitations Prealbumin 16–30 mg/dL Normal Half-life is 2 d. Thus is more sensitive indicator 10–15 mg/dL Mild depletion of acute change in nutritional status than is 5–10 mg/dL Moderate depletion albumin or TFN <5 mg/dL Severe depletion Not routinely available Levels are quickly depleted after trauma or acute infection. Also decreased in response to cirrhosis, hepatitis, and dialysis, and there- fore, should be interpreted with caution Absolute lymphocyte count 1400–2000 Mild depletion May not be valid in cancer patients. Not used (calculated as WBC × % 900–1400 Moderate depletion by some nutritionists lymphocytes) <900 Severe depletion 11 Diets and Clinical Nutrition 213 “Rule of Thumb” Method • Maintenance of the patient’s nutritional status without significant metabolic stress requires 25–30 Cal/kg body weight/d. • Maintenance needs for the hypermetabolic, severely stressed patient or for support- ing weight gain in the underweight patient without significant metabolic stress re- quires 35–40 Cal/kg body weight/d. • Greater than 40 Cal/kg body weight/d may be needed to meet the needs of severely burned patients. Protein Needs Maintenance requirements for nonstressed patients are 0.8 g of protein per kilogram of body weight. Repletion requirements of the nutritionally compromised patient are 1.2–2.5 g of protein per kilogram of body weight. DETERMINING THE ROUTE OF NUTRITIONAL SUPPORT Once nutritional support is indicated, the route for administration is chosen. Enteral supple- mentation by mouth or tube and parenteral nutrition are the main routes for providing nutri- tional support. Enteral Supplementation and Tube Feeding 11 Enteral nutrition encompasses both supplementation by mouth and feeding by tube into the GI tract. If the patient’s oral intake is inadequate, every effort should be made to increase in- take by providing nutrient-dense foods, frequent feedings, or oral supplements. If such at- tempts are unsuccessful, tube feeding may be indicated. In addition, patients who have a functioning GI tract but for whom oral nutrition intake is contraindicated should be consid- ered for tube feedings. If the GI tract is functioning and can be used safely, tube feedings should be ordered in- stead of parenteral nutrition when nutrition support is necessary because it • Is more easily absorbed physiologically • Is associated with fewer complications than TPN • Maintains the gut barrier to infection • Maintains the integrity of the GI tract • Is more cost-effective than TPN • Contraindications to tube feeding can be found in Table 11–3. Parenteral Nutrition Parenteral nutrition usually offers no advantage to the patient with a functioning GI tract. Because it does not achieve greater anabolism nor provide greater control over a patient’s nutritional regimen, parenteral

nutrition is indicated only when the enteral route is not us- able; therefore, the following rule applies: If the gut works, use it. Some patients, because of their disease states, cannot be fed enterally and require par- enteral feedings. Enteral nutrition is to be avoided in the situations noted in Table 11–3. TPN is typically used in these patients and is discussed in detail in Chapter 12. Although parenteral nutrition can be given either via central veins (TPN) or by periph- eral veins (PPN), the tonicity of the fluid required to administer all nutritional requirements 214 Clinician’s Pocket Reference, 9th Edition TABLE 11–3 Contraindications to Tube Feeding Complete bowel obstruction GI bleeding High-output (>500 mL/d) enterocutaneous fistula or fistula not located in the proximal or distal GI tract Hypovolemic or septic shock Ileus Inability to obtain safe enteral tube feeding access Poor prognosis not warranting invasive nutritional support Severe acute pancreatitis Severe intractable diarrhea Severe intractable nausea and vomiting Severe malabsorption Anticipated duration of tube feeding therapy <5 d intravenously requires central administration, and thus PPN may be used as a supplement, but is not adequate to provide all nutritional requirements. 11 PRINCIPLES OF ENTERAL TUBE FEEDING The factors involved in choosing the route for enteral nutrition include the projected dura- tion of feeding by this method, GI tract pathophysiology, and the risk for aspiration. Nasally placed tubes are the most frequently used. Patient comfort is maximized by using a small- bore flexible tube. When enteral feedings are started, it is often important to assess gastric residual volumes. The small-bore tubes do not allow for aspiration of residual volumes, however, which may be significant if gastric emptying is questionable. Thus, larger bore tubes are often used to start, and, once feeding tolerance is ensured, the tube is changed to a small-bore tube, which can be left in place comfortably for prolonged periods. Feeding di- rectly into the stomach (as opposed to the bowel) is often preferable because the stomach is the best line of defense against hyperosmolarity. Patients at risk for aspiration require longer tubes into the jejunum or duodenum. Types of feeding tubes and placement procedures are discussed in detail in Chapter 13, page 272. When long-term feeding is anticipated, a tube enterostomy is usually required. PEG tubes can usually be placed without general anesthesia. Patients with tumors, GI obstruc- tion, adhesions, or abnormal anatomy, however, may require open surgical placement. A je- junal feeding tube may be threaded through a PEG for small-bowel feeding. The placement of a needle catheter or Witzel’s jejunostomy during surgery generally allows for earlier post- operative feeding with an elemental formulation than waiting for the return of gastric emp- tying and colonic function. Enteral Products A variety of enteral products and tube feedings are available (see Table 11–4, page 215, for some examples). Check the enteral formulary for the specific products available in your fa- cility. 11 215 TABLE 11–4 Composition of Some Commonly Available Enteral Formulas Component (per 100 kcal) kcal/ Protein Fat Carbohydrates Na+ K+ mOsm/ Product mL (g) (g) (g) (mEq) (mEq) kg Meal replacements Require normal proteolytic and lipolytic function. Contain lactose. Compleat B 1.00 4.00 4.00 12.0 5.20 3.40 390 Lactose-free Provides proximal absorption. Requires normal proteolytic and lipolytic function. Low residue. Ensure 1.06 3.70 3.70 14.5 3.60 4.0 450 Ensure Plus 1.50 5.50 5.30 19.7 4.90 5.90 600 Isocal 1.06 3.70 3.80 14.4 2.40 2.60 300 Magnacal 2.0 3.5 4.0 12.5 2.20 1.60 590 Osmolite 1.06 3.70 3.80 14.4 2.40 2.60 300 Sustacal 1.00 6.10 2.30 13.8 4.10 5.40 620–700 Travasorb MCT 1.00 4.90 3.30 12.2 1.50 4.50 312 Elemental formulas Provide rapid proximal absorption. Indicated for pancreatic-biliary dysfunction, selective malabsorption, fistu- las, and short bowel syndrome (SBS). Low residue. Nutrients predigested. Peptamen 1.0 4.0 3.9 12.7 2.20 3.21 270 Reabilan 1.0 3.15 4.30 13.2 3.05 3.20 350 Reabilan HN 1.33 4.36 4.30 11.9 3.26 3.18 490 Vital HN 1.00 4.20 1.00 18.8 2.70 3.40 450 Vivonex TEN 1.00 3.82 0.28 20.5 2.00 2.00 630 Vivonex 1.00 2.04 0.15 22.6 2.00 3.00 550 (continued) 11 216 TABLE 11–4 (Continued) Component (per 100 kcal) kcal/ Protein Fat Carbohydrates Na+ K+ mOsm/ Product mL (g) (g) (g) (mEq) (mEq) kg Special metabolic May require vitamin-mineral supplement if used as principal source of nutrition. Amin-Aid 2.00 1.90 4.70 37.3 >1 >1 850 Glucerna 1.0 4.18 5.57 9.37 4.03 4.0 375 Pulmocare 1.5 4.17 6.14 7.04 3.80 2.95 490 Hepatic Aid II 1.17 4.30 3.60 16.8 >1 >1 560 Travasorb Hepatic 1.10 2.90 1.40 20.9 1.9 2.9 690 Travasorb Renal 1.35 2.30 1.80 27.1 >1 >1 590 Fiber-containing Nutritionally complete tube feeding that may help maintain normal bowel function and useful in patients who demonstrate intolerance to low-residue feedings. Enrich 1.1 3.62 3.39 14.3 3.35 3.94 480 (1.3 g fiber) Jevity 1.06 4.20 3.48 14.4 3.81 3.77 310 (1.36 g fiber) Note: Formulation of products at the time of publication. Actual components may vary slightly. 11 Diets and Clinical Nutrition 217 To simplify selection, the nutritional components and osmolality of the enteral product are listed and help classify the formulations. The protein component can be supplied as in- tact proteins, partially digested hydrolyzed proteins, or crystalline amino acids. Each gram of protein provides 4 Cal. The carbohydrate source may be intact complex starches, glucose polymers, or simpler disaccharides such as sucrose. Carbohydrates provide 4 Cal/g. Fat in enteral products is usually supplied as long-chain fatty acids. Some enteral products, how- ever, contain MCTs, which are transported directly in the portal circulation rather than via chyle production. Because MCT oil does not contain essential fatty acids, it cannot be used as the sole fat source. Long-chain fatty acids provide 9 Cal/g, and MCT oil provides 8 Cal/g. The osmolality of an enteral product is determined primarily by the concentration of carbohydrates, electrolytes, amino acids, or small peptides. The clinical importance of os- molality is often debated. Hyperosmolal formulations, with osmolalities exceeding 450 mOsm/L, may contribute to diarrhea by acting in a manner similar to osmotic cathartics. Hyperosmolal feedings are well tolerated when delivered into the stomach (as opposed to the small bowel) because gastric secretions dilute the feeding before it leaves the pylorus to traverse the small bowel. Thus, feedings administered directly to the small bowel (eg, via feeding jejunostomy) should not exceed 450 mOsm/L. Oral supplements differ from other enteral feedings in that they are designed to be more palatable so as to improve compliance. Although most enteral products do not contain lactose (Ensure, Osmolite, others), several oral supplements, commonly referred to as “meal replacements” (such as Compleat B) contain lactose and are therefore not appropriate for patients with lactase deficiency and are not normally used for tube feedings. Based on osmolality and macronutrient content, enteral products can be classified into several categories. Low-osmolality formulas are isotonic and contain intact macronutrients. They usually 11 provide 1 Cal/mL and require approximately 2 L to provide the RDA for vitamins. These products are appropriate for the general patient population and include products such as Ensure. High-density formulas may provide up to 2 Cal/mL. These concentrated solutions are hyperosmolar and also contain intact nutrients. The RDA for vitamins can be met with vol- umes of 1500 mL or less. These products are used for volume-restricted patients. Examples are Nutren 2.0 and Ensure Plus HN. Chemically defined or elemental formulas provide the macronutrients in the predi- gested state. These formulations are usually hyperosmolar and have poor palatability. Pa- tients with compromised nutrient absorption abilities or GI function may benefit from elemental type feedings. Vivonex and Peptamen are two such products. Disease-specific (special metabolic) enteral formulas have been developed for various disease states. Products for pulmonary patients, such as Pulmocare, contain a higher per- centage of calories from fat to decrease the carbon dioxide load from the metabolism of ex- cess glucose. Patients with hepatic insufficiency may benefit from formulations (eg, Hepatic-Aid II) containing a higher concentration of the branched-chain amino acids and a lower concentration of aromatic amino acids in an attempt to correct their altered serum amino acid profile. Formulas containing only essential amino acids have been marketed for the patient in renal failure (Amin-Aid). A low-carbohydrate, high-fat product for persons with diabetes (Glucerna) is available that also contains fiber to help regulate glucose control. Other fiber-containing enteral feedings are available to help regulate bowel function (En- rich, Jevity). The clinical utility of many of the specialty products remains controversial. Initiating Tube Feedings Guidelines for ordering enteral feedings are outlined in Table 11–5, page 218. In summary, when using enteral feedings: 218 Clinician’s Pocket Reference, 9th Edition TABLE 11–5 Routine Orders for Enteral Nutrition Administered by Tube Feeding 1. Confirm tube placement. (Usually by x-ray) 2. Elevate head of bed to 30–45 degrees 3. Check gastric residuals in patients receiving gastric feedings. Hold feed- ings if >1.5–2x infusion rate. Significant residuals should be reinstilled and rechecked in 1 h. If continues to be elevated, hold tube feeding and begin NG suction. 4. Check patient weight 3x/wk. 5. Record strict I&O 6. Request routine laboratory studies 1. Determine nutritional needs. 2. Assess GI tract function and appropriateness of enteral feedings. 3. Determine fluid requirements and volume tolerance based on overall status and concur- rent disease states. 4. Select an appropriate enteral feeding product and method of administration. 5. Verify that the regimen selected satisfies micronutrient requirements. 6. Monitor and assess nutritional status to evaluate the need for changes in the selected 11 regimen. The tube feeding can be given into the stomach (bolus, intermittent gravity drip, or continu- ous) or into the small intestine by continuous infusion (Table 11–6, page 219). Enteral nutri- tion is best tolerated when instilled into the stomach because this method produces fewer problems with osmolarity or feeding volumes. The stomach serves as a barrier to hyperos- molarity, thus the use of isotonic feedings is mandated only when instilling nutrients di- rectly into the small intestine. The use of gastric feedings is thus preferable and should be used whenever appropriate. Patients at risk for aspiration or with impaired gastric emptying may need to be fed past the pylorus into the jejunum or the duodenum. Feedings via a je- junostomy placed at the time of surgery can often be initiated on the first postoperative day, obviating the need for parenteral nutrition. Although enteral nutrition is generally safer than parenteral nutrition, aspiration can be a significant morbid event in the care of these patients. Appropriate monitoring for residual volumes in addition to keeping the head of the bed elevated can help prevent this complica- tion. A “significant residual” may be defined as 11⁄2 times the instillation rate. This can be treated in a number of ways. Any transient postoperative ileus can best be treated by waiting for the ileus to resolve. Metoclopramide or erythromycin may be useful pharmacologic ther- apy for postop ileus (Chapter 22). Patients who have been tolerating feedings and develop intolerance should be carefully assessed for the cause. Feeding intolerance is characterized by vomiting, abdominal distention, diarrhea, or high gastric residual volumes. Complications of Enteral Nutrition Diarrhea: Diarrhea occurs in about 10–60% of patients receiving enteral feedings. The physician must be certain to evaluate the patient for other causes of diarrhea. Formula- related causes include contamination, excessively cold temperature, lactose intolerance, os- molality, and an incorrect method or route of delivery. Eliminate potential causes before using antidiarrheal medications. 11 219 TABLE 11–6 Tube Feeding Delivery Methods Delivery Site/ Indication Delivery Method Notes Suggested Feeding Progression INTRAGASTRIC Bolus Rapid infusion of formula into the Typical starter regimen: 60–120 mL of full- Appropriate for alert stomach by syringe or other strength formula is generally provided patients with intact feeding reservoir; generally Typical feeding progression: Volume of for- gag and cough re- 240–480 mL of formula is given mula provided at each feeding may be flexes and for those every 3–6 h increased in 60–120 mL increments with normal gastric Feedings are usually given over a every 12 h or as tolerated emptying period of 5–15 min Associated symptoms of GI distress, such as bloating, nausea, and distention INTRAGASTRIC Intermittent gravity Generally 240–480 mL of formula Typical starter regimen: 60–120 mL of full- drip is allowed to drip from a feeding strength formula is generally provided container through tubing over a

Typical feeding progression: Volume of 30–60 min period four to eight formula provided at each feeding may times per day be increased to 60–120 mL increments Rate of formula administration is every 12 h or as tolerated controlled with a clamp in the tubing May reduce the incidence of GI complications associated with bolus delivery Highly viscous formulas, such as those that contain 2 Cal/mL, may not flow through the tubing (continued) 11 220 TABLE 11–6 (Continued) Delivery Site/ Indication Delivery Method Notes Suggested Feeding Progression More expensive than bolus method because feeding containers are necessary Not recommended for critically ill patients INTRAGASTRIC Continuous Preferred method to administer Typical starter regimen: Full-strength formula formula if gastric feeding is is generally initiated at a rate of 40 or necessary for a critically ill 50 mL/h patient because it reduces Typical feeding progression: Feeding rate is risk of aspiration generally increased in increments of Use of a feeding pump to deliver 10–15 mL/h every 12 h or as tolerated precise volumes of formula until the goal feeding rate is achieved at a constant rate Goal feeding rates are typically between 80 and 125 mL/h, depending on the individual’s nutritional requirements Volume- and rate-controlled delivery minimizes gastric emptying and reduces the incidence of osmotic diarrhea secondary to dumping syndrome (continued) 11 221 TABLE 11–6 (Continued) Delivery Site/ Indication Delivery Method Notes Suggested Feeding Progression In the hospital setting, the formula is usually provided over a 24-h period; home patients may cycle feedings over an 8–14-h period May be necessary to deliver for- mulas with high viscosity Necessity of feeding pump in addition to feeding bag and tubing increases cost Restricts ambulation in patients who are not critically ill INTRAINTESTINAL Continuous Feeding pump required because Typical starter regimen: Full-strength Appropriate for excessively rapid formula formula is generally initiated at a rate patients who are at delivery, as would occur with of 40–50 mL/h; markedly hypertonic high risk for bolus or gravity drip admin- formulas (>600 mOsm/L) occasionally aspiration, including istration, would probably may be diluted to half-strength if dumping those who cannot result in dumping syndrome, syndrome is present or if a prolonged keep the proper allows tube feeding formula period without enteral nutrition has position during to be delivered in a more elapsed feeding (head of physiologic manner Typical feeding progression: Feeding rate is bed 30 degrees upright) Goal rates are usually generally increased in increments of (continued) 11 222 TABLE 11–6 (Continued) Delivery Site/ Indication Delivery Method Notes Suggested Feeding Progression and those without 80–125 mL/h, depending on the 10–12 mL/h every 12 h or as tolerated an intact gag reflex patient’s nutritional needs until the goal feeding rate is achieved; Usually 24-h infusions are given in if hypertonic formula was initially Required feeding route the hospital, but cyclic infusions diluted, the patient can be switched to when proximal (ie, are an option for the ambulatory full-strength formula after the goal feeding oral, esophageal, or or home patient rate is achieved gastric) GI obstruction Associated with high cost because or impairment is of necessity of feeding containers present and infusion pump Continuous infusions may restrict Preferred delivery site patient ambulation for critically ill patients 11 Diets and Clinical Nutrition 223 • Check medication profile for possible drug-induced cause. • Rule out Clostridium difficile colitis in patients receiving antibiotics (see Chapter 7). • Attempt to decrease the feeding rate or try an alternative regimen such as bolus feed- ing. • Change the formulation, for example, limit lactose or reduce the osmolality. • Use pharmacologic therapy only after eliminating treatable causes (eg, give Lacto- bacillus powder [one packet tid to replenish gut flora]; most effective in patients on antibiotics) or antidiarrheal medications (loperamide [Lomotil], calcium carbonate). Constipation: Although less common than diarrhea, constipation can occur in the enter- ally fed patient. Check to ensure that adequate fluid volume is being given. Patients with ad- ditional requirements may benefit from water boluses or dilution of the enteral formulation. Fiber can be added to help regulate bowel function. Aspiration: Aspiration is a serious complication of enteral feedings and is more likely to occur in the patient with diminished mental status. The best approach is prevention. Ele- vate the head of the bed and carefully monitor residual fluid volume. Further evaluate any patient who may have aspirated or who is assessed as being at increased risk for aspiration prior to instituting enteral feedings. Such patients may not be candidates for gastric feed- ings, and small-bowel feedings may be necessary. Drug Interactions: The vitamin K content of various enteral products varies from 22 to 156 mg/1000 Cal. This can significantly affect the anticoagulation profile of a patient re- ceiving warfarin therapy. Tetracycline products should not be administered 1 h before or 2 h 11 after enteral feedings to avoid the inhibition of absorption. Similarly, enteral feedings should be stopped 2 h before and after the administration of phenytoin. POSTOPERATIVE NUTRITIONAL SUPPORT Most patients can be started on oral feedings postoperatively, the question is when to begin them. Begin feedings once the bowel recovers motility. Motility is delayed in patients un- dergoing laparotomy, whereas feedings begin fairly quickly for patients who undergo surgery on other parts of the body, once they recover consciousness sufficiently to protect their airway. Remember that the gut recovers motility as follows: The small intestine never loses motility (peristalsis is observed in the OR), the stomach regains motility about 24 h postoperatively, and the colon is the last to recover at 72–96 h postoperatively. Thus, by the time a patient reports flatus, one can assume that the entire gut has regained motility. Feed- ings then begin, depending on the exact operation performed and the resulting gastrointesti- nal anatomy. Patients who are to begin oral feedings are usually started on clear liquids (see Table 11–1). As long as the patient is willing to eat regular food, there is no reason not to progress to a regular diet rapidly (after one meal of clear liquids), and there is no need to step through a progression from clear liquids to full liquids to a regular diet. INFANT FORMULAS AND FEEDING Bottle feeding is often chosen by the mother and, in general, commercially available formu- las are recommended over homemade formulas because of their ease of preparation and their standardization of nutrients. Occasionally, special formulas are medically indicated and can only be supplied by commercially available formulas. Commonly used formulas are outlined in Table 11–7. 224 Clinician’s Pocket Reference, 9th Edition TABLE 11–7 Commonly Used Infant Formulas Formula Indications* Human milk Donor Preterm infant <1200 g Maternal All infants Breast milk fortifiers Standard formulas Isoosmolar Enfamil 20 Full-term infants: as supplement to breast milk Similac 20 Preterm infants >1800–2000 g SMA 20 Higher Osmolality Enfamil 24 Term infants: for infants on fluid restriction or Similac 24 & 27 who cannot handle required volumes of 20-Cal SMA† 24 & 27 formula to grow Low Osmolality Similac 13 Preterm and term infants: for conservative initial feeding in infants who have not been fed orally for several days or weeks. Not for long-term use. 11 Soy formulas ProSobee (lactose- Term infants: milk sensitivity, galactosemia, carbo- and sucrose-free) hydrate intolerance. Do not use in preterm in- Isomil (lactose-free) fants. Phytates can bind calcium and cause Nursoy (lactose-free) rickets Protein hydrosylate formulas Nutramigen Term infants: Gut sensitivity to proteins, multiple food allergies, persistent diarrhea, galac- tosemia. Pregestimil Preterm and term infants: disaccharidase defi- ciency, diarrhea, GI defects, cystic fibrosis, food allergy, celiac disease, transition from TPN to oral feeding Alimentum Term infants: protein sensitivity, pancreatic insuf- ficiency, diarrhea, allergies, colic, carbohydrate and fat malabsorption Special formulas Portagen Preterm and term infants: pancreatic or bile acid insufficiency, intestinal resection Similac PM 60/40 Preterm and term infants: problem feeders on standard formula; infants with renal, cardio- vascular, digestive diseases that require de- creased protein and mineral levels, breast- feeding supplement, initial feeding (continued) 11 Diets and Clinical Nutrition 225 TABLE 11–7 (Continued) Formula Indications* Premature formulas Low osmolality Similac Special Premature infants (<1800–2000 g) who are Care 20 growing rapidly. These formulas promote Enfamil Premature 20 growth at intrauterine rates. Vitamin and Preemie SMA 20 mineral concentrations are higher to meet the needs of growth. Usually started on 20 Cal/oz and advanced to 24 Cal/oz as toler- ated. Isoosmolar Similac Special Same as for low-osmolality premature formulas Care 24 Enfamil Special Care 24 Preemie SMA 24 *Multivitamin supplementation such as Polyvisol (Mead Johnson) ¹₂ mL/d may be needed for commercial formulas if baby is taking <2 oz/d. 11 †SMA has decreased sodium content and can be used in patients with congestive heart failure, bronchopulmonary dysplasia, and cardiac disease. Modified and produced with permission from Gomella, TL (ed) Neonatology, 4th ed. Norwalk, CT, Appleton & Lange, 1999 Principles of Infant Feeding Criteria for Initiating Infant Feeding: Most normal full-term infants are fed within the first 4 h after birth. The following criteria should usually be met before initiating infant feedings. • The infant should have no history of excessive oral secretions, vomiting, or bile- stained gastric aspirate. • An examination should have been performed with particular attention to the ab- domen. The examination should be normal with normal bowel sounds and a nondis- tended, soft abdomen. • The infant should be clinically stable. • At least 6 h should pass before recently extubated infants are fed. The infant should be tolerating extubation well and have little respiratory distress. • The respiratory rate should be <60 breaths/min for oral feeding and <80 breaths/min for gavage (tube) feeding. Tachypnea increases the risk of aspiration. Prematurity: Considerable controversy remains concerning the timing of initial enteral feeding for the preterm infant. For the stable larger (>1500 g) premature infant, the first feeding may be given within the first 24 h of life. Early feeding may allow the release of en- teric hormones that exert a trophic effect on the intestinal tract. On the other hand, appre- 226 Clinician’s Pocket Reference, 9th Edition hension about necrotizing enterocolitis (mostly in very low birth weight infants) in the fol- lowing circumstances often precludes the initiation of enteral feeding: perinatal asphyxia, mechanical ventilation, presence of umbilical vessel catheters, patent ductus arteriosus, in- domethacin treatment, sepsis, and frequent episodes of apnea and bradycardia. No established policies are available, and delay and duration of delay in establishing feeding with those conditions varies for every institution. In general, enteral feeding is started in the first 3 d of life, with the objective of reaching full enteral feeding by 2–3 wk of life. Parenteral nutrition including amino acids and lipids should be started at the same time to provide for adequate caloric intake. Choice of Formula: (See Table 11–7, page 224.) Human breast milk is recommended for feeding infants whenever possible. Breast-feeding has many advantages: It is ideal for virtually all infants, produces fewer infantile allergies, is immunoprotective to the infant due to the presence of immunoglobulins, is convenient and economical, and offers several theo- retical psychologic benefits to both the mother and child. Occasionally, an infant cannot be breast-fed due to extreme prematurity or other problems such as a cleft palate. If commercial infant formula is chosen, no special considerations are needed for normal full-term newborns. Selection of the best formula for preterm infants may require more care. The majority of infant formulas are isoosmolar (Similac 20, Enfamil 20, and SMA 20 with and without iron). These formulas are used most often for healthy infants. Formulas for pre- mature infants, containing 24 Cal/oz (Similac 24, Enfamil 24, “preemie” SMA 24), are also isoosmolar and are indicated for rapidly growing premature infants. Many other “specialty” formulas are available for such conditions as milk and protein sensitivity, among others. 11 Many pediatricians recommend vitamin supplements with some formulas if the infant is taking <32 oz/day. An iron-containing formula is generally recommended. Feeding Guidelines 1. Initial feeding. For the initial feeding for all infants, use sterile water or 5% dextrose in water (D5W) if the infant is not being breast-fed. Ten % dextrose in water (D10W) should not be used because it is a hypertonic solution. 2. Subsequent feedings. There is controversy over whether infant formulas should be di- luted for the next several feedings if the infant tolerates the initial one. Some clinicians advocate diluting formulas with sterile

water and advance as tolerated (eg, ¹₄ strength, increase to ¹₂ and then ³₄ strength). Others feel this is unnecessary and that full- strength formula can be used if infants tolerate the initial feeding without difficulty. Breast milk is never diluted. Oral Rehydration Solutions: Infants with mild or moderate dehydration, often due to diarrhea or vomiting, may benefit from oral rehydration formulas. These solutions typically include glucose, sodium, potassium, and bicarbonate or citrate. Common formulations in- clude Pedialyte, Lytren, Infalyte, Resol and Hydrolyte. 12 TOTAL PARENTERAL NUTRITION Common Indications Fat Emulsions Nutritional Principles Starting TPN Nitrogen Balance Assessing TPN Therapy TPN Solutions Stopping TPN Peripheral Parenteral Nutrition Disease-Specific TPN Formulations TPN Additives Common TPN Complications COMMON INDICATIONS Total parenteral nutrition, also called “hyperalimentation,” is the provision of all essential nutrients—protein, carbohydrates, lipids, vitamins, electrolytes, and trace elements—by the intravenous route. Nutrients may be supplied by either a peripheral or central vein. To pro- vide a patient’s entire nutritional requirement by vein, however, a central venous line must be used because of the tonicity of the fluid required. Peripheral veins simply cannot tolerate 12 these hypertonic fluids, and thus peripheral IV alimentation can be used only as a supple- ment. Parenteral nutrition bypasses the GI tract and should be reserved for patients who are unable to receive nutritional support enterally. The principle of “if the gut works use it” is sound practice. How to determine the route of nutritional support is discussed on page 213. The following indications are appropriate for TPN initiation: • Preoperatively, in the malnourished patient. There is no benefit for patients who are not malnourished. • Postoperatively, for patients with a slow return of GI function or in patients with complications that limit or prohibit the use of the GI tract. The interval between surgery and initiation of nutritional support to prevent complications is not defini- tively known. However, many practitioners wait 7–10 d after surgery, anticipating the return of bowel function. If this does not occur, nutritional support is begun. • Patients with Crohn’s disease, ulcerative colitis, pancreatitis, fistulas, and short- bowel syndrome. • Patients who are malnourished secondary to a disease or injury that results in inade- quate oral intake. This may include patients with organ failure, severe metabolic stress, malignancies, burns, or trauma. NUTRITIONAL PRINCIPLES Nutritional assessment to determine the need for TPN requires a history (which in- cludes weight changes over the previous 6 mo), physical, and laboratory evaluation. In- dicators of long-term nutritional depletion include serum albumin and prealbumin levels, 227 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 228 Clinician’s Pocket Reference, 9th Edition anthropometrics, and total lymphocyte count. Nutritional assessment is presented in detail in Chapter 11, page 206. To establish the appropriate caloric amount for TPN therapy, estimate the patient’s daily nonprotein calories and nitrogen requirements. The best method for calculating the BEE re- quirements for nonprotein calories is the Harris–Benedict equation (Chapter 11, page 209). The weight used in this equation determines the amount of calories needed to maintain that weight; therefore, if the patient is morbidly obese, the ideal weight should be established as a goal. Calculation of Caloric Requirements in Stressed Patients The BEE obtained from the Harris–Benedict equation reflects the number of calories from carbohydrate and fat that should be provided to maintain the patient’s weight under non- stressed conditions. Stress, in nutritional terms, is correlated with the amount of cate- cholamines and cortisol released endogenously. These biochemical mediators promote protein breakdown, which is necessary to provide glucose for the brain and red blood cells. • Mild stress: Supply total calories at approximately 1.2–1.4 × BEE. • Moderate stress: 1.5–1.75 × BEE. • Severe stress: 1.75–2.0 × BEE. • Ideally, 25–35 Cal/kg/d should be the dosing range. Bear in mind the patient’s safety may be of concern should these values exceed a daily intake greater than 3000 Cal. In the event this occurs, dose conservatively until nitrogen balance data confirms the need for more aggressive caloric replacement. Nutritional Component Considerations 12 The fundamental principle of TPN is the administration of sufficient protein to avoid catab- olism of endogenous protein (muscle). Carbohydrates must be given to supply necessary calories (at a ratio of 150 Cal/g of nitrogen) to support these anabolic processes. Fat is given as a source of essential fatty acids. The basis for using TPN explains the necessity for pro- tein, carbohydrate, and fat administration. In addition, TPN includes all necessary fluids, electrolytes, vitamins, and trace elements required to support life. Studies have shown that doses between 4–7 mg/kg/min of carbohydrate (generally, do not exceed 5 mg/kg/min) provide optimal protein sparing with minimal liver toxicity. As- sessment of the carbohydrate intake is important in order to limit complications from TPN. Lipid calories should not exceed 3 g/kg/d due to increased complications. Additionally, no more than 50% of total daily calories should be administered as fat. The best method for establishing a protein need for a given patient is the 24-h urine sample testing for UUN levels. This value reflects the amount of protein catabolism occur- ring daily. Urinary losses of 8–12 g/d are consistent with a mild stress condition, 14–18 g/d moderate stress, and greater than 20 g/d with severe stress. Protein dosing should be modified based on the 24-h UUN and daily nitrogen balance. Initially, however, if the patient is considered mildly stressed, 0.8–1.2 g/kg/d is appropriate. In cases of moderate and severe stress (burned and head injured patients) 1.3–1.75 g/kg/d and 2–2.5 g/kg/d may be required, respectively. (Note: Generally, do not exceed 2.0 g/kg/d.) Several studies suggest that doses of protein in this range exceed the patients utilization ca- pacity and may increase BUN. Adequate renal function must be present to provide such high protein loads. Patients with renal failure who are not receiving dialysis may be dosed at the minimum daily allowance, 0.6 g/kg/d, until a decision for dialysis is made. Once the pa- tient is receiving dialysis, normal dosing may be instituted. 12 Total Parenteral Nutrition 229 NITROGEN BALANCE The best method for determining the adequacy of nutritional support is the calculation of ni- trogen balance. A positive nitrogen balance implies that the amount of protein being admin- istered is sufficient to cover the losses of endogenous protein that occur secondary to catabolism. This is the best therapeutic goal for TPN because it is impossible to determine whether the prescribed protein is preventing muscle breakdown or not. Once positive nitrogen balance has been achieved, however, protein replacement has been optimized. In critical care patients, nitrogen losses may be very high, and an attempt should be made to at least achieve nitrogen equilibrium. This may be impossible in the acute phase of injury, in severe trauma, or in burn cases. Thus, minimizing protein loss (–2 to –4 g/d) may be the goal during this period. A negative nitrogen balance is indicative of insufficient protein replacement for the degree of skeletal muscle loss. Under most circumstances, an attempt to achieve positive ni- trogen balance should be made. Patients with renal dysfunction or those who are severely stressed may not be able to achieve a positive balance due to safety concerns. The efficacy of protein doses exceeding 2.5 g/kg/d has not been established. Investigational agents (growth hormone, IGF-1) and specialized formulas (branched-chain amino acids, essential amino acids, glutamine) are being studied in these populations to assess their potential in improving nitrogen retention under these circumstances. The following are key concepts in determining nitrogen balance: • Nitrogen balance = Nitrogen input – Nitrogen output. • 1 g of nitrogen = 6.25 g of protein. • Nitrogen input = (Protein in grams/6.25 g nitrogen). • Nitrogen output = 24-h UUN + 4 g/d (nonurine loss). • The conditions and disease states that increase the amount of nonurine losses for ni- trogen include high-output fistulas and massive diarrhea. Fecal nitrogen measure- 12 ments can be obtained but are difficult for nursing staff to perform. Sample Determination of Nitrogen Balance A patient is receiving 2 L TPN/24 h with 27.5 g crystalline amino acid (protein) solution per liter. 1. 27.5 g protein/L × 2 L = 55 g protein/24 h. 2. Recall that 1 g of nitrogen = 6.25 g of protein. 3. Nitrogen input = 55 g protein/6.25 g protein per gram N = 8.8 g. 4. Patient voided 22.5 dL urine/ 24 h with UUN 66 mg/dL. 5. Nitrogen lost in urine = 22.5 dL × 66 mg/dL = 1485 mg, or about 1.5 g. 6. Add 4.0 g for nonurine nitrogen loss. 7. Nitrogen output = 1.5 g + 4.0 = 5.5 g. 8. Nitrogen balance = Input − output = 8.8 − 5.5 = +3.3 g nitrogen. TPN SOLUTIONS Different strength CAA solutions are available (Table 12–1) to which the pharmacy can add varying concentrations of dextrose, electrolytes, vitamins, and trace elements. Most hospi- tals supply a “house,” or standard, formula for patients with normal renal and hepatic func- tion. Changes in the standard formulas can be made when necessary while a TPN solution is being infused based on measured laboratory parameters. Administration of TPN is never an emergency and in most cases can be provided within 24 h of prescribing. If a formula change is necessary based on a change in patient status, discontinue the TPN and replace it with D10W at the same rate until a new bag of TPN can be provided. 230 Clinician’s Pocket Reference, 9th Edition TABLE 12–1 Typical TPN Solutions for Adults Component Solution 1 Solution 2 CAA 4.25% (42.5 g/L) 4.25% (42.5 g/L) Dextrose 25% (250 g/L, 12.5% (125 g/L, 850 Cal/L) 425 Cal/L) Na 50 mEq/L 50 mEq/L K 50 mEq/L 50 mEq/L Ca 6 mEq/L 6 mEq/L Mg 6 mEq/L 6 mEq/L PO4 15 mMol/L 15 mMol/L Cl 45 mEq/L 45 mEq/L Abbreviation: CAA = crystalline amino acids. Amino acid formulas are supplied as CAA or SAA in concentrations ranging from 3.5–15%. These are diluted by the pharmacy to varying concentrations to provide for the necessary protein dose (2.75%, 4.25%, etc). The final concentrations of dextrose vary, but are usually either 12.5% or 25%. Examples of typical TPN solutions for adults are provided in Table 12–1. The maximum rate of infusion of solution 1 from Table 12–1 should be 100–125 mL/h 12 to avoid excessive glucose administration (remember to consider the patient’s weight and the dosing guidelines of 4–7 mg/kg/min). Fat emulsions should be given with solution 1 to provide essential fatty acids (10%, 500 mL 3×/wk) or as an additional calorie source. Solu- tion 2 is designed to be given at a maximum rate of 125 mL/h, but this only provides 1275 Cal from dextrose and must be supplemented with a fat emulsion (10% 500 mL = 550 Cal, 20% 500 mL = 1000 Cal). Many hospitals have adopted a “three-in-one” solution for the standard house formula. This involves the administration of protein, carbohydrate, and fat from the same TPN bag over a 24-h period; in other words, the fat is not administered peripherally through a sepa- rate site. Caution should be used when altering the standard formula in this situation be- cause the fat emulsion may be less stable to additives and makes incompatibilities less visible. For example, the solution will be milky in color, and a calcium–phosphate problem, normally easily seen, would not be apparent. Additions to these formulations should be done in conjunction with a pharmacist to ensure that precautions are taken for appropriate addi- tive concentrations. Remember, the solutions described in Table 12–1 contain full concentrations of elec- trolytes and are for patients with normal renal function. For patients with renal impairment, the concentrations of potassium, magnesium, phosphorus, and protein should be reduced (see page 235). PERIPHERAL PARENTERAL NUTRITION If a deep line is contraindicated or impossible, a peripheral TPN solution (<7% dextrose with 2.75% SAA, electrolytes, and vitamins) can be given. The majority of nonprotein calo- ries must be given as an IV fat emulsion. In this case, caloric goals will not be met. A posi- 12 Total Parenteral Nutrition 231 tive nitrogen balance will not be achieved in most patients receiving parenteral nutrition by this route. This is usually used only as a supplement to enteric feedings. TPN ADDITIVES Vitamins are a necessary component to TPN solutions. A product conforming to

recommen- dations of the American Medical Association Nutrition Advisory Group is usually used, such as multivitamin infusion-12 (MVI-12). The contents of 2 vials is added to 1 L of TPN solution daily (Table 12–2). In addition to MVI-12, 5–10 mg of vitamin K (phytonadione) must be given IM weekly. Vitamin K may also be added to the TPN and given as a 1-mg IV dose daily. Several manufacturers sell a trace element supplement that conforms to the AMA group’s guidelines. Each milliliter contains 1.0 mg zinc, 0.4 mg copper, 4.0 mg chromium, and 0.1 mg manganese. Suggested doses for trace elements are listed in Table 12–3, page 232. Trace element deficiencies are rare in hospitalized patients receiving short-term TPN supplements. Supplementation should be routine, however, to ensure trace element avail- ability for cell restoration. In patients receiving long-term support or home TPN, additional trace element supplementation may be necessary. Iron can be given as an injectable iron–dextran complex (Dexferrum, InFeD). Note, however, that owing to the inconvenience of its administration, many clinicians avoid in- jectable iron–dextran. A complete medical and hematologic work-up is often indicated be- fore instituting parenteral iron replacement. Prior to receiving the first dose, a test IV dose of 0.5 mL is recommended. Anaphylaxis is rare, but a period of 1h should elapse before the therapeutic dose of iron is administered. Use the following equation to determine the dose of iron: Total replacement dose (mL) = 0.0476 × Weight (kg) × 12 [Desired hemoglobin (g/dL) − Measured hemoglobin (g/dL)] + 1 mL/5 kg weight (max 14 mL) Maximum Daily Dose: Adults >50 kg: 100 mg iron; Peds <5 kg: 25 mg iron, 5–10 kg: 50 mg iron, 0–50 kg: 100 mg iron The iron–dextran is supplied in an injectable form of 50 mg (Fe)/mL. The calculated dose should be added to TPN at 2 mL/L until the entire dose has been given. TABLE 12–2 Typical Vitamins Provided in 1 L of TPN by Adding 2 Vials of Standard MVI–12 Ascorbic acid 100 mg Pyridoxine (B6) 4 mg Vitamin A 3300 IU Dexpanthenol 15 mg Vitamin D 200 IU Vitamin E (α tocopherol) 10 IU Biotin 60 µg Thiamine (B1) 3 mg Folic acid 400 µg Riboflavin (B2) 3.6 mg Vitamin B12 5 µg Niacin 40 mg Abbreviation: MVI–12 = multivitamin infusion–12. 232 Clinician’s Pocket Reference, 9th Edition TABLE 12–3 Suggested Trace Element Dosing Trace Element Parenteral Dose per Day Zinc 2.5–4.0 mg* Copper 0.5–1.5 mg Selenium 20–40mg Chromium 10–15mg Manganese 0.15–0.8 mg *May be higher, up to 15 mg/d, in severe stress or in patients with high-output fistulas. Insulin, when required, can be given subcutaneously as regular insulin using a sliding scale, as shown in Table 12–4. But the preferred method is to add the insulin directly to the TPN solution. This allows a constant infusion of insulin along with the infusion of dex- trose, which avoids the peaks and valleys in blood glucose that occur when the sliding scale is used. The usual starting dose per liter of TPN is 10 units of regular insulin. Doses from 10 to 90 units/L may often be required. Insulin drips are not advised because TPN can be tem- porarily or permanently discontinued, which would then stop the insulin. Other additives in- clude H2 antagonists and heparin. 12 FAT EMULSIONS Lipid emulsions were initially used only to provide essential fatty acids (linoleic acid, and linolenic acid in children). This could be done with minimal supplementation; as little as 4% of total calories per day would prevent the syndrome of EFAD. Most clinicians prescribe 500 mL of 10% lipid emulsion three times weekly to prevent this syndrome. The signs and symptoms of this deficiency include scaling skin rash, alopecia, and wound healing failure. TABLE 12–4 Sliding Scale for Insulin Orders Regular Insulin Dose Urine Glucose* (Units, given SQ) 0–1+ 0 2+ 5 3+ 10 4+ 15 Any acetone: call house officer *Should be checked every 6 h as part of standing TPN orders. 12 Total Parenteral Nutrition 233 Linoleic acid is a precursor to arachidonic acid, which is essential for prostaglandin and leukotriene synthesis. Once data became available establishing the problems associated with overfeeding of carbohydrate calories, the use of lipid for caloric supplementation became more recognized. Commercially available intravenous fat emulsions are derived from soybean oil, with one product (Liposyn II) combining both soybean and safflower oil. The 10% products pro- vide 1.1 Cal/mL, and the 20% products provide 2.0 Cal/mL. Pediatricians often prefer the Liposyn II product because of its higher percentage of linolenic acid. Because the particle size of these emulsions closely approximates naturally occurring chylomicrons, parenteral infusion is possible. In addition, the emulsions are cleared from the bloodstream in a manner and rate similar to that for chylomicrons. Before beginning the IV fat emulsion, the serum triglyceride level should be checked to ensure that hypertriglyceridemia is not present. Provided that the serum triglyceride level is below 400 mg/dL, the fat emulsion can be given over a 6–12-h period. The longer infusion rate is preferred. The first bottle should be given slowly (1 mL/min for 15 min to check for hypersensitivity reaction). Adverse reactions can include dyspnea, fever, chills, chest tight- ness, wheezing, headaches, and nausea. Currently, the only absolute contraindication to the use of IV fat emulsion is type IV hy- pertriglyceridemia, although isolated cases of nontype IV intolerance to the solution have been reported. To monitor for the clearing of the fat from the bloodstream, a trough serum triglyceride level should be tested 8–12 h following the daily infusion of the fat emulsion. Because fat emulsions are primarily composed of triglycerides (essentially cholesterol free), if the blood is mistakenly drawn while the fat is being infused or shortly thereafter, the serum triglyceride level will be markedly elevated. Other possible contraindications include lipoid nephrosis, severe hepatic failure, and allergy to eggs (egg phosphatides are used as the emulsifying agent). 12 Fat emulsions can be administered through peripheral veins, although the vein may be damaged and cease to be functional in 2–3 days. For this reason, it is usually recommended that the fat emulsion be infused into the central line under strict aseptic technique via a ster- ile Y-connector. As mentioned earlier, some institutions combine the lipid with the TPN for- mula in one bag for 24-h administration. This limits the clinicians ability to validate fat clearance from the blood and makes baseline triglyceride data extremely important. STARTING TPN In general, TPN should not be started until a patient has a stable fluid and electrolyte profile. It is usually unwise to begin TPN in a patient who requires large amounts of fluid, may need resuscitation for trauma, or is septic. Once a patient’s fluid and electrolyte requirements are reasonably stable, TPN can be started safely. The initiation of TPN is never an emergency. Placement of a deep line must be done aseptically, as outlined in Chapter 13, page 253. Infection (bacteremia, fungemia) arising from the catheter or the catheter–skin interface is the most common complication of TPN. Many hospitals now have standardized order forms for starting patients on TPN. 1. Baseline laboratory tests: a. CBC with differential and platelets b. PT and PTT c. SMA-7 and SMA-12; in particular check phosphate, glucose, and routine elec- trolytes (Na, K, Cl) d. Urinalysis e. Baseline weight 234 Clinician’s Pocket Reference, 9th Edition 2. Order the type of TPN desired along with the additives and supplements. Medications are generally not added to TPN solutions except insulin and H2 receptor blockers. A 0.22-µm filter should be used with aqueous TPN (no fat). A 1.2-µm filter should be used with three-in-one TPN. 3. Nursing orders: a. Check urine for sugar and acetone every 6-8 h, house officer should be called if sugar is >2+ or acetone is present. b. Take vital signs every shift. c. Change tubing and deep-line dress every other day (or per hospital procedure). d. Weigh patient every other day. e. Monitor daily fluid balance 4. Laboratory monitoring: a. SMA-7 daily until patient is stable, then every other day. b. CBC with differential, platelets, PT/PTT, twice weekly. c. SMA-12 twice weekly (especially liver function tests). d. Triglyceride trough level (obtained at least 6 h after infusion has stopped, prefer- ably prior to hanging next bottle of fat) once or twice weekly. e. 24-h urine for nitrogen balance determinations and creatinine clearance once or twice weekly. 5. Begin the solution at 25–50 mL/h when using a 25% or 50–75 mL/h when using a 12.5% dextrose solution. Increase by 25 mL/h every 24 h, providing the urine sugar levels are negative. Advance to the maximum rate based on the calculated daily caloric need (page 209). Begin the IV fat emulsion the next day, provided that the serum triglyceride levels are less than 400 mg/dL. Remember that glucose intolerance is the major adverse effect seen during the initial infusion period. Urine sugar and acetone 12 levels should be less than 2+, and serum glucose values less than 180–200 mg/dL. If the sugar level rises above these levels, insulin must be given to achieve the desired level of caloric intake. If glucose intolerance develops when using a 25% dextrose so- lution, consider decreasing the amount of calories from dextrose and increasing the calories from fat. (Be sure to check that overfeeding is not occurring, ie, >4–7 mg/ kg/min, in this case reduce the dose of carbohydrate prior to the addition of insulin). Glucose intolerance arising once the patient has been stabilized may signify sepsis. ASSESSING TPN THERAPY Nitrogen balance is a good measure of the success of the TPN regimen because the goal is protein-sparing (see page 229). Serum albumin will not change appreciably during TPN therapy lasting less than 3 wk. This is due to albumin’s long half-life of 22–24 d. In stressed patients, albumin often falls due to reduced production because the body shifts to increased production of acute-phase reactant proteins. STOPPING TPN TPN can usually be stopped when necessary. Although widely practiced, there is rarely a need for a formal weaning schedule. If there are concerns about hypoglycemia, then a 10% dextrose solution can be administered after cessation of the TPN. 12 Total Parenteral Nutrition 235 DISEASE-SPECIFIC TPN FORMULATIONS Cardiac Failure: In patients with CHF, reduce water from 1 to 0.5 mL/Cal or 500 mL insensible loss plus measured water losses. This limits overloading with water from TPN. Other considerations include providing energy needs at the BEE + 30% for initiation of TPN calories, limiting protein initially to 0.8–1 g/kg and reducing sodium to 0.5–1.5 g/d. Diabetes: Consider increasing the percentage of calories provided from fat. Ideally, blood sugar should be well controlled or at least not >200 when initiating TPN. Remember that no more than 50% of total intake should be from fat and not more than 3 g/kg/d. Fat provides 9 Cal/g. Commercial lipid emulsions provide 1.1 or 2 Cal/mL. Insulin should be added to the solution initially at 5–10 units/bag in patients requiring >20 units of insulin daily. Geriatrics: Patients older than 75 years have a documented need for fewer calories. Use caution in monitoring total fluids to prevent overload. Inflammatory Bowel Disease: TPN can be initiated in these patients at approxi- mately 1.5 × RME at 30 Cal/kg of ideal body weight. Protein needs vary from 1 to 2 g/kg of ideal body weight daily. Dose the protein based on a 24-h UUN. Note: Patients with fistulas lose nitrogen via this route and need additional protein. Zinc losses may be greater in this group of IBD patients also. Liver Disease: Specialized formulas of amino acids that contain primarily branched- chain amino acids (leucine, isoleucine, and valine) are available for use in cases of liver dis- ease. Theoretically, these products may improve arousal from hepatic encephalopathy by competing with the aromatic amino acids that are precursors for some centrally active amines. There is no definitive evidence that branched-chain formulas improve patient out- 12 come. The specialized formulas should only be used in cases of severe hepatic disease ac- companied by encephalopathy. In other clinical conditions of liver disease, standard formulas should be used. Lipid emulsions are not recommended in cases of severe hepatic failure when hypertriglyceridemia is present. Pancreatic Disease: Total energy needs may be high in this disease (35 Cal/kg). Pro- tein should be initiated

at 1.5 g/kg/d. Intravenous fat may be administered in these cases be- cause it is metabolized by peripheral tissue lipases. A reasonable nonprotein system would be 70% carbohydrate and 30% fat. Pulmonary Disease: Carbohydrate metabolism produces higher amounts of CO2 than does fat metabolism. Consequently, the patient with CO2 retention problems often is stressed if overfed with carbohydrates. Increasing the percentage of daily nonprotein calo- ries provided by fat (not >60%) may decrease the CO2 load and assist with ventilator wean- ing. Higher fat percentages influence oxygen diffusion capacity and are not beneficial, especially in cases of mild pulmonary compromise. Phosphate depletion is a second clini- cally relevant concern in this population due to the depression of the hypoxic ventilatory drive. Once patients are started on TPN, PO −2 4 often decreases due to the incorporation into ATP. Adequate supplementation and monitoring is very important in this group of patients. Renal Failure: Several considerations become important in this disease. If a patient is not receiving dialysis or is not a dialysis candidate, protein must be restricted to 0.6–0.8 g/kg/d, and total energy needs must be limited to approximately 30 Cal/kg/d. Weight should be ideal or admission weight, so as to control for the influence of water 236 Clinician’s Pocket Reference, 9th Edition retention. Specialized amino acid formulas have been developed for this group of patients. These products provide higher concentrations of essential amino acids than the standard amino acid products. Theoretically, the nitrogen waste products are recycled to make the nonessential amino acids, thereby reducing the BUN content. Risks exist, however, for ele- vations in ammonia when arginine is not also supplemented. Consequently, manufacturers have modified the original formulas to include several nonessential amino acids. Due to these changes, the renal products provide a very similar amino acid profile to those of the SAA solutions at very low concentrations (2.5%). The cost differential can be significant. It is therefore recommended that patients with renal dysfunction receive SAA formulas at a re- duced concentration to provide the minimum daily allowance of protein. TPN should not be supplemented with potassium or magnesium, and sodium should be reduced to 40–180 mEq/d once the GFR is <10 mL/min. Patients receiving hemodialysis or peritoneal dialysis may be fed protein similarly to patients without renal disease. Doses of 1–1.2 g/kg/d may be used. Nitrogen balance calcu- lations are not useful in this population due to the problem of renal clearance of urea waste inherent to kidney disease. Sepsis or Trauma: Sepsis and trauma causes hypermetabolism and requires greater numbers of calories from nonprotein (30–35 Cal/kg) and protein (2–2.5 g/kg/d) sources. Es- timates of RME should be increased by 50% initially, and some cases may support up to 100%. Note that feeding >3000 Cal/d is not recommended. Specialized amino acid formulas are also available for this group of patients. Again, these formulas include higher concentra- tions of the branched-chain amino acids. The reason for their inclusion in this population is to provide substrate directly to the skeletal muscle undergoing catabolism to provide gluco- neogenic precursors. Although these formulas have been shown to normalize the amino acid 12 profile and in some cases improve nitrogen balance, no studies have demonstrated an im- proved patient outcome. The additional cost of these formulas is a deterrent to their routine use in these populations until further data are available. Additional zinc supplementation is often recommended in this group of patients. Studies have shown losses to be increased in stress; therefore, daily supplementation of up to 15 mg of zinc may be appropriate. COMMON TPN COMPLICATIONS Hyperosmolar Nonketotic Coma: Usually found in improperly monitored patients with impaired insulin responses. Caused by excessive glucose levels, usually corrected by administration of insulin and rehydration. Sustained hyperglycemia (>220 mg/dL) depresses monocyte activity and could compromise the immune defenses. Infection (Sepsis): The care of the deep-line site and tubing must be meticulous. Sus- pect sepsis if a previously stable patient becomes glucose-intolerant. If the patient becomes septic, the deep line should be considered a possible source. If no other source of infection can be identified, the deep line must be removed or changed and the tip sent for routine cul- ture and sensitivity. Candida albicans is the most frequently encountered pathogen on the catheter, followed by Staphylococcus aureus, Staphylococcus epidermidis and gram- negative rods. Hypophosphatemia: Severe hypophosphatemia can occur in patients started on TPN after severe weight loss and those with conditions such as anorexia nervosa (refeeding syn- drome). This may also result from increased metabolic processes requiring phosphate and can significantly hamper weaning from the ventilator. 12 Total Parenteral Nutrition 237 Elevated Liver Function Tests: The usual cause is excessive glucose infusion. When the primary metabolic pathway for glucose becomes saturated, excess glucose is converted to intracellular triglycerides in the liver. This is especially seen when rates exceed 4–7 mg/kg/min. A reduction in carbohydrate calories, supplementing with fat, is recom- mended. Cholestasis: This often occurs secondary to overfeeding of fat calories (>3 g/kg/d or >60% of total nonprotein calories). Hyperkalemia: This is the most common electrolyte disturbance seen with TPN. Most TPN formulations contain potassium 40–50 mEq/L and are intended for patients with nor- mal renal function. Excess potassium over and above that required for maintenance and urine losses (usually 3–5 mEq/g nitrogen) is included. Potassium must be closely followed in the elderly and those with impaired renal function. Additionally, many drugs contribute to potassium balance problems. These include some antibiotics that are potassium salts (eg, penicillins); oral phosphate supplements (Neutra-Phos); ACE inhibitors, which reduce potassium excretion (Captopril, Enalapril); and potassium-sparing diuretics (triamterene, spironolactone). Metabolic Alkalosis: Modern SAAs are present as the acetate salt (80–100 mEq/L), which is converted to bicarbonate in vivo. In postoperative patients with nasogastric tubes, the loss of chloride, together with the high infusion of the acetate, can lead to a metabolic al- kalosis. The increased use of histamine blockers and antacids in intensive care patients has also contributed to a higher incidence of this problem. Treating this condition requires in- creasing the chloride level in the solution and reducing the acetate. Hyponatremia: Serum sodium levels of 127–135 mEq/L are commonly seen in patients on TPN. The cause is controversial but is probably due to mild SIADH; therefore the prob- 12 lem is probably an excess of water and not deficiency of sodium. It is usually asymptomatic and does not require a change in formula unless the sodium drops below 125 mEq/L. Hypermagnesemia: This is usually seen in patients with renal failure. Antacid therapy may also contribute to this condition. If potassium is reduced in the TPN, magnesium should also be reduced. This page intentionally left blank. 13 BEDSIDE PROCEDURES Procedure Basics Intrauterine Pressure Monitoring Amniotic Fluid Fern Test IV Techniques Arterial Line Placement Lumbar Puncture Arterial Puncture Orthostatic Blood Pressure Arthrocentesis (Diagnostic and Measurement Therapeutic) Pelvic Examination Bone Marrow Aspiration and Biopsy Pericardiocentesis Central Venous Catheterization Peripherally Inserted Central Catheter Chest Tube Placement (PICC Line) Cricothyrotomy (Needle and Surgical) Peritoneal Lavage Culdocentesis Peritoneal (Abdominal) Paracentesis Doppler Pressures Pulmonary Artery Catheterization Electrocardiogram Pulsus Paradoxus Measurement Endotracheal Intubation Sigmoidoscopy (Rigid) Fever Work-up Skin Biopsy Gastrointestinal Intubation Skin Testing Heelstick Thoracentesis Internal Fetal Scalp Monitoring Urinary Tract Procedures Injection Techniques Venipuncture 13 PROCEDURE BASICS Universal Precautions Universal precautions should be used whenever an invasive procedure exposes the operator to potentially infectious body fluids. Not all patients infected with transmissible pathogens can be identified at the time of hospital admission or even later in their course. Because pathogens transmitted by bloody and body fluids pose a hazard to personnel caring for such patients, particularly during invasive procedures, certain precautions are now required for routine care of all patients whether or not they have been placed on isolation precautions of any type. For these reasons, the CDC calls these Universal Precautions. 1. Wash hands before and after all patient contact. 2. Wash hands before and after all invasive procedures. 3. Wear gloves in every instance in which contact with blood is certain or likely. For ex- ample, wear gloves for all venipunctures, for all IV starts, for IV manipulation, and for wound care. 4. Wear gloves once and discard. Do not wear the same pair to perform tasks on two dif- ferent patients or two different tasks at different sites on the same patient. 5. Wear gloves in every instance in which contact with any body fluid is likely, including urine, feces, wound secretions, respiratory tract care, thoracentesis, paracentesis, etc. 239 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 240 Clinician’s Pocket Reference, 9th Edition 6. Wear gown when splatter of blood or of body fluids on clothing seems likely. 7. Additional barrier precautions may be necessary for certain invasive procedures when significant splatter or aerosol generation seems likely. This does not occur during most routine patient care activities. It may occur in certain instances in the operating room, emergency room, the ICUs, during invasive procedures, and during cardiopulmonary resuscitation. Always wear masks when goggles are worn and vice versa. Informed Consent Patients should be counseled before any procedure concerning the reason for it and the po- tential risks and benefits from it. Explaining the various steps often can make the patient more cooperative and the procedure easier on both parties. In general, procedures such as bladder catheterization, NG intubation, or venipuncture do not require a written informed consent beyond normal hospital sign in protocols. More invasive procedures, such as thora- centesis or lumbar puncture, for example, require written consent and must be obtained by a licensed physician. Basic Equipment Table 13–1 lists useful collections of instruments and supplies, often packaged together, that aid in the completion of the procedures outlined in this chapter. Local anesthesia is dis- cussed in Chapter 17. The size of various catheters, tubes and needles is often designated by French unit (1 french = ¹₃ mm in diameter) or by “gauge.” Reference listings for these designations can be found in Figure 13–1A. Designations of surgical scalpels, used in the performance of many basic bedside procedures and in the operating room are shown in Figure 13–1B. TABLE 13–1 13 Instruments and Supplies Used in the Completion of Common Bedside Procedures MINOR PROCEDURE TRAY Sterile gloves Sterlile towels/drapes 4×4 gauze sponges Povidone–iodine (Betadine) prep solution Syringes: 5-, 10-, 20-mL Needles: 18-, 20-, 22-, 25-gauge 1% Lidocaine (with or without epinephrine) Adhesive tape INSTRUMENT TRAY Scissors Needle holder Hemostat Scalpel and blade (No. 10 for adult, No. 15 for children or delicate work) Suture of choice (2-0 or 3-0 silk or nylon on cutting needle; cutting needle best for suturing to skin) 13 241 French Catheter Scale in French units (1 French = 1/3 mm diameter) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 34 32 30 28 26 24 22 20 19 18 Needle Gauge 3 French = 1.0 mm = .039 in. 18 French = 6 mm = .236 in. 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 Inches 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Centimeters A FIGURE 13–1A: French catheter guide and needle gauge reference. (Courtesy Cook Urological.) 242 Clinician’s Pocket Reference, 9th Edition FIGURE 13–1B: Commonly used scalpel blades. From left to right: Number 10, 11, 12, 15, and 20. The No. 10 is the standard surgical blade; No. 11 is useful for press cuts into abscesses; No. 12 is used to open tubular structures; No. 15 is 13 widely used for bedside procedures and for more delicate work; the No. 20 blade is used when large incisions are called for. AMNIOTIC FLUID FERN TEST Indication • Assessment of rupture of membranes Materials • Sterile speculum and swab • Glass slide and microscope • Nitrazine paper (optional) Procedure 1. After placing a sterile speculum in the vagina, a sample of fluid which has “pooled” in the vault is swabbed onto a glass slide and allowed to air dry. 2. Amniotic fluid produces a microscopic arborization or “fern” pattern, which may be vi- sualized with 10× magnification. False-positive results may occur if cervical mucus is collected; however, the ferning pattern of mucus is coarser. This test is unaffected by meconium, vaginal pH, and blood-to-amniotic-fluid ratios

≤ 1:10. Samples heavily contaminated with blood may not fern. 13 Bedside Procedures 243 3. An additional test used to detect ruptured membranes entails the use of nitrazine paper, which has a pH turning point of 6.0. Normal vaginal pH in the pregnant woman ranges from 4.5 to 6.0; the pH of amniotic fluid is 7.0–7.5. A positive nitrazine test is mani- fested by a color change in the paper from yellow to blue. False-positive results are more common with the nitrazine paper test because blood, meconium, semen, alkalotic urine, cervical mucus, and vaginal infections can all raise the pH. Complication • Bacteria may be introduced if sterile technique is not used. ARTERIAL LINE PLACEMENT Indications • Continuous blood pressure readings are needed (for patients on pressors, with unstable pressures, etc). • Frequent arterial blood gases are needed. Contraindications • Arterial insufficiency with poor collateral circulation (See Allen test, page 246) • Thrombolytic therapy or coagulopathy (relative) Materials • Minor procedure and instrument tray (page 240) • Heparin flush solution (1:1000 dilution) • Arterial line set-up per local ICU routine (transducer, tubing and pressure bag with preheparinized saline, monitor) • Arterial line catheter kit or 20-gauge catheter over needle, 1¹₂–2 in. (Angiocath) 13 with 0.025-in. guidewire (optional) Procedure (See Fig. 13–2) 1. The radial artery is most frequently used and that approach is described here. Other sites, in decreasing order of preference, are the ulnar, dorsalis pedis, femoral, brachial, and axillary arteries. Never puncture the radial and ulnar arteries in the same hand be- cause this may compromise blood supply to the hand and fingers. 2. Verify the patency of the collateral circulation between the radial and ulnar arteries using the Allen test (page 246) or Doppler ultrasound probe. Have the ICU staff pre- pare the flush bag, tubing, and transducer, paying particular attention to removing the air bubbles. 3. Place the extremity on an armboard with a roll of gauze behind the wrist to hyperex- tend the joint. Prep with povidone–iodine, and drape with sterile towels. Wear gloves and a mask. 4. Carefully palpate the artery, and choose the puncture site where it appears most super- ficial. Raise a very small skin wheal at the puncture site with 1% lidocaine using a 25-gauge needle. 5. a. Standard technique: See Figure 13–2. While palpating the path of the artery with the left hand, advance the 20-gauge (preferably 2 in. long) catheter-over-needle as- sembly into the artery at a 30-degree angle to the skin. Once a “flash” of blood is 244 Clinician’s Pocket Reference, 9th Edition 30–45° Radial artery 13 FIGURE 13–2 Technique for arterial line placement. (Reprinted, with permission, from: Gomella TL [ed]: Neonatology: Basic Management, On-Call Problems, Dis- eases, Drugs, 4th ed. Appleton & Lange, Norwalk CT, 1998.) seen in the hub, advance the entire unit 1–2 mm, so that the needle and catheter are in the artery. If blood flow in the hub stops, carefully pull the entire unit back until flow is reestablished. Once the catheter is in the artery, hold the needle steady, and advance the catheter over the needle into the artery. The catheter should slide smoothly into the artery. Withdraw the needle completely and check for arterial blood flow from the catheter. A catheter that does not spurt blood is not in posi- tion. Briefly occlude the artery with manual pressure while the pressure tubing is being connected. Note: The pressure tubing system must be preflushed to clear all air bubbles prior to connection. b. Alternative procedure (“through-and-through” technique): Use the same ap- proach to the artery as in part a, however, purposely puncture the artery through the anterior and the posterior walls. This method is probably most useful in chil- dren and infants. Once a flash of blood is seen in the hub, advance the entire unit together until blood no longer fills the hub. (This can be done in a single motion.) S-l-o-w-l-y withdraw the entire unit until blood starts to fill the hub, then advance the catheter as the needle is withdrawn. Connect the tubing. 13 Bedside Procedures 245 c. Prepackaged kit technique: Kits, sometimes referred to as “quick catheters” are available with a needle and guidewire that allow the Seldinger technique to be used. Place the entry needle at a 30-degree angle to the skin site and insert until a flash of blood rises in the catheter. The catheter does not need to be advanced, but advance both the guidewire portion (orange handle in some kits) and the catheter into the vessel. Remove the wire and connect it to the pressure tubing. 6. If placement is not successful, apply pressure to the site for 5 min and reattempt one or two more times. If still not successful, move to another site. 7. Suture in place with 3-0 silk, and apply a sterile dressing. Splint the dorsum of the wrist to limit mobility and provide catheter stability. 8. If larger vessels such as the femoral artery are used, the clinician can employ the Seldinger technique for femoral artery cannulation: locate the vessel lumen with a small-gauge, thin-walled needle; pass a 0.035 floppy-tipped J(“J” describes the config- uration of the end of the floppy wire) guidewire into the lumen; and use the guidewire to pass a larger catheter into the vessel. Use a 16-gauge catheter assembly at least 6 in. long for the femoral artery. 9. Replace arterial lines using a different site every 4–7 d to decrease risk of infection. 10. Any amount of heparin can make the results of coagulation studies (PTT) inaccurate. If the blood is drawn from the arterial line and unexpectedly high results are obtained, always repeat the test and consider using standard venipuncture technique (see page 309). Despite the removal of the first 5–10 mL from the line, some of the heparinized flush solution can still get into the lab sample tube, yielding unreliable results. 11. Always compare the arterial line pressure with a standard cuff pressure. An occasional difference is normal (10–20 mm Hg) and should be incorporated when following the blood pressure. Complications Thrombosis, hematoma, arterial embolism, arterial spasm, arterial insufficiency with tissue 13 loss, infection, hemorrhage, pseudo-aneurysm formation ARTERIAL PUNCTURE Indications • Blood gas determinations and when arterial blood is needed for chemistry determi- nations (eg, ammonia levels) Materials • Blood gas-sampling kit or • 3–5-mL syringe • 23–25-gauge for radial; 20–22 acceptable for femoral artery • Heparin (1000 U/mL), 1 mL • Alcohol or povidone–iodine swabs • Cup of ice Procedure 1. Use a “heparinized” syringe for blood gas and a “nonheparinized” syringe for chem- istry determinations. If a blood gas kit is not available, a 3–5-mL syringe can be he- parinized by drawing up 1 mL of 1:1000 solution of heparin through a small-gauge 246 Clinician’s Pocket Reference, 9th Edition needle (23–25-gauge) into the syringe, pulling the plunger all the way back. The heparin is then expelled, leaving only a small amount, which coats the syringe. 2. In order of preference, the arteries are radial, femoral, and brachial. If using the radial artery, perform an Allen test prior to puncture of the artery to verify the patency of the ulnar artery. You do not want to damage the radial artery if there is no flow in the ulnar artery. To perform the Allen test, have the patient make a tight fist. Occlude both the ra- dial and ulnar arteries at the wrist and have the patient open the hand. While maintain- ing pressure on the radial artery, release the ulnar artery. If the ulnar artery is patent, the hand should flush red within 6 s. A radial puncture can then be safely performed. If the color return is delayed on part of the hand or remains pale, do not perform the puncture because the collateral flow is inadequate. Choose an alternative site. 3. If using the femoral artery, use the mnemonic NAVEL to aid in locating the important structures in the groin. Palpate the femoral artery just below the inguinal ligament. From lateral to medial the structures are Nerve, Artery, Vein, Empty space, Lymphatic. 4. Palpate the chosen artery carefully. You may wish to inject 1idocaine subcutaneously for anesthesia (use a small needle such as a 25–27-gauge), but this often turns a “one-stick procedure” into a “two-stick” one. Palpate the artery proximally and distally with two fingers, or trap the artery between two fingers placed on either side of the vessel. Hyper- extension of the joint often brings the radial and brachial arteries closer to the surface. 5. Prep the area with either a povidone–iodine solution or alcohol swab. 6. Hold the syringe like a pencil with the needle bevel up, and enter the skin at a 60– 90-degree angle. Often you can feel the arterial pulsations as you approach the artery. 7. Maintaining a slight negative pressure on the syringe, obtain blood on the downstroke or on slow withdrawal (after both sides of the artery have been punctured). Aspirate very slowly. A good arterial sample, because of the pressure in the vessel, should require only minimal back pressure. If a glass syringe or special blood-gas syringe is used, the barrel usually fills spontaneously and it is not necessary to pull on the plunger. 13 8. If the vessel is not encountered, withdraw the needle without coming out of the skin, and redirect it to the pulsation. 9. After obtaining the sample, withdraw the needle quickly and apply firm pressure at the site for at least 5 min or longer if the patient is receiving anticoagulants. Apply pres- sure even if a sample was not obtained in order to prevent a compartment syndrome from extravasated blood. 10. If the sample is for a blood-gas determination, expel any air from the syringe, mix the contents thoroughly by twirling the syringe between your fingers, remove and dispose of the needle, and make the syringe airtight with a cap. Place the syringe in an ice bath if more than a few minutes will elapse before the sample is processed. Note the in- spired oxygen concentration and time of day on the lab slip. ARTHROCENTESIS (DIAGNOSTIC AND THERAPEUTIC) Indications • Diagnostic. Arthrocentesis is helpful in the diagnosis of new-onset arthritis; to rule out infection in acute or chronic, unremitting joint effusion. • Therapeutic. To instill steroids and maintain drainage of septic arthritis; relief of tense hemarthrosis or effusion Contraindications Cellulitis at injection site. Relative contraindication is a bleeding disorder; use caution if co- agulopathy or thrombocytopenia is present or if the patient is receiving anticoagulants. 13 Bedside Procedures 247 Materials • Minor procedure tray (page 240) (18- or 20-gauge needle (smaller for finger or toe) • Ethyl chloride spray can be substituted for lidocaine. • Two heparinized tubes for cell count and crystal examination • Discuss with your microbiology lab their preference for transporting fluid for bacter- ial, fungal, AFB culture, and Gram’s stain. A Thayer–Martin plate is needed if Neis- seria gonorrhoeae (GC) is suspected. • A small syringe containing a long-acting corticosteroid such as Depo-Medrol or tri- amcinolone is optional for therapeutic arthrocentesis. Procedures, General 1. Obtain the patient’s consent after describing the procedure and complications. 2. Determine the optimal site for aspiration, identify landmarks, and mark site with inden- tation or sterile marking pen. Avoid injection of tendons. 3. When aspiration is to be followed by corticosteroid injection, maintaining a sterile field with sterile implements minimizes the risk of infection. 4. Clean the area with povidone–iodine, dry and wipe over the aspiration site with alco- hol. Povidone–iodine can render cultures negative. Let the alcohol dry before begin- ning procedure. 5. Anesthetize the area with lidocaine using a 25-gauge needle, taking care not to inject into the joint space. Lidocaine is bactericidal. Avoid preparations containing epineph- rine, especially in a small digit. Alternatively, spray the area with ethyl chloride (“freeze spray”) just prior to needle aspiration. 6. Insert the aspirating needle, applying a small amount of vacuum to the syringe. When the capsule is entered, fluid usually flows easily. Remove as much fluid as possible, repositioning the syringe if necessary. 7. If corticosteroid is to be injected, remove the aspirating syringe from the needle, which is still in the joint space. It is helpful to ensure that the syringe can easily be removed 13 from the needle before step 6. Attach

the syringe containing corticosteroid, pull back on the plunger to ensure you are not in a vein, and inject contents. Never inject steroids when there is any possibility of an infected joint. Remove the needle, and apply pres- sure to the area (leakage of subcutaneous steroids can lead to localized atrophy of the skin. Generally, the equivalent of 40 mg of methylprednisolone is injected into large joints such as the knee and 20 mg into medium-size joints such as the ankle or wrist. Warn the patient that a postinjection “flare” characterized by pain several hours after the injection can be treated with ice and NSAIDs. 8. Note the volume aspirated from the joint. As an example, the knee typically contains 3.5 mL of synovial fluid; in inflammatory, septic, or hemorrhagic arthritis, the volume can be much higher. A quick bedside test for viscosity is to allow a drop of fluid to fall from the tip of the needle. Normal synovial fluid is highly viscous and forms a several- inch long string; decreased viscosity is seen in infection. A mucin clot test (normally forms in < 1 min; a delayed result suggests inflammation) was once a standard test for RA, but is not now routinely performed. 9. Joint fluid is usually sent for: • Cell count and differential (purple or green top tube) • Microscopic crystal exam using polarized light microscopy (purple or green top tube); normally no debris, crystals, or bacteria are seen; urate crystals are present with gout; calcium pyrophosphate in pseudo-gout. • Glucose (red top tube) See Table 13–2. 248 Clinicians Pocket Reference, 9th Edition • Gram’s stain, and cultures for bacteria, fungi, and AFB as indicated (check with your lab or deliver immediately in a sterile tube with no additives.) • Cytology if a malignant effusion is suspected clinically. Arthrocentesis of the Knee 1. Fully extended the knee with the patient supine. Wait until the patient has a relaxed quadriceps muscle because its contraction plants the patella against the femur, making aspiration painful. 2. Insert the needle posterior to the medial portion of the patella into the patellar-femoral groove. Direct the advancing needle slightly posteriorly and inferiorly (Fig. 13–3) Arthrocentesis of the Wrist 1. The easiest site for aspiration is between the navicular bone and radius on the dorsal wrist. Locate the distal radius between the tendons of the extensor pollicis longus and the extensor carpi radialis longus to the second finger. This site is just ulnar to the anatomic snuff box. Direct the needle perpendicular to the mark (Fig. 13–4). 13 FIGURE 13–3 Arthrocentesis of the knee. (Reprinted, with permission, from: Haist SA et al [eds]: Internal Medicine on Call, 3rd ed. McGraw-Hill, New York, 2001.) 13 Bedside Procedures 249 Extensor pollicis longus Extensor carpi radialis longus 13 FIGURE 13–4 Arthrocentesis of the wrist. (Reprinted, with permission, from: Haist SA et al [eds]: Internal Medicine on Call, 3rd ed. McGraw-Hill, New York, 2001.) Arthrocentesis of the Ankle 1. The most accessible site is between the tibia and the talus. Position the angle of the foot to leg at 90 degrees. Make a mark lateral and anterior to the medial malleolus and me- dial and posterior to the tibialis anterior tendon. Direct the advancing needle posteriorly toward the heel. 2. The subtalar ankle joint does not communicate with the ankle joint and is difficult to aspirate even by an expert. Be aware that “ankle pain” may originate in the subtalar joint rather than in the ankle (Fig. 13–5). Synovial Fluid Interpretation Normal synovial fluid values and values in disease states are found in Table 13–2. 250 Clinician’s Pocket Reference, 9th Edition Tibialis anterior tendon Medial malleolus FIGURE 13–5 Arthrocentesis of the ankle. (Reprinted, with permission, from: Haist SA et al [eds]: Internal Medicine on Call, 3rd ed. McGraw-Hill, New York, 2001.) 13 Noninflammatory Arthritis: Osteoarthritis, traumatic, aseptic necrosis, osteochondri- tis desiccans Inflammatory Arthritis: Gout (usually associated with elevated serum uric acid), pseudo-gout, RA, rheumatic fever, collagen–vascular disease Septic Arthritis: Pyogenic bacterial (S. aureus GC and S. epidermidis most com- mon), TB Hemorrhagic: Hemophilia or other bleeding diathesis, trauma, with or without fracture Complications Infection, bleeding, pain. Postinjection flare of joint pain and swelling can occur after steroid injection and persist for up to 24 h. This complication is felt to be a crystal-induced synovitis due to the crystalline suspension used in long-acting steroids. BONE MARROW ASPIRATION AND BIOPSY Indications • Evaluation of unexplained anemia, thrombocytopenia, leukopenia • Evaluation of unexplained leukocytosis, thrombocytosis, search for malignancy pri- mary to the marrow (leukemia, myeloma) or metastatic to the marrow (small-cell lung cancer, breast cancer) 13 251 TABLE 13–2 Synovial Fluid Analysis and Categories for Differential Diagnosis* Parameter Normal Noninflammatory Inflammatory Septic Hemorrhagic Viscosity High High Decreased Decreased Variable Clarity Transparent Transparent Translucent-opaque Opaque Cloudy Color Clear Yellow Yellow to opalescent Yellow to green Pink to red WBC (per µL) <200 <3000 3000–50,000 >50,000† Usually <2000 Polymorphonuclear <25% <25% 50% or more 75% or more 30% leukocytes (%) Culture Negative Negative Negative Usually positive Negative Glucose (mg/dL) Approx. serum Approx. serum >25, but <serum <25, serum >25 *See page 249 for additional information. †May be lower if antibiotics initiated. Abbreviation: WBC = white blood cells. 252 Clinician’s Pocket Reference, 9th Edition • Evaluation of iron stores; evaluation of possible disseminated infection (tuberculo- sis, fungal disease) • Bone marrow donor harvesting (aspiration) Contraindications • Infection, osteomyelitis near the puncture site • Relative contraindications include severe coagulopathy or thrombocytopenia (may be corrected by platelet transfusion); prior radiation to the region Materials Commercial kits are usually available that contain all the materials necessary. A technician from the hematology lab or BMT facility is necessary to ensure delivery and processing of specimens. Procedure 1. Explain the procedure to the patient and/or the legally responsible surrogate in detail, and obtain informed consent. 2. Usually local anesthesia is all that is required; however, in extremely anxious patients, premedication with an anxiolytic or sedative such as diazepam (Valium) or midazolam (Versed) or an analgesic is reasonable. 3. Bone marrow can be obtained from numerous sites, the most common being the sternum, the anterior iliac crest, and the posterior iliac crest. The posterior iliac crest is the safest and usually the site of choice and is described here. Position the patient on either the ab- domen or on the side opposite the side from which the biopsy specimen is to be taken. 4. Identify the posterior iliac crest by palpation and mark the desired biopsy site with in- 13 delible ink. 5. Use sterile gloves and follow strict aseptic technique for the remainder of the procedure. 6. Prep the biopsy site with sterile povidone–iodine solution and allow the skin to dry. Then wipe the site free of the povidone–iodine with sterile alcohol. Use surgical drapes to cover the surrounding areas. 7. With a 26-gauge needle, administer 1% lidocaine solution intradermally to raise a skin wheal. Then, with the 22-gauge needle, infiltrate the subcutaneous and deeper tissues with lidocaine until the periosteum is reached. At this point, advance the needle just through the periosteum and infiltrate lidocaine subperiosteally. Infiltrate an area ap- proximately 2 cm in diameter, using repeated periosteal punctures. 8. Once you obtain local anesthesia, use a No. 11 scalpel blade to make 2–3-mm skin in- cision over the biopsy site. 9. Insert the bone marrow biopsy needle through the skin incision and then advance with a rotating motion and gentle pressure until the periosteum is reached. Once it is firmly seated on the periosteum, advance the needle through the outer table of bone into the marrow cavity with the same rotating motion and gentle pressure. Generally, a slight change in the resistance to needle advancement signals entry into the marrow cavity. At this point, advance the needle 2–3 mm. 10. Remove the stylet from the biopsy needle and attach a 10-mL syringe to the hub of the biopsy needle. Withdraw the plunger on the syringe briskly, and aspirate 1–2 mL of marrow into the syringe. This may cause severe, instantaneous pain, but slow with- drawal of the plunger or collection of more than 1–2 mL of marrow with each aspira- tion results in excessive contamination of the specimen with peripheral blood. 13 Bedside Procedures 253 11. The marrow aspiration specimen can be used to prepare coverslips for viewing under the microscope or sent for special studies such as cytogenetics and cell markers or for culture. Repeat aspirations may be required to obtain enough marrow to perform all of the studies needed. Also note that certain studies may require heparin or EDTA for col- lection. Contact the appropriate lab prior to the procedure to ensure that specimens are collected in the appropriate solution. 12. If a biopsy is to be obtained, replace the stylet and withdraw the needle. Then reinsert the needle at a slightly different angle and location, still within the area of periosteum previously anesthetized. Once the marrow cavity has been reentered, again remove the stylet and advance the needle d 5–10 mm, using the same rotating motion with gentle pressure. Withdraw the needle several millimeters (but not outside of the marrow cav- ity) and redirect it at a slightly different angle and then advance again. Repeat this step several times. This should result in 2–3 cm of core material entering the needle. Rotate the needle rapidly on its long axis in a clockwise and then a counterclockwise manner. This severs the biopsy specimen from the marrow cavity. Withdraw the needle com- pletely without replacing the stylet. Some operators prefer to hold their thumb over the open end of the needle to create a negative pressure in the needle as it is withdrawn. This may help prevent loss of the core biopsy specimen. 13. Remove the core biopsy by inserting a probe (provided with the biopsy needle) into the distal end of the needle and gently pushing the specimen the full length of the needle and out the hub end. This is important because attempting to push the specimen out the distal end may damage the specimen. Most biopsy needles are tapered at the distal end, presumably allowing the specimen to expand once in the needle and preventing it from being lost when the needle is withdrawn from the patient. 14. The core biopsy specimen is usually collected in formalin solution. Again, plans for special studies should be made prior to the procedure so that any special handling of the biopsy can be done. 15. Observe the biopsy site for excess bleeding and apply local pressure for several min- utes. Clean the area thoroughly with alcohol and apply an adhesive bandage or gauze 13 patch. Instruct the patient to assume a supine position and place a pressure pack be- tween the bed or table and the biopsy site to apply pressure for 10–15 min. This is not an absolute requirement in patients without an underlying coagulopathy or thrombocy- topenia, but still serves to decrease local hematoma formation. Patients with an under- lying bleeding tendency should maintain pressure for 20–25 min. A patient who is stable at this point may resume normal activities. Complications Local bleeding and hematoma, retroperitoneal hematoma, pain, bone fracture, infection CENTRAL VENOUS CATHETERIZATION Indications • Administration of fluids and medications, especially when no peripheral access is available • Administration of hyperalimentation solutions or other hypertonic fluids (eg, am- photericin B) that damage peripheral veins • Measurement of CVP (See Chapter 20, page 397) • Acute dialysis or plasmapheresis (Shiley catheter) • Insertion of a pulmonary artery catheter or transvenous pacemaker 254 Clinician’s Pocket Reference, 9th Edition Contraindications • Coagulopathy dictates the use of the femoral or median basilic vein approach to avoid bleeding complications. Background A central venous catheter (also known as a “deep line”) is a catheter introduced into the su- perior vena cava, inferior vena cava, or one of their main branches. Generally, two tech- niques are used to place central venous lines. One of these (Seldinger technique) involves puncturing the vein with a relatively small needle through which a thin guidewire is placed in the vein. After the needle has been withdrawn, the intravascular appliance or a sheath through which a smaller catheter will be placed is introduced into the vein over the guidewire. The other technique involves puncturing the vein with a larger bore needle through

which the intravascular catheter will fit. The ensuing discussion focuses on the Seldinger technique and placement of either a triple-lumen catheter or a sheath through which a smaller catheter (eg, a pulmonary artery catheter) will eventually be placed. The in- ternal jugular and subclavian approaches are commonly used, but the femoral approach, al- though infrequently utilized, offers several advantages (see following discussion). Another technique, the PICC line is designed for more long-term outpatient administration of med- ications and is described on page 292. Materials Commercially available disposable trays provide all the necessary needles, wires, sheaths, dilators, suture materials, and anesthetics. If needles, guidewires, and sheaths are collected from different places, it is very important to make sure that the needle will accept the guidewire, that the sheath and dilator will pass over the guidewire, and that the appliance to 13 be passed through the sheath will indeed fit the inside lumen of the sheath. Supplies should include the following items: • Minor procedure and instrument tray (page 240); 1% lidocaine (mixed 1:1 with sodium bicarbonate 1 mEq/L removes the sting) • Guidewire (usually 0.035 floppy-tipped J wire) • Vessel dilator • Intravascular appliance (triple-lumen catheter or a sheath through which a pul- monary artery catheter could be placed) • Heparinized flush solution 1 mL of 1:100 U heparin in 10 mL of NS (to be used to fill all lumens prior to placement to prevent clotting of the catheter during place- ment) • Mask, sterile gown, highly recommended Subclavian Approach (Left or Right) The left subclavian approach affords a gentle, sweeping curve to the apex of the right ventri- cle and is the preferred entry site for placement of a temporary transvenous pacemaker with- out fluoroscopic assistance. Hemodynamic measurements are often easier to record from the left subclavian approach. From the left subclavian vein approach, the catheters do not have to negotiate an acute angle, as is commonly the case at the junction of the right subclavian with the right brachiocephalic vein en route to the superior vena cava. This is also a com- mon site for kinking of the deep line. It also has the lowest risk of infection of various cen- 13 Bedside Procedures 255 tral line sites. However, remember that the thoracic duct is on the left side, and the dome of the pleura rises higher on the left. 1. Use sterile technique (povidone–iodine prep, gloves, mask, and a sterile field) when- ever possible. 2. Place the patient flat or head down in the Trendelenburg position with the head in the center or turned to the opposite side (the “ideal” position is somewhat controversial, and left up to operator preference). It may be helpful to place a towel roll along the pa- tient’s spine. 3. Use a 25-gauge needle to make a small skin wheal 2 cm below the midclavicle with 1% lidocaine (mixed 1:1 with sodium bicarbonate 1 mEq/L to help remove the sting). At this point, a larger needle (eg, 22-gauge) can be used to anesthetize the deeper tis- sues as well as locate the vein. 4. Attach a large-bore, deep-line needle (a 14-gauge needle with a 16-gauge catheter at least 8–12 in. long) to a 10–20-mL syringe and introduce it into the site of the skin wheal. 5. Advance the needle under the clavicle, aiming for a location halfway between the suprasternal notch and the base of the thyroid cartilage. The vein is encountered under the clavicle, just medial to the lateral border of the clavicular head of the sternocleido- mastoid muscle. In most patients this is roughly two finger-breadths lateral to the ster- nal notch. Apply gentle pressure on the needle at the skin entrance site to assist in lowering the needle under the clavicle (Fig. 13–7). 6. Apply back pressure as the needle is advanced deep to the clavicle, but above the first rib, and watch for a “flash” of blood. 7. Free return of blood indicates entry into the subclavian vein. Remember that occasion- ally the vein is punctured through both walls, and a flash of blood may not appear as the needle is advanced. Therefore, if a free return of blood does not occur on needle ad- vancement, withdraw the needle slowly with intermittent pressure. A free return of blood heralds the entry of the end of the needle into the lumen. Bright red blood that 13 forcibly enters the syringe indicates that the subclavian artery has been entered. If the arterial entry occurs, remove the needle. In the majority of patients, the surrounding tissue will tamponade any bleeding from the arterial puncture. Because the artery is under the clavicle, holding pressure has little effect on bleeding. 8. a. If you are using an Intracath, remove the syringe, place a finger over the needle hub, and advance the catheter an appropriate distance through the needle. Then withdraw the needle to just outside the skin and snap the protective cap over the tip of the needle. b. If you are using the Seldinger wire technique, advance the wire through the needle and then withdraw the needle. The pulse or ECG should be monitored during wire passage because the wire can induce ventricular arrhythmias. Arrhythmias usually resolve by calmly pulling the wire out several centimeters. Nick the skin with a No. 11 blade, and advance the dilator approximately 5 cm; remove the dilator and advance catheter in over the guidewire (use the brown port on the triple-lumen catheter). While advancing either the dilator or the catheter over the wire, periodi- cally ensure that the wire moves freely in and out. When placing a Cordis, advance the catheter and dilator over the guidewire as one unit (see Chapter 20 Pulmonary Artery Catheter Insertion, page 402, for more details). If the wire does not move freely, it usually is kinked, and the catheter or dilator should be removed and repo- sitioned. Maintain a firm grip on the guidewire at all times. Remove the wire and attach the IV tubing. Note that the wire used to insert a single-lumen catheter is 256 Clinician’s Pocket Reference, 9th Edition shorter than the wire supplied with the triple-lumen catheter. This is most critical when exchanging a triple-lumen for a single-lumen catheter; use the longer triple- lumen wire and insert the wire into the brown port. Place Shiley catheters using the Seldinger wire technique. 9. Attach the catheter to the appropriate IV solution, and place the IV bottle below the level of the deep-line site to ensure a good backflow of blood into the tubing. If no backflow occurs, the catheter may be kinked or not in the proper position. 10. Securely suture the assembly in place with 2-0 or 3-0 silk. Apply an occlusive dressing with povidone–iodine ointment. 11. Obtain a chest x-ray film immediately to verify placement of the catheter tip and to rule out pneumothorax. Ideally, the catheter tip lies in the superior vena cava at its junction with the right atrium, at about the fifth thoracic vertebra. Malpositioned catheters that go into the neck veins may be used only for saline infusion and not for monitoring or TPN infusion. 12. Catheters that cannot be manipulated at the bedside into the chest can usually be posi- tioned properly in the interventional radiology suite with the aid of fluoroscopy. Right Internal Jugular Vein Approach Three different sites are described and used in accessing the right internal jugular vein: ante- rior (medial to the sternocleidomastoid muscle belly), middle (between the two heads of the sternocleidomastoid muscle belly), and posterior (lateral to the sternocleidomastoid muscle belly). The middle approach is most commonly used and has the advantage of using well- defined landmarks. The major disadvantage of the internal jugular site is the patient discom- fort it causes. The site is difficult to dress and is uncomfortable for patients, particularly when turning the head. Procedure 13 1. Sterilize the site with povidone–iodine, and drape area with sterile towels. 2. Administer local anesthesia with lidocaine in the area to be explored. 3. Place the patient in the Trendelenburg (head down) position. 4. Use a small-bore, thin-walled needle (21-gauge) with syringe attached to locate the in- ternal jugular vein. It may be helpful to have a small amount of anesthetic in the sy- ringe to inject during exploration for the vein if the patient notes some discomfort. Some prefer to leave this needle and syringe in the vein and place the large-bore needle directly over the smaller needle, into the vein. This is commonly called the “seeker nee- dle” technique. 5. The internal diameter of the needle used to locate the internal jugular vein should be large enough to accommodate the passage of the guidewire. 6. Percutaneous entry should be made at the apex of the triangle formed by the two heads of the sternocleidomastoid muscle and the clavicle. (See Fig. 13–6.) 7. Direct the needle slightly lateral toward the ipsilateral nipple and enter at a 45-degree angle to the skin. 8. Often a notch can be palpated on the posterior surface of the clavicle. This actually can help locate the vein in the lateral/medial plane because the vein lies deep to this shal- low notch. 9. Successful puncture of the vein is accomplished usually at an unnerving depth of nee- dle insertion and is heralded by sudden aspiration of nonpulsatile venous blood. Bed- side localizing Doppler ultrasound units are available in most operating rooms or intensive care units. They can aid in localization of the internal jugular vein if the stan- dard techniques fail. 13 Bedside Procedures 257 Internal jugular vein Subclavian vein Clavicle Sternocleidomastoid muscle FIGURE 13–6 Technique for the catheterization of the internal jugular vein. 13 10. Inadvertent puncture of the carotid artery is common if the needle is inserted medial to where it should be on the middle approach and is common with the anterior approach. With arterial puncture, the syringe fills without negative pressure because of arterial pressure, and bright red blood pulsates from the needle after the syringe is removed. In this case remove the needle and apply manual pressure for 10–15 min to ensure ade- quate hemostasis. 11. Follow steps 8–12 as for subclavian line (page 255). On chest x-ray, the catheter tip should lie in the superior vena cava in the vicinity of the right atrium, at about the fifth thoracic vertebra. Complications Overall this is a safe procedure when the small-bore needle is first used to identify the vein. • Pneumothorax can be detected when a sudden gush of air is aspirated instead of blood. A postprocedure chest x-ray should always be done to rule out pneumothorax and check for line placement. A pneumothorax requires chest tube placement in vir- tually all cases, especially when the patient is being supported on a ventilator. The left-sided approach is associated with higher risk for pneumothorax because of the higher dome of the left pleura compared with the right. • Perforation of endotracheal tube cuffs 258 Clinician’s Pocket Reference, 9th Edition • Hemothorax from vascular injury or hydrothorax from administration of IV fluids into the pleural space • Catheter tip embolus: Never withdraw the catheter through the needle. It can shear off the tip. • Air embolus: Make sure that the open end of a deep line is always covered with a finger. As little as 50–100 mL of air in a vein can be fatal. If you suspect that air embolization has occurred, place the patient’s head down and turn on left side to keep the air in the right atrium. Obtain a STAT portable chest film to see if air is present in the heart. Left Internal Jugular Vein Approach The left internal jugular vein is not commonly used for central line placement. Better op- tions exist and should be exhausted before resorting to this approach. The procedure is simi- lar to right internal jugular vein approach. In addition to the usual procedural complications common to central lines, this approach has some unique complications, including inadver- tent left brachiocephalic vein and superior vena cava puncture with intravascular wires, catheters, and sheaths and laceration of the thoracic duct resulting in chylothorax. External Jugular Vein Approach This is a safe approach to central venous catheterization but a very technically demanding procedure due to the difficulty in threading the catheter into the central venous system.

This is also an uncomfortable insertion site for the patient because the dressing and IV tubing is on the neck. If the central venous system cannot be entered, this is also a site of last resort for placing a standard IV catheter (“peripheral”) for the administration of routine nonscle- rosing IV fluids. The external jugular vein is usually visible with the patient in the 30° Trendelenburg position. The vein, located in the subcutaneous tissues, crosses the sterno- 13 cleidomastoid muscle arising from just behind the angle of the jaw inferiorly where it drains into the subclavian vein just lateral to the inferior aspect of the sternocleidomastoid muscle. Procedure 1. Place the patient in the Trendelenburg position with the head turned away from the side of insertion. Prep and drape the neck from the ear to the subclavicular area. 2. Having the patient perform the Valsalva maneuver or gently occluding the vein near its insertion into the subclavian vein will help engorge the vein. 3. At the approximate midportion of the vein, make a skin wheal with a 25-gauge needle and lidocaine solution. Use a 21-gauge needle to anesthetize the deeper subcutaneous tissue and to locate the vein. 4. Remove the syringe from the needle and insert a floppy-tipped J wire into the needle. Use the guidewire with gentle pressure to negotiate the turns into the intrathoracic por- tion of the venous system. If there is difficulty passing the wire, have the patient turn the head slightly to help direct the wire. Never forcibly push the wire. As a last resort, fluroscopy can be used to direct the wire into the superior vena cava. 5. Once a sufficient length of guidewire is passed, the locating needle can be removed. 6. An incision in the skin may have to be made to accommodate the catheter. The catheter is then slid over the guidewire and the guidewire is removed. Aspirate blood from the end of the catheter to confirm that it is in the venous system. 7. Follow steps 8–12 as for placement via the subclavian vein (page 255). 13 Bedside Procedures 259 Finger in suprasternal notch Subclavian vein Clavicle First rib 13 Superior vena cava FIGURE 13–7 Technique for the catheterization of the subclavian vein. Complications See Right Internal Jugular Vein Approach, page 257. Femoral Vein Approach The femoral vein approach has several advantages. The procedure is safe, in that arterial and venous sites are more easily compressible, and it is impossible to cause pneumothorax from this site. Placement can be accomplished without interrupting cardiopulmonary resuscita- tion. This site can be used to place a variety of intravascular appliances, including temporary pacemakers, pulmonary artery catheters (expertise with fluoroscopy may be needed), and 260 Clinician’s Pocket Reference, 9th Edition triple-lumen catheters. The major disadvantages are the high risk of sepsis, the immobiliza- tion it causes, and the occasional need for fluoroscopy to ensure proper placement of pul- monary artery catheters or transvenous pacemakers. Procedure 1. Place the patient in the supine position. 2. Use sterile preparation and appropriate draping. Administer local anesthesia in the area to be explored. 3. Palpate the femoral artery. Use the “NAVEL” technique to locate the vein (see page 246). 4. Guard the artery with the fingers of one hand. 5. Explore for the vein just medial to the operator’s fingers with a needle and syringe. 6. It may be helpful to have a small amount of anesthetic in the syringe to inject with ex- ploration. 7. Direct the needle cephalad at about a 30-degree angle and insert below the femoral crease. 8. Puncture is heralded by the return of venous, nonpulsatile blood on application of nega- tive pressure to the syringe. 9. Advance the guidewire through the needle. 10. The guidewire should pass with ease into the vein to a depth at which the distal tip of the guidewire is always under the operator’s control even when the sheath/dilator or catheter is placed over the guidewire. 11. Remove the needle once the guidewire has advanced into the femoral vein. 12. If the catheter is a French 6 or larger, a skin incision with a scalpel blade and the use of a vessel dilator are generally needed. The catheter can then be advanced along with the guidewire in unison into the femoral vein. Be sure always to control the distal end of the guidewire. 13. Follow steps 13 through 18 for the right internal jugular vein approach. 13 Complications • The femoral deep line has the highest incidence of contamination and sepsis. If an occlusive dressing can remain in place and remain free from contamination, this is a safe option. • DVT has occurred from femoral vein catheterization. The risk for DVT increases if the catheter remains in place for prolonged periods. • Uncontrolled retroperitoneal bleeding can occur if the iliac/common femoral artery is inadvertently punctured above the inguinal ligament. Removal of a Central Venous Catheter 1. Turn off the IV flow. 2. Cut the retention sutures, and gently withdraw the catheter. Visually inspect the catheter to ensure it is intact. 3. Apply pressure for at least 2–3 min, and apply a sterile dressing. CHEST TUBE PLACEMENT (CLOSED THORACOSTOMY, TUBE THORACOSTOMY) Indications • Pneumothorax (simple or tension) 13 Bedside Procedures 261 • Hemothorax, hydrothorax, chylothorax, or empyema evacuation • Pleurodesis for chronic recurring pneumothorax or effusion that is refractory to stan- dard management (eg, malignant effusion) Materials • Chest tube (20–36 French for adults, 12–4 French for children) • Water-seal drainage system (Pleurovac, etc) with connecting tubing to wall suction • Minor procedure tray and instrument tray (see page 240) • Silk or nylon suture (0 to 2-0) • Petrolatum gauze (Vaseline) (optional) • 4 × 4 gauze dressing and cloth tape Background A chest tube is usually placed to treat an ongoing intrathoracic process that cannot be man- aged by simple thoracentesis (see page 304). The traditional methods of chest tube place- ment are described. Percutaneous tube thoracostomy kits are also available based on the Seldinger technique. It can be used in dealing with small pneumothoraces when there is no risk of ongoing air leak, but it should not be used with more significant conditions (empyema, major pneumothorax >20%, tension pneumothorax, chronic effusions) Procedure If a patient manifests signs of a tension pneumothorax (acute shortness of breath, hy- potension, distended neck veins, tachypnea, tracheal deviation) before a chest tube is placed, urgent treatment is needed. Insert a 14-gauge needle into the chest in the sec- ond intercostal space in the midclavicular line to rapidly decompress the tension pneu- mothorax and proceed with chest tube insertion. 13 1. Prior to placing the tube, review the chest x-ray unless an emergency does not allow enough time. For a pneumothorax, choose a high anterior site, such as the second or third intercostal space, midclavicular line, or subaxillary position (more cosmetic). Place a low lateral chest tube in the fifth or sixth intercostal space in the midaxillary line and direct posteriorly for fluid removal. (In most patients this location corresponds to the inframammary crease.) For a traumatic pneumothorax, use a low lateral tube be- cause this condition usually is associated with bleeding. Rarely, a loculated apical pneumothorax or effusion may require placement of an anterior tube in the second in- tercostal space at the midclavicular line. 2. Choose the appropriate chest tube. Use a 24–28 French tube for pneumothorax and 36 French for fluid removal. A “thoracic catheter” has multiple holes and works best for nearly all purposes. The vast majority of tubes can be inserted painlessly with generous use of local anesthetics. If the procedure is elective, the patient is extremely anxious, and the patient’s respiratory status is not compromised, sedation occasionally may be helpful. 3. Prep the area with povidone–iodine solution and drape it with sterile towels. Use 1ido- caine (with or without epinephrine) to anesthetize the skin, intercostal muscle, and pe- riosteum of the rib; start at the center of the rib and gently work over the top. Remember, the neurovascular bundle runs under the rib (Fig. 13–8). The needle then can be gently “popped” through the pleura and the aspiration of air or fluid confirms the correct location for the chest tube. 262 Clinician’s Pocket Reference, 9th Edition Thoracic wall entry site Level of skin incision Intercostal muscles Pleura Intercostal vein, artery and nerve 13 FIGURE 13–8 Chest tube technique demonstrating the procedure for creating a subcutaneous tunnel. Note: The skin incision is lower than the thoracic wall entry site. (Reprinted, with permission, from: Gomella TL [ed]: Neonatology: Basic Man- agement, On-Call Problems, Diseases, Drugs, 4th ed. Appleton & Lange, Norwalk CT, 1998.) 4. Make a 2–3-cm transverse incision over the center of the rib with a No. 15 or 11 scalpel blade. Use a blunt-tipped clamp to dissect over the top of the rib and create a subcuta- neous tunnel (see Fig. 13–8). 5. Puncture the parietal pleura with the hemostat, and spread the opening. BE CAREFUL NOT TO INJURE THE LUNG PARENCHYMA WITH THE HEMOSTAT TIPS. Insert a gloved finger into the pleural cavity to gently clear any clots or adhesions and to make certain the lung is not accidentally punctured by the tube. 6. Carefully insert the tube into the desired position with a hemostat or gloved finger as a guide. Make sure all the holes in the tube are in the chest cavity. Attach the end of the tube to a water-seal or Pleur-Evac suction system. Some chest tubes are provided with sharp trocars that are used to pierce the chest wall and place the chest tube simultane- 13 Bedside Procedures 263 ously with minimal amounts of dissection. These instruments are extremely dangerous and are usually placed in the anterior high position (ie, second, third, or fourth ICS). 7. Suture the tube in place. Place a heavy silk (0 or 2-0) suture through the incision next to the tube. Tie the incision together, then tie the ends around the chest tube. Make sure to wrap around the tube several times. Alternatively, a purse string suture (or “U stitch”) can be placed around the insertion site. Make sure all of the suction holes are in the chest cavity before the tube is secured. 8. Cover the insertion site with plain gauze. Make the dressing as airtight as possible with tape, and secure all connections in the tubing to prevent accidental loss of the water seal. Some physicians still wrap the insertion site with petroleum (Vaseline or Xero- form) gauze; however, these materials are not foolproof: they are not water-soluble (therefore, they act as foreign bodies), inhibit wound healing, and do not actually pro- vide a true seal. 9. Start suction (usually –20 cm in adults, –16 cm in children) and take a portable chest x-ray immediately to check the placement of the tube and to evaluate for residual pneu- mothorax or fluid. 10. To remove a chest tube, make sure the pneumothorax or hemothorax is cleared. Check for an air leak by having the patient cough; observe the water-seal system for bubbling that indicates either a system (tubing) leak or persistent pleural air leak. 11. Take the tube off suction but not off water seal, and cut the retention suture. Have the patient inspire deeply and perform the Valsalva maneuver while you apply pressure with petrolatum gauze or with a sufficient amount of antibiotic ointment on 4 × 4 gauze with additional 4 × 4 gauze squares. Pull the tube rapidly while the patient performs the Valsalva maneuver and make an airtight seal with tape. Check an “upright” exhalation chest x-ray film for pneumothorax. Complications Infection, bleeding, lung damage, subcutaneous emphysema, persistent pneumothorax/he- 13 mothorax, poor tube placement, cardiac arrhythmia CRICOTHYROTOMY (NEEDLE AND SURGICAL) Background Cricothyrotomy is a true emergency procedure that should be performed when obtaining an airway using endotracheal or orotracheal intubation is impossible. Indications • When immediate mechanical ventilation is indicated, but an endotracheal or orotra- cheal tube cannot be placed (eg, severe maxillofacial trauma, excessive oropharyn- geal hemorrhage) Contraindications • Surgical cricothyrotomy is contraindicated in children < 12 y; use needle approach. Basic Materials • Oxygen connecting tubing, high-flow oxygen source (tank or wall) • Bag ventilator 264 Clinician’s Pocket Reference, 9th Edition Needle Cricothyrotomy • 12–14-gauge catheter over needle (Angiocath or other), • 6–12-mL syringe • 3-mm pediatric

endotracheal tube adapter Surgical Cricothyrotomy (minimum requirements) • Minor procedure and instrument tray (page 240) plus tracheal spreader if available • No. 5–7 tracheostomy tube (6–8 French endotracheal tube can be substituted) • Tracheostomy tube adapter to connect to bag-mask ventilator Procedures Needle Cricothyrotomy 1. With the patient supine, place a roll behind the shoulders to gently hyperextend the neck. 2. Palpate the cricothyroid membrane, which resembles a notch located between the cau- dal end of the thyroid cartilage and the cricoid cartilage. Prep the area with povidone–iodine solution. Local anesthesia can be used if the patient is awake. 3. Mount the syringe on the 12- or 14-gauge catheter-over-needle assembly, and advance through the cricothyroid membrane at a 45-degree angle, applying back pressure on the syringe until air is aspirated. 4. Advance the catheter, and remove the needle. Attach the hub to a 3-mm endotracheal tube adapter that is connected to the oxygen tubing. Allow the oxygen to flow at 15 L/min for 1–2 s on, then 4 s off by the use of a Y-connector or a hole in the side of the tubing to turn the flow on and off. 5. The needle technique is only useful for about 45 min because the exhalation of CO2 is suboptimal. 13 Surgical Cricothyrotomy 1. Follow steps 1 and 2 as for needle cricothyrotomy. 2. Make a 3–4-cm vertical skin incision through the cervical fascia and strap muscles in the midline over the cricothyroid membrane. Expose the cricothyroid membrane, and make a horizontal incision. Insert the knife handle, and rotate it 90 degrees to open the hole in the membrane. Alternatively, a hemostat or tracheal spreader can be used to di- late the opening. 3. Insert a small (5–7-mm) tracheostomy tube, inflate the balloon (if present), and secure in position with the attached cotton tapes. 4. Attach to oxygen source and ventilate. Listen to the chest for symmetrical breath sounds. 5. A surgical cricothyrotomy should be replaced with a formal tracheostomy after the pa- tient has been stabilized and generally within 24–36 h. Complications • Bleeding, esophageal perforation, subcutaneous emphysema, pneumomediastinum, and pneumothorax, CO2 retention (especially with the needle procedure) CULDOCENTESIS Indications • Diagnostic technique for problems of acute abdominal pain in the female 13 Bedside Procedures 265 • Evaluation of female patient with signs of hypovolemia and possible intraabdominal bleeding • Evaluation of ascites, especially in possible cases of gynecologic malignancy Materials • Speculum • Antiseptic swabs • Povidone–iodine or chlorhexidine • 1% lidocaine • 18–21-gauge spinal needle • 2 (10 mL) syringes and tenaculum Procedure 1. Culdocentesis should be preceded with a careful pelvic exam to document uterine posi- tion and rule out pelvic mass at risk of perforation by the culdocentesis. 2. After obtaining the patient’s informed consent, the vagina is prepped with antiseptic, such as iodine or chlorhexidine. 3. Inject 1% lidocaine submucosally in the posterior cervical fornix prior to tenaculum application. 4. Traction is improved by application of the tenaculum to the posterior cervical lip. 5. Apply an 18–21-gauge spinal needle to a 10-mL syringe, filled with 1 mL of air. 6. As you move the needle forward through the posterior cervical fornix, apply light pres- sure to the syringe until the air passes. Maintain traction on the tenaculum as you ad- vance the spinal needle to maximize the surface area of the cul de sac for needle entry. 7. After intraabdominal entry, ask the patient to elevate herself on elbows to permit grav- ity drainage into the area of needle entry. Apply negative pressure to the syringe. Slow rotation of the needle followed by slow removal may enable a pocket of fluid to be 13 found and aspirated. 8. If first culdocentesis attempt is not successful, the procedure can be repeated with a dif- ferent angle of approach. 9. Although perforation of viscus is a possibility, the complication rate has been very low. Fresh blood that clots rapidly is probably secondary to traumatic tap, and the procedure can be repeated. 10. If blood is aspirated, it should be spun for hematocrit and placed into an empty glass test tube to demonstrate the presence or absence of a clot. Failure of blood to clot sug- gests old hemorrhage. 11. If pus is aspirated, send specimens for GC, aerobic, anaerobic, Chlamydia, Myco- plasma, and Ureaplasma cultures. 12. If a malignancy is suspected, send fluid for cytologic evaluation. Complications Infection, hemorrhage, air embolus, perforated viscus DOPPLER PRESSURES Indications • Evaluation of peripheral vascular disease (ankle-brachial or ankle-arm index) • Routine blood pressure measurement in infants or critically ill adults 266 Clinician’s Pocket Reference, 9th Edition Materials • Doppler flow monitor • Conductive gel (lubricant jelly can also be used) • Blood pressure cuff Procedure (Ankle-Brachial or Ankle-Arm Index) 1. Determine the blood pressure in each arm. 2. Measure the pressures in the popliteal arteries by placing a BP cuff on the thigh. The pressures in the dorsalis pedis arteries (on the top of the foot) and the posterior tibial ar- teries (behind the medial malleolus) are determined with a BP cuff on the calf. 3. Apply conductive jelly and place the Doppler probe over the artery. Inflate the BP cuff until the pulsatile flow is no longer heard. Deflate the cuff until the flow returns. This is the systolic, or Doppler, pressure. Note: The Doppler cannot routinely determine the di- astolic pressure, and a palpable pulse need not be present to use the Doppler. 4. The A/B or AAI index is often computed from Doppler pressure. It is equal to the pressure in the ankle (usually the posterior tibial) divided by the systolic pressure in the arm. An A/B index of >0.9 is usually normal, and an index of <0.5 is usually associated with significant peripheral vascular disease. ELECTROCARDIOGRAM Basic ECG interpretation can be found in Chapter 19, page 367. Indications • Useful in the evaluation of chest pain and other cardiac conditions Materials 13 • ECG machine with paper and lead electrodes • Adhesive electrode pads Procedure 1. Most hospitals have converted to fully automated ECG machines. It is important to be- come acquainted with your particular machine prior to using it. The following is a gen- eral outline. 2. Start with the patient in a comfortable, recumbent position. Explain the procedure to dispel any myths. Instruct the patient to lie as still as possible to cut down on artifacts in the tracing. 3. Plug in the ECG machine and turn it on. 4. Attach the electrodes as outlined here: a. Patient Cables. The standard ECG machine has five lead wires, one for each limb and one for the chest leads. Newer machines have six precordial electrodes, which are all placed in the proper positions prior to performing the procedure. These may be color-coded in the following fashion: • RA: White—right arm • LA: Black—left arm • RL: Green—right leg • LL: Red—left leg • C: Brown—chest 13 Bedside Procedures 267 b. Limb Electrodes. The limb electrodes are flat, rectangular plates held in place by rubber or Velcro straps that encircle the limb; newer machines may use self-adher- ing electrode pads. Place each electrode on the limb indicated, wrist or ankle, usu- ally on the ventral surface. In case of amputation or a cast, the lead may be placed on the shoulder or groin with almost no effect on the tracing. c. Chest (Precordial) Electrodes. The chest electrode is brown and designated by the letter “C.” It is attached to a suction cup that is attached in sequence to each of the positions on the precordium (see the following description). Newer machines allow all leads to be placed prior to running the ECG with all pads applied at the same time. This makes locating the proper positions much quicker and easier (Fig. 13–9). Precordial leads are placed as follows: • V1 = fourth intercostal space just to the right of the sternal border • V2 = fourth intercostal space just to the left of the sternal border • V3 = midway between leads V2 and V4 • V4 = midclavicular line, above the fifth interspace 13 V1 V2 V6 V3 V5 V4 Midaxillary line Anterior axillary line Midclavicular line FIGURE 13–9 Location of the precordial chest leads used in obtaining a routine ECG. 268 Clinician’s Pocket Reference, 9th Edition • V5 = anterior axillary line at the same level as V4 • V6 = midaxillary line at the same level as leads V4 and V5 4. Once the machine is warmed up and the electrodes are positioned or ready for position- ing, make sure that the paper speed is set at 25 mm/s. When everything is ready, follow the directions for your particular machine to obtain the ECG tracing. It should include 12 different leads, that is, I, II, III, AVR, AVL, and V1–6. 5. Label the tracing with the patient’s name, date, time, and any other useful information, such as medications, and your name. A routine 12-lead ECG should take 4–8 min. Helpful Hints 1. The second rib inserts at the sternal angle, and therefore the second intercostal space is directly inferior to the sternal angle. Feel down two more intercostal spaces and you have the fourth intercostal space to position V1 and V2. 2. When you start seeing a solid blue or red line at the top or bottom of the strip, you are about to run out of paper. Always leave enough paper for the next user. 3. Learn the color scheme for the leads; it could be very useful in an emergency. Some memory aids include a. Red and green go to the legs: “Christmas on the bottom” or “When driving your car you use your left leg to brake (red light) and your right leg to go (green light).” b. Black (left) and white (right) go to the arms: “Remember white is right and black is left.” c. Brown is for the chest. ENDOTRACHEAL INTUBATION Indications • Airway management during cardiopulmonary resuscitation 13 • Any indication for using mechanical ventilation (respiratory failure, coma, general anesthesia, etc) Contraindications • Massive maxillofacial trauma (relative) • Fractured larynx • Suspected cervical spinal cord injury (relative) Materials • Endotracheal tube of appropriate size (Table 13–3) • Laryngoscope handle and blade (straight [Miller] or curved [MAC]; size No. 3 for adults, No. 1–1.5 for small children) • 10-mL syringe, adhesive tape, benzoin • Suction equipment (Yankauer suction) • Malleable stylet (optional) • Oropharyngeal airway Technique 1. Orotracheal intubation is most commonly used and is described here. Orotracheal intu- bation should be done only with great care in cases of suspected cervical spine injuries. In such cases nasotracheal intubation is preferred. 13 Bedside Procedures 269 TABLE 13–3 Recommended Endotracheal Tube Sizes Internal Diameter Patient (mm) Premature infant 2.5–3.0 (uncuffed) Newborn infant 3.5 (uncuffed) 3–12 mo 4.0 (uncuffed) 1–8 y 4.0–6.0 (uncuffed)* 8–16 y 6.0–7.0 (cuffed) Adult 7.0–9.0 (cuffed) *Rough estimate is to measure the little finger. 2. Any patient who is hypoxic or apneic must be ventilated prior to attempting endotra- cheal intubation (bag mask or mouth to mask). Remember to avoid prolonged periods of no ventilation if the intubation is difficult. A rule of thumb is to hold your breath while attempting intubation. When you need to take a breath, so must the patient, and you should resume ventilation, and reattempt intubation in a minute or so. 3. Extend the laryngoscope blade to 90 degrees to verify the light is working, and check the balloon on the tube (if present) for leaks. 4. Place the patient’s head in the “sniffing position” (neck extended anteriorly and the head extended posteriorly). Use suction to clear the upper airway if needed. 5. Hold the laryngoscope in the left hand, hold the mouth open with the right hand, and use the blade to push the tongue to patient’s left while keeping it anterior to the blade. 13 Advance the blade carefully toward the midline until the epiglottis is visualized. Use suction if needed. 6. If the straight laryngoscope blade is used, pass it under the epiglottis and lift upward to visualize the vocal cords (Fig. 13–10). If the curved blade is used, place it anterior to the epiglottis (into the vallecula) and gently lift anteriorly. In either case, do not use the handle to pry the epiglottis open, but

rather gently lift to expose the vocal cords. 7. While maintaining visualization of the cords, grasp the tube in your right hand and pass it through the cords. With more difficult intubations, the malleable stylet can be used to direct the tube. 8. In patients who may have eaten recently, gentle pressure placed over the cricoid carti- lage by an assistant helps to occlude the esophagus and prevent aspiration during intu- bation. “Cricoid pressure” can also help visualize the vocal cords in patients whose larynx is situated more anteriorly than usual. 9. When using a cuffed tube (adult and older children), gently inflate air with a 10-mL sy- ringe until the seal is adequate (about 5 mL). Ventilate the patient while auscultating and visualizing both sides of the chest to verify positioning. If the left side does not seem to be ventilating, it may signify that the tube has been advanced down the right mainstem bronchus. Withdraw the tube 1–2 cm, and recheck the breath sounds. Also auscultate over the stomach to ensure the tube is not mistakenly placed in the esopha- gus. Confirm positioning with a chest x-ray. The tip of the endotracheal tube should be a few centimeters above the carina. 270 Clinician’s Pocket Reference, 9th Edition Epiglottis Glottis Trachea 13 Esophagus FIGURE 13–10 Endotracheal intubation using a curved laryngoscope blade. 10. Tape the tube in position, and insert an oropharyngeal airway to prevent the patient from biting the tube. Consider an orogastric tube to prevent regurgitation. Complications • Bleeding, oral or pharyngeal trauma, improper tube positioning (esophageal intuba- tion, right mainstem bronchus), aspiration, tube obstruction or kinking FEVER WORK-UP Although not a “procedure” in the true sense of the word, a fever work-up involves judicious use of invasive procedures. The true definition of a fever can vary from service to service. General guidelines to follow are a temperature of >100.4°F orally on a medical or surgical 13 Bedside Procedures 271 service, or a temperature greater than or equal to 101°F rectally or 100°F orally in an infant or immunocompromised patient. When evaluating a patient for a fever, consider if the temperature is oral, rectal, tym- panic, or axillary (rectal and tympanic temperatures are about 1° higher and axillary temper- atures are about 1° lower than oral); has the patient drunk any hot or cold liquids or smoked around the time of the determination; and is the patient on any antipyretics. Differential di- agnosis of fever and fever of unknown origin are discussed in Chapter 3. General Fever Work-Up 1. Quickly review the chart and medication record if the patient is not familiar to you. 2. Question and examine the patient to locate any obvious sources of fever. a. Ears, nose, and throat: Especially in children b. Neck: Tenderness or stiffness present c. Nodes: Adenopathy d. Lungs: Rales (crackles), rhonchi (wheezes), decreased breath sounds, or dullness to percussion. Can the patient generate an effective cough? e. Heart: Heart murmur, which may suggest SBE f. Abdomen: Presence or absence of bowel sounds, guarding, rigidity, tenderness, bladder fullness, or costovertebral angle tenderness g. Genitourinary: When a Foley catheter is in place; appearance of the urine, grossly and microscopically h. Rectal Exam: Tenderness or fluctuance to suggest an abscess, or acute prostatitis i. Pelvic Exam: Especially in the postpartum patient j. Wounds: Erythema, tenderness, swelling, or drainage from surgical sites k. Extremities: Signs of inflammation at IV sites. Look for thigh or calf tenderness and swelling. l. Miscellaneous: Consider the possibility of a drug fever (eosinophil count on the CBC may be elevated) or NG tube fever. Do all this before you begin to investi- 13 gate the less common or less obvious causes of a fever 3. Laboratory Studies a. Basic: CBC with differential, urinalysis, cultures as indicated: urine, blood, spu- tum, wound, spinal fluid (especially in children less than 4–6 mo old) b. Other: Studies based on your evaluation: (i) Radiographic: Chest or abdominal films, CT or ultrasound exams (ii) Invasive: LP, thoracentesis, paracentesis are more aggressive procedures that may be indicated. Miscellaneous Fever Facts 1. Causes of Fever in the Postop Patient: Think of the “Six W’s”: a. Wind: Atelectasis secondary to intubation and anesthesia is the most common cause of a fever immediately after surgery. To treat, have the patient up and ambu- lating, getting incentive spirometry, P&PD, etc. b. Water: UTI; may be secondary to a bladder catheter c. Wound: Infection d. Walking: Phlebitis, DVT e. Wonder drugs: Drug fever (common causes are listed on page 46). f. Woman: Endometritis, or mastitis. (These are common only in postpartum patients.) 2. Elevated White Cell Counts: Commonly elevated secondary to catecholamine dis- charge after a stress such as surgery or childbirth 272 Clinician’s Pocket Reference, 9th Edition 3. Temperatures of 103–105°F: In adults, think of lung or kidney infections, or bac- teremia. 4. Lethargy, Combativeness, Inappropriate Behavior: Strongly consider doing an LP to rule out meningitis. 5. Elderly Patients: Can be extremely ill without many of the typical manifestations; they may be hypothermic or deny any tenderness. You must be very aggressive to iden- tify the cause. 6. Infants and Children: Have normally elevated baseline temperatures (up to 3 mo 99.4°F, 1 y 99.7°F, 3 y 99.0°F) GASTROINTESTINAL INTUBATION Indications • GI decompression: ileus, obstruction, pancreatitis, postoperatively • Lavage of the stomach with GI bleeding or drug overdose • Prevention of aspiration in an obtunded patient • Feeding a patient who is unable to swallow Materials • Gastrointestinal tube of choice (see following list) • Lubricant jelly • Catheter tip syringe • Glass of water with a straw, stethoscope Types of Gastrointestinal Tubes 1. Nasogastric Tubes a. Levin: A tube with a single lumen, a perforated tip, and side holes for the aspira- 13 tion of gastric contents. Connect it to an intermittent suction device to prevent the stomach lining from obstructing the lumen. Sometimes it is necessary to cut off the tip to allow for the aspiration of larger pills or tablets. The size varies from 10 to 18 French (1 French unit = ¹₃ mm in diameter, see page 241). b. Salem-sump: A double-lumen tube, with the smaller tube acting as an air intake vent so that continuous suction can be applied. This is the best tube for irrigation and lavage because it will not collapse on itself. If a Salem-sump tube stops work- ing even after it is repositioned, often a “shot” of air from a catheter-tipped syringe in the air vent will clear the tube. Both the Salem-sump and Levin tubes have ra- diopaque markings. 2. Intestinal Decompression Tubes (“long intestinal tubes”) a. Cantor tube: A long single-lumen tube with a rubber balloon at the tip. The bal- loon is partially filled with mercury (5–7 mL using a tangentially directed 21- gauge needle, then the air is aspirated), which allows it to gravitate into the small bowel with the aid of peristalsis. Used for decompression when the bowel is ob- structed distally. b. Miller–Abbott tube: A long double-lumen tube with a rubber balloon at the tip. One lumen is used for aspiration; the other connects to the balloon. After the tube is in the stomach, inflate the balloon with 5–10 mL of air, inject 2–3 mL of mer- cury into the balloon, and then aspirate the air. Functioning and indications are es- sentially the same as for the Cantor tube. Do not tape these intestinal tubes to the patient’s nose or the tube will not descend. The progress of the tube can be fol- lowed on x-ray. 13 Bedside Procedures 273 3. Feeding Tubes Virtually any NG tube can be used as a feeding tube, but it is preferable to place a spe- cially designed nasoduodenal feeding tube. These are of smaller diameter (usually 8 French) and are more pliable and comfortable for the patient. Weighted tips tend to travel into the duodenum, which helps prevent regurgitation and aspiration. Most are supplied with stylets that facilitate positioning, especially if fluoroscopic guidance is needed. Always verify the position of the feeding tube with an x-ray prior to starting tube feeding. Commonly used tubes include the mercury-weighted varieties (Keogh tube, Duo-Tube, Dobbhoff, Entriflex), the tungsten-weighted (Vivonex tube), and the unweighted pediatric feeding tubes. 4. Miscellaneous a. Sengstaken–Blakemore tube: A triple-lumen tube used exclusively for the con- trol of bleeding esophageal varices by tamponade. One lumen is for gastric aspira- tion, one is for the gastric balloon, and the third is for the esophageal balloon. Other types of tubes used to control esophageal bleeding include the Linton and Minnesota tubes. b. Ewald tube: An orogastric tube used almost exclusively for gastric evacuation of blood or drug overdose. The tube is usually double lumen and large diameter (18–36 French). c. Dennis, Baker, Leonard tubes: These are used for intraoperative decompression of the bowel and are manually passed into the bowel at the time of laparotomy. Procedure (For Nasogastric and Feeding Tubes) 1. Inform the patient of the nature of the procedure and encourage cooperation if the pa- tient is able. Choose the nasal passage that appears most open. Have the patient sitting up if able. 2. Lubricate the distal 3–4 in. of the tube with a water-soluble jelly (K-Y Jelly or viscous lidocaine), and insert the tube gently along the floor of the nasal passageway. Maintain 13 gentle pressure that will allow the tube to pass into the nasopharynx. 3. When the patient can feel the tube in the back of the throat, ask patient to swallow small amounts of water through a straw as you advance the tube 2–3 in. at a time. 4. To be sure that the tube is in the stomach, aspirate gastric contents or blow air into the tube with a catheter-tipped syringe and listen over the stomach with your stethoscope for a “pop” or “gurgle.” The position of feeding tubes must be verified by a chest x-ray prior to institution of feedings to prevent accidental bronchial instillation. 5. NG tubes are usually attached either to low wall suction (Salem-sump type tubes with a vent) or to intermittent suction (Levin type tubes). The latter allows the tube to fall away from the gastric wall between suction cycles. 6. Feeding and pediatric feeding tubes in adults are more difficult to insert because they are more flexible. Many are provided with stylets that make their passage easier. Feed- ing tubes are best placed into the duodenum or jejunum in order to decrease the risk of aspiration. Administering 10 mg of metoclopramide (Reglan) IV 10 min before inser- tion of the tube assists in placing the tube into the duodenum. Once the feeding tube is in the stomach, the bell of the stethoscope can be placed on the right side of the pa- tient’s midabdomen. As the tube is advanced, air can be injected to confirm progression of the tube to the right, toward the duodenum. If the sound of the air becomes fainter, the tube is probably curling in the stomach. Pass the tube until a slight resistance is felt, heralding the presence of the tip of the tube at the pylorus. Holding constant pressure and slowly injecting water through the tube is often rewarded with a “give,” which signifies passage through the pylorus. The tube often can be advanced far into the 274 Clinician’s Pocket Reference, 9th Edition duodenum with this method. The duodenum usually provides constant resistance which will give with slow injection of water. Placing the patient in the right lateral decubitus position may help the tube enter the duodenum. Always confirm the location of the tube with an abdominal x-ray. 7. Tape the tube securely in place but do not allow it to apply pressure to the ala of the nose. (Note: Intestinal decompression tubes should not be taped because they are al- lowed to pass through the intestine). Patients have been disfigured because of ischemic necrosis of the nose caused by a poorly positioned NG tube. Complications • Inadvertent passage into the trachea may provoke coughing or gagging in the patient. • Aspiration • If the patient is unable to cooperate, the tube often becomes coiled in the oral cavity. • The tube is irritating and may cause a small amount of bleeding in the mucosa of the nose, pharynx, or stomach. The drying and irritation can be

lessened by throat lozenges or antiseptic spray. • Intracranial passage in patient with a basilar skull fracture • Esophageal perforation • Esophageal reflux caused by the tube-induced incompetence of the distal esophageal sphincter • Sinusitis can result from the tube, causing edema of the nasal passages that blocks drainage from the nasal sinuses. HEELSTICK Indication • Frequently used to collect blood samples from infants 13 Materials • Alcohol swabs • Lancet • Capillary or caraway collection tubes • Clay tube sealer Technique 1. Although called a “heelstick,” any highly vascularized capillary bed can be used (fin- ger, ear lobe, or great toe). The heel can be warmed for 5–10 min by wrapping it in a warm washcloth. 2. Wipe the area with an alcohol swab. Use Figure 13–11 to choose the site for the punc- ture on the foot. Use of these sites helps decrease the incidence of osteomyelitis. 3. Use a 4-mm lancet, and make a quick, deep puncture so that blood flows freely. Auto- mated safety lancets (eg, BD Genie lancet for fingersticks and the BD Quick Heel Lancer for heelsticks) are also available. Wipe off the first drop of blood. Gently squeeze the heel and touch a collection tube to the drop of blood. The tube should fill by capillary action. Seal the end of the tube in clay. 4. Most labs can usually make laboratory determinations on small samples from the pedi- atric age group. A Caraway tube can hold 0.3 mL of blood. One to three caraway tubes can be used for most routine tests. For a capillary blood gas, the blood is usually transferred to a 1-mL heparinized syringe and placed on ice. 5. Wrap the foot with 4 × 4 gauze squares or apply an adhesive bandage. 13 Bedside Procedures 275 FIGURE 13–11 Demonstration of the preferred sites and technique of performing a heelstick in an infant. (Reprinted, with permission, from: Gomella TL [ed]: Neona- tology: Basic Management, On-Call Problems, Diseases, Drugs, 4th ed. Appleton & Lange, Norwalk CT, 1998.) INTERNAL FETAL SCALP MONITORING Indication 13 • To accurately assess FHR patterns during labor to screen for possible fetal distress Contraindications • Presence of placenta previa • When it is otherwise impossible to identify the portion of the fetal body where appli- cation is contemplated Materials • Fetal scalp monitoring electrode • Sterile vaginal lubricant or povidone–iodine spray • Spiral electrode • Leg plate/fetal monitor Technique 1. Position the patient in the dorsal lithotomy position (knees flexed and abducted), and per- form an aseptic perineal prep with sterile vaginal lubricant or povidone–iodine spray. 2. Perform a manual vaginal exam, and clearly identify the fetal presenting part. The membranes must be ruptured prior to attachment of the spiral electrode. 276 Clinician’s Pocket Reference, 9th Edition 3. Remove the spiral electrode from the sterile package and place the guide tube firmly against the fetal presenting part. Electrode should not be applied to fetal face, fontanels, or genitalia. 4. Advance the drive tube and electrode until the electrode contacts the presenting part. Maintaining pressure on the guide tube and drive tube, rotate the drive tube clockwise until mild resistance is met (usually one turn). 5. Press together the arms on the drive tube grip, which releases the locking device. Care- fully slide the drive and guide tubes off the electrode wires while holding the locking device open. 6. Attach the spiral electrode wires to the color-coded leg plate, which is then connected to the electronic fetal monitor. Complications • Fetal or maternal hemorrhage, fetal infection (usually scalp abscess at the site of in- sertion) Interpretation Normal FHR is 120–160 bpm. Accelerations are increases in the FHR, and although they can be associated with fetal distress (usually in association with late decelerations), they are almost always a sign of fetal well-being. Decelerations are transient falls in FHR related to a uterine contraction and are of three types: 1. Early Decelerations: Seen in normal labor, slowing of the FHR clearly associated with the onset of the contraction and the FHR promptly returns to normal after the contraction is over. Usually caused by head compression, and occasionally by cord compression. 2. Late Deceleration: Slowing of the FHR that occurs after the uterine contraction starts and the rate does not return to normal until well after the contraction is over. This type of 13 pattern is often associated with uteroplacental insufficiency (fetal acidosis or hypoxia). 3. Variable Decelerations: Irregular pattern of decelerations unassociated with contrac- tions caused by cord compression. If bradycardia persists, evaluate with scalp pH. Other patterns seen include beat-to-beat variability (small fluctuations in FHR 5–15 BPM over the baseline FHR usually associated with fetal well-being); tachycardia (often an early sign of fetal distress, seen with febrile illnesses, hypoxia, fetal thyrotoxicosis); and brady- cardia (associated with maternal and fetal hypoxia, fetal heart lesions including heart block). Sinusoidal pattern can be drug-induced and is seen occasionally with severe fetal anemia. INJECTION TECHNIQUES Indications • Intradermal: Most commonly used for skin testing • Subcutaneous: Useful for low-volume medications such as insulin, heparin and some vaccines • Intramuscular: Administration of parenteral medications that cannot be absorbed from the subcutaneous layer or of high volume (up to 10 mL) Contraindications • Allergy to any components of the injectate • Active infection or dermatitis at the injection site • Intramuscular injections are generally contraindicated with coagulopathy 13 Bedside Procedures 277 Procedures Intradermal (see Skin Testing, page 303) Subcutaneous 1. Deposit the drug within the fat but above the muscle. With careful placement nerve in- jury is rarely a danger. 2. Choose a site free of scarring or active infection. Injection sites include the outer sur- face of the upper arm, anterior surface of the thigh, and lower abdominal wall. With re- peated injections (diabetics, etc) sites should be rotated. 3. 25–27-gauge ³⁄₄–1-in. needles are most commonly used; volume of medication must not exceed 5 mL. Draw up the medication, making certain to expel any air bubbles. 4. Clean site with an alcohol swab. Bunch up the skin between the thumb and forefinger so that the subcutaneous tissue is off the underlying muscle. 5. Warn the patient that there will be “pinch” or “sting,” and insert the needle firmly and rapidly at a 45-degree angle until a sudden release signifies penetration of the dermis. 6. Release the skin, and aspirate to make certain a blood vessel has not been entered and inject slowly. 7. Withdraw the needle and apply gentle pressure. A dressing is not usually necessary. Apply pressure longer if there is bleeding from the site. Intramuscular 1. Common sites include the deltoid, gluteus, and the vastus lateralis. • Deltoid Muscle: The safe zone includes only the main body of the deltoid muscle lying lateral and a few centimeters beneath the acromion. Low risk of radial nerve injury unless the needle strays into the middle or lower third of the arm. • Gluteus Muscles: This is the preferred site in children > 2 y and in adults. Draw an imaginary line from the femoral head to the posterior superior iliac spine. This site 13 (upper outer quadrant of the buttocks) is safe for injections because it is away from the sciatic nerve and superior gluteal artery. • Vastus Lateralis Muscle (anterior thigh): A very safe site in all patients and the site of choice in infants. The only disadvantage of this site is that the firm fascia lata overlying the muscle can make needle insertion somewhat more painful. 2. A 22-gauge, 1¹⁄₂-in. needle is acceptable for most intramuscular injections. Remove air bubbles from the syringe and needle. Wipe the skin with alcohol. 3. Gently stretch the skin to one side and warn the patient of a sting. Penetrate the skin at a 90-degree angle, and advance approximately 1 in. into the muscle. (Obese patients may require deeper penetration with a longer needle). 4. Aspirate to make sure that you have not entered a vessel. Administer the medication. Gently massage the site with alcohol swab or gauze to promote absorption. Complications • Nerve and arterial injury • Abscesses (sterile or septic). Use good technique and rotate injection sites. • Bleeding can usually be controlled with pressure. INTRAUTERINE PRESSURE MONITORING Indication • To accurately assess uterine contraction during labor 278 Clinician’s Pocket Reference, 9th Edition Contraindication • Presence of placenta previa Materials • Pressure catheter and introducer • Transducer connected to fetal monitor • Sterile gloves, vaginal lubricant, povidone–iodine spray • 10-mL syringe, 30 mL sterile water Procedure 1. Prime the transducer with sterile water. 2. Position the patient in the dorsal lithotomy position (knees flexed and abducted), and per- form an aseptic perineal prep with sterile vaginal lubricant or povidone–iodine spray. 3. Perform a manual vaginal exam, and clearly identify the fetal presenting part. The membranes must be ruptured prior to insertion of catheter. 4. Remove the catheter from the sterile package, and place the guide tube through fingers around the presenting part into the uterine cavity. 5. Prime the catheter with sterile water and thread through the guide tube. 6. Attach the distal catheter to transducer and zero to air. Complications Infection, placental perforation if low lying IV TECHNIQUES Indication 13 • To establish an intravenous access for the administration of fluids, blood, or medica- tions • (Other techniques include Central Venous Catheters, page 253 and PICC lines (page 292) Materials • IV fluid • Connecting tubing • Tourniquet • Alcohol swab • Intravenous cannulas (a catheter over a needle [eg, Angiocath, Insyte] or a butterfly needle) • Antiseptic ointment, dressing, and tape Technique 1. It helps to rip the tape into strips, attach the IV tubing to the solution, and flush the air out of the tubing before you begin. Using a catheter–needle assembly (Angiocath, etc) often helps to “break the seal” between the needle and catheter prior to the time that the catheter is in the vein so that dislodging the catheter is less likely. 2. The upper, nondominant extremity is the site of choice for an IV, unless the patient is being considered for placement of permanent hemodialysis access. In this instance, the 13 Bedside Procedures 279 upper nondominant extremity should be “saved” as the access site for hemodialysis. Choose a distal vein (dorsum of the hand) so that if the vein is lost, you can reposition the IV more proximally. Figure 13–12 demonstrates some common upper extremity Basilic Cephalic vein vein Basilic Cephalic vein vein Accessory cephalic vein Median cubital vein 13 Cephalic Basilic vein vein FIGURE 13–12 Principle veins of the arm used to place IV access and in venipuncture, the pattern can be highly variable. (Reprinted, with permission, from: Stillman RM [ed]: Surgery, Diagnosis, and Therapy, Appleton & Lange, Norwalk, CT, 1989.) 280 Clinician’s Pocket Reference, 9th Edition veins; however, avoid veins that cross a joint space. Also avoid the leg because the inci- dence of thrombophlebitis is high with IVs placed there. 3. Apply a tourniquet above the proposed IV site. Use the techniques described in the sec- tion on venipuncture to help expose the vein (page 309). Carefully clean the site with an alcohol or povidone–iodine swab. If a large-bore IV is to be used (16 or 14), local anesthesia (1idocaine injected with a 25-gauge needle) is helpful. 4. Stabilize the vein distally with the thumb of your free hand. Using the catheter-over- needle assembly (Intracath or Angiocath), either enter the vein directly or enter the skin alongside the vein first and then stick the vein along the side at about a 20-degree angle. Direct entry and side entry IV techniques are illustrated in Figures 13–13 and 13–14. Once the vein is punctured, blood should appear in the “flash chamber” of a catheter-over- needle assembly. Advance a few more millimeters to be sure that both the needle and the tip of the catheter have entered the vein. Carefully withdraw the needle as you advance the catheter into the vein (see Fig. 13–13). Never withdraw the catheter over the needle be- cause this procedure can shear off the plastic tip and cause a catheter embolus. Re- move the tourniquet, and connect the IV line to the catheter. Blood loss can be minimized by compressing the vein with the thumb just proximal to the catheter. 5. With the IV fluid running, observe the site for

signs of induration or swelling that indi- cate improper placement or damage to the vein. See Chapter 9 for choosing IV fluids and how to determine infusion rates. 6. Tape the IV securely in place, apply a drop of povidone–iodine or antibiotic ointment and sterile dressing. Ideally, the dressing should be changed every 24–48 h to help re- duce infections. Arm boards are also useful to help maintain an IV site. 7. “Butterfly” or “scalp vein” needle can sometimes be used (see Fig. 13–14). This is a small metal needle with plastic “wings” on the side. It is very useful in infants, who often have poor peripheral veins but prominent scalp veins, children, and in adults who have small, fragile veins. 13 8. Troubleshooting difficult IV placement • If the veins are deep and difficult to locate, a small 3–5-mL syringe can be mounted on the catheter assembly. Proper positioning inside the vein is determined by aspira- tion of blood. If blood specimens are needed on a patient who also needs an IV, this technique can be used to start the IV and to collect samples at the same time. • Whaid’s maneuver can be attempted (J Emerg Nurs, 1993;19:186). Spend about 1 min using both hands to “milk” blood from the arm toward the forearm. While holding the arm compressed with both hands, place a tourniquet above the elbow. Milk the blood from the fingers to the forearm for 3–5 min. When a vein becomes prominent, wrap your hand around the patient’s wrist and place the IV. • If no extremity vein can be found, try the external jugular. Placing the patient in the head down position can help distend the vein. • If all these fail, the next alternative is a central venous line insertion. LUMBAR PUNCTURE Indications • Diagnostic purposes: Analysis of CSF for conditions such as meningitis, encephali- tis, Guillain-Barré syndrome, staging work-up for lymphoma, others • Measurement of CSF pressure or its changes with various maneuvers (Valsalva, etc) • Injection of various agents: Contrast media for myelography, antitumor drugs, analgesics, antibiotics 13 Bedside Procedures 281 Angiocath Vein Vein Needle 13 Catheter Needle Catheter Vein FIGURE 13–13 To insert a catheter-over-needle assembly into a vein, stabilize the skin and vein with gentle traction. Enter the vein and advance the catheter while re- moving the needle. 282 Clinician’s Pocket Reference, 9th Edition A B FIGURE 13–14 Example of a “butterfly” needle assembly and the two different techniques of entering a vein for intravenous access: A direct puncture; and B side entry. (Reprinted, with permission, from: Gomella TL [ed]: Neonatology: Basic Man- agement, On-Call Problems, Diseases, Drugs, 4th ed. Appleton & Lange, Norwalk CT, 1998.) Contraindications 13 • Increased intracranial pressure (papilledema, mass lesion) • Infection near the puncture site • Planned myelography or pneumoencephalography • Coagulation disorders Materials • A sterile, disposable LP kit or • Minor procedure tray (see page 240) • Spinal needles (21-gauge for adults, 22-gauge for children) Background The objective of an LP is to obtain a sample of CSF from the subarachnoid space. Specifi- cally, during an LP the fluid is obtained from the lumbar cistern, the volume of CSF lo- cated between the termination of the spinal cord (the conus medullaris) and the termination of the dura mater at the coccygeal ligament. The cistern is surrounded by the subarachnoid membrane and the overlying dura. Located within the cistern are the filum terminale and the nerve roots of the cauda equina. When an LP is done, the main body of the spinal cord is avoided and the nerve roots of the cauda are simply pushed out of the way by the needle. The termination of the spinal cord in the adult is usually between L1 and L2, and in the pediatric patient between L2 and L3. The safest site for an LP is the interspace between L4 13 Bedside Procedures 283 and L5. An imaginary line drawn between the iliac crests (the supracristal plane) intersects the spine at either the L4 spinous process or the L4–L5 interspace. A spinal needle introduced between the spinous processes of L4 and L5 penetrates the layers in the following order: skin, supraspinous ligament, interspinous ligament, ligamen- tum flava, epidural space (contains loose areolar tissue, fat, and blood vessels), dura, “po- tential space,” subarachnoid membrane, subarachnoid space (lumbar cistern) (Fig. 13–15). Body Spinal Pedicle canal Transverse process Lamina Spinous process Spinal canal Ligamentum 13 flavum Intervertebral Interspinal disk ligaments Spinous Vertebral process body Supraspinous ligament FIGURE 13–15 Basic anatomy for a lumbar puncture. 284 Clinician’s Pocket Reference, 9th Edition Technique 1. Examine the fundus for evidence of papilledema, and review the CT or MRF of the head if available. Discuss the relative safety and lack of discomfort to the patient to dis- pel any myths. Some clinicians prefer to call the procedure a “subarachnoid analysis” rather than a spinal tap. As long as the procedure and the risks are outlined, most pa- tients will agree to the procedure. Have the patient sign an informed consent form. 2. Place the patient in the lateral decubitus position close to the edge of the bed or table. The patient (held by an assistant, if possible) should be positioned with knees pulled up toward stomach and head flexed onto chest (Fig. 13–16). This position enhances flex- ion of the vertebral spine and widens the interspaces between the spinous processes. Place a pillow beneath the patient’s side to prevent sagging and ensure alignment of the spinal column. In an obese patient or a patient with arthritis or scoliosis, the sitting po- sition, leaning forward, may be preferred. 3. Palpate the supracristal plane (see under Background) and carefully determine the loca- tion of the L4–L5 interspace. 4. Open the kit, put on sterile gloves, and prep the area with povidone–iodine solution in a circular fashion and covering several interspaces. Next, drape the patient. 5. With a 25-gauge needle and 1idocaine, raise a skin wheal over the L4–L5 interspace. Anesthetize the deeper structures with a 22-gauge needle. 6. Examine the spinal needle with a stylet for defects and then insert it into the skin wheal and into the spinous ligament. Hold the needle between your index and middle fingers, with your thumb holding the stylet in place. Direct the needle cephalad at a 30–45-de- gree angle, in the midline and parallel to the bed (see Fig. 13–16). 7. Advance through the major structures and pop into the subarachnoid space through the dura. An experienced operator can feel these layers, but an inexperienced one may need to periodically remove the stylet to look for return of fluid. It is important to always re- place the stylet prior to advancing the spinal needle. The needle may be withdrawn, 13 however, with the stylet removed. This technique may be useful if the needle has passed through the back wall of the canal. Direct the bevel of the needle parallel to the long axis of the body so that the dural fibers are separated rather than sheared. This method helps cut down on “spinal headaches.” 8. If no fluid returns, it is sometimes helpful to rotate the needle slightly. If still no fluid appears, and you think that you are within the subarachnoid space, inject 1 mL of air because it is not uncommon for a piece of tissue to clog the needle. Never inject saline or distilled water. If no air returns and if spinal fluid cannot be aspirated, the bevel of the needle probably lies in the epidural space; advance it with the stylet in place. 9. When fluid returns, attach a manometer and stopcock and measure the pressure. Nor- mal opening pressure is 70–180 mm water in the lateral position. Increased pressure may be due to a tense patient, CHF, ascites, subarachnoid hemorrhage, infection, or a space-occupying lesion. Decreased pressure may be due to needle position or ob- structed flow (you may need to leave the needle in for a myelogram because if it is moved, the subarachnoid space may be lost). 10. Collect 0.5–2.0-mL samples in serial, labeled containers. Send them to the lab in this order: • First tube for bacteriology: Gram’s stain, routine C&S, AFB, and fungal cultures and stains • Second tube for glucose and protein: If a work-up for MS, order electrophoresis to detect oligoclonal banding and assay for myelin basic protein characteristic of MS • Third tube for cell count: CBC with differential 13 Bedside Procedures 285 S1 L4 L5 L4 13 L4 Subarachnoid space L5 Cauda equina FIGURE 13–16 When performing a lumbar puncture, place the patient in the lat- eral decubitus position, and locate the L4–L5 interspace. Control the spinal needle with two hands, and enter the subarachnoid space. 286 Clinician’s Pocket Reference, 9th Edition • Fourth tube for special studies as clinically indicated: VDRL neurosyphilis CIEP (counterimmunoelectrophoresis) for bacterial antigens such as H. influenzae, S. Pneu- moniae, N. meningitidis) PCR assay for tuberculous meningitis or herpes simplex encephalitis (allows rapid diagno- sis) If Cryptococcus neoformans is suspected (most common cause of meningitis in AIDS pa- tients) India ink preparation and cryptococcal antigen (latex agglutination test) Note: Some clinicians prefer to send the first and last tubes for CBC because this procedure permits a better differentiation between a subarachnoid hemorrhage and a traumatic tap. In a traumatic tap, the number of RBCs in the first tube should be much higher than in the last tube. In a subarachnoid hemorrhage, the cell counts should be equal, and xanthochromia of the fluid should be present, indicating the presence of old blood. 11. Withdraw the needle and place a dry, sterile dressing over the site. 12. Instruct the patient to remain recumbent for 6–12 h, and encourage an increased fluid intake to help prevent “spinal headaches.” 13. Interpret the results based on Table 13–4. Complications • Spinal headache: The most common complication (about 20%), this appears within the first 24 h after the puncture. It goes away when the patient is lying down and is aggravated when the patient sits up. It is usually characterized by a severe throbbing pain in the occipital region and can last a week. It is thought to be caused by in- tracranial traction caused by the acute volume depletion of CSF and by persistent leakage from the puncture site. To help prevent spinal headaches, keep the patient re- cumbent for 6–12 h, encourage the intake of fluids, use the smallest needle possible, and keep the bevel of the needle parallel to the long axis of the body to help prevent 13 a persistent CSF leak. • Trauma to nerve roots or to the conus medullaris: Much less frequent (some anatomic variation does exist, but it is very rare for the cord to end below L3). If the patient suddenly complains of paresthesia (numbness or shooting pains in the legs), stop the procedure. • Herniation of either the cerebellum or the medulla: Occurs rarely, during or after a spinal tap, usually in a patient with increased intracranial pressure. This complica- tion can often be reversed medically if it is recognized early. • Meningitis. • Bleeding in the subarachnoid/subdural space can occur with resulting paralysis es- pecially if the patient is receiving anticoagulants or has severe liver disease with a coagulopathy. ORTHOSTATIC BLOOD PRESSURE MEASUREMENT Indication • Assessment of volume depletion Materials • Blood pressure cuff and stethoscope 13 287 TABLE 13–4 Differential Diagnosis of Cerebrospinal Fluid Opening Protein Glucose Pressure (mg/ (mg/ Cells Condition Color (mm H2O) 100 mL) 100 mL) (#/mm3) NORMAL Adult Clear 70–180 15–45 45–80 0–5 lymphocytes Newborn Clear 70–180 20–120 2/3 serum 40–60 lymphocytes INFECTIOUS Viral infection Clear or Normal or Normal or Normal 10–500 (“aseptic meningitis”) opalescent slightly slightly lymphocytes increased increased PMNs Bacterial Opalescent Increased 50–10,000 Increased, 25–10,000 infection yellow, may usually 20 PMNs clot Granulomatous Clear or Often Increased, Decreased, 10–500 infection opalescent increased but usually usually lymphocytes (TB, fungal) 500 20–40 NEUROLOGIC Guillain–Barré Clear or Normal Markedly Normal Normal or Syndrome Cloudy increased increased lymphocytes (continued ) 13 288 TABLE 13–4 (Continued) Opening Protein Glucose Pressure (mg/100 (mg/100 Cells Condition Color (mm H2O) mL) mL) (#/mm3) Multiple sclerosis Clear Normal Normal or Normal 0–20 lymphocytes increased Pseudotumor cerebri Clear Increased Normal Normal Normal MISCELLANEOUS Neoplasm Clear or Increased Normal or Normal or Normal or xanthochromic

increased decreased increased lymphocytes Traumatic tap Bloody, no Normal Normal SI increased RBC = peripheral xanthochromia blood; Less RBC in tube 4 than in tube 1 Subarachnoid Bloody or Usually Increased Normal WBC/RBC hemorrhage xanthochromic increased ratio same after 2–8 h as blood Abbreviations: WBC = white blood cell; RBC = red blood cell; PMNs = polymorphonuclear neutrophils. 13 Bedside Procedures 289 Procedure 1. Changes in blood pressure and pulse when a patient moves from supine to the upright position are very sensitive guides for detecting early volume depletion. Even before a person becomes overtly tachycardic or hypotensive because of volume loss, the demon- stration of orthostatic hypotension aids in the diagnosis. 2. Have the patient assume a supine position for 5–10 min. Determine the BP and pulse. 3. Then have the patient stand up. If the patient is unable to stand, have the patient sit at the bedside with legs dangling. 4. After about 1 min, determine the BP and pulse again. 5. A drop in systolic BP greater than 10 mm Hg or an increase in pulse rate greater than 20 (16 if elderly) suggests volume depletion. A change in heart rate is more sensitive and occurs with a lesser degree of volume depletion. Other causes include peripheral vascular disease, surgical sympathectomy, diabetes, and medications (prazosin, hy- dralazine, or reserpine). PELVIC EXAMINATION Indications • Part of a complete physical examination in the female • Used to assist in the diagnosis of diseases and conditions of the female genital tract Materials • Gloves • Vaginal speculum and lubricant • Slides, fixative (Pap aerosol spray, etc), cotton swabs, endocervical brush and cervi- cal spatula prepared for a Pap smear • Materials for other diagnostic tests: Culture media to test for gonorrhea, Chlamy- 13 dia, herpes; sterile cotton swabs, plain glass slides, KOH, and normal saline solu- tions, as needed Procedure 1. The pelvic exam should be carried out in a comfortable fashion for both the patient and physician. A female assistant must be present for the procedure. The patient should be draped appropriately with her feet placed in the stirrups on the examining table. Pre- pare a low stool, a good light source, and all needed supplies before the exam begins. In unusual situations examinations are conducted on a stretcher or bed; raise the pa- tients buttocks on one or two pillows to elevate the perineum off the mattress. 2. Inform the patient of each move in advance. Glove hands before proceeding. 3. General inspection: a. Observe the skin of the perineum for swelling, ulcers, condylomata (venereal warts), or color changes. b. Separate the labia to examine the clitoris and vestibule. Multiple clear vesicles on an erythematous base on the labia suggest herpes. c. Observe the urethral meatus for developmental abnormalities, discharge, neo- plasm, and abscess of Bartholin’s gland at the 4 or 8 o’clock positions. d. Inspect the vaginal orifice for discharge, or protrusion of the walls (cystocele, rec- tocele, urethral prolapse). e. Note the condition of the hymen. 290 Clinician’s Pocket Reference, 9th Edition 4. Speculum examination: a. Use a speculum moistened with warm water not with lubricant (lubricant will in- terfere with Pap tests and slide studies). Check the temperature on the patient’s leg to see if the speculum is comfortable. b. Because the anterior wall of the vagina is close to the urethra and bladder, do not exert pressure in this area. Pressure should be placed on the posterior surface of the vagina. With the speculum directed at a 45-degree angle to the floor, spread the labia and insert the speculum fully, pressing posteriorly. The cervix should pop into view with some manipulation as the speculum is opened. c. Inspect the cervix and vagina for color, lacerations, growths, nabothian cysts, and evidence of atrophy. d. Inspect the cervical os for size, shape, color, discharge. e. Inspect the vagina for secretions and obtain specimens for a Pap smear, other smear, or culture (see tests for vaginal infections and Pap smear in item 7). f. Inspect the vaginal wall; rotate the speculum as you draw it out to see the entire canal. 5. Bimanual examination: a. For this part, stand up. It is best to use whichever hand is comfortable to do the in- ternal vaginal exam. Remove the glove from the hand that will examine the ab- domen. b. Place lubricant on the first and second gloved fingers, and then, keeping pressure on the posterior fornix, introduce them into the vagina. c. Palpate the tissue at 5 and 7 o’clock between the first and second fingers and the thumb to rule out any abnormality of Bartholin’s gland. Likewise, palpate the ure- thra and paraurethral (Skene’s) gland. d. Place the examining fingers on the posterior wall of the vagina to further open the introitus. Ask the patient to bear down. Look for evidence of prolapse, rectocele, 13 or cystocele. e. Palpate the cervix. Note the size, shape, consistency, and motility, and test for ten- derness (the so-called chandelier sign or marked cervical tenderness, which is positive in PID). f. With your fingers in the vagina posterior to the cervix and your hand on the ab- domen placed just above the symphysis, force the corpus of the uterus between the two examining hands. Note size, shape, consistency, position, and motility. g. Move the fingers in the vagina to one or the other fornix, and place the hand on the abdomen in a more lateral position to bring the adnexal areas under examination. Palpate the ovaries, if possible, for any masses, consistency, and motility. Unless the fallopian tubes are diseased, they usually are not palpable. 6. Rectovaginal examination: a. Insert your index finger into the vagina, and place the well-lubricated middle fin- ger in the rectum. b. Palpate the posterior surface of the uterus and the broad ligament for nodularity, tenderness, or other masses. Examine the uterosacral and rectovaginal septum. Nodularity here may represent endometriosis. c. It may also be helpful to do a test for occult blood if a stool specimen is available. 7. Papanicolaou (Pap) smear: The Pap smear is helpful in the early detection of cervical intraepithelial neoplasia and carcinoma. Endometrial carcinoma is occasionally identified on routine Pap smears. It is recommended that low-risk patients have routine Pap smears done every 2–3 y, but only after three annual Pap smears are negative. High-risk patients such as those exposed to in utero DES, patients with HPV infections, history of cervical dysplasia or cervical intraep- 13 Bedside Procedures 291 ithelial neoplasia, more than two sexual partners in the patient’s lifetime, and intercourse prior to age 20 should obtain an annual Pap smear. a. With the unlubricated speculum in place, use a wooden cervical spatula to obtain a scraping from the squamocolumnar junction. Rotate the spatula 360 degrees around the external os. Smear on a frosted slide that has the patient’s name written on it in pencil. Fix the slide either in a bottle of fixative or with commercially available spray fixative. The slide must be fixed within 10 s or a drying artifact may occur. b. Next, obtain a specimen from the endocervical canal using a cotton swab or com- mercial available endocervical brush and prepare the slide as described in part a. c. Using a wooden spatula, an additional specimen should be obtained from the pos- terior/lateral vaginal pool of fluid and smeared on a slide. d. Complete the appropriate lab slips. Forewarn the patient that she may experience some spotty vaginal bleeding following the Pap smear. 8. Tests for cervical/vaginal infections: a. GC culture: Use a sterile cotton swab to obtain a specimen from the endocervical canal and plate it out on Thayer–Martin medium. b. Vaginal saline (wet) prep: Helpful in the diagnosis of Trichomonas vaginalis or Gardnerella vaginalis. A thin, foamy, white, pruritic discharge is associated with a Trichomonas infection. Mix a drop of discharge with a drop of NS on a glass slide and cover the drop with a coverslip. It is important to observe the slide while it is still warm to see the flagellated, motile trichomonads. If a patient has a thin, watery, gray, malodorous discharge, an infection with Gardnerella vaginalis may be present. Bacterial vaginosis is most often caused by G. vaginalis and can be di- agnosed by the presence of “clue cells,” which represent polymorphonuclear white cells dotted with the G. vaginalis bacteria, a vaginal pH of > 4.5 and a fishy amine odor with addition of KOH to the secretions. Alternatively, these can be seen by using a hanging drop of saline and a concave slide. Lactobacillus is normally the predominant bacteria in the vagina in the absence of specific infection and the nor- 13 mal pH is usually < 4.5. c. Potassium hydroxide prep: If a thick, white, curdy discharge is present, the pa- tient may have a Candida albicans (monilial) yeast infection. Prepare a slide with one drop of discharge and one drop of aqueous 10% KOH solution. The KOH dis- solves the epithelial cells and debris and facilitates viewing of the hyphae and mycelia of the fungus that causes the infection. d. Gram’s stain: Material can easily be stained in the usual fashion (Chapter 7, page 122). Gram-negative intracellular diplococci (so-called GNIDs) are pathogno- monic of Neisseria gonorrhoeae. The most commonly found bacteria in Gram’s stains are large gram-positive rods (lactobacilli), which are normal vaginal flora. e. Herpes cultures: A routine Pap smear of the cervix or a Pap smear of the herpetic lesion (multiple, clear vesicles on a painful, erythematous base) may demonstrate herpes inclusion bodies. A herpes culture may be done by taking a viral culture swab of the suspicious lesion or of the endocervix. f. Chlamydia cultures: Special swabs can be obtained from the microbiology lab for Chlamydia cultures. PERICARDIOCENTESIS Indications • Emergency treatment of cardiac tamponade • Diagnose the cause of pericardial effusion 292 Clinician’s Pocket Reference, 9th Edition Contraindications • Minimal pericardial effusion (< 200 mL) • After CABG due to risk of injury to grafts • Uncorrected coagulopathy Materials • Electrocardiogram machine • Prepackaged pericardiocentesis kit or Procedure and instrument tray (page 240) with pericardiocentesis needle or 16–18-gauge needle 10 cm long Background Cardiac tamponade results in decreased cardiac output, increased right atrial filling pres- sures, and a pronounced pulsus paradoxus. Procedure 1. If time permits, use sterile prep and draping with gown, mask, and gloves. 2. Draining the pericardium can be approached either through the left para xiphoid or the left parasternal fourth intercostal space. The para xiphoid is safer, more commonly used, and described here (Fig. 13–17). 3. Anesthetize the insertion site with lidocaine. Connect the needle with an alligator clip to lead V on the ECG machine. Attach the limb leads, and monitor the machine. 4. Insert the pericardiocentesis needle just to the left of the xiphoid and directed upward 45 degrees toward the left shoulder. 5. Aspirate while advancing the needle until the pericardium is punctured and the effusion is tapped. If the ventricular wall is felt, withdraw the needle slightly. Additionally, if the 13 needle contacts the myocardium, pronounced ST segment elevation will be noted on the ECG. 6. If performed for cardiac tamponade, removal of as little as 50 mL of fluid dramatically improves blood pressure and decreases right atrial pressure. 7. Blood from a bloody pericardial effusion is usually defibrinated and will not clot, whereas blood from the ventricle will clot. 8. Send fluid for hematocrit, cell count, or cytology if indicated. Serous fluid is consistent with CHF, bacterial infection, TB, hypoalbuminemia, or viral pericarditis. Bloody fluid (HCT >10%) may result from trauma; be iatrogenic; or due to MI, uremia, coagulopa- thy, or malignancy (lymphoma, leukemia, breast, lung most common) 9. If continuous drainage is necessary, use a guidewire to place a 16-gauge intravenous catheter. Complications Arrhythmia, ventricular puncture, lung injury PERIPHERALLY INSERTED CENTRAL CATHETER (PICC LINE) Indications • Home infusion of hypertonic or irrigating solutions and drugs • Long-term infusion of medications (antibiotics, chemotherapeutics) • TPN • Repetitive venous blood sampling 13 Bedside Procedures 293 Parasternal approach 1 2 3 4 5 6 To ECG, 7 V lead To ECG, V lead 13 Paraxyphoid approach FIGURE 13–17 Techniques for pericardiocentesis. The paraxiphoid approach is the most popular. (Reprinted, with permission, from: Stillman RM [ed]: Surgery, Diag- nosis, and Therapy, Appleton &

Lange, Norwalk CT, 1989.) Contraindications • Infection over placement site • Failure to identify veins in an arm with a tourniquet in place Materials • PICC catheter kit (contains most items necessary including the silastic long arm line) • Tourniquet, sterile gloves, mask, sterile gown, heparin flush, 10-mL syringes 294 Clinician’s Pocket Reference, 9th Edition Background Installation of a PICC allows for central venous access through a peripheral vein. Typically, a long-arm catheter is placed into the basilic or cephalic vein (See Fig. 13–12) and is threaded into the subclavian vein/superior vena cava. PICCs are useful for long-term home infusion therapies. The design of PICC catheters can vary, and the operator should be famil- iar with the features of the device (attached hub or detachable hub designs). Procedure 1. Explain the procedure to the patient and then obtain informed consent. Position the pa- tient in a sitting or reclining position with the elbow extended and the arm in a depen- dent position. The arm should be externally rotated. 2. Using a measuring tape, determine the length of the catheter required. Measure from the extremity vein insertion site to the subclavian vein. 3. Wear mask, gown, protective eyewear, and sterile gloves. Prep and drape the skin in the standard fashion. Set up an adjacent sterile working area. 4. Anesthetize the skin at the proposed area of insertion. Apply a tourniquet above the proposed IV site. 5. Trim the catheter to the appropriate length. Most PICC lines have an attached hub, and the distal end of the catheter is cut to the proper length. Flush with heparinized saline. 6. Insert the catheter and introducer needle (usually 14-gauge) into the chosen arm vein as detailed in the section on IV techniques (page 279). Once the catheter is in the vein, re- move the introducer needle. 7. Place the PICC line in the catheter and advance (use a forceps if provided by the manu- facturer of the kit to advance the PICC line). Remove the tourniquet and gradually ad- vance the catheter the requisite length. Remove the inner stiffening wire slowly once the catheter has been adequately advanced. 13 8. Peel away the introducer catheter. Attach the Luer-lock, and flush the catheter again with heparin solution. Attempt to also aspirate blood to verify patency. 9. Attach the provided securing wings, and suture in place. Apply a sterile dressing over the insertion site. 10. Confirm placement in the central circulation with a chest x-ray. Always document the type of PICC, the length inserted, and the site of its radiologically confirmed place- ment. 11. If vein cannulation is difficult, a surgical cutdown may be necessary to cannulate the vein. If the catheter will not advance, fluoroscopy may be helpful. 12. Instruct the patient on the maintenance of the PICC. The PICC should be flushed with heparinized saline after each use. Dressing changes should be performed at least every 7 d under sterile conditions. Patient must be instructed to evaluate the PICC site for signs and symptoms of infection. Patient must also be instructed to come to the emer- gency room for evaluation of any fevers. 13. For venous samples, a specimen of at least the catheter volume (1–3 mL) must first be withdrawn and then discarded. The PICC must always be flushed with heparinized saline after each blood draw. PICC Removal Position the patient’s arm at a 90-degree angle to his body. Remove the dressing and gently pull the PICC out. Apply pressure to site for 2–3 min. Always measure the length of the catheter and check prior documentation to ensure that the PICC line has been removed in its 13 Bedside Procedures 295 entirety. If a piece of a catheter is left behind, an emergency interventional radiology consult is in order. Complications Site bleeding, clotted catheter, subclavian thrombosis, infection, broken catheter (leakage or embolization), arrhythmia (catheter inserted too far) PERITONEAL LAVAGE Indications • Diagnostic peritoneal lavage (DPL) is used in the evaluation of intraabdominal trauma (bleeding, perforation) (Note: Spiral CT of the abdomen has largely replaced this as an initial screening for intraabdominal trauma in the emergency setting.) • Acute peritoneal dialysis and the treatment of severe pancreatitis Contraindications • None are absolute. Relative contraindications include multiple abdominal proce- dures, pregnancy, known retroperitoneal injury (high false-positive rates) cirrhosis, morbid obesity and any coagulopathy. Materials • Prepackaged diagnostic peritoneal lavage or peritoneal dialysis tray Procedure 1. A Foley catheter and a nasogastric or oro gastric tube must be in place. Prep the ab- domen from above the umbilicus to the pubis. 13 2. The site of choice is in the midline 1–2 cm below the umbilicus. Avoid the site of old surgical scars (danger of adherent bowel). If a subumbilical scar or pelvic fracture is present, a supraumbilical approach is recommended. 3. Infiltrate the skin with 1idocaine with epinephrine. Incise the skin in the midline verti- cally, and expose the fascia. 4. Either pick up the fascia and incise it, or puncture it with the trocar and peritoneal catheter. Caution is needed to avoid puncturing any organs. Use one hand to hold the catheter near the skin and to control the insertion while using the other hand to apply pressure to the end of the catheter. After entering the peritoneal cavity, remove the tro- car and direct the catheter inferiorly into the pelvis. 5. During a diagnostic lavage, gross blood indicates a positive tap. If no blood is encoun- tered, instill 10 mL/kg (about 1 L in adults) of lactated Ringer’s solution or NS into the abdominal cavity. 6. Gently agitate the abdomen to distribute the fluid and after 5 min, drain off as much fluid as possible into a bag on the floor. (Minimum fluid for a valid analysis is 200 mL in an adult.) If the drainage is slow, try instilling additional fluid, carefully reposition- ing the catheter. 7. Send the fluid for analysis (amylase, bile, bacteria, hematocrit, cell count). See Table 13–5 for interpretation. 8. Remove the catheter and suture the skin. If the catheter is inserted for pancreatitis or peritoneal dialysis, suture it in place. 296 Clinician’s Pocket Reference, 9th Edition TABLE 13–5 Criteria for Evaluation of Peritoneal Lavage Fluid Positive >20 mL gross blood on free aspiration (10 mL in children) ≥100,000 RBC/mL ≥500 WBC/mL (if obtained >3 h after the injury) ≥175 units amylase/dL Bacteria on Gram’s stain Bile (by inspection or chemical determination of bilirubin content) Food particles (microscopic analysis of strained or spun specimen) Intermediate Pink fluid on free aspiration 50,000–100,000 RBC/mL in blunt trauma 100–500 WBC/mL 75–175 units amylase/dL Negative Clear aspirate ≤ 100 WBC/mL ≤ 75 units amylase/dL Source: Reprinted, with permission, from: Way L (ed): Current Surgical Diagnosis and Treatment, 10e. Appleton and Lange, Norwalk CT. 1994. Abbreviations: RBC = red blood cells; WBC = white blood cells. 13 9. A negative DPL does not rule out retroperitoneal trauma. A false-positive DPL can be caused by a pelvic fracture or bleeding induced by the procedure (eg, laceration of an omental vessel). Complications Infection/peritonitis or superficial wound infection, bleeding, perforated viscus (bladder, bowel) PERITONEAL (ABDOMINAL) PARACENTESIS Indications • To determine the cause of ascites • To determine if intraabdominal bleeding is present or if a viscus has ruptured (Diag- nostic peritoneal lavage is considered a more accurate test. See preceding proce- dure.) • Therapeutic removal of fluid when distention is pronounced or respiratory distress is associated with it (acute treatment only) Contraindications • Abnormal coagulation factors • Bowel obstruction, pregnancy 13 Bedside Procedures 297 • Uncertainty if distention is due to peritoneal fluid or to a cystic structure (ultrasound can usually differentiate) Materials • Minor procedure tray (see page 240) • Catheter-over-needle assembly (Angiocath, Insyte 18–20-gauge with a 1¹₂-in. needle) • 20–60-mL syringe • Sterile specimen containers Procedure Peritoneal paracentesis is surgical puncture of the peritoneal cavity for the aspiration of fluid. Ascites is indicated by abdominal distention, shifting dullness, and a palpable fluid wave. 1. Explain the procedure and have the patient sign an informed consent form. Have the patient empty the bladder, or place a Foley catheter if voiding is impossible or if signif- icant mental status changes are present. 2. The entry site is usually the midline 3–4 cm below the umbilicus. Avoid old surgical scars because the bowel may be adhering to the abdominal wall. Alternatively, the entry site can be in the left or right lower quadrant midway between the umbilicus and the anterior superior iliac spine or in the patient’s flank, depending on the percussion of the fluid wave (Fig. 13–18). 3. Prep and drape the patient appropriately. Raise a skin wheal with the lidocaine over the proposed entry site. 4. With the catheter mounted on the syringe, go through the anesthetized area carefully at an oblique angle while gently aspirating. You will meet some resistance as you enter 13 the fascia. When you get free return of fluid, leave the catheter in place, remove the needle, and begin to aspirate. Sometimes it is necessary to reposition the catheter be- cause of abutting bowel. 5. Aspirate the amount of fluid needed for tests (20–30 mL). If the tap is therapeu- tic, 10–15 L can be safely removed. This large volume must be removed relatively slowly. 6. Quickly remove the needle, apply a sterile 4 × 4 gauze square, and apply pressure with tape. 7. Depending on the clinical picture of the patient, send samples for total protein, specific gravity, LDH, amylase, cytology, culture, stains, or CBC. Complications Peritonitis, perforated viscus, hemorrhage, precipitation of hepatic coma if patient has se- vere liver disease, oliguria, hypotension Diagnosis of Ascitic Fluid A complete listing is found in Chapter 3, page 43. Transudative ascites is found with cir- rhosis, nephrosis, and CHF. Exudative ascites is found with tumors, peritonitis (TB, perfo- rated viscus), hypoalbuminemia. See Table 13–6 to interpret the results of fluid analysis. 298 Clinician’s Pocket Reference, 9th Edition 13 FIGURE 13–18 Preferred sites for abdominal (peritoneal) paracentesis. Be sure to avoid old surgical scars. (Reprinted, with permission, from: Krupp MA [ed]: The Physician’s Handbook, 21st ed. Lange Medical Publications, Los Altos CA, 1985.) PULMONARY ARTERY CATHETERIZATION (See Chapter 20, page 399) PULSUS PARADOXUS MEASUREMENT (See also Chapter 20, page 393) Indication • Used in the evaluation of cardiac tamponade and other diseases Materials • Blood pressure cuff and stethoscope 13 Bedside Procedures 299 TABLE 13–6 Differential Diagnosis of Ascitic of Pleural Fluid Lab Value Transudate Exudate Appearance Clear yellow Clear or turbid Specific gravity 1.016 1.016 Absolute protein 3 g/100 mL 3 g/100 mL Protein (ascitic or pleural to 0.5 0.5 serum ratio) LDH (ascitic or pleural to 0.6 0.6 serum ratio) Absolute LDH 200 IU 200 IU Glucose (serum to ascitic or 1 1 pleural ratio) Fibrinogen (clot) No Yes WBC (ascitic) 500/mm3 1000/mm3 WBC (pleural) Very low 2500/mm3 Differential (pleural) PMNs early, monocytes later RBC (ascitic) 100 RBC/mm3 OTHER SELECTED TESTS Cytology: Bizarre cells with large nuclei may represent reactive mesothelial cells and not a malignancy. Malignant cells suggest a tumor. pH (pleural): Generally 7.3. If between 7.2 and 7.3, suspect TB or ma- lignancy or both. If 7.2, suspect empyema. 13 Glucose (pleural): Normal pleural fluid glucose is ²₃ serum glucose. Pleural fluid glucose is much lower than serum glucose in effusions due to rheumatoid arthritis (0–16 mg/100 mL); low 40 mg/100 mL in empyema. Triglycerides and positive Sudan stain (pleural fluid): Chylothorax. Food fibers (ascitic): Perforated viscus. Abbreviations: LDH = lactate dehydrogenase; WBC = white blood cells; RBC = red blood cells; PMNs = polymorphonuclear neutrophils; TB = tuberculosis. Background Pulsus paradoxus is an exaggeration of the normal inspiratory drop in arterial pressure. Inspi- ration decreases intrathoracic pressure. The result is increased right atrial and right ventricu- lar filling with an increase in right ventricular output. Because the pulmonary vascular bed also distends, these changes lead to a delay in left ventricular filling and subsequently a de- creased left ventricular output. This drop in systolic blood pressure is usually <10 mm Hg. In the case of cardiac compression (eg, acute asthma or pericardial tamponade), the right side of the heart fills more with inspiration and decreases the left ventricular volume to even greater degree as a result of compression of the pericardial sac. This exaggerated 300 Clinician’s Pocket Reference, 9th Edition decrease in left ventricular output

drops the systolic pressure >10 mm Hg. See Figure 20–1 (page 394) for a graphic representation of a paradoxical pulse. Procedure 1. A simple, qualitative method involves palpating the radial pulse, which “disappears” on normal inspiration. 2. A more precise quantitative method requiring that the patient take a breath, let it out, and hold it. Determine the systolic BP. 3. Ask the patient to breathe again. Once the patient is breathing normally, drop the pres- sure in the cuff slowly until you hear the pulse during inspiration. 4. The difference in systolic pressure should be <10 mm Hg. If not, a so-called paradox exists. 5. Differential diagnosis includes pericardial effusion, cardiac tamponade, pericarditis, COPD, bronchial asthma, restrictive cardiomyopathies, hemorrhagic shock SIGMOIDOSCOPY (RIGID) Indications • Diagnosis and treatment of lower gastrointestinal problems • Part of the standard work-up of blood in the stool Materials • Examination gloves, lubricant, tissues • Occult blood stool test kit (Hemoccult paper and developer) • Sigmoidoscope with obturator and light source 13 • Insufflation bag • Long (rectal) swabs and suction catheter • Proctologic examination table (helpful but not essential) Procedure 1. Several techniques can be used to examine the distal large bowel. These include rigid sigmoidoscopy (endoscopic examination of the last 25 cm of the GI tract), flexible sig- moidoscopy (examination up to 40 cm from the end of the GI tract), proctoscopy (roughly synonymous to sigmoidoscopy, but technically means examination of the last 12 cm), and anoscopy (examination of the anus and most distal rectum). 2. Enemas and cathartics are not routinely given before sigmoidoscopy, although some people prefer to give a mild prep such as a Fleet’s enema just before the exam. Explain the procedure, and have the patient sign a consent form. 3. Sigmoidoscopy can be performed with the patient in bed lying on side in the knee–chest position, but the best results are obtained with the patient in the “jackknife” position on the procto table. Do not position the patient until all materials are at hand and you are ready to start. 4. Converse with the patient to create distraction and to relieve apprehension. Announce each maneuver in advance. Glove before proceeding. 5. Observe the anal region for skin tags, hemorrhoids, fissures, and so on. Do a careful rectal exam with a gloved finger and plenty of lubricant, and check for fecal occult blood (Hemoccult test) on the stool recovered on the glove. 13 Bedside Procedures 301 6. Lubricate the sigmoidoscope well with water-soluble jelly, and insert it with the obtura- tor in place. Aim toward the patient’s umbilicus initially. Advance 2–3 cm past the in- ternal sphincter, and remove the obturator. 7. Always advance under direct vision and make sure that the lumen is always visible (Fig. 13–19). Insufflation (introducing air) may be used to help visualize the lumen, but remember this may be painful. It is necessary to follow the curve of the sigmoid to- ward the sacrum by directing the scope more posteriorly toward the back. A change from a smooth mucosa to concentric rings signifies entry into the sigmoid colon. The scope should reach 15 cm with ease. Use suction and the rectal swabs as needed to clear the way. 8. At this point, the sigmoid curves to the patient’s left. Warn the patient that he or she may feel a cramping sensation. If you ever have difficulty negotiating a curve, do not force the scope. 9. After advancing as far as possible, slowly remove the scope; use a small rotary motion to view all surfaces. Observation here is critical. Remember to release the air from the colon before withdrawing the scope. 10. Inform the patient that he or she may experience mild cramping after the procedure. Sacrum Sigmoid colon Rectum A 13 B Umbilicus FIGURE 13–19 The sigmoidoscope is advanced under direct vision as shown. 302 Clinician’s Pocket Reference, 9th Edition Complications • Bleeding, bowel perforation (rare) SKIN BIOPSY Indications • Any skin lesion or eruption for which the diagnosis is unclear • Any refractory skin condition Contraindications • Any skin lesion that is suspected to be a malignancy (eg, melanoma) should be referred to a plastic surgeon or dermatologist for excisional biopsy rather than a punch biopsy. Materials • 2-, 3-, 4-, or 5-mm skin punch • Minor procedure tray (page 240) • Curved iris scissors and fine-toothed forceps (Ordinary forceps may distort a small biopsy specimen and should not be used.) • Specimen bottle containing 10% formalin • Suturing materials (3-0 or 4-0 nylon) Procedure 1. If more than one lesion is present, choose one that is well developed and representative of the dermatosis. For patients with vesiculobullous disease, an early edematous lesion should be chosen rather than a vesicle. Avoid lesions that are excoriated or infected. 13 2. Mark the area to be biopsied with a skin-marking pen. Inject the lidocaine to form a skin wheal over the site of the biopsy. 3. After putting on sterile gloves and preparing a sterile field, take the punch biopsy speci- men. First, immobilize the skin with the fingers of one hand, applying pressure perpen- dicular to the skin wrinkle lines with the skin punch. Core out a cylinder of skin by twirling the punch between the fingers of the other hand. As the punch enters into the subcutaneous fat, resistance will lessen. At this point, the punch should be removed. The core of tissue usually pops up slightly and can be cut at the level of the subcuta- neous fat with curved iris scissors without using forceps. If a tissue core does not pop up, it may be elevated by use of a hypodermic needle or fine-toothed forceps. Be sure to include a portion of the subcutaneous fat in the specimen. 4. Place the specimen in the specimen container. 5. Hemostasis can be achieved by pressure with the gauze pad. 6. Defects from 1.5 and 2-mm punches usually do not require suturing and heal with very minimal scarring. Punch defects that are 2–4 mm can generally be closed with a single suture. 7. A dry dressing should be applied and removed the following day. 8. Sutures can be removed as early as 3 d from the face and 7–10 d from other areas. Complications Infection (unusual); hemorrhage (usually controlled by simple application of pressure); keloid formation, especially in a patient with a prior history of keloid formation 13 Bedside Procedures 303 SKIN TESTING Indications • Screening for current or past infectious agent (TB, coccidioidomycosis, etc) • Screening for immune competency (so-called anergy screen) in debilitated patients Materials • Appropriate antigen (usually 0.1 mL)(eg, 5 TU PPD) • A small, short needle (25-, 26-, or 27-gauge) • 1-mL syringe • Alcohol swab Procedure 1. Skin tests for delayed type hypersensitivity (type IV, tuberculin) are the most com- monly administered and interpreted. Delayed hypersensitivity (so called because a lag time of 12–36 h is required for a reaction) is caused by the activation of sensitized lym- phocytes after contact with an antigen. The inflammatory reaction results from direct cytotoxicity and the release of lymphokines. Allergy tests (immediate wheal and flare) are rarely performed by the student or house officer. 2. The most commonly used site is the flexor surface of the forearm, approximately 4 in. below the elbow crease. 3. Prep the area with alcohol. With the bevel of the 27-gauge needle up, introduce the nee- dle into the upper layers of skin, but not into the subcutis. Inject 0.1 mL of antigen such as the PPD. The goal is to inject the antigen intradermally. If done properly, you will raise a discrete white bleb, approximately 10 mm in diameter (known as the Mantoux test). The bleb should disappear soon, and no dressing is needed. If a bleb is not raised, move to another area and repeat the injection. 13 4. Mark the test site with a pen, and if multiple tests are being administered, identify each one. Also, document the site in the patient’s chart. 5. To interpret the skin test, examine the injection site at 48–72 h. If nonreactive, check again at 72 h. Measure the area of induration (the firm raised area), not the erythe- matous area. Use a ballpoint pen held at approximately a 30-degree angle and bring it lightly toward the raised area. Where the pen touches is the area of induration. Measure two diameters and take the average. 6. It is important to check the PPD and other tests at intervals. If the patient develops a se- vere reaction to the skin test, apply hydrocortisone cream to prevent skin sloughing. Specific Skin Tests TST (Tuberculin Skin Testing): Routine TST in low-risk individuals is not currently recommended. High-risk individuals should undergo periodic TST (CXR findings suspi- cious for TB, recent contact of known or suspected TB cases, [includes health care work- ers], high-risk immigrants [Asia, Africa, Middle East, Latin America] , medically underserved (IV drug abusers, alcoholics, homeless), chronically institutionalized, HIV-in- fected or immunosuppression) The Mantoux test is the standard technique for TST and relies on the intradermal injec- tion of PPD. The tine test for TB is no longer recommended by the CDC. The PPD comes in three tuberculin unit “strengths”: 1 TU (“first”), 5 TU (“intermediate”), and 250 TU (“second”). 1 TU is used if the patient is expected to be hypersensitive (history of a positive 304 Clinician’s Pocket Reference, 9th Edition skin test); 5 TU is the standard initial screening test. A patient who has a negative response to a 5-TU test dose may react to the 250-TU solution. A patient who does not respond to the 250-TU is considered nonreactive to PPD. A patient may not react if he or she has not been exposed to the antigen or if the patient is anergic and unable to respond to any antigen chal- lenge. A positive TST indicates the presence of M. tuberculosis infection, either active or past (dormant) and an intact cell-mediated immunity. Interpretation of a positive PPD test is based on the clinical scenario. Patients who have been previously immunized with percutaneous BCG may give a false-positive PPD, usually 10 mm or less. • 0–5 mm induration: Negative response • ≥5 mm: Considered positive in contacts of known TB cases, CXR findings consis- tent with TB infection, HIV infection or in patients who are immunosuppressed, oc- casionally in non-TB mycobacterial infection due to cross reactivity • ≥10 mm induration: Considered positive in patients with chronic diseases (diabetics, alcoholics, IV drug abusers, other chronic diseases), homeless, immigrants from known TB regions, children <4 y • >15 mm induration: Positive in individuals who are healthy and otherwise do not meet the preceding risk categories Anergy Screen (Anergy Battery): An anergy screen is based on the assumption that a patient has been exposed in the past to certain common antigens and a healthy patient is able to mount a reaction to them. To perform the screen, antigens such as mumps, or Can- dida. These are generally applied and read just like the PPD test (a reaction of >5 mm in- duration is considered a positive test and indicates intact cellular immunity). Anergy screens are sometimes used to evaluate a patient’s immunological status and in the following spe- cific situations: If you suspect a patient is PPD-positive, and the patient does not react to the test, do an anergy screen along with the PPD test to see if the patient has any cellular 13 immune response. THORACENTESIS Indications • Determining the cause of a pleural effusion • Therapeutically removing pleural fluid in the event of respiratory distress • Aspirating small pneumothoraces where the risk of recurrence is small (ie, postoper- ative without lung injury) • Instilling sclerosing compounds (eg, tetracycline) to obliterate the pleural space Contraindications • None are absolute (pneumothorax, hemothorax, or any major respiratory impairment on the contralateral side, or coagulopathy) Materials • Prepackaged thoracentesis kit with either needle or catheter (preferred) or • Minor procedure tray (page 240) • 20–60 mL syringe, 20- or 22-gauge needle 1¹₂-in. needle, three-way stopcock • Specimen containers 13 Bedside Procedures 305 Procedure Thoracentesis is the surgical puncture of the chest wall to aspirate fluid or air from the pleural cavity. The area of pleural effusion is dull to percussion with decreased whisper or breath sounds. Pleural fluid causes blunting of

the costophrenic angles on chest x-ray. Blunting usually indicates that at least 300 mL of fluid is present. If you suspect that less than 300 mL of fluid is present or you suspect that the fluid is loculated (trapped and not free-flowing), a lateral decubitus film is helpful. Loculated effusions do not layer out. Tho- racentesis can be done safely on fluid visualized on lateral decubitus film if at least 10 mm of fluid is measurable on the decubitus x-ray. Ultrasound may also be used to localize a small or loculated effusion. 1. Explain the procedure, and have the patient sign an informed consent form. Have the patient sit up comfortably, preferably leaning forward slightly on a bedside tray table. Ask the patient to practice increasing intrathoracic pressure using the Valsalva maneu- ver or by humming. 2. The usual site for thoracentesis is the posterior lateral aspect of the back over the di- aphragm but under the fluid level. Confirm the site by counting the ribs based on the x- ray and percussing out the fluid level. Avoid going below the eighth intercostal space because the risk of peritoneal perforation is great. 3. Use sterile technique, including gloves, povidone–iodine prep, and drapes. Thoracente- sis kits come with an adherent drape with a hole in it. 4. Make a skin wheal over the proposed site with a 25-gauge needle and 1idocaine. Change to a 22-gauge, 1¹₂-in. needle and infiltrate up and over the rib (Fig. 13–20); try 13 Pleura Lung tissue Local 1 anesthetic 2 Rib Effusion Neurovascular bundle (nerve, artery, vein) FIGURE 13–20 When performing a thoracentesis, the needle is passed over the top of the rib to avoid the neurovascular bundle. 306 Clinician’s Pocket Reference, 9th Edition to anesthetize the deeper structures and the pleura. During this time, you should be as- pirating back for pleural fluid. Once fluid returns, note the depth of the needle and mark it with a hemostat. This gives you an approximate depth. Remove the needle. 5. Use a hemostat to measure the 14–18-gauge thoracentesis needle to the same depth as the first needle. Penetrate through the anesthetized area with the thoracentesis needle. Make sure that you “march” over the top of the rib to avoid the neurovascular bun- dle that runs below the rib (see Fig. 13–20). With the three-way stopcock attached, ad- vance the thoracentesis catheter through the needle, withdraw the needle from the chest, and place the protective needle cover over the end of the needle to prevent injury to the catheter. Next, aspirate the amount of pleural fluid needed. Turn the stopcock, and evacuate the fluid through the tubing. Never remove more than 1000–1500 mL per tap! This may result in hypotension or the development of pulmonary edema due to reexpansion of compressed alveoli. 6. Have the patient hum or do the Valsalva maneuver as you withdraw the catheter. This maneuver increases intrathoracic pressure and decreases the chances of a pneumotho- rax. Bandage the site. 7. Obtain a chest x-ray to evaluate the fluid level and to rule out a pneumothorax. An ex- piratory film may be best because it helps reveal a small pneumothorax. 8. Distribute specimens in containers, label slips, and send them to the lab. Always order pH, specific gravity, protein, LDH, cell count and differential, glucose, Gram’s stain and cultures, acid-fast cultures and smears, and fungal cultures and smears. Optional lab studies are cytology if you suspect a malignancy, amylase if you suspect an effusion secondary to pancreatitis (usually on the left) or esophageal perforation, and a Sudan stain and triglycerides (>110 mg/dL) if a chylothorax is suspected. Complications • Pneumothorax, hemothorax, infection, pulmonary laceration, hypotension, hypoxia 13 due to ventilation–perfusion mismatch in the newly aerated lung segment Differential Diagnosis of Pleural Fluid For a more complete differential, see Chapter 3. Transudate is usually associated with nephrosis, CHF, cirrhosis; an exudate is associated with infection (pneumonia, TB), malig- nancy, empyema, peritoneal dialysis, pancreatitis, chylothorax. See Table 13–6, page 299, for the differential diagnosis. URINARY TRACT PROCEDURES Bladder Catheterization Indications • Relieving urinary retention • Collecting an uncontaminated urine specimen for diagnostic purposes • Monitoring urinary output in critically ill patients • Performing bladder tests (cystogram, cystometrogram) Contraindications • Urethral disruption, often associated with pelvic fracture • Acute prostatitis (relative contraindication) 13 Bedside Procedures 307 Materials • Prepackaged bladder catheter tray (may or may not include a Foley catheter) • Catheter of choice (see Fig. 13–21): Foley: Balloon at the tip to keep it in the bladder. Use a 16–18 French for adults (the higher the number, the larger the diameter). Irrigation catheters (“three-way Foley”) should be larger (20–22 French). Coudé (pronounced “COO-DAY”): An elbow-tipped catheter useful in males with prostatic hypertrophy (the catheter is passed with the tip pointing to 12 o’clock). Red rubber catheter (Robinson): Plain rubber or latex catheter without a balloon, usually used for “in-and-out catheterization” in which urine is removed but the catheter is not left indwelling. Procedure 1. Each insertion of a catheter implants bacteria into the bladder, so strict aseptic tech- nique is mandatory. 2. Have the patient lie supine in a well-lighted area; females with knees flexed wide and heels together to get adequate exposure of the meatus. 3. Get all the materials ready before you attempt to insert the catheter. Open the kit, and put on the gloves. Open the prep solution, and soak the cotton balls. Apply the sterile drapes. 13 FIGURE 13–21 Types of bladder catheters include (from the top) the straight “Robinson” catheter [or red rubber catheter], Foley catheter with standard 5-mL bal- loon, the Coudé catheter, and “three-way” irrigating catheter with 30-mL balloon. Catheters have been shortened for illustrative purposes. 308 Clinician’s Pocket Reference, 9th Edition 4. Inflate and deflate the balloon of the Foley catheter to ensure its proper function. Coat the end of the catheter with lubricant jelly. 5. In females, use one gloved hand to prep the urethral meatus in a pubis-toward-anus di- rection; hold the labia apart with the other gloved hand. With uncircumcised males, re- tract the foreskin to prep the glans; use a gloved hand to hold the penis still. 6. The hand used to hold the penis or labia should not touch the catheter to insert it; a dis- posable forceps in the kit can be used to insert it. Or the forceps can be used to prep, then the gloved hand can insert the catheter. 7. In the male, stretch the penis upward perpendicular to the body to eliminate any inter- nal folds in the urethra that might lead to a false passage. Use steady, gentle pressure to advance the catheter. The bulbous urethra is the most likely part to tear. Any signifi- cant resistance encountered may represent a stricture and requires urological consulta- tion. In males with BPH, a Coudé tip catheter may facilitate passage. Some tricks used to get a catheter to pass in a male are to make sure that the penis is well stretched and to instill 30–50 mL of sterile water-based surgical lubricant (K-Y jelly) into the urethra with a catheter-tipped syringe prior to passage of the catheter. Viscous lidocaine jelly for urologic use can help lubricate and relieve the discomfort of difficult catheter place- ment. Allow at least 5 min after instillation of the lidocaine jelly for the anesthetic ef- fect to take place. 8. In both males and females, insert the catheter to the hilt of the drainage end. Compress the penis toward the pubis. These maneuvers ensure that the balloon inflates in the bladder and not in the urethra. Inflate the balloon with 5–10 mL of sterile water or, oc- casionally, air. After inflation, pull the catheter back so that the balloon comes to rest on the bladder neck. There should be good urine return when the catheter is in place. If a large amount of lubricant jelly was placed into the urethra, the catheter may need to be flushed with sterile saline to clear the excess lubricant. A catheter that will not irri- gate is in the urethra, not the bladder. 13 9. Any male who is uncircumcised should have the foreskin repositioned to prevent mas- sive edema of the glans after the catheter is inserted. 10. Catheters in females can be taped to the leg. In males, the catheter should be taped to the abdominal wall to decrease stress on the posterior urethra and help prevent stricture formation. The catheter is usually attached to a gravity drainage bag or some device for measuring the amount of urine. Many new kits come with the catheter already secured to the drainage bag. These systems are considered “closed” and should not be opened if at all possible. “In-and-Out” Catheterized Urine 1. If urine is needed for analysis or for culture and sensitivity, especially in a female pa- tient, a so-called in-and-out cath can be done. This is also useful for measuring residual urine in males or females. The incidence of inducing infection with this procedure is about 3%. 2. The procedure is identical to that described for bladder catheterization. The main dif- ference is that a red rubber catheter (no balloon) is often used and is removed immedi- ately after the specimen is collected. Clean-Catch Urine Specimen 1. A clean-catch urine is useful for routine urinalysis, is usually good for culturing urine from males, but is only fair for culturing urine from females because of the potential for contamination. 13 Bedside Procedures 309 2. For males: a. Expose the glans, clean with a povidone–iodine solution and dry it with a sterile pad. b. Collect a midstream urine in a sterile container after the initial flow has escaped. 3. For females: a. Separate the labia widely to expose the urethral meatus; keep the labia spread throughout the procedure. b. Cleanse the urethral meatus with povidone–iodine solution from front to back, and rinse with sterile water. c. Catch the midstream portion of the urine in a sterile container. Percutaneous Suprapubic Bladder Aspiration Indications (Used most frequently in young children) • When urine cannot be obtained by a less invasive method • In the presence of urethral abnormalities • In the presence of a refractory UTI Contraindications • If the child has voided within the last hour, or if the bladder cannot be percussed Procedure 1. This procedure is almost exclusively limited to the very young pediatric patient (usu- ally <6 months). 2. Immobilize the child. Do not attempt this procedure if the child has voided within the 13 last hour. 3. Palpate the bladder above the pubic symphysis (the bladder sticks high above the pubis in a young child when it is full). Some suggest occluding the urethra by holding the penis in a male and by inserting a finger in the rectum to exert pressure in the female. Percuss out the limits of the bladder. 4. Obtain a 20-mL syringe with a 23- or 25-gauge, 1¹₂-in. needle. Prep with povidone–io- dine and alcohol 0.5–1.5 cm above the pubis. Anesthesia is not routinely used. 5. Insert the needle perpendicular to the skin in the midline; maintain negative pressure on the downstroke and on withdrawal until urine is obtained (Fig.13–22). 6. If no urine is obtained, wait at least 1 h before reattempting the procedure. VENIPUNCTURE Materials • A tourniquet (a 1¹₂-in. Penrose drain or glove is acceptable) • Alcohol prep sponge • Proper specimen tubes for desired studies (red top, purple top, etc.) (Table 13–7) • Appropriate-sized syringe for volume of blood needed (5 mL, 10 mL, etc), or a Va- cutainer tube and appropriate needle and Vacutainer holder • A 20–22-gauge needle (Larger needles are uncomfortable, and smaller ones can cause hemolysis or clotting; the higher the gauge number, the smaller the needle.) 310 Clinician’s Pocket Reference, 9th Edition Pubic Bladder symphysis Uterus 13 Rectum FIGURE 13–22 The technique and anatomic structures in suprapubic bladder as- piration. (Reprinted, with permission, from: Gomella TL [ed]: Neonatology: Basic Management, On-Call Problems, Diseases, Drugs, 4th ed. Appleton & Lange, Nor- walk CT, 1998.) Procedure Venipuncture (phlebotomy) is the puncture of a vein to obtain a sample of venous blood for analysis. Blood cultures, IV techniques, and arterial punctures are discussed in other sec- tions of the chapter. 13 311 TABLE 13–7 Tube Guide for Venipuncture Using the Vacutainer System* Number or

Vacutainer Inversions at Vacutainer Hemogard Blood Collection Tubes Closure Additive (Invert gently, do not shake) Laboratory Use Black/red marbled Gold Clot activator and 5 SST brand tube for serum demon- (“Tiger Top”) gel for serum strations in chemistry. Tube inversions separation ensure mixing of clot activator with blood and clotting within 30 min Green/red marbled Light green Lithium heparin and 8 PST brand tube for plasma gel for plasma determinations in chemistry. Tube separation inversions prevent clotting Red Red None 0 For serum determinations in chemistry, serology, and blood banking. Yellow/black Orange Thrombin 8 For stat serum determinations in marbled chemistry. Tube inversions prevent clotting, usually in less than 5 min Royal blue Royal blue Sodium heparin 8 For trace element, toxicology, and Na EDTA 8 nutrient determinations. Special None 0 stopper formulation offers the lowest verified levels of trace elements available. (See package insert) Green Green Sodium heparin 8 For plasma determinations in Lithium heparin 8 chemistry. Tube inversions prevent Ammonium heparin 8 clotting (continued ) 13 312 TABLE 13–7 (Continued) Number or Vacutainer Inversions at Vacutainer Hemogard Blood Collection Tubes Closure Additive (Invert gently, do not shake) Laboratory Use Gray Gray Potassium oxalate/ 8 For glucose determinations. Tube Sodium fluoride inversions ensure proper mixing of Sodium fluoride 8 additive and blood. Oxalate and Lithium iodoacetate 8 heparin, anticoagulants, will give 8 samples that are serum Brown Brown Sodium heparin 8 For lead determinations. This tube is certi- fied to contain less than .01 µ/mL (ppm) lead. Tube inversions prevent clotting Yellow Yellow Sodium 8 For blood culture specimen polyanetholesulfonate collections in microbiology. Tube (SPS) inversions prevent clotting. Lavender Lavender Liquid EDTA 8 For whole blood hematology Freeze-dried 8 determinations. Tube inversions Na EDTA prevent clotting Light blue Light blue 0.105 M sodium 8 For coagulation determinations on citrate (3.2%) plasma specimens. Tube inversions 0.129 M sodium 8 prevent clotting. Note: Certain tests citrate (3.8%) require chilled specimens. Follow recommended procedures for collection and transport of specimen *Based on products from Becton-Dickinson. Abbreviation: EDTA = ethylene diamine tetraacetic acid. 13 Bedside Procedures 313 1. Collect the necessary materials before you begin. 2. The most commonly used sites for routine venipuncture are the veins of the antecubital fossa (see Fig. 13–12, page 279). Other sites that can be used include the dorsum of the hand, the forearm, the saphenous vein near the medial malleolus, or the external jugular vein. If all the routine peripheral sites are unacceptable, the femoral vein can be used. Never draw a blood sample proximal to an IV site. The high concentration of IV fluid in the veins at this location may make the laboratory studies invalid. 3. Apply the tourniquet at least 2–3 in. above the venipuncture site. Have the patient make a fist to help engorge the vein. If veins are difficult to locate, some helpful techniques include slapping the vein to cause reflex dilation, hanging the extremity in a dependent position, wrapping the extremity in a warm soak, substituting a blood pressure cuff for the standard tourniquet, or applying nitroglycerin paste below and over the area may help dilate the veins. 4. Swab the site with the alcohol prep pad, and allow the alcohol to evaporate. 5. Use the syringe and needle with the bevel up and puncture the skin alongside the vein. After the needle is through the skin, use the thumb of your free hand to stabilize the vein and prevent it from rolling. 6. Enter the vein on the side at about a 30-degree angle while applying gentle back pres- sure on the syringe. Withdraw the sample slowly to prevent the vein from collapsing. An alternative acceptable technique is to enter both the skin and vein in one stick, how- ever this maneuver requires practice because the vein is often stuck through and through. 7. The Vacutainer system is a very useful means of collecting blood, especially if several different sample tubes need to be filled. Mount a 20–22-gauge Vacutainer needle on the Vacutainer cup. Enter the vein as directed previously. Advance the collection tube onto the needle inside the Vacutainer. The vacuum inside the tube automatically collects the sample. If you hold the Vacutainer steady, several tubes can be collected in this fashion. 8. After the blood is collected (by whatever method), remove the tourniquet, withdraw the needle, and apply firm pressure with the alcohol swab or sterile gauze for 2–3 min. Ele- 13 vation of the extremity is helpful. Current evidence indicates that bending the arm actu- ally increases the size of the venipuncture site and should be discouraged. 9. If a needle and syringe are used, distribute the samples to the blood tubes. The best technique is to insert the needle into the tube and allow the vacuum to draw in the ap- propriate volume of blood for a given tube (this is most critical for coagulation studies). Distribute the blood to the coagulation and CBC tubes first because clotting of the blood in the syringe can invalidate the results. Mix the tubes thoroughly. Blood drawn for typing and cross-matching usually has special labels that require signature of the person that obtained the sample. 10. If no peripheral veins can be located, puncture of the femoral vein can be attempted. Locate the femoral artery. The mnemonic of lateral to medial structures in the groin is NAVEL: Nerve, Artery, Vein, Empty space, Lymphatic. The femoral vein should be just medial to the femoral artery. After prepping the skin, insert the needle perpendicu- lar to the skin, and gently aspirate. The vein should be about 1–1¹₂ in. below the skin. Apply firm pressure after the collection of the sample because hematomas are frequent complications of femoral venipunctures. Should you accidentally enter the femoral artery, it is acceptable to collect the sample. Apply pressure for a longer period if the artery is entered. 11. In children and the elderly with fragile veins, a butterfly (21–25-gauge) can be used to obtain a sample (see Fig. 13–12). 314 Clinician’s Pocket Reference, 9th Edition 12. When completed with the venipuncture needle, follow the CDC recommendations and DO NOT reshield the needle with the protective cap. Whenever possible, dispose of the needle immediately into the sharps collection container located on each hospital unit where blood is routinely drawn. Newer “safe needles” (Safety-Lok, ProGuard, Puncture-Guard, etc) are designed to attach to the Vacutainer system and have mecha- nisms to help protect the tip and hopefully diminish the incidence of accidental needle- sticks. 13 14 PAIN MANAGEMENT Terminology Evaluation of Patient with Pain Classification of Pain Pain Measurement Adverse Physiologic Effects of Pain Practical Pain Management Principles of Pain Control Patient-Controlled Analgesia “Men do not fear death, they fear the pain of dying.” TERMINOLOGY Pain is the most common symptom that brings patients to see a physician, and it is fre- quently the first alert of an ongoing pathologic process. The International Association for the Study of Pain defines pain as: An “unpleasant sensory and emotional experience associ- ated with actual or potential tissue damage.” Acute pain is common postoperatively and in acute injury. Chronic pain can be associated with conditions such as cancer and arthritis. Oligoanalgesia is the failure to recognize or properly treat pain. This may result because the physician makes a judgment without asking the patient if she or he hurts or discredits the patient’s response and bases the determination of pain severity on the physician’s own sub- jective past pain experience. Accordingly, the gold standard for determining if a patient is in pain is to ask the patient if he or she hurts and then to attempt to objectively verify the report with monitors, touch, or direct vision. 14 Nociception is derived from the Latin word noci, meaning “harm or injury.” It refers to the detection, transduction, and transmission of noxious stimuli. Stimuli generated from thermal, mechanical, or chemical tissue damage may activate nociceptors, which are free nerve endings. (All nociception produces pain but not all pain results from nociception.) CLASSIFICATION OF PAIN Pain can be broadly divided into acute and chronic pain. Acute Pain Acute pain is caused by noxious stimulation due to injury or disease process or abnormal function of muscle or viscera. It is a manifestation of autonomic, psychologic, and behavior responses, which can be self-limited and resolve with treatment (eg, after trauma, after surgery, MI, pancreatitis, or renal calculi). Acute pain is further classified as • Superficial. Nociception from skin, subcutaneous tissue, or mucous membrane. Lo- calized, sharp, pricking, throbbing, or burning • Deep somatic. From muscle, tendon, joints, bones. Less localized, dull aching in character 315 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 316 Clinician’s Pocket Reference, 9th Edition • Visceral. From internal organ or coverings (parietal pleura, pericardium, or peri- toneum). Can be localized or referred. May accompany sympathetic or parasympa- thetic manifestations as changes in BP or heart rate, nausea and vomiting Chronic Pain The determination of whether pain is chronic should not be based on its duration, but rather on the substantial damage it causes to an individual in terms of functional loss, psychologic distress (sleep and affective disturbances), and social and vocational dysfunction. This pain usually results from peripheral nociception of the peripheral or central nervous system, and it usually lacks neuroendocrine stress response: musculoskeletal disorders, chronic visceral disorders, lesions of peripheral nerves, nerve root, dorsal root ganglia (causalgia, phantom limb pain, postherpetic neuralgia), stroke, spinal cord injury, MS, or cancer. ADVERSE PHYSIOLOGIC EFFECTS OF PAIN This is usually associated with acute pain and is proportional to pain intensity. (Table 14–1) PRINCIPLES OF PAIN CONTROL • Proper patient evaluation, including pain measurement • Good physician–patient relationship built on trust • Consideration of both psychologic and emotional aspects • Acknowledgment that treatment depends on patient’s compliance, understanding, and cooperation • Possible combination therapy • Explanation of side effects (if unavoidable) and how to treat them • Proper follow-up EVALUATION OF PATIENT WITH PAIN 14 1. Ask the patient if she or he is in pain? Bear in mind that you must trust and believe what the patient says. 2. Obtain a detailed history of this pain: • Character of the pain (dull, colicky, sharp) • Duration of pain 3. Is the pain referred to other sites of the body (eg, ureteral calculi may be referred to the ipsilateral testicle)? 4. What relieves the pain: Rest, position? 5. What makes it worse: Movement, positions, activities? 6. Are there any accompanying symptoms: Nausea, vomiting, headache? • Perform a physical examination and request imaging studies if an organic cause is suspected. • Chronic pain frequently affects daily activity and social interaction so psychosocial evaluation may be indicated. PAIN MEASUREMENT The most commonly used two methods of pain measurement are Visual Analogue Scale (VAS) and McGill Pain Questionnaire (MPQ). 14 317 TABLE 14–1 Adverse Physiologic Sequelae of Pain Organ System Adverse Effect RESPIRATORY Increased skeletal muscle tension Hypoxia, hypercapnia Decreased total lung compliance Ventilation–perfusion abnormality, atelectasis, pneumonitis ENDOCRINE Increased adrenocorticotropic hormone Protein catabolism, lipolysis, hyperglycemia Decreased insulin, decreased testosterone Decreased protein anabolism Increased aldosterone, increased antidiuretic hormone Salt and water retention, congestive heart failure Increased catecholamines Vasoconstriction Increased angiotensin II Increased myocardial contractility CARDIOVASCULAR Increased myocardial work Dysrhythmias, angina IMMUNOLOGIC Lymphopenia, depression of reticuloendothelial system, Decreased immune function leukocytosis Reduced killer T-cell cytotoxicity (continued ) 14 318 TABLE 14–1 (Continued) Organ System Adverse Effect COAGULATION EFFECTS Increased platelet adhesiveness, diminished fibrinolysis Increased incidence of thromboembolic phenomena Activation of coagulation cascade GASTROINTESTINAL Increased sphincter tone Ileus Decreased smooth muscle tone GENITOURINARY Increased sphincter tone Urinary retention Decreased smooth muscle tone 14 Pain Management 319 Visual Analogue Scale The VAS is a 10-cm horizontal line with the words NO PAIN at one end and WORST PAIN IMAGINABLE at the other end. The patient is asked to put a mark on this line at the point that identifies the intensity but not quality of his or her pain. This has been called the “fifth vital sign” and is commonly used in the hospital setting to guide pain management. 0 1 2 3 4 5 6 7 8 9 10 No pain Worst possible pain McGill Pain Questionnaire The MPQ (Melzack R: The McGill Pain Questionnaire: Major properties and scoring meth- ods. Pain 1975;1:277–299.) is a checklist of words describing symptoms. Scores are then analyzed

in various dimensions (sensory and affective) to identify the quality of pain. This tool is usually used in the detailed management of pain syndromes. Psychologic Evaluation: A psychologic evaluation is indicated if medical examina- tion fails to reveal any apparent cause for the patient’s pain. The Minnesota Multiple Per- sonality Inventory (Hathaway SR and McKinley JC: MMPI. University of Minnesota Press, Minneapolis, 1989.) and Beck Depression Inventory (Beck AT, Steer RA: Internal consis- tencies of the original and revised Beck Depression Inventory. J Clin Psychol 1984;40(6): 1365–1367.) are two commonly used tools for evaluating chronic pain and depression. These questionnaires should not only determine the patient’s psychologic status but also evaluate his or her behavior and response to pain and its management. Electromyography and Nerve Conduction Testing: This method differentiates be- tween neurogenic and myogenic causes and confirms diagnoses of nerve entrapment, neural trauma, and polyneuropathies. Thermography: Normally, heat from body surfaces is emitted in the form of infrared energy; this emission is symmetrical in homologous areas. Neurogenic pathophysiologic changes result in asymmetry. This infrared energy can be measured and displayed; hypere- 14 mission indicates an acute stage and hypoemission a chronic stage. Diagnostic and Therapeutic Neural Blockade: Neural blockade with local anes- thetics can be used to diagnose and manage both acute and chronic pain. PRACTICAL PAIN MANAGEMENT The goal of pain management is to provide the patient adequate relief with minimum side effects (eg, drowsiness). Always begin therapy with the lowest dose of any medication that provides relief. Oral, rectal, or transdermal routes are preferred over parental therapy. Pain management can be generally divided into • Pharmacologic • Nonpharmacologic • Combinations according to the patient response and compliance Pharmacologic The World Health Organization has made specific recommendations concerning pain man- agement. These principles apply primarily to cancer pain but can be used in any clinical set- ting. Start at step 1 and advance to the next level based on patient response. 320 Clinician’s Pocket Reference, 9th Edition Step 1: Nonopioid agents (NSAIDs, acetaminophen, etc) Step 2: Weak opioids (codeine, oxycodone) Step 3: Strong opioids (morphine, fentanyl) Specific pharmacologic agents are reviewed in the following section and in Table 14–2. Supplements can enhance the effects of analgesics and allow dose reduction of some agents. Nonopioid Analgesics: Aspirin, acetaminophen, and NSAIDs are the principal nono- pioid analgesics used to treat mild and moderate pain. NSAIDs are primarily cyclooxyge- nase inhibitors that prevent prostaglandin-mediated amplification of chemical and mechanical irritants of the sensory pathways. Short-term perioperative use of ketorolac (Toradol) can reduce pain medication requirement. Side effects: Possible hepatotoxicity (large doses of acetaminophen); stomach upset, nausea, dyspepsia, ulceration of gastric mu- cosa, dizziness, platelet dysfunction, exacerbation of bronchospasm, and acute renal insuffi- ciency (aspirin and NSAIDs). Opioids: These drugs attach to opioid receptors, which are responsible for the analgesia. Side effects: Sedation, dizziness, miosis, nausea, vomiting and constipation from smaller doses, to respiratory depression, apnea, cardiac arrest and circulatory collapse, coma, and death after high intravenous doses. Opioids can be taken orally, parenterally, or neuroaxially (intrathecal/epidural). They are available in short- (q4h) and long-duration forms (eg, q12h, q24h). Opioids can also be given as a patient-controlled analgesia (PCA) (see section with that title). Comparison of the different opioid narcotic can be found in Table 14–2. Antidepressants: The analgesic effect produced by antidepressants is due to reuptake of serotonin and norepinephrine. Side effects: Antimuscarinic effects (dry mouth, impaired visual accommodation, urinary retention), antihistaminic (sedation), and alpha adrenergic blockage (orthostatic hypotension). Neuroleptics: Useful in patients with agitation and psychologic symptoms. Side ef- fects: Extrapyramidal, mask-like facies, festinating gait, cogwheel rigidity (bradykinesia). 14 Anticonvulsants: These medications act by suppressing the spontaneous neural dis- charge. Side effects: Bone marrow depression, hepatotoxicity, possible ataxia, dizziness, confusion, and sedation (at toxic doses). Corticosteroids: These are antiinflammatory analgesics. Side effects: HTN, hyper- glycemia, and increased tendency to infection, peptic ulcer, osteoporosis, myopathies, and Cushing’s syndrome. Systemic Local Anesthetics: These drugs produce sedation and central analgesia. Side effects from excessive dosing: Toxicity with cardiovascular collapse and CNS symp- toms in the form of tonic–clonic seizures. Respiratory arrest usually follows. Nonpharmacologic • Nerve blocks or neurolysis (destruction of the nerve) • Radiation: Useful for cancer pain (ie, bony metastasis) • Psychologic intervention: Using cognitive therapy, behavioral therapy or biofeed- back relaxation technique and hypnosis • Physical therapy: Heat and cold can provide pain relief by alleviating muscle spasm. Heat decreases joint stiffness and increases blood flow; cold vasoconstricts and reduces tissue edema. 14 321 TABLE 14–2 Selected Agents Commonly Used in Pain Initial Maximum Route Onset Duration Dose Dose NON-OPIOID Acetaminophen (Tylenol, Datril) PO 0.5 h 4 h 500–1000 mg 1200 mg Aspirin/sodium salicylate PO 0.5–1 h 4 h 500–1000 mg 3600 mg Celecoxib (Celebrex) PO 3 h 12 h 100 mg 400 mg Diclofenac sodium (Voltaren) PO 1 h 4–6 h 25–75 mg 200 mg Ibuprofen (Motrin, Rufen) PO 0.5 h 4–6 h 400 mg 3200 mg Indomethacin (Indocin) PO 0.5 h 4–6 h 25–50 mg 200 mg Piroxicam (Feldene) PO 1 h 48–72 h 10–20 mg 20 mg Rofecoxib (Vioxx) PO 2–3 h 24 h 12.5 mg 50 mg OPIOID Codeine IM 0.25–0.5 h 4–6 h 15 mg 60 mg PO 0.25–1 h 3–4 h 15 mg 60 mg Fentanyl IV 1.7–2.3 h 1 h 1–1.5 µg/kg 150 TD 1.7–2.3 h 1 h 25 mg/h 100 µg/h Meperidine (Demorol) PO 0.5–1 h 2–3 h 1–1.5 mg/kg 50–100 mg IM 0.12–0.5 h 2–4 h 1–1.5 mg/kg 50–100 mg Methadone (Dolophine) PO 0.5–1 h 4–8 h 2.5–10 mg 160 mg IM 0.25 h 4–6 h 2.5–10 mg 160 mg Morphine (various) IV Rapid 1–2 h 0.1–15 mg/kg 2.5–15 mg IM 0.3 h 3–4 h 0.1–0.15 mg/kg 10–15 mg (continued ) 14 322 TABLE 14–2 (Continued) Initial Maximum Route Onset Duration Dose Dose Nalbuphine (Nubain) IV — — 1–5 mg 160 mg IM 0.25 h 3–6 h 10–20 mg 160 mg OTHER/SUPPLEMENTS Amitriptyline PO 7–21 d — 25–150 mg 300 mg Carbamazepine PO — — 200 mg 1600 mg Dexamethasone PO — — 0.5–9 mg/kg 20–100 mg IV — — 0.5–9 mg/kg 20–100 mg Haloperidol (Haldol) PO — — 0.5–5 mg 100 mg IV 1 h 3 wk 0.5–5 mg 100 mg Abbreviations: PO = by mouth; IV = intravenous; IM = intramuscular; TD = transdermal. 14 Pain Management 323 • Acupuncture: Needles inserted into discrete anatomically defined points and stimu- lated by mild electric current. Believed to release endogenous opioids • Electrical stimulation of the nervous system: Can produce analgesia. The three methods are 1. Transcutaneous electrical stimulation (TENS) with electrodes applied to skin 2. Spinal cord stimulation by inserting electrodes epidurally connected to external generator 3. Intracerebral stimulation with electrodes implanted in the periaqueductal or periventric- ular area PATIENT-CONTROLLED ANALGESIA Most commonly used after surgery, allows the patient to self-administer the dose of narcotic via an IV pump. The patient treats the pain as soon as he or she feels necessary, thus avoid- ing the peak and trough of a narcotic dosing regimen that may lead to extremes of pain and potential oversedation. The pain management team can titrate the dose of the drug as re- quired using a computerized system that controls the total dose and the interval between each dose with the use of a continuous basal infusion dosage. PCA duration varies based on procedure and patient response (eg, gyn 1–2 d, bowel 2–5 d, thoracotomy 4–6 d). Reduce dose in elderly (¹₃–²⁄₃). Consider discontinuation of PCA when patients are able to take analgesics PO. PCA Parameters • Dose: Number of mL (typically morphine concentration, page 321) given on activa- tion of button by patient • Lockout: Minimum interval of time in minutes between PCA doses • Hourly Max: Maximum volume (mL) that machine administers in an hour • Basal Rate: Continuous infusion rate (not required on all patients) The following table shows examples of PCA orders: 14 Hourly Dose Lockout Max Typical Procedure (mL) (min) (mL) Basal Moderately painful (lower abdominal, 1 6 8 None incisions, minor orthopedic procedures) Fairly painful (upper abdominal 1.5 6 10 None–1 incisions) mL/h Very painful (thoracotomy, total 1.5 6 10 0.5– knee replacement) 1.5 mL This page intentionally left blank. 15 IMAGING STUDIES X-Ray Preparations Spiral (Helical) CT Scan Common X-Ray Studies: Noncontrast Magnetic Resonance Imaging (MR Common X-Ray Studies: Contrast or MRI) Ultrasound Nuclear Scans CT Scans How to Read a Chest X-Ray X-RAY PREPARATIONS In general, follow this rubric: plain films before contrast, contrast before barium. Each hos- pital has its own guidelines for patient x-ray preps. Consult the radiology department prior to ordering. Examinations that require no specific bowel preparation include routine chest x- rays, flat and upright abdominal films, T-tube cholangiograms, cystograms, C-spines, skull series, extremity films, CT scan of the head or chest, and many others. Studies that usually require such preps as enemas, laxatives, oral contrast agents, or those that require that the patient be NPO prior to the examination include oral cholecys- togram, upper GI series, SBFT, barium enema, IVP, CT scan of the abdomen or pelvis, and many others. COMMON X-RAY STUDIES: NONCONTRAST Chest Chest X-Ray (Routine): Includes PA and lateral chest films. Used in the evaluation of 15 pulmonary, cardiac and mediastinal diseases, and traumatic injury. See page 335 on How to Read a Chest X-Ray. Expiratory Chest: Used to help visualize a small pneumothorax Lateral Decubitus Chest: Allows small amounts of pleural effusion or suspected sub- pulmonary effusion to layer out and permits diagnosis of as little as 175 mL of pleural fluid Lordotic Chest: Allows better visualization of apices and lesions of the right and left upper lobes. Often used in the evaluation of TB Portable Chest and AP Films: Cannot be used to accurately evaluate heart size or widened mediastinum but can be used to detect effusions, pneumonia, edema, and to verify line or tube placement Rib Details: Special views that more clearly delineate rib pathology; useful when plain chest radiogram or bone scan suggests fractures or other metastatic lesions. 325 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 326 Clinician’s Pocket Reference, 9th Edition Abdominal Abdominal Decubitus: Used in debilitated patients instead of an upright abdominal film. The left side should be down to find free air outlining the liver and right lateral gutter. Acute Abdominal Series (“obstruction series”): Includes a flat and upright ab- dominal (KUB) and chest x-ray. Good for initial evaluation of an acute abdomen (See KUB.) Cross-Table Lateral Abdominal: Used in debilitated patients to look for free air or to identify an aortic aneurysm. KUB, Supine and Erect: Short for “kidneys, ureter, and bladder” and also known as “flat and upright abdominal,” “scout film,” or “flat plate.” Useful when the patient com- plains of abdominal pain or distension, and for initial evaluation of the urinary tract (80% of kidney stones and 20% of gallstones are visualized on these films). To read, look for calcifi- cations, foreign bodies, the gas pattern, psoas shadows, renal and liver shadows, flank stripes, the vertebral bodies, and pelvic bones. On the upright, look for air–fluid levels of an adynamic ileus or mechanical obstruction and for free air under the diaphragm, which sug- gests a perforated viscus or recent surgery; however, the upright chest x-ray (especially the lateral view) is often best to spot a pneumoperitoneum. Other Noncontrast X-Rays C-Spine: Usually includes PA, lateral, and oblique films. Useful for the evaluation of trauma, neck pain, and neurologic evaluation of the upper extremities. All seven cervical vertebrae must be seen for this study to be acceptable. DEXA: Measures bone mineral density at a variety of sites (femur/lumbar spine); used in the diagnosis and monitoring of response to treatment of osteoporosis Mammography: Detects cancers greater than 5 mm in size. Two forms equal in diag- nostic quality: • Screen film. Produces standard black and white x-ray via a specially designed mam- mographic machine; 3–5× smaller radiation dose 15 • Xeromammography. Blue and white paper image using a general x-ray machine. Delivers a higher radiation dose. Current American Cancer Society screening guide- lines for asymptomatic women: 35–39 baseline, 40–49 every 1–2 years, >50 every year. Sinus Films (Paranasal Sinus Radiographs): Used to evaluate sinus trauma, si- nusitis, neoplasms, or congenital disorders Skull Films: Used to detect fractures and aid in the identification of pituitary tumors or

congenital anomalies Vertebral Radiography: Used to evaluate fractures, dislocations, subluxations, disk disease, and the effects of arthritic and metabolic disorders of the spine COMMON X-RAY STUDIES: CONTRAST An agent, such as barium or Gastrografin, or an intravenous contrast agent is used for these studies. If a GI tract fistula or perforation is suspected, inform the radiologist because this may affect the choice of contrast agent (ie, water-soluble contrast [eg, Gastrografin] instead 15 Imaging Studies 327 of barium). Standard IV contrast media are ionic and may be associated with rare contrast reaction when administered systemically (see following section). The use of nonionic con- trast media may limit these side effects. Ionic Contrast Media • Oral cholecystographic agents: Telepaque etc • GI contrast agents: Barium sulfate—Baro-CAT, Tomocat, etc • Injection: Diatrizoate meglumine—Hypaque Meglumine, Urovist Meglumine, An- giovist, etc Diatrizoate sodium: Hypaque Sodium, Urovist Sodium, etc Gadopentetate dimeglumine: Magnevist, etc Iodamide meglumine: Renovue, etc Iothalamate meglumine: Conray, etc Iothalamate sodium: Angio Conray, etc Diatrizoate meglumine and diatrizoate sodium: Angiovist, Hypaque-M, etc • Not for intravascular use, for instillation into cavities: Diatrizoate meglumine, Cystografin, etc Diatrizoate meglumine and diatrizoate sodium Gastrografin, etc Diatrizoate sodium: Oral or rectal (Hypaque sodium oral) Iothalamate meglumine: Cysto-Conray urogenital Diatrizoate meglumine and iodipamide meglumine: Sinografin intrauterine instillation Nonionic Contrast Media Injectable • Iohexol: Omnipaque • Iopamidol: Isovue • Ioversol: Optiray • Metrizamide: Amipaque Contrast reactions to IV agents may occur; severe reaction occurs 1/1000 and death due to 15 anaphylaxis 1/40,000. Reactions include hives, bronchospasm, or pulmonary edema. (Pre- medication with steroids may not prevent a reaction.) Vagal reactions (hypotension and bradycardia) are another adverse effect. A history of asthma is a risk factor and a previous reaction to contrast does not necessarily preclude using IV contrast (allergy to seafood or io- dine is no longer considered an important risk factor). Premedication with two doses of PO methylprednisolone, once at 12 h prior and then 2 h prior to IV contrast, is effective in re- ducing the incidence of reactions. Alternatively, use of new and more expensive (up to 10× the cost) nonionic contrast agents, lessens pain and cardiac dysfunction, with a possible overall decrease in adverse reactions. Angiography: A rapid series of films obtained after a bolus contrast injection via percu- taneous catheter. Used to image the aorta, major arteries and branches, tumors, and venous drainage via late “run-off” films. Helical CT scans are now capable of generating angio- graphic images. • DSA. This is the latest enhancement of this study, allows reverse negative views and requires less contrast load 328 Clinician’s Pocket Reference, 9th Edition • Cardiac angiography. Definitive study for diagnosis and assessment of severity of CAD. Significant (>70% occlusion) stenotic lesions seen: 30% involve single ves- sels, 30% involve two, and 40%, three vessels. Can discriminate angina secondary to aortic valve disease and that from CAD • Cerebral angiography. Evaluation of intra- and extracranial vascular disease, ather- osclerosis, aneurysms, and A-V malformations. Not used for detection of cerebral structural lesions (use MRI or CT instead) • Pulmonary angiography. Visualization of emboli, intrinsic or extrinsic vascular ab- normalities, A-V malformations, and bleeding due to tumors. Most accurate diag- nostic procedure for PE but only used if lung V/Q scan is not diagnostic BE: Examining the colon and rectum. Indications include diarrhea, crampy abdominal pain, heme-positive stools, change in bowel habits, and unexplained weight loss • Air-contrast BE. Done with the “double contrast” technique (air and barium) to bet- ter delineate the mucosa. More likely to show polyps • Gastrografin enema. Similar to the barium enema, but water-soluble contrast is used (clears colon more quickly than barium). If the Gastrografin leaks from the GI tract, it is less irritating to the peritoneum (does not cause “barium peritonitis”). Therapeutic in the evaluation of severe obstipation, colonic volvulus, perforation, di- verticulitis, or postop anastomotic leak Barium Swallow (Esophagogram): Evaluating the swallowing mechanism and in- vestigating esophageal lesions or abnormal peristalsis Cystogram: Bladder filled and emptied and a catheter in place. Used to evaluate bladder filling defects (tumors, diverticulum) and bladder perforation. Can also be done using CT scanning (see also VCUG) Enteroclysis: Selective intubation of the proximal jejunum and rapid infusion of con- trast. Better than an SBFT in evaluating polyps or obstruction (adhesions, internal hernia, etc). May be used to evaluate small-bowel sources of chronic bleeding after negative upper and lower endoscopy ERCP: Contrast endoscopically injected into the ampulla of Vater to visualize the com- 15 mon bile and pancreatic ducts in evaluating obstruction, stones, and ductal pattern Fistulogram (Sinogram): Injection of water-soluble contrast media into any wound or body opening to determine the connection of the wound or opening with other structures HSG: Evaluating uterine anomalies (congenital, fibroids, adhesions) or tubal abnormalities (occlusion or adhesion) often as part of infertility evaluation. Contraindicated during menses, undiagnosed vaginal bleeding, acute PID, or if pregnancy suspected. Patient in pelvic exam position, speculum placed and uterine os cannulated; then contrast injected ExU or IVP: Contrast study of the kidneys and ureters. Limited usefulness for evaluat- ing bladder abnormalities. Indications include flank pain, kidney stones, hematuria, UTI, trauma, and malignancy. Bowel prep helpful but not essential. Verify recent creatinine level. Nephrotomograms often included that include cuts of the kidney to further define the three-dimensional location or nature of renal lesions or stones Lymphangiography. Iodinated oil injected to opacify lymphatics of the leg, inguinal, pelvic, and retroperitoneal areas. Used to test the integrity of the lymphatic system or evalu- ate for metastatic tumors (testicular, etc) or lymphoma 15 Imaging Studies 329 Myelogram: Evaluating the subarachnoid space for tumors, herniated disks, or other cause of nerve root injury. Using LP technique, contrast injected in the subarachnoid space OCG: Visualizing gallbladder in the evaluation of cholelithiasis or cholecystitis. Patient given oral contrast pills 12–24 h before the study. Serum bilirubin should be <2 mg/100 mL. Used infrequently PTHC: Visualizing biliary tree in a patient unable to concentrate the contrast media (bilirubin >3 mg/100 mL). Percutaneous needle inserted into a dilated biliary duct; contrast injected. Percutaneous Nephrostogram: In the management of renal obstruction, percuta- neous placement through the renal parenchyma and into the collecting system to relieve and or evaluate the level and cause of obstruction RPG: Contrast material injected into the ureters through a cystoscope. Indications in- clude allergy to IV contrast, a kidney or ureter that cannot be visualized on an IVP, filling defects in the collecting system, renal mass, and ureteral obstruction RUG: Demonstrates traumatic disruption of the urethra and urethral strictures SBFT: Usually done after a UGI series. A delayed film shows the jejunum and ileum. Used in the work-up of diarrhea, abdominal cramps, malabsorption, and UGI bleeding T-Tube Cholangiogram: Resolution of swelling in some patients who have a T-tube placed in the common bile duct for drainage after gallbladder and common bile duct surgery. To evaluate the degree of swelling, look for residual stones, and evaluate patency of bile duct drainage UGI Series: Includes the esophagogram plus the stomach and duodenum. Useful for vi- sualizing ulcers, masses, hiatal hernias, and in the evaluation of heme-positive stools and upper abdominal pain VCUG: Bladder filled with contrast through a catheter, then catheter is removed, and the 15 patient allowed to void. Used for diagnosis of vesicoureteral reflux and urethral valves and in the evaluation of UTI Venography, Peripheral: Contrast slowly injected into small foot or ankle vein to evaluate patency of deep veins of leg and calf. Look for a filling defect or outline of a thrombus. Noninvasive exams for DVT, such as Doppler ultrasound and impedance plethys- mography, often used together before invasive venography and are highly sensitive (>90%) for proximal thrombi. A radionuclide scan with technetium-99-labeled RBCs sometimes used but is less sensitive and specific than the previously mentioned tests. ULTRASOUND Abdominal: Gallbladder (95% sensitivity in diagnosing stones), thickening of wall, bil- iary tree (obstruction), pancreas (pseudo-cyst, tumor, pancreatitis), aorta (aneurysm), kid- neys (obstruction, tumor, cyst), abscesses, ascites Endovaginal: Most useful in the diagnosis of gynecologic pathology (uterus, ovaries) 330 Clinician’s Pocket Reference, 9th Edition Pelvic (A full bladder is desirable) • Pregnancy. Fetal dating (biparietal diameters); diagnosis of multiple gestations; de- termination of intrauterine growth retardation, hydrocephalus, and hydronephrosis; localization of the placenta • Gynecology. Ovarian and uterine masses (tumors, cysts, fibroids, etc.), ectopic preg- nancy, abscesses Thyroid: Evaluate thyroid nodules (cyst versus solid) and to direct biopsies. Ultrasound alone cannot usually differentiate benign from malignant lesions. Transrectal: Most useful in the diagnosis of prostate pathology and directing prostate biopsies Echocardiograms • M-mode. Valve mobility, chamber size, pericardial effusions, septal size • Two-dimensional. Valvular vegetations, septal defects, wall motion, chamber size, pericardial effusion, valve motion, wall thickness • Doppler. Cross-valvular pressure gradients, blood flow patterns, and valve orifice areas in the work-up of cardiac valvular disease Other Ultrasound Uses Testicular (identify and characterize masses, eg, hydrocele versus tumor), intraoperative, de- termine bladder emptying CT SCANS Computerized tomography (also called CAT for computerized axial tomography) can be performed with or without intravenous contrast. A dilute oral contrast agent administered prior to abdominal or pelvic scans helps delineate the bowel. IV contrast is used to provide vascular and tissue enhancement for some CT scans; a current creatinine level should be available to determine suitability of IV contrast administration. Virtually any body part can be scanned depending on the indications, but it is most helpful in evaluating the brain, lung, mediastinum, retroperitoneum (pancreas, kidney, nodes, aorta), and liver, and to a lesser ex- 15 tent in the pelvis, colon, or bone. CT scans allow for the use of density measurements (also known as Hounsfield units) to differentiate cysts, lipomas, hemochromatosis, vascular (“enhancing”) and avascular (“nonenhancing”) lesions. In Hounsfield units, bone is +1000, water is 0, fat is −1000, and other tissues fall within this scale, depending on the machine settings. Metal and barium can cause distortion of the image. Head: Evaluation of tumors, subdural and epidural hematomas, atrioventricular (A-V) malformations, hydrocephalus, and sinus and temporal bone pathology. Initial test of choice for trauma; may be superior to MRI in detecting hemorrhage within first 24–48 h Abdomen: Images virtually all intraabdominal and retroperitoneal organs or disease processes. Good accuracy with abscesses, but ultrasound may show smaller collections ad- jacent to the liver, spleen, or bladder. Surgical clips or barium in the gut may cause artifacts. IV contrast usually given, so check creatinine level; when using a water-soluble contrast (Tomocat, others) to visualize the gut, the patient must receive an oral contrast beforehand. Retroperitoneum: Useful for evaluating pancreatitis and its complications; pancreatic masses; nodal metastasis from colon, prostate, renal, or testicular tumors; adrenal masses (>3 cm suggestive of carcinoma); psoas masses; aortic aneurysms 15 Imaging Studies 331 Pelvis: Staging and diagnosis of bladder, prostate, rectal, and gynecological carcinoma Mediastinum: Masses, ectopic parathyroids Neck: Work-up of neck masses, abscesses, and other diseases of the throat and trachea Chest: Able to find 40% more nodules than whole lung tomograms, which demonstrate 20% more nodules than plain chest x-ray. Although calcification is suggestive of benign dis- ease (eg, granuloma), no definite density value can reliably separate malignant from benign lesions. Useful in differentiating hilar adenopathy from vascular structures seen on plain chest x-ray Spine: MRI generally preferred over CT. However, rare conditions, contraindication to MRI, or artifact from metal may make the CT the preferred test. SPIRAL (HELICAL) CT SCAN Spiral CT can be used for any type of imaging (not commonly used for brain). It relies on rapid scan acquisition (ie, >8 frames/s). This minimizes motion artifact and allows for cap- turing a bolus of contrast at peak levels in the region being scanned. Standard CT is too slow to capture this peak flow. These contrast-enhanced scans allow detailed 3-D reconstruction and angiographic evaluations. Bony structures can also be visualized and do not require contrast. The term spiral/helical is derived from the fact that the tube spins around the pa- tient while the table moves. Spiral CT can compensate for “streak” artifact due to implanted metallic devices. Examples for uses of this technology include diagnosis of PE, pretrans- plant angiography, evaluation of flank pain and determination of kidney stones (largely re- placing emergency IVP), and rapid evaluation of trauma. MAGNETIC RESONANCE IMAGING (MR OR MRI) How It Works Although the physics of MRI is beyond the scope of this section, certain key concepts are essential

to interpreting studies generated by this technology. MRI uses measurements of the magnetic movements of atomic nuclei to delineate tissues. Specifically, when nuclei, such as hydrogen, are placed in a strong magnetic field, they resonate and emit radio signals when pulsed with radio waves. A defined sequence of magnetic pulses and interval pauses 15 produces measured changes in the tissue’s magnetic vectors, which results in an MRI image. T1, or longitudinal relaxation time, is the measurement of magnetic vector changes in the z axis during the relaxation pause. T2, or transverse relaxation time, is the magnetic vector changes in the x-y plane. Each tissue, normal or pathologic, has a unique T1 and T2 for a given MRI field strength. In general T1 > T2. T1 = 0.1–2 s and T2 = 0.03–0.6 s. The inherent tissue differ- ences between various T1’s and T2’s give the visual contrast seen between tissues on the MRI image. An image is T1-weighted if it depends on the differences in T1 measurements for visual contrast, or T2-weighted if the image depends on T2 measurements. The most common pulse sequence is called spin echo (SE). Partial saturation (PS) and inversion recovery (IR) are two newer pulse sequences. Available MRI views are transverse, sagittal, oblique, and coronal. How to Read an MRI SE T1-Weighted Images: Provide good anatomic planes due to the wide variances of T1 values among normal tissues. 332 Clinician’s Pocket Reference, 9th Edition • Brightest (high signal intensity): Fat • Dark or black: Pathological tissues, tumor or inflammation, fluid collections • Black (low signal intensity): Respiratory tract, GI tract, calcified bone and tissues, blood vessels, heart chambers, and pericardial effusions SE T2-Weighted Images: Pathology prolongs T2 measurements, and normal tissues have a very small range of T2 values. T2-weighted images provide the best detection of pathology and a decreased visualization of normal tissue anatomy. Tumor surrounded by fat may be lost on T2 imaging. • Brightest : Fat and fluid collections • Bright : Pheochromocytomas When to Use MRI In general, MRI imaging is at least equal to CT imaging. MRI is superior to CT for imaging of brain, spinal cord, musculoskeletal soft tissues, adrenal and renal masses, and areas of high CT bony artifact. However, spiral CT may now have overcome some of these disadvan- tages. Advantages • No ionizing radiation • Display of vascular anatomy without contrast • Visualization of linear structures: Spine and spinal cord, aorta, and cava • Visualization of posterior fossa and other hard to see CT areas • High-contrast soft tissue images Disadvantages • Claustrophobia due to confining magnet (Newer open MRI scanners may obviate this problem.) • Longer scanning time resulting in motion artifacts • Unable to scan critically ill patients requiring life support equipment • Metallic foreign bodies: Pacemakers, shrapnel, CNS vascular clips, metallic eye fragments, and cochlear implants are contraindications 15 MRI Contrast: Gadolinium (gadopentetic dimeglumine) is an ionic contrast agent that acts as a paramagnetic agent and enhances vessels or lesions with abnormal vascularity. Uses of MRI MRI is very sensitive to motion artifact; anxious or agitated patients may require sedation. Intramuscular glucagon may be used to suppress intestinal peristalsis on abdominal studies. If metallic eye fragments are possible, a screening CT of the orbits should be obtained prior to any MRI examination. It is generally contraindicated in patients with intracranial aneurysm clips, intraocular metallic fragments, and pacemakers. Dental fillings and dental prostheses have thus far not been a problem. Abdomen: Useful for differentiating adrenal lesions, staging tumors (renal, GI, pelvic), evaluation of abdominal masses, and virtually all intraabdominal organs and retroperitoneal structures. Useful in differentiating benign adenomas from metastasis Chest: Mediastinal masses, differentiates nodes from vessels, cardiac diseases, tumor staging, aortic dissection or aneurysm 15 Imaging Studies 333 Head: Analysis of all intracranial pathology may identify demyelinating diseases; some conditions are better evaluated by CT (see previous section), including acute trauma. MRS may increase the sensitivity of diagnosis of many neurologic diseases by providing a bio- chemical “fingerprint” of tissues in the brain. Performed in conjunction with an MRI equipped with the MRS capability. Some uses include differentiating dementias, tumors, MS, and many others. Musculoskeletal System: Bone tumors, bone and soft tissue infections, evaluation of joint spaces (except if a prosthesis is in place), marrow disorders, aseptic necrosis of the femoral head Pelvis: Evaluation of all pelvic organs in males and females. Differentiates endometrium from myoma and adenomyosis. Diagnosis of congenital uterine anomalies (eg, bicornuate, septate). Endorectal surface coil allows enhanced imaging of structures such as the prostate. Spine: Diseases of the spinal column (herniated discs, tumors, etc) NUCLEAR SCANS The following is a listing of some of the more commonly used nuclear scans and their pur- poses. Most are contraindicated in pregnancy; check with your nuclear medicine depart- ment. Adrenal Scan: Used to accurately localize a pheochromocytoma when MRI or CT is equivocal. Uses labeled MIBG ; patient must return several days later for imaging after ad- ministration. Bleeding Scan: Used to detect the source of GI tract bleeding. • 99mTC (technetium-99m) sulfur colloid scan. Used to detect bleeding of 0.05–0.1 mL/min. • 99mTC (technetium-99m)-labeled red cell scan. Same as sulfur colloid scan, but may be superior for localizing intermittent bleeding Bone Scan: Metastatic work-ups (cancers most likely to go to bone: prostate, breast, kidney, thyroid, lung); evaluation of delayed union of fractures, osteomyelitis, avascular necrosis of the femoral head, evaluation of hip prosthesis, to distinguish pathological frac- 15 tures from traumatic fractures Brain Scan: Metastatic work-ups, determination of blood flow (in brain death or ather- osclerotic disease), evaluation of space-occupying lesions (tumor, hematoma, abscess, [A- V] malformation), and encephalitis Cardiac Scans: Diagnosis of MI, stress testing, ejection fractions, measurement of car- diac output, diagnosis of ventricular aneurysms • Thallium-201 (201Tl). Examines myocardial perfusion via uptake of 201Tl by normal myocardium. Normal myocardium appears hot, and ischemic or infarcted areas cold. AMI (<12 h) seen as a hotspot, old MI (scar) seen as cold on both resting and exer- cise scans, and ischemia is cold on exercise scan and returns to normal after rest. • Technetium-99m pyrophosphate. Recently damaged myocardium concentrates 99mTc pyrophosphate, producing a myocardial hotspot. Most sensitive 24–72 h after AMI • Technetium-99m ventriculogram. 99mTc-labeled serum albumin or RBCs are used. Demonstrates abnormal wall motion, cardiac shunts, size and function of heart 334 Clinician’s Pocket Reference, 9th Edition chambers, cardiac output, and ejection fraction. Another form of this study is MUGA scan, data collection from which is synchronized to ECG, and selected aspects are used to create a “moving picture” of cardiac function. May be done at rest or during exercise stress test. Gallium Scans: Location of abscesses (5–10 d old), chronic inflammatory lesions, original lymphoma staging or follow-up for disease detection, lung cancer, melanoma, other neoplastic tissues Hepatobiliary Scans (HIDA-Scan, BIDA-Scan): Differential diagnosis of biliary obstruction (when bilirubin >1.5 and <7 mg/100 mL), acute cholecystitis, diagnosis of bil- iary atresia; NOT good for stones unless cystic duct is completely occluded and acute chole- cystitis present Indium-111 octreotide (OctreoScan): Imaging method for tumors with somato- statin receptors (pheochromocytoma, gastrinomas, insulinomas, small-cell lung cancer) I125 (Iodine-125) Fibrinogen Scanning: Used to detect venous thrombosis in the lower extremities. After injection of the tracer, the patient is scanned several hours and for several days after. Most useful to identify clot at or below the knees. False-positives with varicosities, cellulitis, incisions, arthritis, hematomas and with recent venography. Product availability is a problem at present. Liver–Spleen Scan: Estimation of organ size, parenchymal diseases (hepatitis, etc), abscess, cysts, primary and secondary tumors Lung Scan (V/Q Scan): Used along with a chest x-ray for evaluation of PE (a normal scan rules out a PE, an indeterminate scan requires further study via a pulmonary an- giogram, and a clear perfusion deficit coupled with a normal ventilation scan is highly probable for a PE). V/Q scans can provide evidence of pulmonary disease, COPD, and em- physema. Renal Scans: Agents are generally classified as functional tracers or morphologic tracers. 15 • 131I Hippuran. Primarily a renal function agent; useful in renal insufficiency for evaluation of function; visualization is poor, and radiation dose can be high • Technetium-99m glucoheptonate. Useful as a combination renal cortical imaging agent and renal function agent; primarily used to evaluate overall function, but can be used to determine vascular flow and to visualize the renal parenchyma and col- lecting system • Technetium-99m DMSA (dimercaptosuccinic acid). Used only as a renal cortical imaging agent • Technetium-99m DTPA (diethylenetriamine pentaacetic acid). Primarily a renal function agent; useful for renal blood flow studies, estimation of GFR, evaluation of the collecting system • Technetium-99m mercaptoacetyltriglycine (MAG3). A relatively new agent, pri- marily a functional agent, very good imaging of the renal parenchyma can be ob- tained within minutes of injection and a low radiation dose. May eventually replace all other renal agents. Strontium-89 (Metastron): Not technically an imaging agent, but used in the pallia- tive therapy of multiple painful bony metastasis (ie, prostate or breast cancer). Because this 15 Imaging Studies 335 is a pure beta emitter, the radioactivity remains in the body so no special precautions (other than blood and urine analysis) are needed. SPECT Scan: Single-photon emission-computed tomography, a technique whereby multiple nuclear images are sequentially displayed similar to a CT scan; can be applied to many nuclear scans. Thyroid Scan: Most commonly with technetium-99m pertechnetate. Useful for evalua- tion of nodules (solitary cold nodules require a tissue diagnosis because 25% are cancer- ous). Scan patterns in correlation with lab tests may help diagnose hyperfunctioning adenomas, Plummer’s and Graves’ diseases, and multinodular goiters; localize ectopic thy- roid tissues (especially after thyroidectomy for cancer); and identify superior mediastinal thyroid masses HOW TO READ A CHEST X-RAY Determine the Adequacy of the Film • Inspiration: Diaphragm below ribs 8–10 posteriorly and 5–6 anteriorly • Rotation: Clavicles are equidistant from the spinous processes • Penetration: Disc spaces are seen but bony details of spine cannot be seen PA Film Remember, the film is on the patient’s chest and the x-rays are passing from back (posterior) to front (anterior). The structures described in the following material are shown in Figure 15–1. Soft Tissues: Check for symmetry, swelling, loss of tissue planes, and subcutaneous air. Skeletal Structures: Examine the clavicles, scapulas, vertebrae, sternum, and ribs. Look for symmetry. In a good x-ray, the clavicles are symmetrical. Check for osteolytic or osteoblastic lesions, fractures, or arthritic changes. Look for rib-notching. Diaphragm: Sides should be equal and slightly rounded, although the left may be slightly lower. Costophrenic angles should be clear and sharp. Blunting suggests scarring or fluid. It takes about 100–200 mL of pleural fluid to cause blunting. Check below the di- 15 aphragm for the gas pattern and free air. A unilateral high diaphragm suggests paralysis (ei- ther from nerve damage, trauma, or an abscess), eventration or loss of lung volume on that side because of atelectasis or pneumothorax. A flat diaphragm suggests COPD. Mediastinum and Heart: The aortic knob should be visible and distinct. Widening of the mediastinum is seen with traumatic disruption of the thoracic aorta. In children, do not mistake the normally prominent thymus for widening. Mediastinal masses can be associated with Hodgkin’s disease and other lymphomas. The trachea should be in a straight line with a sharp carina. Tracheal deviation suggests a mass (tumor), goiter, unilateral loss of lung vol- ume (collapse), or tension pneumothorax. The heart should be less than one-half the width of the chest wall on a PA film. If greater than one-half, think of CHF or pericardial fluid. Hilum: The left hilum should be up to 2–3 cm higher than the right. Vessels are seen here. Look for any masses, nodes, or calcifications. Lung Fields: Note the presence of any shadows from CVP lines, NG tubes, pulmonary artery catheters, etc. The fields should be clear with normal lung markings all the way to the periphery. The vessels should taper to become almost invisible at the periphery. 336 Clinician’s Pocket Reference, 9th Edition 2 2 1 3 3 14 4 7 6 5 8 9 9 10 11 9 19 9 12 13 18 15 18 16 17 15 Posteroanterior Chest X-ray 1. Trachea 10. Left atrium 2. First rib 11. Right atrium 3. Clavicle 12. Right ventricle 4. Aortic knob 13. Left ventricle

5. Left pulmonary artery 14. Superior vena cava 6. Right pulmonary artery 15. Inferior vena cava 7. Carina 16. Gastric air bubble 8. Pulmonary trunk 17. Splenic flexure air 9. Pulmonary veins 18. Costophrenic angles 19. Descending aorta FIGURE 15–1 Structures seen on a posteroanterior (PA) chest x-ray film. 15 Imaging Studies 337 1 6 5 9 2 7 8 20 10 11 4 13 12 3 14 15 19 18 15 17 16 15 Lateral Chest X-ray 1. Manubrium 11. Left mainstem bronchus 2. Body of sternum 12. Right ventricle 3. Xiphoid process 13. Left atrium 4. Breast shadow 14. Left ventricle 5. Trachea 15. Right diaphragm 6. Scapula 16. Left diaphragm 7. Left pulmonary artery 17. Gastric air bubble 8. Ascending aorta 18. Costophrenic angles 9. Aortic arch 19. Inferior vena cava 10. Right pulmonary artery 20. Retrosternal clear space FIGURE 15–2 Structures seen on a lateral chest x-ray film. 338 Clinician’s Pocket Reference, 9th Edition Vessels in the lower lung should be larger than those in the upper lung. A reversal of this difference (called cephalization) suggests pulmonary venous hypertension and heart failure. Kerley’s B lines, small linear densities found usually at the lateral base of the lung, are associated with CHF. Check the margins carefully; look for pleural thickening, masses, or pneumothorax. If the lungs appear hyperlucent with a relatively small heart and flattening of the diaphragms, COPD is likely. Thin plate-like linear densities are associated with atelectasis. To locate a lesion, do not forget to check a lateral film and remember the “silhouette sign.” Obliteration of all or part of a heart border means the lesion is anterior in the chest and lies in the right middle lobe, lingula, or anterior segment of the upper lobe. A radiopac- ity that overlaps the heart but does not obliterate the heart border is posterior and lies in the lower lobes. Examine carefully for the following: 1. Coin lesions: Causes are granulomas (50% which are usually calcified), (histoplasmo- sis 25%, TB 20%, coccidioidomycosis 20%, varies with locale); primary carcinoma (25%), hamartoma (<10%), and metastatic disease (<5%). 2. Cavitary lesions: Causes are abscess, cancer, TB, coccidioidomycosis, Wegener’s gran- ulomatosis. 3. Infiltrates: Two major types a. Interstitial pattern. “Reticular.” Causes are granulomatous infections, miliary TB, coccidioidomycosis, pneumoconiosis, sarcoidosis, CHF. “Honeycombing” represents end-stage fibrosis caused by sarcoid, RA, and pneumoconiosis. b. Alveolar pattern. Diffuse, quick progression and regression. Can see either “but- terfly” pattern or air bronchograms. Causes are PE, pneumonia, hemorrhage or PE associated with CHF. Lateral Film Examine the structures shown in Figure 15–2. Use this study to check for the three-dimen- sional location of lesions. Pay close attention to the retrosternal clear space, costophrenic angles, and the path of the aorta. 15 16 INTRODUCTION TO THE OPERATING ROOM Sterile Technique Draping the Patient Entering the OR Finding Your Place The Surgical Hand Scrub Universal Precautions Preparing the Patient Latex Allergy Gowning and Gloving Working in the OR can be the best or worst experience of a clinical rotation. However, fa- miliarity with OR procedure is crucial to the success of any such experience. Preparing yourself before you get to the OR by knowing the patient thoroughly and having a basic un- derstanding of what is planned will greatly enhance your OR experience. Don’t fall into the trap of stereotyping the nurses as cranky, the surgeons as egotistical, and the medical stu- dents as stupid. Avoid this by learning the routine of the OR. Be alert, attentive, and, above all, patient. Soon, the routine should become second nature. Most importantly, don’t be afraid to admit to the scrub nurse and the circulating nurse that you’re new at this. They are usually more than happy to help you follow correct procedures. STERILE TECHNIQUE Members of the OR team, which includes the surgeon, assistants, students, and scrub nurse (the one who is responsible for passing the instruments and gowning the OR team), main- tain a sterile field. The circulating nurse acts as a go-between between the sterile and non- sterile areas. Sterile areas include • Front of the gown to the waist 16 • Gloved hands and arms to the shoulder • Draped part of the patient down to the table level • Covered part of the Mayo stand • Back table where additional instruments are kept The sides of the back table are not considered sterile, and anything that falls below the level of the patient table is considered contaminated. ENTERING THE OR From the moment you enter the OR, everything is geared toward maintaining a sterile field. The use of sterile technique begins in the locker room. Change into scrub clothing (remem- ber to remove T-shirts and tuck the scrub shirt into the pants). Be sure that the ties of the scrub pants are also tucked inside the pants. Scrub clothes may occasionally be worn on the wards, provided that they are covered by a clinic coat or some other form of gown, but you 339 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 340 Clinician’s Pocket Reference, 9th Edition need to check your hospital or departmental requirements on this. If you do wear scrub clothing out of the OR, be sure that it is not bloodstained. Pass into the anteroom to get your mask, cap, and shoe covers. The mask should cover your entire nose and mouth. Full hoods are necessary for men with a beard. The cap must cover all of your hair. Because of universal precautions, OR staff are now required to use protective eyewear while at the operative field. While wearing glasses, it is helpful to tape the mask to the bridge of your nose to prevent fogging during the surgery. Special masks are also available with self-adhesive strips to help prevent fogging of glasses. Tape the glasses to your forehead if you think they may be loose enough to fall onto the table during the op- eration! Do not wear nail polish, and remove any loose jewelry, watches, and rings before scrubbing. Make sure that shoelaces are tucked inside the shoe covers. The mask does not need to be worn in the hall of the OR suite (but everything else does) at most hospitals. The mask must be worn in the OR itself, near the scrub sinks, and in the substerile room between ORs. Find the operating room where the patient is located, and assist in transport, if neces- sary. Introduce yourself to the intern or resident and nurse, and try to get an idea of when to begin scrubbing (usually when the first surgeon starts to scrub). If you have a pager, follow the OR procedures and remove the pager if you are going to be scrubbed into the case. THE SURGICAL HAND SCRUB The purpose of a surgical hand scrub is to decrease the bacterial flora of the skin by me- chanically cleansing the arms and hands before the operation. Key points to remember: (1) If contamination occurs during the scrub, it is necessary to start over, and (2) In emer- gency situations exceptions are made to the time allowed for scrubbing (as in obstetrics, when the baby is brought out from the delivery room and the student is still scrubbing!). Caps and masks should be properly positioned before the start of the scrub. Povidone–Iodine (Betadine) Hand Scrub Scrubbing technique depends somewhat on local custom. Some ORs want a timed scrub in which the duration of scrubbing is determined by watching the clock. Other ORs use an “anatomic” scrub in which the duration of scrubbing is determined by counting strokes. Either is acceptable, and you should find out what the custom is at your institution. Timed Scrub 16 1. Perform a general prewash, with surgical soap and water, up to 2 in. above the elbows. 2. Use disposable brushes if available. Aseptically open one brush and place it on the ledge above the sink for the second half of the scrub. Open another brush and begin the scrub with Betadine. Use the nail cleaner to clean under all fingernails. 3. Scrub both arms during the first 5 min. Start at the fingertips and end 2 in. above the el- bows; pay close attention to the fingernails and interdigital spaces. Discard the brush and rinse from fingertips to elbows. 4. Take the second brush and repeat step 3. Always start at the fingertips and work up to the elbows. 5. Always allow water to drip off the elbows by keeping the hands above the level of the elbows. 6. Move into the OR to dry your hands and arms (back into the room to push the door open). 7. Scrubbing times: a. Ten minutes at the start of the day or with no previous scrub within the last 12 h and on all orthopedic cases 16 Introduction to the Operating Room 341 b. Five minutes with a previous scrub or between cases if you have not been out of the OR working with other patients Chlorhexidine (Hibiclens) 6-Min Hand Scrub (Timed) 1. Wet your hands and forearms to the elbows with water. 2. Dispense about 5 mL of Hibiclens into your cupped hands and spread it over both hands and arms to the elbows. 3. Scrub vigorously for 3 min without adding water. Use a sponge or brush for scrubbing, and pay particular attention to fingernails, cuticles, and interdigital spaces. 4. Rinse thoroughly with running water. 5. Dispense another 5 mL of Hibiclens into your cupped hands. 6. Wash for an additional 3 min. There is no need to use a brush or sponge at this point. Rinse thoroughly. Move into the OR back first to dry your hands. Anatomic Scrub 1. Perform a general prewash, with surgical soap and water, up to 2 in. above the elbows. 2. Use disposable brushes if available. Aseptically open one brush and place it on the ledge above the sink for the second half of the scrub. Open another brush and begin the scrub with Betadine. Use the nail cleaner to clean under all fingernails. 3. Each surface is to be scrubbed vigorously 10 times. Start with each finger (each of which has four surfaces), proceeding to the hand, the forearm, and the arm above the elbow. After finishing one extremity, do the other from fingers to above the elbow. Be sure to include all parts of your hand, especially the interdigital spaces. 4. Rinse both arms thoroughly. 5. Now rescrub each extremity, this time not going above the elbow. This is done in a sim- ilar fashion, 10 times on each surface from fingers to elbow. 6. Rinse thoroughly and proceed into the OR. PREPARING THE PATIENT The exact technique may vary in different medical centers, but the patient prep involves me- chanically cleansing the patient’s skin in the region of the surgical site to reduce bacterial flora. Ask the intern or resident to guide you through the procedure the first time, and con- 16 sider doing it yourself thereafter. It is always better to prep a wider area than you think nec- essary. For example, for a midline laparotomy, the patient is prepped from nipples to pubis, and from the flank at table level on one side to the table level on the other side. Materials Usually, a small prep table is present containing the following: • Gloves • Towels • Betadine or other scrub soap (optional) • Betadine or other paint solution • 4 × 4 gauze squares or sponges • Ring forceps (optional) 342 Clinician’s Pocket Reference, 9th Edition Technique 1. Patient prep is usually done before putting on the sterile gown. Don a pair of gloves, and scrub the area designated by the intern or resident for 4–6 min. Use the 4 × 4s (or sponges) and the soap solution. This is generally done three times with a gauze or sponge in each hand for a total of 4–6 min. Note, however, many times this traditional wound scrubbing is no longer performed routinely and is used only in specific circum- stances, such as, contaminated wounds. 2. Drape the area with a towel, and then gently pat the area dry if the wound was scrubbed. Taking care not to contaminate the area, gently peel off the towel from one side, being careful not to allow the

towel to fall back on the prepped area. Also be careful not to con- taminate your own arms, so that you do not have to rescrub before gowning. 3. Use 4 × 4s or sponges to paint the exposed area with the Betadine or other provided so- lution, using the proposed incision site as the center. Move circumferentially away from the incision site. Never bring the 4 × 4s back to the center after they have painted more peripheral areas. This is done as a series of concentric circles. Some centers will only “paint” and not “scrub” with the soap solution. Some surgeons want the paint dried with a towel at the end, and others like to leave it “wet.” Check with the resident or attending physician before you start, to find out exactly what is wanted. 4. When you are finished with the prep, remove your gloves in a sterile manner and pro- ceed to get your gown on. GOWNING AND GLOVING 1. If you have just completed the hand scrub, back into the room to push the door open; keep your hands above your elbows. 2. Ask the scrub nurse for a towel. Do not be impatient because the scrub nurse is often very busy. Stick out one hand, palm up and well away from the body. The nurse will drape the towel over your hand. 3. Bend at the waist to maintain sterility of the towel. It should never touch your clothing. 4. With one-half of the towel, dry one arm, beginning at the fingers; change hands and dry the other arm with the remaining half of the towel. Never go back to the forearm or hands after drying your elbows. 5. Drop the towel in the hamper. Again, remember to keep your hands above your elbows. 6. Ask for a gown and hold your arms out straight. The scrub nurse will place the gown 16 on you, and the circulator will tie the back for you. 7. The nurse will usually hold out a right glove with the palm toward you. Push your hand through the glove. Gloves come in different sizes–small (5¹₂–6¹₂), medium (7–7¹₂), and large (8–8¹₂)–and different materials: standard latex gloves, hypoallergenic (powder-free), reinforced (orthopedic), and latex-free. Ask the resident or scrub nurse for guidance on the type of glove to request. It is good form to ask the circulating nurse to open your gloves before you actually begin to scrub. 8. Repeat the procedure with the left glove. It is easier if you use two fingers of your gloved right hand to help hold the left glove open. 9. Visually inspect the gloves for any holes. 10. Give the scrub nurse the long string of your front gown-tie. Hold the other string your- self and turn around in place. Tie the strings. 11. The nurse may offer you a damp sponge to clean the powder off the gloves (the powder has been implicated in some postoperative complications, eg, adhesions). This varies by locale. 16 Introduction to the Operating Room 343 12. Now wait patiently; stay out of the way, and keep your hands above your waist. Hold them together to prevent yourself from accidentally dropping them or touching your mask. This is one of the most difficult things for the neophyte to remember. Be atten- tive. The only things that are sterile are your chest to your waist in the front and your hands to the shoulders. Your back is not sterile, nor is your body below the waist. Avoid crossing your arms. DRAPING THE PATIENT Draping the patient is usually done by the surgeon and assistants. Watch how they do it, and consider helping at a later date. It is harder to keep sterile than it looks. FINDING YOUR PLACE The medical student is often the “low man or woman on the totem pole” and, initially, usu- ally stands down by the patient’s feet. Ask the senior surgeon where you should stand. The first thing to remember is that once you are scrubbed, you must not touch anything that is not sterile. Put your hands on the sterile field and do not move about unnecessarily. If you need to move around someone else, pass back to back. When passing by a sterile field, try to face it. When passing a nonsterile field, pass it with your back toward it. If you are ob- serving a case and are not scrubbed in, do not go between two sterile fields, and stay about 1 ft away from all sterile fields to avoid contamination (and condemnation!). When not scrubbed in, it often helps to keep your hands behind your back, being careful not to back into the instrument table. Do not drop your hands below your waist or the table level. Do not grab at anything that falls off the side of the table–it is considered contaminated. If something falls, you can qui- etly inform the circulating nurse. Do not reach for anything on the scrub nurse’s small in- strument stand (the Mayo stand). You may ask for the instrument to be given to you. If someone says that you have contaminated a glove or anything else, do not move and do not complain or disagree. Remember that the focus of the OR is maintaining a sterile field, so if anyone says, “You’re contaminated,” accept the statement and change gloves, gown, or whatever is needed. If a glove alone is contaminated, hold the hand out away from the sterile field, fingers extended and palms up, and a circulating nurse will pull the glove off. The same is true if a needle sticks you or if a glove tears. Tell the surgeon and scrub nurse that you are contaminated and change gloves. If you have to change your gown, step away from the table. The circulator will remove first the gown and then the gloves. This procedure prevents the contaminated inside of the 16 gown from passing over the hands. Regown and reglove without scrubbing again. Always be aware of “sharps” on the field. When passing a potentially injurious instru- ment, always make the other members of the team aware that you are passing a sharp (ie, “needle back.” “knife back,” etc). Attempt to learn the names and functions of the common instruments. A knowledgeable student may be more likely to actively participate in the case. At the end of the case (once the dressing is on the wound), you may remove the gown and gloves but not the mask, cap, or shoe covers. To protect yourself, remove your gown first, and remove your own gloves last. This keeps your hands clean of any blood or fluids that got onto your gown during the procedure. Assist in the transfer of the patient to the postoperative recovery room. Postop orders and a brief operative note are written immedi- ately. See Chapter 2 (page 36) on how to write postop notes and orders. It is good form to offer to write the postop note and orders if you are comfortable with the process. Due to governmental regulations that affect attending physicians at teaching hospitals, the attending must often write the note him or herself. At the very least, the attending of record will 344 Clinician’s Pocket Reference, 9th Edition annotate an “attestation” to your note saying that the surgeon was “personally present dur- ing the critical portions of the procedure.” UNIVERSAL PRECAUTIONS All operating room personnel are at risk for infection with blood-borne agents responsible for such diseases as AIDS and hepatitis. To reduce the incidence of such transmission, a set of guidelines called Universal Precautions has been developed by the CDC. The underlying principle is that, because patients cannot be routinely tested for HIV and are rarely tested preoperatively for hepatitis, the safest policy is to treat all patients as though they are in- fected with these agents. This approach ensures evenhanded treatment of all patients and the safest work environment for those who are exposed to the blood of others. Minimizing the risks to all who are in the OR requires constant vigilance. Movements must be coordinated among surgeon, assistant, and technician. Fingers are never used to pick up needles; this is done only with another instrument. Fingers and hands should not be used as retractors. Two people should never be holding the same sharp instrument. Placing a sharp instrument down or handing it to another member of the team is always preceded by a verbal warning that notifies the recipient that a sharp object is about to be passed. Protective eyewear must be worn by all members of the operating team. The practice of “double gloving” is often reserved for cases in which the patient is known to carry a transmissible agent. This technique definitely reduces the incidence of blood–skin contact, especially in light of the extraordinarily high incidence of unrecognized glove perforations. Until puncture-resistant gloves are developed, this is the best approach we have. LATEX ALLERGY Individuals with certain medical conditions or occupations that are heavily exposed to prod- ucts containing natural rubber latex may became sensitized to it and develop allergic reac- tions. Up to 7% of health care workers can have allergic reactions. Certain conditions (ie, spina bifida, cerebral palsy) predispose patients to an 18–40% incidence of allergy. Reac- tions can vary from mild rash and itching to anaphylaxis. Latex products are found in a wide array of products, from gloves and drapes, to IV tubing. Occasionally patients will have documented latex allergy. Hospitals have latex allergy protocols, and hospitals maintain an inventory of latex-free products. 16 17 SUTURING TECHNIQUES AND WOUND CARE Wound Healing Surgical Knots Suture Materials Suture Removal Suturing Procedure Tissue Adhesives Suturing Patterns WOUND HEALING The process of wound healing is generally divided into four stages: inflammation, fibroblast proliferation, contraction, and remodeling. There are three different types of wound healing: primary intent (routine primary suturing); secondary intent (the wound is not closed with su- ture and closes by contraction and epithelialization, most often used for wounds that are in- fected and packed open); and tertiary intent (also called delayed primary closure; the wound is left open for a time and then sutured at a later date, used often with grossly contaminated wounds). SUTURE MATERIALS Suture materials can be broadly defined as absorbable and nonabsorbable. Absorbable su- tures can be thought of as temporary; these include plain gut; chromic gut; and synthetic materials such as polyglactin 910 (Vicryl), polyglycolic acid (Dexon), and poliglecaprone (Monocryl). These are resorbed by the body when left internally after a variable period (Table 17–1). Polydioxanone (PDS) is a long-lasting absorbable suture. Nonabsorbable su- tures can be thought of as “permanent” unless they are removed; these include silk, stainless steel wire, polypropylene (Prolene), and nylon (see Table 17–1). The size of a suture is defined by the number of zeros. The more zeros in the number, the smaller the suture. For example, a 5-0 suture (00000) is much smaller than a 2-0 (00) suture. 17 Most sutures come prepackaged and mounted on needles (“swaged on”). Cutting nee- dles are used for tough tissues such as skin, and tapered needles are used for more delicate tissues such as the intestine. The most common needle for skin closure is the ³₈ in. circle cutting needle. SUTURING PROCEDURE The following guidelines cover the repair of lacerations in the emergency setting. Similar principles hold true for closure of wounds in the operating room. The choice of appropriate suture material is based on many factors, including location, extent of the laceration, strength of the tissues, and preference of the physician. 345 Copyright 2002 The McGraw-Hill Companies, Inc. Click Here for Terms of Use 17 346 TABLE 17–1 Common Suture Materials ABSORBABLE Suture (Brand Name) Description Tensile Strength* Absorbed† Common Uses Fast catgut Twisted/Fast 3–5 d 30 d Facial lacerations in children absorption Plain catgut Twisted/Rapidly 7–10 d 70 d Vessel ligation, subcutaneous absorbable tissues Chromic catgut Twisted/absorbable 10–14 d 90 d Mucosa Polyglycolic acid Braided/Absorbable 14–21 d 60–90 d GI, subcutaneous tissues (Dexon) Polyglactin 910 (Vicryl Braided/Absorbable 5 d 42 d Skin repair needing rapid Rapide) absorption Polyglactin 910 (Vicryl) Braided/Absorbable 21 d 56–70 d Bowel, deep tissue Poliglecaprone 25 Monofilament/ 7–14 d 91–119 d Skin, bowel (Monocryl) Absorbable Polydioxanone (PDS) Monofilament/ 28 d 6 mo Fascia, vessel