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CLIN.CHEM. 26/6,691-699 (1980) The Expanding Role of Microsomal Enzyme Induction, and Its Implications for Clinical Chemistry David M. Goldberg Microsomal enzyme induction, a term denoting the ability of the substrate for a microsomal enzyme to enhance the activity of that enzyme and frequently of related enzymes, has been demonstratQd in a wide range of tissues, notably the liver, placenta, small intestinal mucosa, and peripheral lymphocytes. The major agents that cause microsomal enzyme induction are drugs and xenobiotics. Factors modulating the extent of enzyme induction by a given agent include age and nutrition, and wide species variations are encountered with different inducing agents. Markers for microsomal enzyme induction include determination of the plasma half-life for conveniently measured drugs, and the measurement of endogenous metabolites such as 6flhydroxycortisol and D.glucaric acid in 24-h urine collections. While these are valuable for monitoring enzyme induction in healthy patients, they are altered in certain forms of liver disease, and results must then be interpreted with caution. Microsomal enzyme induction may interfere with reference values, particularly for membrane-bound enzymes, in otherwise healthy populations, and may play a role in metabolic bone disease, drug interactions, carcinogenesis, and hypertriglyceridemia. Drug therapy of the neonatal and congenital hyperbilirubinemias has been inspired by the mechanism of hepatic microsomal enzyme induction, and “markers” for enzyme induction can be used to monitor drug compliance. The activity of serum y-glutamyltransferase seems to be especially valuable for this purpose. Additional Keyphrases: aminopyrine anticonvulsants antipyrine half-life aryl hydrocarbon hydroxylase ‘ 3,4benzpyrene ‘ carcinogenesis cigarette smoking ‘ cytochrome P450 - phenytoin . drug interaction epilepsy epoxide hydrase ‘ ethanol D-glucaric acid #{149} y -glutamyltransferase 63-hydroxycortisoI hyperbilirubinemia hypertriglyceridemia . metabolic bone disease phenobarbital . polycyclic hydrocarbons theophylline tricyclic antidepressants What Is “Microsomal Enzyme Induction”? The term “microsomal enzyme induction” denotes the ability of a substrate for a microsomal enzyme to enhance the activity of that enzyme-and frequently of related enzymes-by promoting their de novo synthesis (J_3),1 This, The Department of Biochemistry, The Hospital for Sick Children, 555 University Ave., Toronto; and the Department of Clinical Biochemistry, The University of Toronto, Toronto, Ontario, Canada. 1 Ed. note: History repeats itself. There was a large flurry of interest, some 150 papers, in “induced” enzymes in the first half of this century (cf, e.g., the reviews by Abderhalden: Abwehrfermente, Verlag Steinkopff, Dresden, 1944, and Ergeb. Enzymforsch. 11: 1, 1950). Then it died away, although there w,as later work on “adaptive” enzymes, principally in microbiology. Received and accepted Feb. 12, 1980. in turn, may lead to a dramatic increase in the endoplasmic reticulum of the affected cells, classically seen in the liver of the phenobarbital-treated rodent (4), although extensive morphological changes accompanying microsomal enzyme induction are the exception rather than the rule. Most of the agents recognized as inducers of microsomal enzymes are foreign to the body and are knowingly ingested as drugs or are fortuitously ingested as environmental and food contaminants. The number of compounds in both categories is increasing at an alarming rate as our society proliferates the use of therapeutic and abused drugs and of chemical substances in industrial processes and agriculture that can enter the human food chain. Current research is uncovering the enzyme-inducing properties of these agents as awareness of the problem by government authorities and the scientific community expands. Some representative examples are shown in Table 1. Where Does It Occur? Although most of our knowledge of microsomal enzyme induction has come from recognition of its effects upon the liver, other tissues such as placenta, small intestinal mucosa, and peripheral lymphocytes may show similar responses to environmental agents. The availability of the latter cells for study without the need for invasive biopsy procedures has stimulated interest in their diagnostic potential as “markers” of the body’s exposure and response to carcinogenic hydrocarbons. The clinical chemist is being challenged on many fronts to an awareness of the phenomenon and its scope. He should understand the subtle as well as the more obvious implications this has for his interpretation of laboratory data and his role in developing new test procedures to evaluate enzyme induction where this is of help in the diagnosis and management of patients. The Microsomal “Drug Hydroxylation System” For an understanding of microsomal enzyme induction, it is necessary to describe the hepatic drug hydroxylation system as a prototype. This is shown schematically in Figure 1. The reaction involves molecular oxygen, one atom of which is used to hydroxylate the drug, while the other atom is used to generate water from reduced pyridine nucleotides. The electrons required to reduce the free atom of oxygen generally come from NADPH, but under certain circumstances NADH can also provide the necessary reducing equivalents. Where NADP is the source of electrons, the flow is through the enzyme cytochrome c reductase (EC 1.6.99.3) to a group of cytochromes that are characterized by their spectral properties and that also differ in enzyme specificity. The best characterized are cytochrome P450, the terminal electron-transport system for barbiturate hydroxylation, and cytochrome P448, the terminal electron-transport protein for hydroxylation of aromatic hydrocarbons, especially those that have carcinogenic properties. The function of this system in general terms is to detoxify drugs and foreign compounds and to render them more water-soluble, to facilitate their excretion by the kidney. A CLINICAL CHEMISTRY, Vol. 26, No. 6, 1980 691 NADPH NADH - Cytochrome reductase c Cytochrome reductase b5 Lipid e.-Cytochrome Cytochrome P-450 P-448 _____ b5 --CN- sensitive factor -.02 Fig. 1. Pathwaysand components of hepatic microsomal electron-transport system utilized in drug hydroxylation reactions From ret. 1 with kind permission of the authors and publishers (Karger, Basel) number of qualifications must be made, to put this statement into proper perspective: (a) The chemical reactions of which this system is capable go far beyond simple hydroxylation and include demethylation, de-ethylation, epoxidation, deamination, and dehalogenation, although all these reactions can be generically regarded as catabolic. (b) Metabolism of a drug or carcinogen by this system may actually enhance its biological potency. (c) Other chemical reactions that do not primarily involve the hepatic cytochrome P450 drug-hydroxylation system, such as sulfation and glucuronidation, may be used by the liver to detoxify foreign compounds. (d) The cytochrome P450 system and its congener hemoproteins are found in tissues other than the liver, in organelles other than the microsomes, and are involved in the hydroxylation of important endogenous compounds such as cholesterol and the steroid hormones (for example, by adrenal mitochondria), and of heme itself by the heme oxygenase system of macrophages, which converts heme to biliverdin and has been shown to be substrate-inducible. Drugs and Enzyme Induction Compounds metabolized ylation system are capable system, and thereby the by the microsomal drug-hydroxof inducing the components of the metabolism of many other com- Table 1. Range of Microsomal Enzyme-Inducing Agents Demonstrated in Man pounds that act as substrates for the same metabolic pathway. Administration of phenobarbital induces the enzyme hexobarbital oxidase and cytochrome P450, as well as many other enzymes that feed into the cytochrome P450 system. The carcinogen 3,4-benzpyrene induces the enzyme aryl hydrocarbon hydroxylase and cytochrome P448, as well as the activity of many other enzymes which feed into the cytochrome P448 pathway. Compounds that are strong inducers of cytochrome P450-related enzymes are weak inducers-or even inhibitors-of cytochrome P448-related enzymes. However, many of these interactions vary with the agent and show marked differences among animal species. The mechanism of this induction is not known in detail. It seems to depend upon de novo protein synthesis, because it can be blocked by agents such as actinomycin D, cycloheximide, and puromycin (1), which inhibit one or other of the steps in transcription or translation of the genetic code. One proposal is that the inducing agent acts to relieve repression of enzyme synthesis; another suggestion is that binding of the exogenous inducer at the active site blocks the binding of an endogenous inducer (possibly a lipid peroxide), which thus accumulates and stimulates synthesis of the enzyme until a new steady-state is reached (3). It is even more difficult to explain the increase in non-heme microsomal enzymes such as glucuronyl transferases, and in plasma membrane enzymes such as y-glutamyltransferase (EC 2.3.2.2), as well as the generalized increase in microsomal protein that occurs with some inducing agents, notably phenobarbital. How Specific Are the Microsomal Hemoproteins? Relaxant (muscle) Sedatives Phenylbutazone ‘iseofuIvin Rifampicin Warfarin Alcohol Marijuana Polychlorinated and polybrominated biphenyls Dioxane Progesterone DDT Tobacco smoke Coal tar paste Barbecued foods Meprobamate Barbiturates The relationship between the number of cytochrome P450 enzyme proteins and the wide range of activities catalyzed by the microsomal drug-hydroxylation system is not yet resolved. It is inherently unlikely that there is a specific hemoprotein enzyme available for each metabolizable drug or chemical substance, because most of these are compounds to which the body has not previously been exposed. Although the number of individually recognized cytochrome P450 congeners is approaching double figures, this still falls considerably short of the number required to support the known specific reactions of the drug hydroxylation system. An alternative possibility is that there are different binding proteins for each compound or class of compound, and that these binding proteins in turn react with only one of the known forms of cytochrome P450. Many fascinating reconstitution experiments have been performed with animal materials to obtain deeper insight into this issue. Studies of this type are now becoming feasible with human liver. Cytochrome P450 from adult (5, 6) and from fetal (7) human liver was purified by using chromatography on Octyl-Sepharose 4B as the initial step. The fetal hemoprotein differed from the adult form in that it alone catalyzed hydroxylation of aniline in a reconstituted system. A com- Methaqualone ponent Stimulants Glutethimide Nikethamide (P450) reductase (EC 1.6.2.4) from human liver; this enzyme proved capable of using cytochrome P450 and cytochrome P448 for different catalytic reactions (5), and it therefore Class of compounds Anticonvulsants Some Individual agents Phenobarbital Anti-inflammatory Phenytoin Aminopyrine Antifungal Ant!biotlcs Anticoagulants Agents of addiction Industrial products Oral contraceptives Pesticides Polycyclic hydrocarbons 692 CLINICAL CHEMISTRY, Vol. 26, No. 6, 1980 of this system was purified NADPH-cytochrome c seems that substrate specificity and hemoprotein specificity are not mediated through different reductase enzymes. Another achievement has been the purification of epoxide hydratase (EC 3.3.2.3) from human liver microsomes, although some minor contaminants were still present (8). The enzyme preparation was active with a wide range of alkene and arene oxides. What Factors Affect Enzyme Induction? Enzyme inducibility shows marked species-related differences and, in man, genetic factors exercise a paramount influence (9). Dependency upon age has also been clearly demonstrated. Pregnancy and seasonal factors modulate the response to enzyme-inducing agents. So do nutritional factors, with fat (10), protein (11), and carbohydrate (12) all being important and relatively independent of each other or of their caloric equivalents. Of those vitamins so far examined, only vitamin E has been shown to exercise-a role in enzyme induction in the experimental animal (13). How Can We Recognize Induction? Microsomal Enzyme Recognition of hepatic microsomal enzyme induction stems from the availability of some analytical tools for studying this process. The activity of drug hydroxylation reactions can be indirectly assessed by measuring the plasma half-life of the drug after its injection. After equilibration in the appropriate distribution space, its concentration in the plasma decays according to first-order kinetics. Although several factors such as renal and biliary excretion contribute to this clearance, in most instances it is the activity of the liver drug-metabolizing system that is rate limiting. Measurement of drug half-lives involves the semi-invasive technique of injection followed by frequent blood sampling, although in the case of some drugs, such as aminopyrine, the availability of a 14C-labeled form of the drug enables an index of its metabolism to be obtained by measuring the excretion of labeled carbon dioxide in the breath (14, 15). Discrepancies are seen between disappearance rate of 14cJO2 from the breath and aminopyrine clearance from plasma because the aminopyrine is labeled at two methyl groups, which are demethylated at different rates (16). For this reason, [14C-methoxy]glycodiazine (glymidine) has been proposed as a more reliable compound because its plasma clearance correlates well with terminal disappearance of ‘4C02 from the breath. With antipyrine and with some other drugs, salivary measurements can be used instead of blood measurements to follow drug clearance (17). This has been validated by comparison of the clearance rates of the drug and its three major metabolites in plasma, saliva, and urine (18). However, for drugs in general, the pKa and the extent of protein binding must be considered in deciding the suitability of substituting salivary for plasma clearance measurements. Endogenous factors that can be measured are the urinary excretion of 6$-hydroxycortisol and D-glucaric acid. Cortisol is mainly metabolized to 17-hydroxy-corticoids, but a small fraction is converted in liver microsomes to the alternative excretory product, 6f3-hydroxycortisol, and this fraction is greatly increased during enzyme induction. There is growing interest in this compound as an index to steroid hormone metabolism as well as a marker of microsomal enzyme induction (19), and its study has been greatly facilitated by the development of radioimmunoassays for its measurement in urine and plasma (20,21). D-Glucaric acid is the main excretory product of the glucuronic acid pathway, which in mammals other than primates and guinea pigs goes on to ascorbic acid. This pathway, and therefore formation of its terminal product D-glucaric acid, is greatly stimulated in situations where microsomal enzyme induction occurs, for reasons that are not entirely clear, aside from its known location in the endoplasmic reticulum. There is a need for better methods than those currently used and which are predominantly based upon the ability of the lactone formed from D-glucaric acid (by heating the urine after acidification) to inhibit the enzyme /3-D-glucuronidase (EC 3.2.1.31) (22). A method based on gas-liquid chromatography suffers the disadvantage of converting D-glucaric acid into a series of different lactones, which then have to be individually quantitated (23); thus the procedure is inherently a complicated one. As Table 2 demonstrates, the normal ranges for these tests are not very reproducible and are based on small numbers. Closest agreement among different laboratories exists for antipyrine half-life, which currently has to be #{235}onsideredas the reference procedure for demonstrating microsomal enzyme induction. However, the increases in excretion of 6/3hydroxycortisol and D-glucaric acid usually exceed the decreases in drug half-lives. Although reference ranges for these chemical markers of enzyme induction are not very useful, important information can be obtained by sequentially measuring these constituents in the same individual. The most direct and unequivocal evidence of microsomal enzyme induction is afforded by demonstrating increased activity of the enzyme responsible for metabolizing the inducing agent or the appropriate form of cytochrome P450 to which it is related. This is seldom possible in human patients. It is important to Table 2. Values for Indicators of Hepatic Microsomal Enzyme Induction Reported In Healthy Untreated Control Subjects (mean ± SD) Antipyrine Indicator8 half-life (h)” Reference no. 11.5 ± 2.1 (n = 15) 14.1 ± 2.7 (n = 7) 18 24 11.4± 3.0(n = 10.9 ± 1.5 (n = 25 26 27 8) 16) 14) 15) 31) 6) 12.1 ± 3.9 (n = 11.3 ± 3.1 (n = 12.0 ± 4.3 (n = 10.8 ±1.1 (n = Paracetamol half-life 28 29 30 (h) 2.5 ± 0.7 (n = 31) Phenylbutazone 29 half-life (h) 81 ± 16 (n = 18) 64 ± 12 (n = 12) 6/3-Hydroxycortisol 185 ± 72 (n excretion (t 9/ 24 h) 32 18) = 320 ± 160 (n 32 33 = 34 20 14) 286 ± 54 (n = 10 males) 233 ± 80 (n = 8 females) 487 ± 145 (n = 6) o-Glucaric acid excretion 20 21 (mol/24 34.3 ± 16.0 (n = 18) 53.0 ± 10.0 (n = 16 males) 41.0±8.0(n= l4females) 56.1 ± 15.6 (n = 38 males) 32.5 ± 4.9 (n = 19 males) 9.9 ± 3.7 (n = 17) a b h)C 35 36 36 37 38 39 Based on mixed male and female populations unless otherwise stated. All resultsbased on serum clearance except first, which Is based on salivary clearance. C Some discrepancies due to difterences arising from standardization with D-glucaric acid or with its lactone. CLINICAL CHEMISTRY, Vol. 26. No. 6, 1980 693 Table 3. Influence of Alcohol on t1/ Clearance of, Drugs from the Plasma (mean ± SD, in hours; from refs. 60 and 61) Alcoholics Tolbutamide 2.8 ± 0.5 (n31) Non-alcoholics 5.8 ± 2.2 (n13) Warfarin 26.5 ± 13.3 (n = 15) 41.4 ± 19.2 (n = 11) Phenytoin 16.3 ± 6.8 (n = 15) 23.5±11 (n = 76) note that the procedures listed in Table 2 do not always show a close correlation with induction of the specific enzyme in animal experiments, or amongst each other in human studies. This is due to several factors: (a) Each procedure probably is subject to a different dose relationship. (b) Each is subject to a different time course and may show increase early, late, or only for a relatively limited period after exposure to the inducing agent. (c) All are indirect and therefore are subject to influences of the inducing agent other than that of microsomal enzyme induction alone. (d) They may be differentially affected by disease, or physiological states such as age or pregnancy. What Are the Effects of Disease on Enzyme Induction? This point has received much attention recently because clinicians have become interested in examining drug metabolism as an index of hepatic function. By use of antipyrine clearance, hepatic drug metabolism was shown to be impaired in cholestatic patients, but the impairment was reversible and the liver remained sensitive to microsomal enzyme inducers (27). Patients with advanced cirrhosis and chronic active hepatitis (but not compensated cirrhosis or acute hepatitis) manifested reduced antipyrine clearance, which correlated with and frequently appeared earlier than abnormalities in prothrombin time (28, 40). However, in line with a study on malnourished children (41), antipyrine clearance in these patients was found to be greatly prolonged if the diet was inadequate. Although the authors failed to find an effect exercised by other environmental factors such as use of alcohol and cigarette smoking, this is contradicted by another report in which the [t4Claminopyrine breath test was used, where clearance was found to be faster in cirrhotic patients who were consuming alcohol or enzyme-inducing drugs (42). By contrast, oral contraceptives and therapeutic doses of propranolol seem to slow the clearance rate of antipyrine (30, 31), while the delayed clearance of three model analgesics in a group of cirrhotic patients was shown to be associated with portalsystemic shunting of blood, although this was not established as the sole contributing factor (43). Again, ethanol enhances acetylation of sulfamethazine in man and may obfuscate the patient’s true “acetylator status” (44). With the above complexities in mind, microsomal enzyme induction clearly is one of several phenomena that may prevent accurate assessment of the intrinsic ability of the liver to metabolize drugs in health and disease. Because hepatic disease alters drug metabolism, this in turn may lead to erroneous conclusions when drug clearances are used to monitor enzyme induction rather than to test hepatic function. In both endeavors, the need for a detailed nutritional, social, and drug 694 CLINICAL CHEMISTRY, Vol. 26, No. 6, 1980 history of each patient hardly requires emphasis. Nevertheless, an experimental study recently reported in this journal (45) claimed that antipyrine half-life was a more sensitive index of hepatic damage caused by cytotoxic drugs than were several serum enzyme assays, including alkaline phosphatase (EC 3.1.3.1), y-glutamyltransferase, aspartate aminotransferase (EC 2.6.1.1), and glutamate dehydrogenase (EC 1.4.1.3). Not only drug metabolism but also the excretion of D-glucane acid is affected by disease. Apart from the obvious errors of interpretation likely in patients with renal disease, significantly increased excretion has been reported in all jaundiced patients, most notably where this is obstructive (39), and also in patients with certain forms of porphyria (46). What Are the Consequences Enzyme Induction? of Microsomal (a) Interference with reference values. Microsomal enzyme induction increases the activities in serum of some enzymes commonly used in diagnosis, especially in diagnosis of liver disease (46-52). Gamma-glutamyltransferase is the enzyme in serum that is most frequently and dramatically affected, and this poses problems in defining reference ranges for this enzyme because even moderate drinking of alcohol can increase its activity (53). Other membrane-associated enzymes such as alkaline phosphatase and 5’-nucleotidase (EC 3.1.3.5) may be more active in the serum of patients who are taking such drugs (47). It is important to recognize this relationship and to avoid the over-investigation of such abnormalities in the absence of clinical corroboration of liver disease. Serum ‘y-glutamyltransferase activity can serve as a convenient marker of microsomal enzyme induction (51, 54-56). It is simpler to measure than the urinary constituents and the results are promptly available. It is the most convenient and cheapest index of microsomal enzyme induction currently available, although it will become more abnormal in hepatobiliary disease than will the other indices described above. (b) Metabolic bone disease. The relationship between metabolic bone disease and anticonvulsant therapy has been recognized for the past decade. It may depend upon the rapid metabolism of vitamin D by hepatic hydroxylating enzymes to inactive metabolites. Patients receiving anticonvulsants show a much faster half-life of vitamin D3 than do normal subjects (57) and their serum calcium concentrations are lower (58). In such patients, there is significant inverse correlation between serum calcium concentration and serum y-glutamyltransferase activity (59), convincing evidence that the enzyme induction and the hypocalcemia are directly connected. (c) Drug interactions. This is well illustrated by the effects of chronic alcohol consumption on drug metabolism. Table 3 shows that the clearance of three drugs known to be metabolized by the hepatic drug-hydroxylation system is enhanced because of the powerful enzyme-inducing effect exercised by ethanol (60, 61). The data were obtained when the patients were not actively drinking alcohol. When alcohol and the drug are given simultaneously, drug clearance is prolonged because the alcohol acts as a competitive substrate inhibitor of the drug-metabolizing system. This explains why it is dangerous to take barbiturates along with alcohol, although the chronic alcoholic is resistant to barbiturate as long as he is not drinking alcohol at the same time. Plasma warfarin half-life is dramatically lowered by various enzyme-inducing drugs (62). Patients receiving such drugs will require larger doses of warfarmn to achieve the desired change in prothrombin time. If the enzyme-inducing drug is withdrawn while the warfarin is continued at the same dosage, the increase in half-life that follows will lead to the dose being inappropriately high, with risk of fatal hemorrhage. j 11l.T.J 2000 %..:.I9RI1Qtct LHNo8ARBITONE180mDAfl.,Y1 30 F. 25 15 10 5 0 0 2 4 6 8 10121416 1820 TIME (DAYS) Fig. 2. Response to calorie restriction and to phenobarbital (phenobarbitone)of serum bilirubin concentration in patient with Criggler-Naj jar disease Type II From ref. 91 with kind permission of the authors andof the publishersof Gas- troenterology (d) Cigarette smoking. It has been known for some time that cigarette smoking affects the disposition of certain drugs by enhancing their clearance through microsomal enzyme induction (9, 63), theophylline being a striking example (64). Smoking halves the mean plasma concentration of propanolol for a constant drug dose, but this effect diminishes with age due to an age-dependent resistance to enzyme inducibility (65). A similar age-dependent resistance to the enzyme-inducing properties of cigarette smoke has been observed with antipyrine clearance (66). The enzyme-inducing properties of tobacco smoke are predominantly ascribable to its content of 3,4-benzpyrene, and because this compound is also generated by the charcoal broiling of meat, persons eating such food also have faster rates of drug clearance (67). Moreover, it is present in coal tar, and when this is applied to human skin in usual therapeutic doses, it increases by two- to five-fold the activity of the enzyme aryl hydrocarbon hydroxylase (AHH; EC 1.14.14.1), which catalyzes the initial step in benzpyrene metabolism (68). This enzyme is present in the epidermis rather than the dermis, and it increases with age in healthy humans (69). Activities of this enzyme in the placentas of women who smoked during pregnancy were more than 25-fold those of non-smokers, and the effect seemed to be dose-dependent in that activities were greatest among mothers who smoked the most (70, 71). Indeed, placental AHH can be used as a measure of fetal exposure to maternal cigarette smoking (72). Much effort is being expended in measuring the AHH activity of peripheral blood lymphocytes, monocytes, and macrophages, directly as well s in cultured and mitogen-stimulated preparations of these cells (73, 74). A good correlation with cigarette consumption is revealed by these studies. AHH activity in lymphoblasts showed high absolute induction in 39% of patients with untreated lung cancer but in only 15% of normal subjects, and the frequency in patients with other malignancies was not different from that of controls (75). Indices of enzyme induction are thus being used as a measure of cancer risk among smokers, although the current procedures need to be greatly refined to achieve acceptable predictive value. (e) Carcinogenesis. The relationship between carcinogenesis and enzyme induction is very complex. Many aromatic hydrocarbons that can induce experimental cancer in animals are metabolized by the microsomal hydroxylating system to inactive compounds. If the hydroxylating system has already been activated by enzyme-inducing drugs, conversion to inactive metabolites will be faster and tumor development will be slower (76, 77). The opposite situation may also apply. Compounds such as polycyclic hydrocarbons are metabolized by microsomal enzymes to active carcinogens (78), and this conversion will be accelerated where microsomal enzyme induction has occurred. Environmental agents may be responsible for as many as 90% of human cancers (79), and because the metabolism of these agents is highly dependent upon microsomal enzymes, the interaction between carcinogenesis and microsomal enzyme induction could be the most fruitful area of cancer research in the next decade. (I) Hypertriglyceridemia. A relationship between microsomal enzyme induction and hypertniglyceridemia has been proposed in several clinical states, including hypertension (80), diabetes mellitus (81), and use of oral contraceptives (82). Support has been provided by other authors (83,84) and has been extended by the original authors (85, 86). Because hypertniglycenidemia is a risk factor for ischemic heart disease, this association could represent an unfavorable influence of microsomal enzyme induction upon survival in human populations. What Are the Uses of Microsomal Induction? Enzyme (a) Treatment of hyperbilirubinemia. Enzyme induction has been used in the treatment of subjects with unconjugated hyperbilirubinemia. These included neonates (87), patients with Gilbert’s disease (88), and patients with Criggler-Naj jar Type II disease (89). Enzyme-inducing drugs increase not only the cytochrome P450 content of human liver (90), but also its ability to conjugate bilirubin (since this is an enzymic function of the microsomes), which leads to a dramatic decrease in serum bilirubin concentration (91). This was valuable in treating neonatal hyperbilirubinemia before phototherapy was introduced for this condition. It still has a role in the treatment of Cniggler-Najjar Type II patients. Microsomal enzyme induction has been used prophylactically in mothers whose infants are expected to suffer from hyperbilirubinemia. Phenobarbital (92) and ethanol (93) have been given to mothers for this purpose, and lead to lower bilirubin concentrations in the serum of their offspring than those in control infants. When the carbohydrate content of the diet is drastically decreased, the concentration of unconjugated bilirubin in serum increases. The increase is greater in patients with Gilbert’s disease, readily distinguishable from the much smaller increase in normal subjects (Figure 2), and this provides a useful diagnostic test for Gilbert’s disease, particularly when the patient is anictenic (94). Recent experimental work has shown that fasting diminishes the supply of UDP-glucuronic acid available for bilirubin conjugation (95) and this stresses the already compromised bilirubin UDPglucuronosyltransferase system of patients with Gilbert’s disease, whereas the normal subject is better able to cope with this unfavorable situation. (b) Monitoring of drug compliance. Enzyme induction as an index of compliance with prescribed therapy has been well defined in the treatment of alcoholism. This is related to the very high serum y-glutamyltransferase activity occurring in CLINICAL CHEMISTRY, Vol. 26, No. 6, 1980 695 100 100S . 2 90- S 90 0 . S E S . N 0 60- 0 E a S 0 . ‘in 0 30S S a C S a > a 2 30- : :. bDEQUAT Too ioo DOSAGE MUCH LITTLE I (CONTROLS) (EPILEPTICSON ANTICONVULSANTS) I : Fig. 3. Urinary o-glucaric acid excretion in control subjects and in epileptic patients short’y after starting anticonvulsant therapy on a standard dose In relation to control of seizures and presence of side effects From ref. 102,with kind permissionof Masson Publishing Co chronic alcoholics, which declines towards normal on withdrawal from alcohol. Thus programs for the rehabilitation of alcoholic patients can be devised in which these measurements are used as a valuable aid in judging the effectiveness of the regime (96-98). However, recent reports have emphasized the need for caution in using such protocols, because the decline in serum ‘y-glutamyltransferase activity on withdrawal from alcohol may not be as consistent or as prompt as the earlier authors suggested (99, 100). The role of y-glutainyltransferase as an index of microsomal enzyme induction has been emphasized in many recent publications (38-42,54-56), and, as I indicated earlier, this strongly limits its utility asa diagnostic test in hepatobiliary disease (101). By measuring drug concentrations in blood or urine, one cannot invariably assess a patient’s compliance with therapy. I have therefore examined the role of enzyme-induction parameters in this context. Epileptic patients given a standard dose of anticonvulsants on a body-weight basis had extremely variable serum drug concentrations when measured shortly after therapy was begun (102). Those with lower values were mcre poorly controlled clinically than those with higher serum values. High D-glucaric acid excretion was observed in patients whose therapy did not seem to control seizures, suggesting that these patients had responded to the drug by rapidly inducing microsomal enzymes, leading to rapid drug metabolism, and that they required a higher drug dose (Figure 3). Patients who displayed side effects from the standard dose had lower Dglucaric acid excretion, presumably because their enzyme systems were not induced sufficiently to prevent accumulation of the drug to higher concentrations than were therapeutically desirable. This information can be obtained by measurements of drug metabolism and clearance, but these are more complicated than measurement of some acceptable index to microsomal enzyme induction-which therefore can serve as the initial step in evaluating such patients. Epileptic patients stabilized after three to six months on a suitable dosage of phenytoin, individualized for each subject, demonstrated an apparent correlation between the dose required for stabilization and the urinary D-glucaric acid excretion (Figure 4). The extent of enzyme induction as reflected 696 A Daily phenytoin CLINICAL CHEMISTRY, Vol.26,No. 6,1980 8 6 dosage, mg/kg Fig. 4. Relationship between urinary D-glucaric acid excretion and daily dose of phenytoin (diphenylhydantoin) in patients after several months of stabilization with control of seizures and absence of side effects From ref. 102,with kind permission of MassonPublishingCo by D-glucaric acid seemed to determine the dose of phenytoin required to suppress seizures reliably. Similar observations have been made with other enzyme-induction indices and other enzyme-inducing drugs; in general, patients who demonstrate a sharp enzyme-induction response can be expected to require more drug to attain a therapeutic response. It is more meaningful to evaluate the change in the measured variable above the basal value before initiating therapy, because the variance in reference values makes it essential to use each subject as his own reference. Urinary excretion of D-glucaric acid and ‘y-glutamyltrans- U #{149} 100- U . #{149} U . . U U 4 E -j 0 E #{149} 90- U 0 U U #{149} . #{149} N 0 E . 0 a #{149} . . 60. U U . U 9) 0 > . a #{149} . #{149} .2 . C J #{149} 301 2. 3. .1 4#{149} .3 POOR RESPONDERS GOOD RESPONDERS Fig. 5. Urinary D-glucaric acid excretion and serum ‘y-glutamyltransferase activity in psychiatric patients receiving tricyclic antidepressants, graded according to response to therapy From ref. 102, with kind permission of Masson Publishing Co ferase activity in serum were measured in psychiatric patients receiving tricyclic antidepressant drugs who were classified as poor responders and good responders (Figure 5). Both indices of microsomal enzyme induction were higher in good responders than in poor responders. Among poor responders, enhanced drug metabolism was unlikely to be a factor since the response to microsomal enzyme induction was also poor. Patients labeled 1, 2, 3, and 4 in Figure 5 finally admitted to taking the drugs irregularly. Monitoring indices of microsomal enzyme induction can therefore provide guidance concerning decisions about increasing the dose of a drug, switching to a different drug, or, in the event of total noncompliance, changing to some therapy other than one requiring drugs (102). Concluding Remarks Microsomal enzyme induction is a phenomenon that has excited widespread interest since its existence was first recognized. Its impact upon biochemical pharmacology and environmental medicine has been explosive, and it is now beginning to have important implications for clinical medicine of which the clinical chemist should be aware. This review has attempted to focus attention on these latter aspects, with special emphasis upon the more recent literature. References to fundamental biochemical mechanisms will be found in earlier reviews (1-3). It is reasonable to assume that clinical laboratories will be increasingly required to implement and develop analytical procedures, stimulated by the need to gain a keener insight into drug metabolism by individual patients. This process will accelerate as the function of clinical chemistry changes from a diagnostic role to one primarily devoted to the monitoring of therapy. Chemical indices of microsomal enzyme induction will then assume prominence in the laboratory armamentarium of the future. References 1. Lu, A. Y. H., Kuntzman, R., and Conney, A. H., The liver microsomal hydroxylation enzyme system. Induction and properties of the functional components. In Frontiers of Gastrointestinal Research, 2, L. Van der Reis, Ed., S. Karger, Basel, 1976, pp 1-31. 2. 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