Pharmacogenetics 1998, 8:28 3-289 Review Hypothesi: Comparisons of inter- and intra-individual variations can substitute for twin studies in drug research W. Kalow, B-K. Tang and L, Endrenyi Deparement of Pharmacology. University of Toronta, Toronto. Ontario, Canada Received 16 July 1997; accepted 21 January 1998 ‘Twin studies are useful devices to deten istics that tend to differ among indi temporary characteristics. One therefore may ask whether twin studies are nec the geneti ‘element in phi g (0 thei and between indi ine the heritability of persistent but variable character- rug responses are not pet cent affairs; they are scems logical (o investigate the response vari dividuals, and to compare the va duals, We attempt here to describe a theoretical background of this venture. of the responses wi and to show some results of the exercise. Potential sources of error or uncertainty are discussed. Keywords: heritability, genetic component, variations, repeat studies The established value of twin studies is to measure the contribution of heredity to a variable trait in a human population (Galton, 1876; Weinberg. 1901; Thompson and Thompson, 1986: Vogel and Motulsky, 1986). A twin study measures a given feature in groups of ider tical (monozygotic, MZ) and fraternal (dizygotic, DZ) twins and compares the within pair variances between these two types of twins: this comparison is mathemati- cally translated into heritability data. If the measured feature is a persistent characteristic, such as a person's height or intelligence. a twin study is— outside of family investigations or DNA studies the only means to obtain heritability data. For the purpose of twin studies, persistent character- istics of a person differ fundamentally trom temporary characteristics. such as the response to a given drug. which disappears after elimination of the drug. Hence, a drug response can be repeatedly produced in a given individual. We therefore want to raise the question of whether twins are necessary for estimating the genetic component in more transient events such as the Correspndsnce to De W Kula Depart of Parmacsig: Mil Scences Bulking. Universtiy of front Torta, Ontario MS 188, Cais Ta 141697827 Meta 1 416978 6595: ema halon doronto.ce (9960-314. © 1998 ippineot-Raven Publishers determination of the fate or effect of a drug (Kalow, 1996). Instead of administering a drug once to each ‘member of a pair of twins (Vesell, 1992), is it possible to give the drug twice (o the same person and obtain similar information (Kalow, 1996)? Can a group of people be given a drug at intervals, two or three times to cach person in that group, and the within individual and the between individual variabilities in terms of drug response or pharmacokinetic parameters be compared? Consideration of this procedure seems to be worth- while because a comparison of between and within individual variabilites is simpler and less costly than a twin study, ‘Theoretical considerations Genetic aspects Ia given drug response is much influenced by genetic variation, it is logical to assume that the dillerences between individuals must be larger than the time-to- time differences within individuals. On the other hand, environmental determinants should lead to similar magnitudes of inter- and intra-individual variation. at Jeast within groups of people —even if an environmental influence can vary with an individual's genotype.284 ‘Assume that we measure, in a conventional way. the variation in drug response shown by individuals within a group. There are mine the variation, Variation caused by the genotype of each individual (Vc). ‘This determines the fundamental. responsiveness. Geneticists often distinguish between additive and domi- nant components of the genotype (Vogel and Motulsky. 1986}: we will neglect this possible distinction, ‘Temporary variability. ‘This can represent environmental effects upon the individual (V,). We will neglect the possibility of correlations between V,, and V, that may occur if the same environmental factor has different effects on different genotypes. The error of measurement Vy. Let the between individual variation in response to the drug be measured in terms of the standard deviation (SD,}. The square of any stan- dard deviation is the variance: in the absence of interac- tions (eg. as an approximation) variances can be treated ‘as additive components. Hence: sp,2= V, +2 + Vag possible to determine the error of measurement Vy by repeatedly measuring the same sample or samples. Assume that we measure repeatedly the drug. response in a group of individuals and determine the within individual standard deviation SD,. We thereby assume that the genetic components that contribute to the first, second, and each further drug response are identical and therefore cancel out. Hence: 2+ Vy2.and thus SD,2 sp2= Ve With this subtraction, we are making the assumption. that Vj2 and Vy? are identically contributing to the between and within individual variances. If we take the ratio V,2/SD,2, we may assume to have determined heritability in the broad sense. However, because we have introduced several shortcuts and ‘omissions in our definitions, we will consider our deter- mination an estimate, and we will therefore designate the ratio (SD,2 — SD,2/SD,2 not as heritability. but simply as an estimate of the genetic component that contributes to a pharmacodynamic or pharmacokinetic parameter, In this review. when using the terms "genetic component’ and ‘r,, (lor the ratio in the estimated ‘genetic component’), w referring to the equation: Tec = SD,2- $D,2)/SD,? ‘The equation proposed to calculate genetic components is similar (o the intraclass correlation as formulated by Snedecor and Cochran (1967): (Sb2-SD,2/[SD2 + UH1)SD,2 , Kalow et al where 1 is the number of exposures of each individual Here, the denominator becomes a weighted combina- jon of the between and within individual variations ‘The intraclass correlation is widely used to analyse twin for heritability measurements (Vogel and 1. 1986). Because its outcome is more conserv- ative than that of the genetic component, and because it is modified by the number of repeats. we will use both, equations for comparative purposes. The number of individuals tested in this way is important. For example. it is easy to show that a comparison between only two individuals, each tested twice. could give random and therefore nonsensical answers, Because the accuracy of a measured standard deviation doubles with the square of the number of test items, we recommend the inclusion of 16 individuals for a good estimation of the genetic component in a drug response. Because each individual is tested at least twice, itis possible to base the estimate of SD,? on the first set of tests, the second set of tests. or on the average between them, We recommend use of the average as the most reliable measurement Targets of measurement A study may aim to estimate the genetic component in cither a pharmacokinetic or pharmacodynamic para- meter. In either case, the characteristics of the measured parameter may be a function of the dose of the drug, In pharmacodynamics, a high dose may stimulate a toxic response. a low dose a therapeutic response, Its easy t0 imagine different genetic components in these two kinds of responses. ‘The nature of a pharmacokinetic measurement may also be fundamentally dose-dependent. Assume that the measurement represents mainly the consequences of an enzymatic metabolic step. At low substrate concentration. the reaction rate may be essentially determined by the strength of binding of a drug to the enzyme, that is, by Ky. @ feature mainly dependent on enzyme structure, At high concentrations, the major rate-determining characteristic may be the maximal reaction rate, the Vj,q. & feature much dependent on enzyme concentration. It is not likely that Ky, and Vig are equally dependent on genetic determinants, Furthermore, the metabolism of a given drug could be caused by different enzymes, depending on drug concentration. ‘These considerations regarding drug dosage should affect twin studies as much as the repeat studies proposed here, However, we are not aware whether any twin study to determine heritability of a drug response emphasized the potentially crucial effect of drug dose. Thus, it is likely that the nature and emphasis of studies based on between and within individualSimplified estimation of heritability differences will emphasize thi has been neglected in twin studi pect of the topic that Sources of pertinent data Looking through the pharmacological literature, almost all measurements reported in terms of means and stan- dard deviation (mean + SD) refer to data [rom different individuals, Thus, the standard deviation is usually an indication of overall between individual differences. Systematic studies of within individual differences (ie. of differences between repeated responses of persons to a given drug) are common only in studies of bioavailability. Most of these data are in pharmaceutical archives and are unpublished. Another source of data on intra-individual variation are studies of population pharmacokinetics that include repeat measurements of drug or metabolite concentrations in given subjects (Sheiner and Ludden, 1992: Fernandez De Gatta et al., 1996), For example, the NONMEM computer program allows the calcula- tion of intra-individual and interindividual variabili ties (where the varlabilities relate to the predictions of a model}: nevertheless, published data on this subject which display this variability information in a directly usable form are also rare. ‘Thus, in many or most cases. one has to deliberately create data that allow comparisons of intra- and inter- individual vari Examples of case stu: Urinary caffeine metabolite ratios Study 1. A paper by Denaro etal, (1996) quotes caffeine metabolite ratios, comparing between and within, ividual variations. The authors did not subject their data to the inquiry that is the essence of the present article. ‘There are two sets of data, one set consisting of two tests in 12 individuals, the other of three tests in eight individuals: our analysis of these two sets of tests yielded results of sufficient similarity that we show only the analysis of the 12 individual test. The interval between repeat caffeine administrations was 1 week. All volunteers were cigarette smokers ‘The data and our treatment of the data are shown in Table 1. The first column in all but the last row shows different ratios of five major caffeine metabolites in urine: the last row is based on measurements of caffeine in blood. The second column lists the enzyme activities which are pointed to by the different metabo- lite ratios. The different magnitudes of the mean are the averages of the metabolite ratios: they are not indicative of amounts or concentrations of enzymes. ‘The stan- dard deviations (SD,) after each mean are measures of 285 the variation of each enzyme activity between individ- uals while SD, indicates the within individual varta- tion, ‘The results differ much between the indicated enzymes, ‘The largest values are associated with the ‘Nacetyltransferase/2 (NAT2), an enzyme very well known to be genetically polymorphic (Price Evans. 1992): it is logical that the inter-individual differences are much larger than the intra-individual differences for this enzyme. It is interesting to note this result: however. the distribution curve of the urinary meta- bolite ratio is usually bimodal (Tang et al., 1991) so that expression of variability in terms of SD and variance is not strictly appropriate. ‘The lowest values are associated with xanthine oxidase activity. This is an enzyme that is known to have disease-associated rare variants but that is not expected to show genetic variation under most circumstances {Simmonds et al.. 1995), although in a study by Kalow and ‘Tang (1991), four of 176 healthy individuals showed unexpectedly low activity: their data did not fit the otherwise normal distribution. It meets logical expectation to find comparatively low values for an enzyme with apparently little genetic but mostly environmentally controlled variation. ‘The 17U/17X metabolite ratio cannot be precisely defined in enzymological terms, The hydroxylation of paraxanthine (17X) is known to depend somewhat more on CYP2A6 than on CYPIA2 but the 17X demethylation by CYP1A2—1X is faster than its hydro- xylation (Gu et «l.1992). Furthermore. the urinary excretion of 17X depends also on urinary flow (Tang et al,, 1994), However, CYP2A6 is a genetically variable enzyme (Fernandez-Salguero ct al., 1995), and the rela- tively high values may be a reflection of that fact. ‘This leaves. as ratios indicative of the nature of CYPIA2 variation, a set of intermediate valu: suggesting some genetic component in this ratio. CYPIA2 activity inhuman liver has a constitutive element and it is inducible via the Ah-receptor (Okey, 1992; Kalow and Tang, 199 3), Because the volunteers were smokers, some induction of CYP1A2 activity in this sample can be assumed. In our experience. caffeine clearance is a better indicator of CYP1A2 activity than are the urinary metabolite ratios shown in their data (Kalow and ‘Tang, 1993). Hence, the somewhat larger values associated with caffeine clearance than with urinary metabolite determinations are a genuine indication that CYP1A2 activity has a distinct genetic component, although the environmental influence is substantial. Shahidi (1967) found an apparently rare familial deficiency of phenacetin O-deethylation, a reaction now known to be catalysed by CYP1A2 (Sesardic et al. 1988). There are two twin studies from different286 Kalow et al. ‘Table 1. Caffeine metabolism: genetic admixture and intraclass correlations of betuven/within person variations. affine metabotie ratio or clearance Encume Observations Lnterpretcions Mean SD, SDy (AAMU+UFIXV/17X cyp1az B88H4S2 £252 0.69 (AAMU+IU41XV/17U cyp1a2 10274457 £2.54 0.69 AUX. CYP2A6 + CYPIA2 0934046 £016 oss AAMU/(AAMU+101X) NacetyHransferase-2 (NAT2) 0364013 £003 0.95 wax Xanthine oxidase L89L071 062 024 Caffeine clearance (Wh/kg) cyp1az 014#007 £003 0.82 Observations by Denaro etal. (1996) Caffeine metabolites in urine: 17, 1.7-dimethylxanthine; 17U, :methyluracl,a stabilised conversion product of the ucetylated metabolite surements In blood, Mean +8D,, mean and standard deviation of betwet individual variation; standard deviation $D,, of within-individual variation. Genetic component = Fe s AAMU, 5-acetylamino-6-amino~ Caffeine clearance determined via Intraclass correlation 2yISD,2 + (1-1)SD, 7}. laboratories (Miller et al., 1984: Miller et al., 1985) exploring the fate of theophylline: the main metabolite of theophylline, 3-methyl-xanthine is catalysed by CYPIA2 (Gu et al..1 992). Both papers reported distinct heritabilities of 3-methylxanthine formation from theo- phylline: for example, Miller et al. (1985) reported a heritability of 0.61 for the formation of this metabolite. In summary, the repeat data with caffeine make sense in comparison with known genetic data, Study 2, Zhou (1995) investigated the metabolism of caffeine in a group of students by testing urinary metabolites, In contrast to the above cited study, the individuals were nonsmokers. At 8 a.m. after an overnight fast. each individual drank a cup of coffee containing 140 mg of caffeine. Urines collected 16-25 h after caffeine intake were utilized. Twenty-one students were available for a repeat study. The metabolite ratios used for enzyme assessments were the same as in the study by Denaro et al. (1996). However, the repeat measures were carried out a year later, not 1 week later as by Denaro and co-workers. ‘The enzymes assessed by Zhou were NAT2. CYP1A2 and XO. Zhou calculated: genetic component: Foe = 0.92 (NAT2); 0.61 (CYPLA2): 0.51 (XO). Intraclass correlation: r,= 0.80 (NAT2); 0.35 (CYP1A2): 0.37 (XO) Consistent with the Data by Denaro et al, (1996) are the values for NAT2, the enzyme known to be polymor~ phically variable, and for CYPIA2. A difference between the two sets of data is the suggestion of some genetic element in the control of xanthine oxidase (XO) activity. As indicated in the discussion of Study 1, the difference could represent chance. dependent on the selection of participating volunteers. limethylurate: 1X. I-methybsanthine: 11), 1-methylurate: (SDz-SD,2/8D, Study 3. In order to demonstrate the magnitudes of fluc- tuation of caffeine-clearance and halflife, Balogh et al (1993) measured the caffeine decay curves in blood of 12 healthy men, and they compared the inter-individual, and intra-individual variations. They reported a vari- ance distribution of 21.4% for intra-individual and of 78.6% of inter-individual variation. Using these data, yc = 0.7 3. while r, = 0.57..These data compare reason- ably well with the values of 0.82 and 0.70, respectively, calculated from the data by Denaro et al. (1996) and reported here in Table 1 Ghucuronidation of oxazepaan Work in this laboratory (Patel et a. 1995: Patel, 1997) showed that different glucuronyl tranferases catalyse the glucuronidation of the enantiomers S-oxazepam and R-oxazepam. The ghicuronidation of S-oxazepam is caused by UGT2B7, which occurs in at least two allelic forms. The T to C at nucleotide 802 variant binds to S-oxazepam with so low affinity (high K,) to be catalyt- ically almost inactive, The presence of this variant becomes clinically visible by measuring the ratio of S- oxazepam to R-oxazepam in blood or urine: thus the S/R ratio of oxazepam has become a means of phenotyping, individuals for the presence of this UGT2B7 variant, Figure 1 shows comparisons of the S/R ratio in 11 individuals, all healthy students, including men and women (unpublished data).'The much larger inter-indi- vidual than intra-individual variability of this ratio is obvious. This observation initiated our successful search for the variant, The results of our calculations 98; r,= 0.95. Both numbers are exceed- ingly high and confirm that the S/R ratio is predomi- nantly under genetic control. To date, genetic control isSimplified estimation of heritability ‘WeTin SUBJECT VARIATION Fig. 1. Illustration of within and between subject variation of the ratio of $ to R oxazepam glucuronide. The enantiomers of oxazepam glucuronide were measured three times at weekly intervals in 8 h urine of 11 healthy volunteers. Oxazepam was administered orally at a dose of 15 mgoof the racemate, Note the consistently lower values in two of the individuals, and the very much larger imter- Individual than intea-individual variation. known only of the transferase affecting S-oxazepam (Patel, 1997). but the very high ratios might be taken to ‘suggest that there is also genetic control of the enzyme affecting R-oxazepam: this will be a subject of further study. Extensive metabolisers of dextromethorphan Ereshefsky ¢t al. (1995) studied the urinary metabolic ratio of dextromethorphan three times in seven individ- uals. The purpose of their study was to assess the reliability of intra-individual variation as the basis for investigations of drug-drug interaction. The metabolic ratio. the dextromethorphan:dextrorphan ratio. is typically used to define the variability of CYP2D6 by distinguishing between extensive and genetically poor metabolisers; extensive metabolisers may be either homozygotes or heterozygotes with enzyme activity, while poor metabolisers are homozygotes without CYP2D6 activity. The coefficients of variation were 30% for intra-individual and 163% for inter-individual variation. ‘This means the genetic component is estimated to be 0.97, while the intraclass correlation is 0.90. The data clearly support the thesis of the present paper. Pharmacokinetics of ethanol In an attempt to find information in the literature concerning drugs for which both twin studies and data on intra-individual variation have been reported, we found a study on ethanol by Kopun and Propping (1977) who combined both kinds of investigation. ‘These authors tested 19 identical and 21 fraternal twin pairs and 11 single individuals. The latter were tested twice with a 2 month interval. All individuals received a high dose of 1.2 g/kg of ethanol. ‘The 287 ‘Table 2. Analysis of ethanol data of Kopun and Propping, 1197) Repeat study ‘Twin study heritability) Fs Absorption rate 057 060033, (mg/mal x 30 min) Rate of metabolism O41 037030 (mg/kg x h) Elimination rate 0.46 o7 045 (engi x hy ‘The heritability tests involved 80 twin individuals. There were 11 single individuals, each tested twice. The ethanol dose was 1.3 glkg. rye = (SD,? -SD,?V/SD,? = genetic component: = (SD,2- SD,2)/ISD,2 + (n-1)8D,2] = intraclass correla- tion, authors determined the heritabilities of the rates of absorption, metabolism and elimination of ethanol, and they listed the magnitudes of inter- and intra- individual variation of the 11 individuals from which we calculated the pertinent ratios, The results are shown in Table 2. The two tests may be judged as showing reasonable agreement. It seems appropriate to contrast this confirmatory observation with some discrepant data reported in the literature. Fraser et al. (1995) conducted a study of 22 healthy young men who each received four doses of ethanol, two of these under fasting conditions, and two after a standard meal. Each dose of ethanol was 0.3 g/kg. a low dose. All pharmacokinetic values differed between fasted and fed individuals, thereby directly indicating the importance of food intake as at least one environmental modifier. A remarkable feature of their study is the observation that several of the coefficients of variation are slightly larger within than between individuals, giving negative values to the calculated genetic components, and strongly suggesting a lack of genetic determinants and virtually complete environmental control of the system. Pethaps the low dose of ethanol used by these authors contributed to this result which is fundamentally different from the results of all other studies. For example, Passananti et al. (1990) gave 1 g/kg ethanol four times over 4 weeks under carefully stan- dardized conditions to eight fasted individuals. The slopes of the linear decay curves showed coefficients of variation that averaged 8% for the intra-individual and 14% for the interindividual differences. Hence, the genetic component was calculated to be 0.67. while the intraclass correlation was 00.34, a low value reflecting the four separate exposures.288 Vesell etal. (1971) conducted a twin study to deter- mine the genetic component in ethanol metabolism. Alter administration of 1 g/kg ethanol to sets of seven identical and seven fraternal twins, they arrived at a heritability of 0.98, indicating almost complete genetic control of ethanol metabolism. Martin et al, (1978) calculated that the 95% confi- dence limits of both tests (Vesel et al. 1971; Kopun and Propping, 1977) included the values of 0.0 and 1.0, indicating lack of statistical significance. ‘They concluded that the number of twin pairs had been far too small to allow a useful conclusion. Martin ¢t al. (1985) therefore repeated the study using 206 pairs of twins and arrived at a heritability of 0.62 + 0.06 for peak blood alcohol levels. and a heritability of 0.49 £ 0,07 for the rate of ethanol elimination. Thus, the data obtained in the largest set of twin studies were similar to those of Kopun and Propping. ‘Thus, several data agree that there is an approximate 500% heritability in the control of ethanol elimination if the dose is high enough. With high dose, cofactor supply is the rate limiting factor besides enzyme activity (Kalant, 1996). With low dose. cofactor supply ample. Liver blood flow. first-pass metabolism. rate of absorption, gastric emptying, alcoholism, exercise and time of day, are all known to affect ethanol elimination (Wilkinson, 1980; Kalant, 1996). It would not be surprising to find that there is a genetic element controlling the cofactor supply and enzyme activity but not, for example, the liver blood flow. Discussion ‘The equations that we took to indicate the genetic components and the intraclass correlations, comparing vidual variations from repeat studies of drug metabolism. appear to be able to Indicate the presence or absence of substantial genetic contributions to variation, Despite these encouraging indications, both theory and observations call for addi- tional, systematic comparison of the results of twin-and repeat-studies in pharmacology. Heritability is always a relative and not an absolute value and. thus. should be the concept of genetic components. Heritability describes a fraction of total variation, Assuming a given extent of genetic control. the measured heritability tends to be larger the more uniform the tested population: heritability would be higher in a healthy population than a patient popula- tion, In other words, a given value of heritability applies only to the population in which it was established. This rule applies to twin studies, and obviously also affects conclusions from comparison of intra~ and inter- individual variation. Kalow et al, ‘The relativity of heritability measurements can also be shown by imagined data: let repetition of drug administration be produced once at a weekly interval and another time at a yearly interval. ‘This difference of liming may not affect any genetic difference but the repeats may show lesser person-to-person differences with the smaller interval than the larger interval. If so, for example, the calculated genetic component would change with the interval and would be smaller with the longer interval. ‘The important point remains that a clear interval between two measurements isa precondi- tion for the determination of intra-individual vari- ability. A systematic error in the assessment of high her tability is possible if an environmental determinant affects both the initial and the repeat administrations to 4 person. Imagine. for example, a study containing a group of heavy smokers with a smoking-induced enzyme tested twice, and a group of twice-tested non- smokers: the smoke-induced enzyme activity would appear mathematically to be indistinguishable from a genetically controlled high activity. This. possibility implies the need for special attention to uniformity of environmental factors during such studies. 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