The effects of N-B transition of human serum albumin on the specific drug-binding sites.

In the pH range 6-9, human serum albumin undergoes a conformational change termed the neutral-base (N-B) transition. Recently, it has been shown that the N-B transition causes enhanced binding at the warfarin-binding site (site I). The present study used fluorescence and equilibrium dialysis to investigate the effects of the N-B transition, chloride, calcium and fatty acids on the specific binding sites I and II on human serum albumin. The effect of the N-B transition of human serum albumin provides a further distinction between site I and II binding characteristics. The N-B transition of albumin caused a change in conformation at site I which resulted in increased binding of drugs and fluorescent probes at this site, whereas there was no effect on acidic drug binding at site II. These effects on site I and II are qualitatively similar to those induced by fatty acids (increased drug binding at site I and no change at site II). However, the effects of increasing pH and fatty acids were additive, showing that they were caused by two different conformational changes. The effect of Cl- on site I binding was pH-dependent and was abolished by the presence of fatty acid. Ca2+ reduced the fluorescence of site I probes but had no effect on a site II fluorescent probe. Effects of pH were also investigated with drugs not binding to site I or II. Increasing pH caused a decrease in binding to indomethacin, increases in binding of L-tryptophan, tolmetin and quinidine and no change in the binding of salicylic acid, diflunisal and phenytoin.

Allelic and functional variability of cytochrome P4502C9.

Single nucleotide substitutions are known to result in a different amino acid at one of four sites in cytochrome P4502C9 (CYP2C9) namely: residue 144: Arg/Cys; residue 358: Tyr/Cys; residue 359: Ile/Leu and residue 417: Gly/Asp. Polymerase chain reaction (PCR)-based amplification of the nucleotide fragments encompassing the four residues (144, 358-359 and 417) in 18 samples of human genomic DNA from a liver bank and one sample of DNA extracted from the blood of a known poor metabolizer of tolbutamide has been carried out. The products of PCR amplification were analysed by either allele-specific restriction endonucleases or probed with radioactively labelled allele-specific oligonucleotides in dot blot hybridizations. Fourteen individuals were homozygous for Arg144 and four were heterozygous Arg/Cys144. All individuals analysed were homozygous for Tyr358 (n = 17) and for Gly417 (n = 18). With the exception of one heterozygote the other 17 subjects were homozygous for Ile359. The genotype of the known poor metabolizer of tolbutamide was homozygous for Arg144, Leu359 and Gly417. The relative levels of expression of the Cys and Arg144 alleles was studied in the heterozygotes. A relative 5- to 10-fold greater expression of the Cys- over the Arg144 allele was noted in two heterozygotes. There was no apparent correlation of genotype to the hydroxylation of the known CYP2C9 substrates phenytoin, tolbutamide, torasemide and diclofenac. Apparent K(m) values for the cDNA-expressed Arg144/Ile359, Cys144/ Ile359 and Arg144/Leu359 variants towards tolbutamide were 91 microM, 62 microM and 229 microM, respectively. It is likely that functional changes occurring as a result of the Ile359Leu transition are responsible for the tolbutamide poor metabolizer phenotype.

Tolbutamide hydroxylation in humans: lack of bimodality in 106 healthy subjects.

The tolbutamide hydroxylation capacity was studied in 106 healthy unrelated volunteers from the Australian population. Following a 500 mg oral dose of tolbutamide, the ratio of metabolites (hydroxytolbutamide plus carboxytolbutamide) to unchanged tolbutamide excreted in urine from 6 to 12 h post-dose (urinary metabolic ratio, MR) was determined. Metabolic ratio values did not appear bimodally distributed, even following various transformations of the data (i.e. Log10, inverse, Log10 inverse). A poor metabolizer (PM) subject from a previous clinical study, however, could be distinguished (MR value 159) from the above subjects (MR value range 324-3033), particularly from the histogram plot of inverse tolbutamide metabolic ratio. The poor metabolizer's parents had metabolic ratio values (526 and 478) that were at the lower end of the range of metabolic ratios obtained from the population study, and may indicate that they both have a heterozygous genotype and that a recessive form of inheritance is most likely. As the hydroxylations of tolbutamide and phenytoin are closely linked, the incidence of slow tolbutamide metabolizers is likely to be similar to that for phenytoin (about 1:500) and this is consistent with the failure to detect a single poor tolbutamide metabolizer in our random sample of 106 individuals.

Caffeine as a probe for human cytochromes P450: validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques.

The molecular basis for the use of caffeine (CA; 1,3,7-trimethylxanthine) as a probe for specific human cytochromes P450 has been investigated. The CA 1-, 3- and 7-demethylations (to form theobromine, paraxanthine and theophylline, respectively) all followed biphasic kinetics in human liver microsomes. Mean apparent Km values for the high- and low-affinity components of the demethylations ranged from 0.13-0.31 nM and 19.2-30.0 mM, respectively. cDNA-expressed CYP1A2 catalysed all three CA demethylations, and the apparent Km for CA 3-demethylation (the major metabolic pathway in humans) by the expressed enzyme was similar to the Km for the high-affinity liver microsomal CA 3-demethylase. IC50 values for inhibition of the CA demethylations by alpha-naphthoflavone were similar for both expressed CYP1A2 and the high-affinity microsomal demethylases. Moreover, CA was a competitive inhibitor of expressed CYP1A2 catalysed phenacetin O-deethylation, with the apparent Ki (0.080 mM) closely matching the apparent Km (0.082 mM) for CA 3-demethylation by the expressed enzyme. Expressed CYP1A1 was additionally shown to catalyse the 3-demethylation of CA, although activity was lower than that observed for CYP1A2. While these data indicate that CYP1A2 is responsible for the high-affinity component of human liver CA 3-demethylation, two limitations associated with the use of CA as an in vitro probe for CYP1A2 activity have been identified: (i) CA 3-demethylation reflects hepatic CYP1A2 activity only at appropriately low substrate concentrations; and (ii) CA is a non-specific CYP1A substrate and CYP1A1 may therefore contribute to CA 3-demethylase activity in tissues in which it is expressed. An anti-CYP3A antibody essentially abolished the 8-hydroxylation of CA to form trimethyluric acid, suggesting formation of this metabolite may potentially serve as a marker of CYP3A isozyme(s) activity.

The role of the CYP2C9-Leu359 allelic variant in the tolbutamide polymorphism.

Tolbutamide undergoes hydroxylation in humans via a cytochrome P450-mediated pathway. The primary P450 isozyme responsible for this metabolism is thought to be CYP2C9. Population studies have indicated the existence of slow metabolizers of tolbutamide (approximately 1 in 500) suggesting a rare polymorphism associated with 2C9. Several allelic variants of 2C9 have been identified; however, the effect of these allelic variations on metabolism in vivo is not established. In the present study, the coding regions, intron-exon junctions, and upstream region of CYP2C9 were amplified by PCR and sequenced in two slow metabolizers. One individual was homozygous for Leu359/Leu359 and the other individual was heterozygous for Arg144/Cys144 and for Ile359/Leu359. No other genetic variations in 2C9 were detected in these individuals. PCR-RFLP tests showed that Arg144 Tyr358 Ile359 Gly417 is the principle CYP2C9 allele. Frequencies of the rarer Leu359 and Cys144 alleles were 0.06 and 0.08, respectively, in a Caucasian-American population and 0.005 and 0.01 respectively in African-Americans. The frequency of the Leu359 allele was 0.026 in Chinese-Taiwanese, but the Cys144 allele was not detected in this population. Studies in a recombinant yeast expression system showed that the Leu359 variant had the highest Km and the lowest Vmac for hydroxylation of tolbutamide of all the CYP2C9 allelic variants. This allelic variant also had the highest Km for the 7-hydroxylation of S-warfarin. The present data suggest that the incidence of the Leu359 allelic variant of CYP2C9 may account for the occurrence of poor metabolizers of tolbutamide.

Effect of age on patterns of theophylline metabolism.

The pattern of excretion of urinary theophylline and theophylline metabolites were studied in three groups of patients (premature neonates, children, and adults). When adjusted to a standard dose of 10 mg/kg/day to allow valid comparison between groups, differeces in mean corrected trough plasma theophylline concentrations (corrected Cp) were found that were of similar magnitude to differences reported in theophylline clearance in these groups. Despite the lower corrected Cp in children, urinary theophylline metabolite patterns were similar in adults and children, indicating that the increased clearance in children is not associated with induction of a specific metabolic pathway. In premature neonates, only unchanged theophylline and caffeine were found in urine, indicating the absence of oxidative pathways for theophylline metabolism. In both adults and children, there was high positive correlation between urinary excretion of 3-methylxanthine (3MX) and 1-methyluric acid (1MU). Both 3MX and 1MU correlated negatively with urinary excretion of 1.3.-dimethyluric acid (DMU). It is suggested that N-demethylation of theophylline to 3MX and 1MU is catalyzed by a different form of cytochrome P-450 than that involved in the 8-hydroxylation to DMU.

Cigarette smoking and theophylline clearance and metabolism.

Differences in plasma theophylline clearance (ClT) and metabolism between smoking and nonsmoking normal subjects were examined by analysis of plasma and urinary theophylline concentrations and of urinary metabolite concentrations under steady-state oral dosing conditions. ClT in smokers (0.053 +/- 0.006 1 . hr-1 . kg-1) was greater than in nonsmokers (0.032 +/- 0.002 l . hr-1 . kg-1, p less than 0.005). Analyses of urinary metabolites revealed that clearance to l-methyluric acid (Cl1MU) and clearance to 3-methylxanthine (Cl3MX) were increased in smokers 1.99-fold and 2.10-fold over nonsmoking controls (P less than 0.005). Clearance to 1.3-dimethyluric acid (ClDMU) was also enhanced in smokers 1.68-fold compared to controls (P less than 0.01). The positive relationship between Cl1MU and Cl3MX in both smokers and nonsmokers (r = 0.98, P less than 0.001) supports the concept that two N-demethylation pathways for theophylline metabolism are under common regulatory control and involve a form of cytochrome P450 distinct from that mediating 8-hydroxylation of theophylline to DMU. These results suggest that cigarette smoking induces both of the cytochrome P450-mediated pathways of theophylline metabolism but that N-demethylation may be increased to a greater extent than 8-hydroxylation.

Determinants of acetaminophen metabolism: effect of inducers and inhibitors of drug metabolism on acetaminophen's metabolic pathways.

Acetaminophen metabolism and clearance after a single 1 gm oral dose of the drug was investigated in 12 healthy men, six of whom were cigarette smokers, and in six men who were receiving anticonvulsant drugs for epilepsy. The 12 healthy subjects were studied before and after 1 wk of pretreatment with cimetidine (1 gm/day) or sulfinpyrazone (800 mg/day). There was no significant difference in acetaminophen clearance (ClAP) between nonsmokers and smokers; cimetidine pretreatment had no effect on ClAP. Neither cigarette smoking nor cimetidine pretreatment had a significant effect on any of the metabolic pathways of acetaminophen. In contrast, sulfinpyrazone pretreatment increased ClAP by 23% (from 5.70 +/- 0.21 to 7.00 +/- 0.39 ml/min/kg) and ClAP was 46% greater in the epileptic subjects who received anticonvulsant drugs than in the control group (8.32 +/- 0.45 and 5.70 +/- 0.21 ml/ml/kg). In both cases the increase in ClAP was a result of induction of acetaminophen glucuronidation and oxidation; clearance of the glucuronic acid conjugate was 26% and 59% greater and clearance of the glutathione-derived conjugates (reflecting the activity of the oxidative pathway) was 43% and 60% greater in the groups given sulfinpyrazone and anticonvulsants, respectively.

Effects of allopurinol on theophylline metabolism and clearance.

The effects of allopurinol on the plasma clearance and metabolism of theophylline in man were investigated under single-dose and multiple-dosing conditions. No change in theophylline clearance was found with the concomitant use of allopurinol but 1-methylxanthine (1MX), a theophylline metabolite not previously described in man, was detected in urine during control and allopurinol treatment phases under both single- and multiple-dosing conditions. 1MX excretion increased at the expense of 1-methyluric acid (1MU) during the allopurinol treatment phase. It is proposed that the secondary biotransformation of 1MX to 1MU is mediated by xanthine oxidase.

Mechanisms of inhibition of tolbutamide metabolism: phenylbutazone, oxyphenbutazone, sulfaphenazole.

Tolbutamide half-life was increased by chronic administration of sulfaphenazole (9.5 hr to 28.6 hr, n = 2), phenylbutazone (7.9 hr to 23.1 hr, n = 8), and oxyphenbutazone (8.1 hr to 30.2 hr, n = 2). The rate of elimination of tolbutamide was decreased within 1 to 2 hr of a single dose of sulfaphenazole and the tolbutamide half-life was increased from 9.2 hr to 25.7 hr (n = 2). In contrast, phenylbutazone and oxyphenbutazone, administered as single oral doses of 800 mg, had no immediate effect on tolbutamide elimination. At times greater than 20 to 30 hr after the single dose of phenylbutazone or oxyphenbutazone the rate of tolbutamide elimination was decreased. It is suggested that phenylbutazone and oxyphenbutazone act by inducing form of cytochrome P-450 with low activity for tolbutamide hydroxylation, whereas sulfaphenazole acts by direct inhibition of the microsomal mixed function oxidase system.

Assessment of caffeine exposure: caffeine content of beverages, caffeine intake, and plasma concentrations of methylxanthines.

The caffeine content of all tea or coffee beverages consumed by 17 healthy adults over 24 hours was measured. Plasma caffeine, theophylline, theobromine, and paraxanthine concentrations were determined over the same 24 hours. The average caffeine content per drink was 60.4 +/- 21.8 mg for instant coffee (14-fold range), 80.1 +/- 19.2 mg for brewed coffee (2.8-fold range), and 28.8 +/- 13.7 mg for tea (5.5-fold range). The number of drinks of coffee and tea consumed was a poor index of actual caffeine intake (r2 = 0.42). Caffeine intake correlated poorly with the 24-hour average caffeine concentration (r2 = 0.41), but there was a very good correlation between a single plasma caffeine concentration measured at 5 PM and the 24-hour average concentration (r2 = 0.94). The same was true for paraxanthine (r2 = 0.86). Paraxanthine accounted for 67.3% of the total dimethylxanthines in plasma, while theobromine and theophylline accounted for 24.4% and 8.3%, respectively. Mean caffeine clearance was 1.2 +/- 0.3 ml/min/kg. Plasma caffeine concentration before the first drink in the morning correlated very poorly with caffeine clearance (r2 = 0.07), even when adjusted for caffeine intake (r2 = 0.21).

Lidocaine disposition--sex differences and effects of cimetidine.

The effects of pretreatment with cimetidine (1 gm daily for 3 days) on the disposition of lidocaine were examined in 18 healthy subjects, seven of whom were chronic cigarette smokers. Each subject received separate oral (200 mg) and intravenous (75 or 100 mg) doses of lidocaine before and after cimetidine. After cimetidine, lidocaine apparent oral clearance (Clo) reduced 42% +/- 7%; oral bioavailability increased 35% +/- 10%; and systemic clearance (Cls) and volume of distribution at steady-state (Vdss) decreased 21% +/- 6% and 20% +/- 7%. Elimination t 1/2 was unchanged. Calculated values for Cls after cimetidine based on reduction in Clo and consequent decrease in hepatic extraction ratio correlated significantly with observed values for Cls (r = 0.67). Women had larger Vdss (64% +/- 19%), longer t 1/2 (50% +/- 18%), and greater oral lidocaine bioavailability (63% +/- 29%) than did men. There was no sex difference in and no influence of cigarette smoking on the effects of cimetidine. The results indicate that cimetidine reduces metabolic clearance of lidocaine. The consequent reduced hepatic extraction explains the observed reduction in Cls.

Theophylline metabolism in relation to antipyrine, debrisoquine, and sparteine metabolism.

Theophylline plasma clearance (Clp) and clearance to its metabolites ( Clm ), as well as antipyrine saliva clearance ( Clsal ) and its Clm were compared in a crossover study in 25 healthy subjects. They were selected with regard to smoking status (nine smokers, 16 nonsmokers) and oxidation phenotype of debrisoquine and sparteine (six poor metabolizers [PMs] and 19 extensive metabolizers [EMs]). Clm of theophylline (1,3-dimethyluric acid, 1-methyluric acid, and 3-methylxanthine) correlated (r greater than or equal to 0.92) to each other and to total theophylline Clp (r greater than or equal to 0.97). Smokers had higher Clm to all metabolites, particularly by the N-demethylation pathways. After correction for the effect of smoking, there was no difference between EMs and PMs with regard to theophylline Clp or Clm . Antipyrine clearances by EMs and PMs ( Clsal and Clm of 4-OH-antipyrine, 3-OH- methylantipyrine , or norantipyrine) also did not differ. Antipyrine Clsal and Clm correlated to theophylline Clp (r between 0.50 and 0.69). It is concluded that theophylline metabolism (N-demethylations and C-oxidation) is not under the same genetic control as sparteine and debrisoquine oxidations, and that there may be a partial overlap in factors that regulate the metabolism of theophylline and antipyrine.

Validation of the tolbutamide metabolic ratio for population screening with use of sulfaphenazole to produce model phenotypic poor metabolizers.

The present study has validated kinetically a convenient method to measure tolbutamide hydroxylation capacity in human beings by use of urinary metabolic ratios. The known in vivo and in vitro inhibitory properties of sulfaphenazole were used to convert control phase subjects to phenotypically "poor" metabolizers of tolbutamide. Six healthy subjects were given a single 500 mg oral dose of tolbutamide with and without sulfaphenazole, 500 mg every 12 hours. Tolbutamide, hydroxytolbutamide, and carboxytolbutamide in urine were determined by newly developed HPLC procedures. Plasma tolbutamide clearance and half-life were measured, as were the metabolic ratio (hydroxytolbutamide + carboxytolbutamide/tolbutamide) in successive 6-hour urine collections. The mean tolbutamide plasma clearance decreased from 0.196 +/- 0.026 ml/min/kg without sulfaphenazole to 0.039 +/- 0.009 ml/min kg with sulfaphenazole, and the mean half-life of tolbutamide increased from 7.28 +/- 0.89 hours to 38.76 +/- 13.30 hours. The metabolic ratio determined in the 6 to 12 hour urine collection period decreased from 794.0 +/- 86.6 to 126.0 +/- 79.3, and this collection period also gave the best separation of subjects between phases. There was a good correlation between tolbutamide plasma clearance and metabolic ratio (rs = 0.853, p less than 0.01, n = 12) and between the percentage decrease in plasma tolbutamide clearance and the percentage decrease in metabolic ratio (r = 0.932, p less than 0.01, n = 6). The tolbutamide urinary metabolic ratio therefore effectively distinguishes tolbutamide hydroxylase activity in "normal" subjects and in those converted to model phenotypically "poor" metabolizers by sulfaphenazole.

Effects of tricyclic antidepressants on drug metabolism.

The effects of chronic treatment with amitriptyline and nortriptyline on the elimination from plasma of warfarin, dicumarol, phenytoin, and tolbutamide were examined in man. No alteration of plasma half-life of warfarin, phenytoin, or tolbutamide was observed following dosage with the tricyclic antidepressants used. There was no consistent effect on the metabolism of dicumarol following treatment with amitriptyline or nortriptyline although the bioavailability of dicumarol appeared to be increased. In some subjects, this increased bioavailability was associated with significant prolongation of the plasma half-life of dicumarol due to its dose-dependent kinetics.

Effects of time of dose in relation to food on the bioavailability of Theo-Dur Sprinkle at steady state in asthmatic children.

The effects of administration of Theo-Dur Sprinkle in the fasting state (phase A), 10 minutes before food (phase B), and immediately after food (phase C) were investigated in 12 children with asthma aged 5 to 9 years at steady state. The AUC during the dosing interval was significantly reduced in phases B and C compared with phase A, and bioavailability relative to the fasting state was reduced to 77% +/- 15% (range 61% to 104%) in phase B and 70% +/- 16% (range 40% to 103%) in phase C. The average plasma theophylline concentration during the dosing interval and Cmax were also significantly reduced in phases B and C compared with phase A. The morning predose plasma theophylline concentration in phase B was 22% lower than the predose concentration with Theo-Dur tablets taken 10 minutes before breakfast. The diurnal variation in predose plasma theophylline concentrations was increased from 28% in phase A to 63% in phase C. There was no significant difference in any parameter between administration 10 minutes before food and immediately after food.

Protein binding of some non-steroidal anti-inflammatory drugs in rheumatoid arthritis.

The protein binding of 4 non-steroidal anti-inflammatory drugs and warfarin was investigated in 10 patients with rheumatoid arthritis and in a matched control group of patients with osteoarthritis. There were no differences between the groups in the free fractions of the 5 drugs studied. Fluorescent probe studies also showed only minor differences between groups. The results do not support previous suggestions that drug protein binding is altered in rheumatoid arthritis. Albumin concentration was positively correlated with the binding of salicylate, but not with binding of the other 4 drugs. Fatty acid concentration was directly correlated with the binding of warfarin and indomethacin. Correlations between drugs with respect to protein binding were investigated, but only that between salicylate and ibuprofen was significant.

Mechanism of action of paracetamol protective agents in mice in vivo.

The mechanism of action of cysteine, methionine, N-acetylcysteine (NAC) and cysteamine in protecting against paracetamol (APAP) induced hepatotoxicity in male C3H mice in vivo has been investigated by, characterising the effect of the individual protective agents on the metabolism of an hepatotoxic dose of APAP, and determining the efficacy of the protective agents in animals treated with buthionine sulphoximine (BSO), a specific inhibitor of glutathione (GSH) synthesis. Co-administration of cysteine, methionine or NAC increased, while co-administration of cysteamine decreased, the proportion of GSH-derived conjugates of APAP excreted in the urine of mice administered APAP, 300 mg/kg. Pretreatment of animals with BSO abolished the protective effect of cysteine, methionine and NAC, whereas cysteamine still afforded protection against APAP after BSO treatment. In conjunction with other data, these results suggest the most likely mechanism for the protective effect of cysteine, methionine and NAC is by facilitating GSH synthesis, while the most likely mechanism for the protective effect of cysteamine is inhibition of cytochrome P-450 mediated formation of the reactive metabolite of APAP.

Protein binding of GP53,633: a basic non-steroidal anti-inflammatory drug.

The protein binding of GP53,633 [2-tert. butyl-4(5)-phenyl-5(4)-(3-pyridyl)-imidazole], a basic non-steroidal anti-inflammatory drug (NSAID), has been investigated. Although GP53,633 is a base (PKa 6.4) rather than an acid, the binding in plasma or serum was totally accounted for by binding to albumin. Scatchard analysis of the binding to albumin suggested the presence of one high affinity site and a number of low affinity sites. GP53,633 and its major metabolite, CGP8716, displaced site-I fluorescent probes (DNSA and warfarin) but not the site-II probe dansylsarcosine. Binding studies by equilibrium dialysis showed that GP53,633 and its metabolite displaced site-I drugs but not site-II drugs, and 14C-GP53,633 was itself displaced by site-I but not site-II drugs. As with other site-I drugs, the binding of GP53,633 was enhanced by addition of oleic acid at molar ratios of up to 2:1 with albumin. Albumin binding of GP53,633 was markedly increased by raising the pH from 6.0 to 8.5 suggesting that only the unionised drug can bind at site-I. The data are consistent with the major part of the binding energy at site-I being due to hydrophobic interactions and also suggest that there is a cationic centre on the protein at or near site-I which precludes the binding of positively charged drugs.

In vitro evidence for the involvement of at least two forms of human liver UDP-glucuronosyltransferase in morphine 3-glucuronidation.

Morphine 3-glucuronidation kinetics and the inhibitory effects of a number of xenobiotics on morphine glucuronidation in human liver microsomes have been investigated. In both native and detergent-activated microsomes morphine glucuronidation exhibited biphasic kinetics, with a high affinity, low capacity component and a low affinity, high capacity component. These data suggest the involvement of at least two forms of human liver UDP-glucuronosyltransferase (UDPGT) in morphine glucuronidation. The high affinity morphine-UDPGT activity is likely to be of most importance in morphine glucuronidation in vivo. Chloramphenicol, 4-hydroxybiphenyl, 4-methylumbelliferone, 1-naphthol and 4-nitrophenol were all shown to inhibit the low affinity morphine-UDPGT activity, but only chloramphenicol and 1-naphthol were competitive inhibitors. Each of these xenobiotics were shown to be a non-inhibitor of the high affinity morphine-UDPGT activity, or at least to have considerably lower affinity for this enzyme form(s) than morphine itself. Overall the results provide further evidence for the heterogeneity of human liver UDPGT and, in conjunction with other recent studies (Miners JO et al., Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes, Biochem Pharmacol 37: 665-671, 1988) of the kinetics of human liver glucuronidation reactions, indicate that xenobiotic substrates such as morphine, 4-methylumbelliferone and 1-naphthol may be used to differentiate UDPGT isozyme activities in human liver microsomes.