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Caffeine (5 mg kg-1) was administered orally to two healthy, non- smoking subjects on three separate occasions-before, and during therapy with the xanthine oxidase inhibitor allopurinol at doses of either 300 or 600 mg daily. Plasma and urinary levels of methylxanthines, endogenous oxypurines and allopurinol and its metabolite oxypurinol were measured using h.p.l.c. analyses. Allopurinol treatment caused a specific, dose-dependent inhibition of the conversion of the caffeine metabolite 1-methylxanthine (1X) to 1- methyluric acid (1U). A good correlation was observed in both subjects between the urinary 1U/1X molar ratio and the ratio of endogenous urate to hypoxanthine + xanthine at the different allopurinol doses, supporting the proposal that the 1U/1X molar ratio after caffeine intake provides an in vivo index of xanthine oxidase activity in man. [ABSTRACT FROM AUTHOR]

A method is presented for the use of caffeine, in the forms commonly ingested by a large proportion of the world's population, to test for the clinically important acetylation polymorphism. Each of 146 subjects provided a spot sample of urine between 2 and 6 h after coffee, tea or cola soft drink consumption, and the molar ratio of 5-acetylamino-6- formylamino −3-methyluracil (AFMU) to 1-methylxanthine (1X) was determined by a simple h.p.l.c. assay. The ratio afforded segregation of three apparent modes of acetylation capacity in this population, in concordance with a standard sulphamethazine phenotyping procedure and with other methods using controlled caffeine intake and urine collections. The day-to-day consistency of the method was established in eight selected subjects. [ABSTRACT FROM AUTHOR]

Domestic abuse often begins or escalates during the perinatal period, increasing the risk of adverse pregnancy outcomes and death of the woman and infant. The hidden nature of domestic abuse, compounded by barriers to disclosure, means many clinicians are likely to have unknowingly encountered a patient who is being abused and missed a vital opportunity for intervention. This educational article presents the experience of a woman who was abused during pregnancy. It describes how to facilitate a disclosure and conduct an assessment and illustrates safeguarding duties alongside interventions.

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG -encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria., Competing Interests: The authors declare no conflict of interest.

Objective: To conduct the cloning identification and characterization of the sequence of human IL-17A promoter so as to analyze the regulatory mechanism of the gene expression of IL-17., Methods: First of all, the potential promoter region of IL-17A was found by means of the bioinformatics methods. Then, it was cloned into the reporter vector with PCR technique. Finally, the activity of the test promoter was determined by dual luciferase reporter system., Results: Two transcriptional start points of the upper region, 600 bp and 1000 bp, of IL-17A were obtained by PCR clone and proved to have certain activities by dual luciferase reporter system. Also, they could be activated by IL-17A activator STAT3, which could start the expression of the reported gene., Conclusions: Clone established the regulatory region of human IL-17A promoter, which provided bases to the subsequent function research., (Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.)

DNA in dried blood spots of 39 vivax malaria patients (2009-2010) from Anhui (Bengbu urban district and counties of Wuhe, Huaiyuan, Mengcheng and Lixin) was extracted. The Plasmodium vivax LDH (PvLDH) gene was amplified, cloned and sequenced. The sequences were subjected to NCBI Blast program. The results showed that the targeted DNA fragment size was 951 bp without difference among the 39 samples (accession No. GU078391), and was more than 99% homologous to the PvLDH sequences in other strains from GenBank. There was only one different amino acid in the protein sequences between the isolates from Anhui and EJEU60134 or MIA061251 strains.

Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.

Hypnosis has been shown to be efficacious in a range of clinical conditions, including the management of chronic pain. However, not all individuals are able to enter a hypnotic state, thereby limiting the clinical utility of this technique. We sought to determine whether hypnotic susceptibility could be increased using three methods thought to facilitate relaxation, with particular interest in an EEG neurofeedback protocol which elevated the theta to alpha ratio. This was compared with progressive muscle relaxation and self-hypnosis. Ten subjects with moderate levels of susceptibility (2-7/12) were randomly assigned to each condition and assessed for hypnotic susceptibility prior to and upon completion of 10 sessions of training. Hypnotic susceptibility increased post-training in all groups, providing further evidence that operant control over the theta/alpha ratio is possible, but contrary to our predictions, elevation of the theta/alpha ratio proved no more successful than the other interventions. Nonetheless, all three techniques successfully enhanced hypnotic susceptibility in over half of the participants (17/30), a similar incidence to that reported using other methods. As previously reported, the majority who were not susceptible to modification were at the lower levels of susceptibility, and the greater increases tended to occur in the more susceptible subjects. However, here enhancement was disclosed in some at low levels, and capability was found of reaching high levels, both features not typically reported. Further research is warranted.

Basidiomycete PM2, a lignin-degrading white rot fungus, produces lgnin peroxidase (Lip) and manganese peroxidase (Mnp) in nutrient nitrogen limited liquid cultures. This fungus was selected for its ability to decolorize azo group of dyes. In order to improve production of the peroxidases and rapid dye decolorizing activity by basidiomycete PM2, the addition of veratryl alcohol or Tween 80 to nutrient nitrogen limited liquid cultures were tested. It was found to have a large stimulatory effect on Mnp activities and decolorization rate of azo dyes. A maximum Mnp activities of 254.2 u/L with veratryl alcohol and 192.2 u/L with Tween 80 were achieved respectively. These values were about 3.4-fold and 2.5-fold higher than that obtained in the control cultures (without alcohol or Tween 80), whereas the levels of Lip activity detected were very low (about 12 u/L)in all the cultures. In further experiments using three kinds of azo dyes of congo red, orange G and orange IV, enzyme activities and dye decolorization were investigated in the above-mentioned cultures. The results showed that Mnp activities and decolorization were notably higher than those obtained in the control cultures in the presence of azo dyes. Cultures supplemented with Tween 80 were more adequate for dye decolorization. The rates of the decolorization with Tween 80 of congo red (95.4%), orange G (98.5%) and orange IV (54.4%) after 24 hours of dye incubation were higher than that supplemented with veratryl alcohol. According to the results, Mnp activities secreted by basidiomycete PM2 play an essential role in the process of dye decolorization. Tween 80 was the main factor affecting the decolorization. The analysis of structure of the three kinds of azo dyes indicats that the extent of decolorization is affected by the dye molecular structure. The types and quantity of the substituted groups on the aromatic ring of azo dyes have effect on the percentage of biological decolorization.

Introduction: Mammographically dense breast tissue is a strong predictor of breast cancer risk, and is influenced by both mitogens and mutagens. One enzyme that is able to affect both the mitogenic and mutagenic characteristics of estrogens is cytochrome P450 1A2 (CYP1A2), which is principally responsible for the metabolism of 17beta-estradiol., Methods: In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity, malondialdehyde (MDA) levels, and mammographic density. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Levels of serum and urinary MDA, and MDA-deoxyguanosine adducts in DNA were measured. Mammograms were digitized and measured using a computer-assisted method., Results: CYP1A2 activity in postmenopausal women, but not in premenopausal women, was positively associated with mammographic density, suggesting that increased CYP1A2 activity after the menopause is a risk factor for breast cancer. In premenopausal women, but not in postmenopausal women, CYP1A2 activity was positively associated with serum and urinary MDA levels; there was also some evidence that CYP1A2 activity was more positively associated with percentage breast density when MDA levels were high, and more negatively associated with percentage breast density when MDA levels were low., Conclusion: These findings provide further evidence that variation in the activity level of enzymes involved in estrogen metabolism is related to levels of mammographic density and potentially to breast cancer risk.

Introduction: Breast cancer risk may be determined by various genetic, metabolic, and lifestyle factors that alter sex hormone metabolism. Cytochrome P450 1A2 (CYP1A2) is responsible for the metabolism of estrogens and many exogenous compounds, including caffeine., Methods: In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity and known or suspected risk factors for breast cancer. Blood levels of sex hormones, lipids, and growth factors were measured. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Stepwise and maximum R regression analyses were used to identify covariates related to CYP1A2 activity after adjustment for ethnicity., Results: In both menopausal groups CYP1A2 activity was positively related to smoking and levels of sex hormone binding globulin. In premenopausal women, CYP1A2 activity was also positively related to insulin levels, caffeine intake, age, and plasma triglyceride levels, and negatively related with total cholesterol levels and body mass index. In postmenopausal women CYP1A2 activity was positively associated with insulin-like growth factor-1, and negatively associated with plasma triglyceride, high-density lipoprotein cholesterol, and age at menarche., Conclusion: These results suggest that CYP1A2 activity is correlated with hormones, blood lipids, and lifestyle factors associated with breast cancer risk, although some of the observed associations were contrary to hypothesized directions and suggest that increased CYP1A2 function may be associated with increased risk for breast cancer.

Aims: To determine whether the variability in cytochrome P(450) (CYP)3A4 metabolic function is exhibited at both transcription and translation levels and to examine the population distribution of CYP3A4 enzyme kinetics, protein, and mRNA., Methods: Enzyme kinetics of testosterone 6beta-hydroxylation, immunoblot CYP3A4 protein, and CYP3A4 mRNA were determined in a microsomal bank of human livers. The distribution of these determinations was analyzed using cumulative distribution (probit) plots and normality test variable (NTV) to detect deviation from normality., Results: Mean hepatic CYP3A4 protein and relative CYP3A4 mRNA were 35+/-23 pmol/mg and 79+/-59 (CYP3A4/beta-actin), respectively. Kinetic parameter estimates of testosterone 6beta-hydroxylation were 611+/-684 pmol/mg/min for maximum rate of the reaction (V(max)) and 206+/-48 micro M for the Michaelis constant (K(m)). The CYP3A4 gene expression and its activity exhibited a relatively high degree of interindividual variability. Furthermore, significant correlation between CYP3A4 protein and V(max) of testosterone 6beta-hydroxylation (r=0.82, P<0.001) as well as CYP3A4 protein and its mRNA (r=0.52, P<0.01) was observed. Cumulative distribution plots and histograms for the CYP3A4 protein, its mRNA, and maximum testosterone 6beta-hydroxylation exhibited evidences of deviation from log-normal distribution. The minimum NTV value for the distribution of CYP3A4 protein corresponding to the inflection point in the probit occurred at approximately 10% cumulative frequency. The percentage of low CYP3A4 protein phenotype was consistent for CYP3A4 activity and its mRNA. In contrast, the distribution of K(m) of testosterone 6beta-hydroxylation does not show evidence of bimodality., Conclusions: The distribution of CYP3A4 metabolic function, protein, and mRNA is non-normal and may represent a regulatory polymorphism in hepatic CYP3A4 gene expression. In contrast, assessment of the possibility of a structural variant of CYP3A4 through evaluation of the standard deviation relative to the mean kinetic constant value suggests that structural mutation of CYP3A4 may not be a major factor affecting interindividual variation in CYP3A4 metabolic function.

Clozapine is an atypical antipsychotic drug and displays efficacy in 30% to 60% of patients with schizophrenia who do not respond to traditional antipsychotics. A clozapine concentration greater than 1,150 nmol/L increases the probability of antipsychotic efficacy. However, plasma clozapine concentration can vary more than 45-fold during long-term treatment. The aim of this study was to assess the contribution of CYP1A2 to variability in steady-state concentration of clozapine and its active metabolite norclozapine. Patients with schizophrenia or schizoaffective disorder were prospectively monitored during clozapine treatment (N = 18). The in vivo CYP1A2 activity was measured using the caffeine metabolic ratio (CMR) in overnight urine. Trough plasma samples were drawn after at least 5 days of treatment with a constant regimen of clozapine. A significant negative association was found between the CMR and the dose-corrected clozapine (r(s) = -0.87,p < 0.01) and norclozapine (r(s) = -0.76,p < 0.01) concentrations. Nonsmokers displayed a higher clozapine (3.2-fold) and norclozapine (2.3-fold) concentration than smokers (p < 0.05). Furthermore, there was marked person-to-person variation in CYP1A2 activity during multiple-dose clozapine treatment (coefficient of variation = 60%). Age, weight, serum creatinine, and grapefruit juice consumption did not significantly contribute to variability in clozapine and norclozapine concentration (p > 0.05). In conclusion, CYP1A2 is one of the important contributors to disposition of clozapine during multiple-dose treatment. Although further in vitro experiments are necessary, the precise metabolic pathways catalyzed by CYP1A2 seem to be subsequent to the formation of norclozapine, hitherto less recognized quantitatively important alternate disposition routes, or both. From a clinical perspective, an environmentally induced or constitutively high CYP1A2 expression can lead to a decrease in steady-state concentration of clozapine as well as its active metabolite norclozapine. Thus, interindividual variability in CYP1A2 activity may potentially explain treatment resistance to clozapine in some patients. CYP1A2 phenotyping with a simple caffeine test may contribute to individualization of clozapine dosage and differentiate between treat ment noncompliance and high CYP1A2 activity.

Objective: To characterize the distribution of mathematically derived human hepatic CYP1A1 activity using differential inhibition of ethoxyresorufin O-deethylation (EROD) by fluvoxamine., Methods: Quantitative CYP1A1- and CYP1A2-mediated EROD activities were determined in 42 human livers using differential inhibition of EROD by fluvoxamine. CYP1A2-specific activity was also measured by phenacetin O-deethylation and caffeine 3-demethylation. Distributions of CYP1A1-mediated EROD and CYP1-A2 probe activities were analyzed using cumulative distribution (probit) plots and the Kolgomorov-Smirnov test. Age effect on CYP1A1- and CYP1A2-mediated EROD activities was evaluated using descriptive statistics and analysis of variance., Results: The derived CYP1A1 protein concentration of 0.58 +/- 1.04 pmol/mg was only 4% of the derived CYP1A2. Since CYP1A1 is intrinsically far more active than CYP1A2 in mediating EROD, contribution of CYP1A1 to EROD represented approximately 25-40% of CYP1A2 contribution. Three of the 42 livers exhibited no CYP1A1-mediated EROD. Approximately 8% of the individuals showed high CYP1A1 activity phenotype based on cumulative distribution curve analysis. Hepatic CYP1A1 activity was more variable than that of CYP1A2. The variance of CYP1A1-mediated EROD was significantly different from that of CYP1A2, using the Kolgomorov-Smirnov statistical test. Even though not statistically significant, an age-related pattern in CYP1A1-mediated activity was identified: activity was high in the pre-puberty group, then decreased in the young/mature adult group and, finally, a slight increase was observed in old age., Conclusions: Distribution pattern in CYP1A1-mediated EROD suggests that the low derived CYP1A1 expression is most likely induced rather than constitutive. CYP1A1 activity deviates from log-normal distribution; the variations in hepatic CYP1A1 activity may affect the conversion of procarcinogens to carcinogens. The age-related trend in CYP1A1-mediated EROD activity hints that CYP1A1 responsiveness to inducers may change with age as well as with exposure to environmental inducers. These findings prompt (1) future genotyping studies to determine whether increased CYP1A1 inducibility is a result of genetic factors and (2) studies to address whether CYP1A1 inducibility changes with age.

The CYP3A4 enzyme contributes to the disposition of more than 60 therapeutically important drugs and displays marked person-to-person variability of the catalytic function. However, the extent of genetic contribution to variability in CYP3A4 activity remains elusive. Recently, we showed that a comparison of between- (SDb2) and within-person (SDW2) variances provides an estimate of the genetic component of variability in drug disposition. The aim of the present analysis was to assess the genetic control of CYP3A4 activity in vivo. A computerized literature search was conducted covering 1966 to September 1999 to identify studies reporting repeated administration of CYP3A4 substrates. The genetic contribution (rGC) to disposition of each CYP3A4 substrate was obtained by the formula (SDb2-SDW2)/SDb2. The rGC values approaching 1.0, point to overwhelming genetic control, whereas those close to zero suggest that environmental factors dominate. A total of 16 studies with 10 different CYP3A4 substrates were identified (n = 161 subjects). The rGC for hepatic CYP3A4 activity as measured by midazolam plasma clearance or the erythromycin breath test was 0.96 (0.92-0.98) (95% Cl) and 0.89 (0.65-0.98), respectively (P < 0.05). The point estimates of rGC for composite (hepatic + intestinal) CYP3A4 activity measured after oral administration of cyclosporine, ethinylestradiol, ethylmorphine, nifedipine and nitrendipine, ranged from 0.66-0.98 (median: 0.83) (P < 0.05). Cyclosporine data suggested a higher genetic control of CYP3A4 at night than during the day. These data indicate that further molecular genetic investigations are warranted to identify genetic variants at CYP3A4 or elsewhere in the genome which contribute to regulation of CYP3A4 activity.

1. Caucasian liver samples were used in this study. N-demethylation of trimethadione (TMO) to dimethadione (DMO) was monitored in the presence of chemical inhibitors of CYPs, such as fluconazole, quinidine, dimethyl-nitrosamine, acetaminophen, phenacetin, chlorzoxazone and mephenytoin. Trimethadione N-demethylation was selectively inhibited by dimethylnitrosamine and chlorzoxazone (> 50%) and weakly inhibited by tolbutamide (12%) and fluconazole (22%), whereas other inhibitors showed no effect. This result suggested that TMO metabolism to DMO is mainly mediated by CYP2E1 and marginally by CYP2C and CYP3A4. 2. Fifteen human livers were screened and interindividual variability of TMO N-demethylation activity was 3-fold. Chlorzoxazone 6-hydroxylation activity was also measured and both activities were significantly correlated (r=0.735, p < 0.01). 3. DMO production by human cDNA expressed CYP enzymes was observed mainly for CYP2E1 (10.8 nmol/tube), marginally for CYP2C8 (0.22 nmol/tube) and not detectable for other CYP enzymes. 4. These results indicate that TMO metabolism is primarily catalysed by CYP2E1 and that trimethadione would be a suitable selective probe drug for the estimation of human CYP2E1 activity in vivo.

It has been difficult to study the regulation of cytochrome P4501A2 (CYP1A2) because expression of this enzyme is reported to be limited or absent in cell culture. We found that CYP1A2 can be induced significantly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), or benz[a]anthracene in the human colon carcinoma cell line LS180. TCDD and MC each caused a dramatic elevation of CYP1A2 mRNA, as assessed by reverse transcription-polymerase chain reaction or by northern blot analysis. TCDD also increased immunoreactive CYP1A2 protein and the activity of phenacetin-O-deethylase, a diagnostic catalytic marker for CYP1A2. The induction of CYP1A2 at all levels (mRNA, protein, catalytic activity) was concentration- and time-dependent: the EC50 for mRNA induction by TCDD = 0.5 nM, and by MC = 1.4 microM. Inducible CYP1A2 mRNA also was detected at lower levels in two other human cell lines, the hepatoma cell line HepG2 and the breast carcinoma cell line MCF-7. CYP1A1 and CYP1B1, additional CYP1 enzymes regulated by the aryl hydrocarbon receptor (AHR), also were inducible by TCDD and MC in LS180 cells; their concentration-dependent induction was highly correlated with induction of CYP1A2 at mRNA, protein, and catalytic levels. CYP1B1 was constitutively expressed and inducible in the LS180, MCF-7, and HepG2 cell lines as well as in the human choriocarcinoma cell line JEG-3 and the squamous cell carcinoma line A431. CYP1A2 was neither constitutively expressed nor inducible in A431 or JEG-3 cells. The expression of mRNAs encoding the regulators of CYP1 enzymes-the AHR and its heterodimerization partner, the ARNT (AH receptor nuclear translocator) protein-was not altered by treatment with TCDD or MC. However, the cytosolic content of AHR protein and ARNT protein was depleted substantially following treatment with TCDD. The LS180 cell line should constitute a good model for further mechanistic studies on AHR-regulated CYP1A2 expression.

Aims: To study the potential utility of caffeine based probes of CYP1A2 enzyme activity in predicting the pharmokinetics of tacrine in patients with Alzheimer's disease., Methods: The pharmokinetics of a single 40 mg oral dose of tacrine were measured in 19 patients with Alzheimer's disease. Each patient also received 2 mg kg(-1) [13C-3-methyl] caffeine orally and had breath and urine samples collected., Results: Tacrine oral clearance (CL F(-1) kg(-1)), which varied 15-fold among the patients, correlated significantly with the 2 h total production of 13CO2 in breath (r=0.56, P=0.01), and with each of two commonly used urinary caffeine metabolite ratios: the 'paraxanthine/caffeine ratio' (1,7X + 1, 7U)/1,3,7X) (r=0.76, P=0.0002) and the 'caffeine metabolic ratio' (AFMU + 1X + 1U)/1, 7U)(r=0.76, P=0.0001)., Conclusions: These observations support a central role for CYP1A2 in the in vivo disposition of tacrine and the potential for drug interactions when tacrine treated patients receive known inducers or inhibitors of this enzyme. The magnitude of the correlations we observed, however, are probably not sufficient to be clinically useful in individualizing tacrine therapy.

Twin studies are useful devices to determine the heritability of persistent but variable characteristics that tend to differ among individuals. Drug responses are not persistent affairs; they are temporary characteristics. One therefore may ask whether twin studies are necessary to assess the genetic element in pharmacological responsiveness. To measure the genetic component contributing to their variability, it seems logical to investigate the response variation by repeated drug administration to given individuals, and to compare the variability of the responses within and between individuals. We attempt here to describe a theoretical background of this venture, and to show some results of the exercise. Potential sources of error or uncertainty are discussed.

Ethoxyresorufin O-deethylation (EROD) has been used as a specific probe for CYP1A1 and CYP1A2. Selective inhibition of one of these cytochromes P450 may differentiate their activity in human liver. Four inhibitors were chosen to examine the selective inhibition of EROD activity, using cDNA of CYP1A1 and CYP1A2. The two flavones, alpha-naphthoflavone and apigenin, while differing in potency, inhibited expressed human CYP1A1, CYP1A2, and human liver microsomes to a similar extent. Isosafrole and fluvoxamine were found to inhibit CYP1A2 selectively, with Ki values of 14 and 800 times, respectively, lower than those for CY1A1. A set of equations was developed to estimate both CYP1A1 and CYP1A2 activity. Levels of CYP1A2 in four human liver specimens ranged from 44.4 to 76.7 pmol/mg protein, which significantly correlated with phenacetin O-deethylase activity (r = 0.99; P < 0.001). Low levels of CYP1A1 activity were present in all four investigated livers, ranging from 0.4 to 2.7 pmol/mg protein.

Objective: The therapeutic response to PGE1 is highly variable, and a contribution by variable formation of its active tertiary metabolite PGE0 is in question. Hence, the objective of this study was to assess the person-to-person variation of the reduction of the inactive intermediate metabolite 15-KD PGE1 by human liver and human erythrocytes in forming the active metabolite PGE0., Methods: Source of enzyme was lysed erythrocytes from 29 donors, and a bank of 37 donor livers including specimens from 15 children. Tritium-labelled 13,14-dihydro-15-keto-prostaglandin E1 (15-KD PGE1) was used at low nanomolar concentrations and found to be converted almost exclusively to the more polar compound 13,14-dihydro-prostaglandin E1 (PGE0) by an NADPH-dependent carbonyl reductase. The identity of the product PGE0 was established by comparison of its chromatographic and mass spectral characteristics with authentic PGE0., Results: Lysed erythrocytes had readily measurable enzymatic activity; differences between the preparations from 29 subjects were very small with only a twofold range of variation. In contrast to lysed erythrocytes, intact erythrocytes did not catalyse the reaction so that the erythrocyte activity should be medically immaterial. 15-KD PGE1 15-ketoreductase activity of liver cytosol averaged 61.1 fmol.min-1.mg-1 protein in preparations from 37 human livers. Individual activities varied over an almost tenfold range, with indications of a non-normal distribution. Kinetic studies of selected specimens showed substantially different Vmax values but indistinguishable kM values, suggesting that the individual variation in 15-KD PGE1 15-ketoreduction is the result of differences in enzyme concentration rather than of structural enzyme variations. The activity in 15 livers from children was significantly lower than in those from adults. Inhibition data suggest that both the liver and the erythrocyte enzymes belong to the class of carbonyl reductases., Conclusions: The variations in hepatic enzyme activity may be expected to affect the transformation of 15-KD PGE1 to the active metabolite PGE0 in vivo. The clinical significance remains to be explored.

We investigated the involvement of CYP1A2 in the pharmacokinetics and metabolism of caffeine using mice lacking its expression (CYP1A2 -/-). The half-life of caffeine elimination from blood was seven times longer in the CYP1A2 -/- than wild-type mice. The clearance was concomitantly eight times slower. No parameter that could affect the pharmacokinetics differed between CYP1A2-/-and wild-type mice such as creatinine for kidney function; alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin for liver function; or albumin for protein binding. Other P450s CYP2A, 2B, 2C, 2EI, and 3A were also unchanged in the knockout animals. Caffeine 3-demethylated metabolites thought previously to be characteristic of CYP1A2 (especially 1-methylxanthine and I-methylurate) were also found in the urines of the CYP1A2-/-animals, although at 40% of the level found in wild-type mice. These data indicate that the clearance of caffeine in wild-type mice is primarily determined by CYP1A2.

Previous studies have shown that bupranolol, a beta-adrenoceptor blocker, is a substrate of cytochrome P4502D6 (CYP2D6). A sensitive in vitro assay was developed to quantify the formation of hydroxybupranolol using HPLC. A TLC method, using radiolabeled bupranolol, was also developed to test the reproducibility of the two methods. Both of them gave virtually identical results; however, the HPLC method was sensitive to 20 pmol and the TLC with radiolabeled substrate to <1 pmol of hydroxybupranolol. The KM value for bupranolol was lower than that reported for any other substrate of CYP2D6. The KM value in microsomes of a typical human liver (L-1) was 0.272 +/- 0.02 (SE) mu M and the Vmax was 360 +/- 10 (SE) pmol/mg/min (0.83 +/- 0.02 pmol/pmol cytochrome P450/min). The KM value for the CYP2D6 expressed in yeast was 0.076 +/- 0.003 (SE) mu M, and the Vmax was 43 +/- 1 (SE) pmol/mg/min (0.64 +/- 0.01 pmol/pmol cytochrome P450/min). Quinidine competitively inhibited the formation of hydroxybupranolol, with Ki values of 5 nM in expressed CYP2D6 and 14.05 nM in human liver (L-1).

Previously, one of us (B. K. T.) developed an assay that measures levels of free ethanol and ethanol conjugates in urine and showed that the mean levels of these ethanol markers in confirmed alcoholics were at least 20-fold higher than those levels in control subjects. In this study, we assessed the relationship of these biomarkers with self-reported levels of alcohol intake in a multiethnic sample of Los Angeles County residents who were male and over the age of 35 years (n = 128; 40 non-Hispanic whites, 46 blacks, 17 Chinese, and 25 Japanese). Regardless of race, the mean levels of free, bound, and total (free plus bound) ethanol were lowest in nondrinkers, intermediate in weekly drinkers, and highest in daily drinkers (P = 0.0001 in all three statistical tests of differences in the three biomarkers). Stepwise discriminant analysis showed that of the three potential biomarkers, total ethanol best discriminates between the three classes of drinkers (non, weekly, and daily), and that additional inclusion of either free or bound ethanol in the discriminant function had negligible effect. Overall, mean level of total ethanol was 2.2 times higher in weekly than in nondrinkers; daily drinkers, in turn, showed a 4.2-fold increase in mean total ethanol relative to weekly drinkers. However, there was no correlation between any of the three biomarkers and self-reported level (in grams of ethanol) of average consumption in either weekly or daily drinkers whose mean intake was about 13 and 42 g of ethanol/day, respectively. As the level of urinary free ethanol and ethanol conjugates showed extraordinary differences among racial groups for a given level of self-reported ethanol intake, the data suggest possible interracial differences in the in vivo elimination rate of ethanol; this latter finding needs to be confirmed in larger studies.

Although conjugation with glucuronic acid is a major process for converting many xenobiotics into hydrophilic, excretable metabolites, relatively little has been reported concerning interindividual variability of glucuronidation in human populations. Oxazepam, a therapeutically active metabolite of diazepam, is one of a number of C3-hydroxylated benzodiazepines for which glucuronide conjugation is the predominant pathway of biotransformation. The drug is normally formulated as a racemic mixture of inactive (R) and active (S) enantiomers. In the present study we have investigated the use of oxazepam as a potential probe drug for studying the variability of glucuronide conjugation, and for demonstrating the extent to which genetic factors may be responsible. In preliminary studies we determined oxazepam pharmacokinetics metabolite profiles after administration of racemic (R,S) oxazepam to eleven human volunteers. The (S) glucuronide was preferentially formed and excreted in nine of the eleven subjects. The ratios of (S) to (R) glucuronide metabolites (S/R ratios) were 3.87 +/- 0.79 (mean +/- SD) and 3.52 +/- 0.60 in urine and plasma, respectively. However, both ratios were significantly lower in two subjects (p < 0.01). In these two atypical subjects, the half-life of (R,S) oxazepam was also markedly longer (14.7 and 15.9 h) than in the other subjects (8.1 +/- 3.2 h). A good correlation (rs = 0.90) between the S/R-glucuronide ratio in urine and the plasma clearance of (R,S) oxazepam suggested that a low S/R ratio may be a marker of poor elimination of oxazepam. In further investigations, the drug was administered to 66 additional subjects. The S/R-glucuronide ratio in 8 h pooled urine was bimodally distributed, with 10% of all subjects possessing ratios below an apparent antimode of 1.9. A survey of the in vitro formation of oxazepam glucuronides by microsomes from 37 human livers also showed that 10% of the livers displayed an abnormally high apparent Michaelis constant (Km) for the formation of the (S) glucuronide, but not of the (R) glucuronide. These results suggest that the glucuronidation of the pharmacologically active (S) enantiomer of oxazepam is decreased in a significant percentage (10%) of Caucasian individuals. The observed in vitro differences in apparent kinetics of the S-glucuronidation reaction may reflect defects at the genetic level, leading to structural changes in the isozyme(s) of UDP-glucuronyltransferase that catalyse this reaction.

Objectives: To examine the effects of occupational exposure to substances contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cytochrome P-4501A2 activity in a cross sectional medical survey., Methods: The exposed workers had been employed at two chemical plants > 15 years earlier in the manufacture of 2,4, 5-trichlorophenol and its derivatives. The control group consisted of people with no occupational exposure to phenoxy herbicides and who lived within the communities of the exposed workers. A total of 58 workers and 125 unexposed controls participated in the analysis. Cytochrome P-450 activity was assessed with test that measures caffeine metabolites in the urine. A ratio of metabolites of caffeine (CMR) constituted a measure of P-4501A2 activity., Results: Compared with the control group in multivariate logistic regression, raised non-significant associations were found for three of four categories of TCDD in exposed workers (TCDD < 20 pg/g, odds ratio (OR) 1.7, 95% confidence interval (95% CI) 0.6 to 5.0, TCDD 20-66, OR 0.3, 95% CI 0.0 to 1.7; TCDD 67-147, OR 2.3, 95% CI 0.6 to 8.8; TCDD > or = 148, OR 3.1, 95% CI 0.8 to 12.5). We found a strongly significant association of CMR and urinary cotinine, a measure of smoking, and urinary free ethanol. We found weak non-significant associations between P-4501A2 activity and increased serum TCDD among workers., Conclusions: The absence of an association between serum TCDD and cytochrome P-4501A2 may be due to the size of the study, insensitivity of the CMR to assess cytochrome P-4501A2 activity, or inadequate levels of exposure, although these were among the highest in human groups tested.

1,4-Benzodiazepine anxiolytics such as diazepam and halazepam are converted in vivo to oxazepam, an active metabolite with a hydroxyl group at the asymmetric C3 position. D-glucuronic acid couples with the C3 hydroxyl group of oxazepam to form pharmacologically inactive diastereomeric glucuronide conjugates. Conjugation with glucuronic acid is catalysed by the microsomal UDP-glucuronosyltransferase (UGT) enzyme system, which includes an undetermined number of isozymes. Although 1,4-benzodiazepines are ultimately cleared as oxazepam glucuronide, little is known about the particular UGT isozyme(s) responsible for the conjugation at the C3 position of these molecules. Microsomal preparations from three human livers were used to study the glucuronidation of (R,S)oxazepam in vitro. The predominant formation of the S- over the R-glucuronide was reflected by the kinetic parameters: For (S)oxazepam glucuronide, the constants were Km = 0.18 +/- 0.02 mM and Vmax = 202.6 +/- 25.0 nmol min-1 per mg protein; for (R)oxazepam glucuronide, they were Km = 0.22 +/- 0.02 mM, Vmax = 55.4 +/- 9.5 nmol min-1 per mg protein. Inhibition studies suggest that the two diastereomeric glucuronidations are catalysed by different UGT isozymes. That is, there was competitive inhibition of (S)oxazepam glucuronidation by non-steroidal anti-inflammatory drugs (NSAIDs), including ketoprofen while (R)oxazepam glucuronidation was not equally inhibited by these compounds. The order of potency of inhibitors of (S)oxazepam glucuronidation in this study was the same as the rank order of substrates conjugated by UGT2B7; hyodeoxycholic acid, estriol, (S)naproxen, ketoprofen, ibuprofen, fenoprofen, clofibric acid, and morphine (in descending order). The inhibition profile of (S)oxazepam glucuronidation suggests that UGT2B7 is the catalysing enzyme.

Lovastatin, widely used to lower cholesterol, is a pro-drug that requires metabolic activation through hydrolysis by carboxyesterases. There appear to be at least three distinct esterases in humans capable of catalysing this reaction, one in plasma and two in the liver. The rate of lovastatin hydroxy acid formation was measured as 15.8 pmol.ml-1.min-1 in plasma, 2.13 pmol.mg-1 protein.min-1 in hepatic microsomes and 0.92 pmol.mg-1 protein.min-1 in cytosol. The data suggest that on average the three esterases together are capable of activating about 220 nmol (90 micrograms) lovastatin per minute per person, to which the esterases of plasma, liver microsomes and liver cytosol contribute approximately 18, 15 and 67%, respectively. All three esterases showed evidence of inter-individual variability. In one of 17 livers, both cytosolic and microsomal esterase activity was completely missing, while two other liver specimens lacked one esterase. Such variability must be expected to influence the therapeutic efficacy of the drug, and they might be related to its occasional toxicity.

Caffeine is increasingly used as a biochemical probe for liver function, in cancer epidemiology, and in pharmacogenetics, with its recognized ability to assess the activities of CYP1A2, xanthine oxidase, and N-acetyltransferase-2. The activity of these hepatic enzymes was tested in 45 Shona children from a rural area of Zimbabwe with use of caffeine as a probe. Many of these rural black children had lower indexes of CYP1A2 activity than otherwise on our extensive records; the average value (3.78 +/- 2.9) was significantly (p < 0.001) lower than that of healthy white urban children from Zimbabwe (8.86 +/- 3.36) or from Canada (7.92 +/- 1.88), or that of healthy Canadian adults (5.96 +/- 2.4). A higher CYP1A2 activity in children than in adults is usual. The low CYP1A2 activity of the children from rural Zimbabwe calls for medical studies and suggests a widespread and perhaps serious impairment of certain liver functions. Causes could be parasitic infections with Schistosoma mansoni, causing schistosomiasis, which are endemic, in addition to generally poor nutrition and frequent iodine deficiency. By contrast, the xanthine oxidase activity in rural Shona children was slightly higher than that reported for a healthy Canadian adult population. The N-acetyltransferase activities were comparable in both the rural and urban children and were also similar to those reported in a population study of healthy adult Canadians.

Acetohexamide, an oral antidiabetic agent, is metabolized by carbonyl reductase to hydroxyhexamide, which has a higher hypoglycemic potency than the parent compound. In the present study, interindividual variability of carbonyl reductase activity in erythrocyte was examined. Enzyme activity in 31 healthy subjects (23.9 plus minus 3.4 years, mean plus minus SD) was monitored by measuring formation of hydroxyhexamide using HPLC methods. Using 0.5 mM acetohexamide as substrate, reductase activity of 6.06 plus minus 0.06 nmol min(minus sign1) gHb(minus sign1) (range: 5.9--6.2) with a coefficient of variation of 15% was observed in erythrocytes. Acetohexamide-reducing activity in erythrocytes showed a normal distribution and the interindividual variability of the reductase activity was found to be small, implying that the large variability reported for the acetohexamide plasma half-life is not caused by the amount of reductase enzyme in erythrocytes.

Caffeine biotransformation was demonstrated in three novel human hepatocyte cell lines established from normal liver tissue and cultured continuously for 19 to 30 months. Caffeine and its metabolites were identified and quantified by high performance liquid chromatography. Without induction, caffeine was metabolized to the four primary metabolites [theobromine (37X), paraxanthine (17X), theophylline (13X), 1,3,7-trimethylurate (137U)]. Under these basal conditions 137U was the predominant metabolite. The actual pattern of metabolite production was a reproducible characteristic of each line. After induction with dibenz(a,h)anthracene, the formation of 17X was increased 4-17 fold. Induction with phenobarbital did not change the metabolic profile. These human hepatocyte lines can reproduce in vitro metabolism of caffeine observed in man in vivo.

Two established caffeine-based urinary methods for measuring CYP1A2 activity were compared with each other, and also with the systemic clearance of caffeine which served as a standard of reference for such activity. Following a standardized dose, caffeine (137X) and its metabolites were measured in urine and plasma of 39 healthy subjects. The measurements allowed determinations of: (1) systemic caffeine clearance (CL(caff)); (2) the caffeine metabolite ratio (AFMU + 1X + 1U)/17U determined in an overnight-urine specimen and referred to as CMR, and (3) the ratio (17X + 17U)/137X measured in urine collected between 4 and 5 h after caffeine intake and referred to as PCUR for 'paraxanthine-caffeine urinary ratio'. The PCUR showed a bimodal distribution and a relatively wide variation, CL(caff) and CMR were both normally distributed. The correlation between CL(caff) and CMR was r = 0.77 (p < 0.001), between CLcaff and PCUR r = 0.46 (p < 0.01), and between CMR and PCUR r = 0.40 (p < 0.02). The difference between the correlation coefficients 0.77 and 0.46 was statistically significant (z-test; p < 0.05). The well established decrease of caffeine metabolism by oral contraceptive use was observed with both CL(caff) and CMR but not with PCUR. Examination of possible explanations for the differences between PCUR and CMR led to the finding of a correlation between PCUR and the renal clearance of caffeine (CLr) with r = -0.47 (p < 0.01). Further scrutiny demonstrated that a bimodal or non-normal frequency distribution as shown by PCUR was also shown by CLr and by urine flow rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Six human cytochrome P450s expressed in HepG2 cells using vaccinia virus cDNA-directed expression, were used to study the biotransformation of caffeine and its metabolites. CYP1A2 alone was responsible for caffeine 3-demethylation and paraxanthine 7-demethylation; in addition, 1A2 catalysed virtually all reactions related to caffeine and its metabolites. The metabolic profile of caffeine biotransformation by CYP1A2 averaged 81.5% for paraxanthine, 10.8% for theobromine and 5.4% for theophylline formation. It remained quite uniform when caffeine concentrations were varied. The most striking finding was that CYP2E1 (the ethanol-inducible form) had major influences upon caffeine metabolism: in particular, it catalysed the formation of theophylline and theobromine from caffeine. Thus, the in vivo metabolite profiling of caffeine may reveal CYP2E1 activities in addition to the previously documented activities of CYP1A2, polymorphic N-acetyltransferase and xanthine oxidase.

Acetaminophen (paracetamol) is extensively conjugated with glucuronic acid and sulfate prior to renal excretion. A minor metabolic route involves microsomal oxidation of acetaminophen to a hepatotoxic reactive intermediate, which subsequently undergoes glutathione (GSH) conjugation, yielding cysteine and mercapturate conjugates, both of which are excreted in the urine (Slattery et al., 1987). Data collected by de Morais et al. (1989) indicated that in comparison with normal subjects, glucuronidation of acetaminophen was impaired in subjects with Gilbert's syndrome, a genetically-based impairment of bilirubin glucuronidation. Thus, inter-subject and ethnic differences in acetaminophen disposition have pharmacogenetic and toxicological implications. This study was conceived to explore these differences. Urinary excretion of acetaminophen and its metabolites was observed in 125 Caucasian and 33 Oriental subjects. No appreciable difference was noted in the mean fraction of drug excreted as glucuronide between the two groups (51.5% in Caucasians vs 51.8% in Orientals). However, the data strongly indicated that the excretion of acetaminophen glucuronide was not normally distributed. Bimodality was apparent in both groups, with 20% of Caucasian and 33% of Oriental subjects displaying relatively extensive glucuronidation. In addition, glucuronidation displayed a strong negative correlation with sulfation (r = -0.97), suggesting the existence of a compensatory mechanism between the two metabolic pathways. The mean fractional excretions of cysteine and mercapturate conjugates did show significant differences between Caucasians and Orientals (p < 0.005). In addition, the ratio of mercapturate to total GSH-derived conjugates recovered appeared to be bimodal, indicating possible heterogeneity in the conversion of the cysteine conjugate to mercapturate via N-acetylation.

Caffeine was used as a metabolic probe to screen healthy subjects for their activities of two enzymes, deduced to be CYP1A2 (an inducible cytochrome P450) and xanthine oxidase. A longitudinal study revealed modest effects of caffeine dose, ethanol intake, and time-of-day on the CYP1A2 index, without any effect on the xanthine oxidase index. The coefficients of intraindividual variation not accounted for were 5.0% for the xanthine oxidase and 17.2% for the CYP1A2 index. In a population study, both indexes showed a log normal distribution, with CYP1A2 values of most subjects covering a 6.3-fold range but only a 1.7-fold range with xanthine oxidase. The CYP1A2 index was 33% decreased in women who used oral contraceptives and substantially increased in cigarette smokers. Neither the CYP1A2 nor the xanthine oxidase index differed between volunteers of Chinese and European extraction. Four of 178 subjects showed unexplained low xanthine oxidase values (i.e., values several standard deviations below the mean).

A gas chromatographic mass spectrometric procedure is described for the measurement of free ethanol and conjugates of ethanol and acetaldehyde. It was demonstrated that conjugates of ethanol and acetaldehyde were present in urine of alcoholics. Urinary levels of free ethanol and the conjugates in abstaining alcoholics were higher than those of social drinkers. On the average, alcoholics excreted equal amounts of free ethanol and ethanol conjugates while controls eliminated three parts of ethanol conjugates to one part of free ethanol. After 14 days of abstinence, 16/23 (70%) of alcoholics had levels of free ethanol and acetaldehyde conjugates higher than the mean +2 SD of social drinkers for these substances. When the molar ratio of ethanol conjugates/free ethanol was considered, five more subjects who were alcoholics could be classified as being alcoholics. There was one false positive among 46 control subjects.

The use of two caffeine metabolite ratios for acetylator phenotyping was validated by demonstrating concordance with two sulfamethazine tests in 178 unrelated healthy subjects. The caffeine metabolites used for this purpose were 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U). The ratio AAMU/(AAMU + 1X + 1U), referred to as molar ratio or N-acetyltransferase, was compared with the ratio AAMU/1X. The results indicated that, for screening purposes, the acetylator phenotype can be determined by analysis of a 6-hour urine sample after a cup of coffee or strong tea or a can of caffeine-containing soft drink. The ratio AAMU/1X is the ratio of choice for the study of subjects in whom variability of xanthine oxidase can be neglected; use of the ratio AAMU/(AAMU + 1X + 1U) appears appropriate for special purposes. Gender, ethnic origin, habitual or moderate consumption of coffee, tea, soft drinks, or ethanol, or cigarette smoking have little if any effect on the caffeine tests for acetylator phenotyping.

Delayed hypersensitivity reactions are among the most severe adverse effects of the sulfonamides in current clinical use. These reactions appear to occur because of differences in the metabolism and detoxification of reactive metabolites of the sulfonamides. N-Acetylation is a major metabolic pathway for the sulfonamides. Slow acetylation phenotype might be a risk factor for the development of these reactions. We determined the acetylation phenotype of 21 patients who had suffered hypersensitivity reactions to the sulfonamides. There were 11 females and 10 males in the group, with a mean age of 15 years (age range, 1.8 to 50 years). Their acetylator phenotype was determined by determining the ratio of urinary caffeine metabolites (1-methylxanthine to 5-amino-6-formylmethyluracil after an oral dose of 50 mg caffeine). Nineteen (90%) of the patients were slow acetylators compared to a 55% incidence of slow acetylators in a race-matched control population (p less than 0.008). This suggests that a slow acetylator phenotype is a risk factor for the development of sulfonamide hypersensitivity reactions and provides further support for the role of imbalances in genetically determined pathways of metabolism and detoxification of the sulfonamides in the pathogenesis of these reactions.

It has been realized recently that the primary metabolism of caffeine in humans is catalyzed by P-450IA2 and that the rate of caffeine metabolism can be estimated from a metabolic ratio in a single urine sample. A population of 178 students including 19 smokers were subjected to this caffeine test to establish their P-450IA2 index. Both stated numbers of cigarettes smoked per day and urinary cotinine levels as a confirmatory measure correlated significantly with enzyme activity showing dose-effect relationships (r = 0.62 and 0.89, respectively). Nevertheless, more nonsmokers than smokers had the highest enzyme indexes, suggesting that dietary elements or other factors may determine P-450IA2 activities in populations. Because P-450IA2 is a monooxygenase that may be confined to the liver, caffeine reveals directly the Ah-receptor-dependent enzyme induction only in the liver, but it may also be a signal of induction elsewhere.

Gentisamide (GAM), the hydroxylated metabolite of salicylamide (SAM), underwent sequential metabolism, and GAM-5-glucuronide was the only metabolite detected in the single-pass in situ rat liver preparation (10 ml/min/liver) perfused at varying SAM steady-state input concentrations (CIn). The exclusive formation of GAM-5-glucuronide was unexpected, because GAM, when administered to the rat liver, formed predominantly monosulfate conjugates at the 5- and 2-positions (Morris et al., J. Pharmacol. Exp. Ther. 245: 614-624, 1988). Kinetic parameters obtained from single-pass studies for SAM sulfation (CIn increasing from 31 to 347 microM) and glucuronidation and hydroxylation (CIn decreasing from 1383 to 130 microM, with 0.85 mM SO4(2-), and CIn increasing from 32 to 800 microM in an absence of SO4(2-), with 2,6-dichloro-4-nitrophenol, a sulfation inhibitor) revealed sulfation as a high-affinity, high-capacity pathway, glucuronidation as a lower-affinity, high-capacity pathway and hydroxylation as a low-affinity, low-capacity pathway. However, these parameters would not explain the exclusive glucuronidation of GAM when generated from SAM. Rather the zonal localization of metabolizing activities [a periportal sulfation, evenly distributed glucuronidation, and perivenous hydroxylation system (Xu and Pang, J. Pharmacokinet. Biopharm. 17: 645-671, 1989; Morris et al., J. Pharmacokinet. Biopharm. 16: 633-656, 1988)] could explain the complete lack of sulfation in SAM sequential metabolism. The different metabolite patterns arising from the administrations of a metabolite precursor (SAM) versus a preformed metabolite (GAM), due to the proximity of enzymes for formation and sequential metabolism, may serve to explain the differential toxic or pharmacologic effects observed in other precursor-metabolite pairs.

A number of studies have shown that drug glucosidation occurs in vertebrates. This review summarizes information on the chemical, physical and biological properties of eleven drug glucosides. Three out of two hundred individuals exhibited a complete lack of amobarbital-N-glucoside formation. The data from a pair of identical twins who were deficient in this metabolic pathway suggested the presence of a recessive trait controlled by a single pair of autosomal genes which regulate N-glucoside formation. In addition, average values seemed to be subject to interethnic variation.

The two main metabolites of amobarbital excreted in urine are 3'-hydroxyamobarbital (C-OH) and 1-(beta-D-glucopyranosyl) amobarbital (N-glu). When testing the metabolite ratio in small single samples of urine, it was found that the urine in a Caucasian population contained about one-third glucose conjugation and two-thirds hydroxylation product, while an Oriental population excreted both metabolites in equal proportion. Attempts to learn the causes for the different metabolite ratios led to an investigation of metabolite concentrations in urine. The sums of the average urinary concentration of C-OH was greater in Caucasians than in Orientals, no matter how the data were expressed; the reverse was true for the N-glu metabolite. C-OH data was scattered more widely among Orientals than Caucasians; this might indicate bimodality of the distribution curves. There also was a trend toward more N-glu metabolite in urine of females than of males. Measuring the metabolite/creatinine ratios narrowed the distribution range of the data, particularly after correction for sex difference in creatinine, but population differences were not changed. Expected relationships between metabolite content of urine, sampling times, and plasma half-life (t1/2) were established by calculation. A Caucasian female with no capacity for N-glucosidation was found during the first part of this population survey. An Oriental male with only trace capacity for amobarbital hydroxylation was found in the second part.