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Biomedical subjects

B K Tang

Publications and source records attributed to B K Tang.

At least 19 recordsLinked to original sources
PubMed1997

Distinction of CYP1A1 and CYP1A2 activity by selective inhibition using fluvoxamine and isosafrole.

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.

Cell Line↗
PubMed1997

Interindividual variation in the enzymatic 15-keto-reduction of 13,14-dihydro-15-keto-prostaglandin E1 in human liver and in human erythrocytes.

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.

Adolescent↗
PubMed1996

Role of CYP1A2 in caffeine pharmacokinetics and metabolism: studies using mice deficient in CYP1A2.

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.

Animals↗
PubMed1996

A sensitive method for determination of cytochrome P4502D6 activity in vitro using bupranolol as substrate.

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).

Adrenergic beta-Antagonists↗
PubMed1995

Variable activation of lovastatin by hydrolytic enzymes in human plasma and liver. 4.

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.

Biotransformation↗
PubMed1995

Low CYP1A2 activity in rural Shona children of Zimbabwe.

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.

Acetyltransferases↗
PubMed1995

(S)oxazepam glucuronidation is inhibited by ketoprofen and other substrates of UGT2B7.

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.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1995

Interindividual variability in the glucuronidation of (S) oxazepam contrasted with that of (R) oxazepam.

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.

Adult↗
PubMed1995

Induction of P-450 in workers exposed to dioxin.

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.

Adult↗
PubMed1995

A urinary marker of alcohol intake.

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.

Adult↗
PubMed1994

Caffeine biotransformation in human hepatocyte lines derived from normal liver tissue.

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.

Adult↗
PubMed1994

Caffeine as a probe for CYP1A2 activity: potential influence of renal factors on urinary phenotypic trait measurements.

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)

Adult↗
PubMed1992

Variability of acetaminophen metabolism in Caucasians and Orientals.

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.

Acetaminophen↗
PubMed1992

Biotransformation of caffeine, paraxanthine, theobromine and theophylline by cDNA-expressed human CYP1A2 and CYP2E1.

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.

Biotransformation↗