Interindividual variability of coumarin 7-hydroxylation in a Turkish population

One hundred healthy Turkish volunteers (70 male, 30 female) aged from 19 to 56 years were given 5 mg coumarin p.o. after an overnight fast. Urine samples were collected before and 2, 4 and 8 h after drug administration. The extent and rate of formation of 7-OH-coumarin (7OHC) was determined by the urinary excretion of the metabolite as measured with the fluorometric method.On average, 80% of 7OHC formed was excreted in 2 h. The total amount of 7OHC formed was 59.8% (21.5%) (mean and SD, n=100, range 17–100%) of the given dose. The percentage of 7OHC excreted during the first 2 h compared with the 7OHC excretion at 8 h was a constant and stable individual characteristic for the rate of the formation of 7OHC (2 h coumarin test).Although four individuals had relatively slow coumarin test values (34–40%), no clear-cut polymorphism in the rate of 7OHC formation was found. However, 7OHC formation was lower in males and in cigarette smokers.The scientific contents of this paper contribute to the goals of the COST B1 project.     

Proguanil metabolism in relation to S-mephenytoin oxidation in a Turkish population

The oxidation of proguanil was studied in 89 unrelated healthy Turkish volunteers after administration of proguanil (single dose, 200  mg, orally). Based on the distribution of the ratio of proguanil to cycloguanil excreted in urine, and using an antimode value of 15, the prevalence of poor metabolizers in a Turkish population was estimated to be 5.6% (95% confidence interval 2.0%–17.3%) which was similar to that in the other Caucasian populations. The relationship between the oxidative capacities of CYP2C19 for the two substrates, proguanil and mephenytoin, was studied in 39 subjects (two poor and 37 extensive metabolizers of proguanil). The two poor metabolizers of proguanil were also identified as poor metabolizers of S-mephenytoin and no misclassification by the two phenotyping methods was observed. The correlation between the metabolic ratio of proguanil to cycloguanil and the S/R-mephenytoin ratio as assessed by Spearman's rank test, was statistically significant ( r _s=0.50, P <0.001).     

Genetic variation in the metabolism of coumarin in mouse liver.

The metabolism of 50 microM [3-14C] coumarin to polar products separated by high performance liquid chromatography (HPLC) and covalently bound metabolites in liver microsomes was compared in a series of inbred strains of mice. Coumarin metabolism to total polar products was higher in female than male mice. In all strains, the coumarin 3,4-epoxidation pathway was the major route of metabolism with o-hydroxyphenylacetaldehyde (o-HPA) as the major metabolite. However, in females, there was a major strain difference in the degree of metabolism to coumarin 7-hydroxylase with DBA/2 and 129 having high 7-hydroxycoumarin formation, CBA/Ca having intermediate levels and the other strains low levels. The differences between the strains was much less pronounced in the male mice. There was also evidence for strain variation in metabolism in the quantities of a number of other coumarin metabolites as detected by HPLC analysis of incubate extracts. However, this variation was of a quantitative nature and relatively small. The metabolism of B6C3F1 hybrid mice, in which coumarin had been identified as carcinogenic in a long-term cancer bioassay, was qualitatively similar to that of the other genotypes. The DBA/2 mouse has been suggested as a model for the metabolism of coumarin in humans. The pattern of metabolism found in this strain is different from most other strains. However, the pattern found for all the mouse strains, including DBA/2, differed appreciably from the profiles for other species including humans in the extent of 7-hydroxylation.     

Paraoxonase 1 mutations in a Turkish population.

Human serum paraoxonase 1 (PON1) catalyzes the hydrolysis of certain organophosphate pesticides and nerve gases and so may alter significantly an individual's susceptibility to the toxicity of these chemicals. Moreover, PON1 hydrolyzes lipid peroxides complexed to low density lipoproteins (LDL) and therefore it was suggested that PON1 may be one of the genes that is involved in the pathogenesis of cardiovascular diseases. Its activity shows interindividual and interethnic variability. At least two mutation sites, namely Gln192Arg (Q/R) and Leu55Met (L/M) were reported responsible for the variations in enzyme activity. The aim of the present study was to determine the frequency of these mutations in Turks and compare the results with other European and Oriental populations. A total of 381 unrelated Turkish individuals were genotyped for Gln192Arg and Leu55Met polymorphisms by PCR-RFLP using AlwI and NlaIII, respectively. Genotype distribution was QQ = 0.49, QR = 0.40, RR = 0.11, and LL = 0.52, LM = 0.39, MM = 0.09. Thus frequencies of high activity alleles R (Arg) and L (Leu) were found as 0.31 and 0.72, respectively. The frequency of these alleles was slightly higher in Turkish subjects than other Caucasian populations but much lower compared to Oriental populations.     

Flavin-containing monooxygenase 3 gene polymorphisms in Turkish population

Flavin-containing monooxygenases (FMOs) represent the second most important human monooxygenase system, after cytochrome P450s (CYPs) and catalyze the oxygenation of many chemicals containing nitrogen-, sulphur-, phosphorous-, selenium- and other nucleophilic heteroatoms. FMO3 is the prominent FMO form in adult human liver. For FMO3, both interindividual variability within a single ethnic group and variability between ethnic groups have been reported. In our study, three prevalent functional FMO3 variants (E158K, V257M, and E308G) were genotyped in healthy Turkish people by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. The frequencies of alleles and haplotypes were compared with those obtained from different populations. It was found that FMO3 158K, 257M and 308G alleles, demonstrate impaired metabolism toward many FMO3 substrates, were observed frequently in Turkish population similar to the other populations. Also, the frequencies of haplotypes were determined based on individual allelic frequencies and it was observed that the most common haplotypes were haplotip EVE and KVE (E158K/V257M/E308G), which together accounted for 80% of all haplotypes. The obtained data from the present study could be useful for further studies assessing sensitivity to therapeutic drugs, environmental toxicants and common disease.     

N-acetylation phenotyping with sulphadimidine in a Turkish population

Summary The distribution of acetylator phenotypes was studied in 244 unrelated Turkish subjects. Sulphadimidine and its acetylated metabolite were measured in 6 h plasma and 0–6 h urine samples after an oral dose of 10 mg/kg. Subjects with 37.5% or less acetylsulphadimidine in plasma were regarded as slow acetylators and the others as rapid acetylators.The mean plasma concentration of acetylsulphadimidine was about 2.5-times lower in slow acetylators. Urinary excretion of total sulphadimidine (free + acetylated) was also significantly lower in slow acetylators compared to rapid acetylators. The frequency of slow acetylators was 60.7% in the population (95% confidence interval 54.3% to 66.8%).Sulphadimidine acetylation showed no variation due to sex, age, body weight or pre-existing disease.This study is supported by Hacettepe University Research Fund 84-01-011-01     

Flavin-containing monooxygenase 3 gene polymorphisms in Turkish population

Flavin-containing monooxygenases (FMOs) represent the second most important human monooxygenase system, after cytochrome P450s (CYPs) and catalyze the oxygenation of many chemicals containing nitrogen-, sulphur-, phosphorous-, selenium- and other nucleophilic heteroatoms. FMO3 is the prominent FMO form in adult human liver. For FMO3, both interindividual variability within a single ethnic group and variability between ethnic groups have been reported. In our study, three prevalent functional FMO3 variants (E158K, V257M, and E308G) were genotyped in healthy Turkish people by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) methods. The frequencies of alleles and haplotypes were compared with those obtained from different populations. It was found that FMO3 158K, 257M and 308G alleles, demonstrate impaired metabolism toward many FMO3 substrates, were observed frequently in Turkish population similar to the other populations. Also, the frequencies of haplotypes were determined based on individual allelic frequencies and it was observed that the most common haplotypes were haplotip EVE and KVE (E158K/V257M/E308G), which together accounted for 80% of all haplotypes. The obtained data from the present study could be useful for further studies assessing sensitivity to therapeutic drugs, environmental toxicants and common disease.     

TYMS and DPYD polymorphisms in a Turkish population

Objective: The thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) enzymes affect the outcome of 5-fluorouracil (5-FU)-based chemotherapy. Genetic polymorphisms of the thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) genes that may affect chemotherapy are described. The aim of this study was to determine the frequencies of TYMS and DPYD polymorphisms in healthy Turkish individuals. Methods: Genotyping analyses of the promoter enhancer region of TYMS (TSER) and the exon 14-skipping mutation of the DPYD (DPYD*2A) genes were conducted in 250 unrelated, healthy volunteers from the central region of Turkey using a PCR-based assay. Results: The distribution of theTSER*2/*2, *2/*3 and *3/*3 genotypes were 17.6%, 48.8%, and 33.6%, respectively. The frequencies of the TSER*2 and *3 alleles in the Turkish population were 0.42 and 0.58, respectively. No individuals with the variant DPYD*2A allele were identified in the study group. Conclusion: The frequency of the TSER*3 allele among members of the Turkish population was similar to frequencies observed in other Caucasian populations but was lower than those found in Japanese and Chinese populations.     

Comparative metabolism and kinetics of coumarin in mice and rats.

Coumarin, a well recognized rat hepatotoxicant, also causes acute, selective necrosis of terminal bronchiolar Clara cells in the mouse lung. Further, chronic oral gavage administration of coumarin at 200 mg/kg, a dose that causes Clara cell death, resulted in a statistically significant increased incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. In contrast, mouse lung tumors were not observed at the 100 and 50 mg/kg dose levels in the oral gavage study, or in CD-1 mice following chronic intake of coumarin at levels equivalent to 276 mg/kg in diet. The current studies were designed to determine the impact of oral gavage vs dietary administration on the pharmacokinetics and metabolism of coumarin in CD-1 and B6C3F1 mice and F344 rats. Following the administration of 200 mg/kg 14C-coumarin via oral gavage, lung C(max) values (total 14C-associated radioactivity) were five- and 37-fold greater than those resulting from a 50 mg/kg oral gavage dose or 1000 ppm in diet, respectively. Coumarin (200 mg/kg) pharmacokinetics and metabolism was also examined in F344 rats following oral gavage dosing. Total 14C-coumarin associated radioactivity in plasma was 3.5-fold lower than in the mouse, and the plasma half-life in rats was five-times longer than in mice. Using non-radiolabeled compound (200 mg/kg), coumarin and products of the coumarin 3,4-epoxidation pathway were quantitated in plasma and urine after oral gavage administration to mice and rats. 7-Hydroxycoumarin (7-HC) was quantitated in mouse plasma and urine. o-Hydroxyphenylacetic acid (o-HPAA) reached a concentration of 37 microg/ml in plasma, and accounted for 41% of the dose in the urine, whereas the C(max) for 7-hydroxycoumarin was 3 microg/ml, and represented 7% of the administered dose. In the rat, the plasma C(max) for o-HPAA was 6 microg/ml, and accounted for 12% of the dose. The coumarin C(max) in rat plasma was comparable to that in mouse. Coumarin 3,4-epoxide (CE) and its rearrangement product o-hydroxyphenylacetaldehyde (o-HPA) and o-hydroxyphenylethanol (o-HPE), were not detected at any time point in plasma or urine. This analysis of coumarin and CE pharmacokinetics in rodents suggests that the differential tumor response in the mouse oral gavage and dietary bioassays is a function of the route of exposure, whereas species differences in lung toxicity between mice and rats result from heightened local bioactivation in the mouse lung.     

Consumption of watercress fails to alter coumarin metabolism in humans.

Watercress is an excellent source of phenethyl isothiocyanate (PEITC), an effective inhibitor of nitrosamine carcinogenesis in rodents. The mechanism of inhibition is believed to be due in part to inhibition of cytochrome P450 (P450) enzymes. P450 2A6 is a catalyst for the metabolic activation of several nitrosamines. In this study, we investigated the effect of watercress consumption on coumarin 7-hydroxylation, a P450 2A6-specific reaction, in a group of 15 nonsmoking, healthy volunteers. The urinary excretion of 7-hydroxycoumarin (7OHC) was determined. For 6 of the 15 subjects, watercress consumption decreased the amount of 7OHC excreted in the first 2 h following coumarin administration. However, the mean 0- to 2-h excretion of 7OHC for all 15 subjects was not significantly lowered by the consumption of watercress, 2.8 +/- 0.78 versus 3.1 +/- 0.53 mg (+/-S.D.). The mean 7OHC excreted from 2 to 4 h (1.1 +/- 0.50 mg) was significantly higher (P = 0.027) during watercress consumption than before (0.77 +/- 0.22 mg), suggesting a delay in coumarin metabolism. Total excretion of 7OHC was unaffected by watercress consumption. Therefore, under the conditions of our study, PEITC and other constituents of watercress had at most a marginal inhibitory effect on P450 2A6-catalyzed coumarin 7-hydroxylation.     

GSTT1 null genotype frequency in a Turkish population

In humans, glutathione-S-transferases (GSTs) are involved in the detoxification of xenobiotics. A deletion polymorphism in the glutathione S-transferase Theta 1 gene (GSTT1 null genotype) is associated with an increased risk of certain forms of cancer. The distribution of this polymorphism in 240 healthy individuals has been examined, using a polymerase chain reaction (PCR)-based method. The GSTT1 null genotype frequency was 20% in a Turkish Population. Received: 18 March 1998 / Accepted: 8 April 1998     

Characteristics of coumarin metabolism by liver microsomes from cobalt-pretreated mice

1. Cobalt pretreatment of male mice leads to increased liver microsomal mixed-function oxidation of various coumarin derivatives; 7-ethoxycoumarin, 7-hydroxycoumarin and coumarin are metabolized three to four times more rapidly than in control microsomes.

2. In Arrhenius plots of 7-ethoxycoumarin de-ethylation, the temperature at which phase transition occurs is not changed by cobalt pretreatment. However, at high temperature, above the transition point, the slope indicates that cobalt lowers the apparent activation energy of the reaction to 65% of control value.

3. Metabolism of 7-hydroxycoumarin by hepatic microsomes from cobalt-treated mice gave 3,7-, 6,7- and 7,8-dihydroxycoumarin, identified by g.l.c. and mass spectrometry.     

Interspecies differences in coumarin metabolism in liver microsomes examined by capillary electrophoresis

1. A simple, rapid method was developed for studying xenobiotic metabolism by cytochrome P450 in liver microsome preparations. Capillary electrophoresis was used to separate the metabolite from the metabolic mixture.

2. Coumarin is metabolized to 7-hydroxycoumarin by a cytochrome P450 isoenzyme. Human, bovine, gerbil, mouse (Schofield, CD1), rat, rabbit, porcine, and cynomologus monkey microsomal preparations were investigated for coumarin metabolism by determining the content of 7-hydroxycoumarin present after metabolism.

3. Separation of 7-hydroxycoumarin from the reaction mixture was carried out in 50 mM phosphate buffer, pH 6.8, on a fused silica capillary at 25°C and 15 kV. The metabolic matrix consisted of an NADPH regeneration system, 205.5 μM coumarin, and the microsomal preparation. Standard curves were prepared in the microsomal preparation and the limit of quantification was 6.17 μM, with a linear range from 0 to 308.5 μM.

4. The reaction was initiated by the addition of the microsomes. An aliquot of the reaction mixture was removed at specific timed intervals over 2?h and injected directly onto a capillary electrophoresis column and the concentration of 7-hydroxycoumarin determined. The metabolism of coumarin to 7-hydroxycoumarin is greatest in human and monkey microsomes.     

HPLC测定没食子中没食子酸的含量

目的　建立没食子中没食子酸的含量测定方法。方法　采用HPLC法测定没食子酸的含量 ,色谱柱为WatersSymmetryR○C18柱 (5 μm ,4 .6mm× 15 0mm) ;流动相为甲醇 - 0 .0 5 %磷酸溶液 (5∶95 ) ;流速 0 .8ml·min-1;检测波长 2 73nm。结果　没食子酸进样量在 0 .0 5 06～ 0 .4 0 6 0 μg范围内线性关系良好 ,平均加样回收率为 98.99% ,RSD =1.5 %。 结论　所用方法简便、快速、准确。     

Catechol-O-methyltransferase (COMT) genetic polymorphism in a Turkish population

Catechol-O-methyltransferase (COMT) inactivates neurotransmitters, catechol hormones and drugs such as levodopa and methyldopa. A low activity allele has been demonstrated at codon 108/158 of the soluble and membrane-bound COMT, respectively, whereby a G to A transition results in a valine to methionine substitution. Ethnic and inter-individual differences in red blood cell COMT activity have been observed in the different populations studied so far. Since, no information is available on inter-individual variability of COMT genotype in Turkish population, we genotyped 217 healthy, unrelated Turkish individuals. The allelic frequencies of COMT gene in the Turkish population were found to be the same as has been observed in Caucasians, but different from Orientals.     

Paracetamol metabolism in Indian population

To investigate the profile of paracetamol (CAS 103-90-2, Calpol) metabolism in Indian population and to compare with the profiles of studies conducted in other populations, a study was conducted in 100 healthy male human volunteers. After an overnight fast, the volunteers were administered an oral dose of 1 g of paracetamol, urine was collected up to 8 h and samples were analyzed by high performance liquid chromatography for the estimation of urinary recovery of paracetamol and its metabolites, i.e. sulphate, glucuronide, cysteine and mercapturate conjugates. 25.29 +/- 5.5 (mean +/- S.D.)% of sulphate conjugate, 60.55 +/- 8.5% of glucuronide conjugate, 5.05 +/- 2.1% of unchanged paracetamol, 2.76 +/- 2.4% of cysteine conjugate and 6.37 +/- 3.8% of mercapturate conjugate were recovered. The mean combined recovery of glutathione conjugates (9.13%) in Indians is found to be very high and is equal to that in Caucasians of Scotland (9.3%), which indicates that Indians are equally predisposed to hepatotoxicity as Caucasians.     

紫外分光光度法测定川白芷中总香豆素类成分的含量

目的　建立川白芷药材中总香豆素的含量测定方法。方法　采用紫外分光光度法,测定波长30 0nm。结果　线性范围2 .2～11.0 μg·ml-1(r=0 .9999) ,回收率为10 1.32 % ,RSD =3.34% (n =6 )。结论　所用方法准确、快速、重复性好,可用于川白芷药材中总香豆素的测定。     

Determination of coumarin and umbelliferone mixtures in whole blood by spectrophotofluorometry.

A spectrophotofluorometric method is described for the quantitative analysis of coumarin, umbelliferone, and mixtures thereof in whole blood. The two drugs were selectively isolated from blood by solvent extraction. Analysis of the isolated coumarin was based on the measurement of the fluorophore at activation and emission wavelengths of 361 and 491 nm, respectively. The fluorophore was obtained by irradiating an alkaline methanolic solution of the drug with UV light. A linear relationship between fluorescence and concentration existed over the concentration range of 0.02-0.2 mug of coumarin/ml. A mean recovery value of 94.8% was obtained from whole blood. The isolated umbelliferone was determined according to established methods at activation and emission wavelengths of 370 and 450 nm, respectively, and the limit of detection was 10 times more sensitive than previously reported. A linearity response was obtained between 1 and 10 ng of umbelliferone/ml. Good recovery data for mixtures of coumarin and umbelliferone in whole blood were obtained.