Investigating the Relations Between Caffeine-Derived Metabolites and Plasma Lipids in 2 Population-Based Studies.

OBJECTIVE: To investigate the relations between caffeine-derived metabolites (methylxanthines) and plasma lipids by use of population-based data from 2 European countries. METHODS: Families were randomly selected from the general population of northern Belgium (FLEMENGHO), from August 12, 1985, until November 22, 1990, and 3 Swiss cities (SKIPOGH), from November 25, 2009, through April 4, 2013. We measured plasma concentrations (FLEMENGHO, SKIPOGH) and 24-hour urinary excretions (SKIPOGH) of 4 methylxanthines-caffeine, paraxanthine, theobromine, and theophylline-using ultra-high-performance liquid chromatography-tandem mass spectrometry. We used enzymatic methods to estimate total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels and the Friedewald equation for low-density lipoprotein cholesterol levels in plasma. We applied sex-specific mixed models to investigate associations between methylxanthines and plasma lipids, adjusting for major confounders. RESULTS: In both FLEMENGHO (N=1987; 1055 [53%] female participants) and SKIPOGH (N=990; 523 [53%] female participants), total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels increased across quartiles of plasma caffeine, paraxanthine, and theophylline (total cholesterol levels by caffeine quartiles in FLEMENGHO, male participants: 5.01±0.06 mmol/L, 5.05±0.06 mmol/L, 5.27±0.06 mmol/L, 5.62±0.06 mmol/L; female participants: 5.24±0.06 mmol/L, 5.15±0.05 mmol/L, 5.25±0.05 mmol/L, 5.42±0.05 mmol/L). Similar results were observed using urinary methylxanthines in SKIPOGH (total cholesterol levels by caffeine quartiles, male participants: 4.54±0.08 mmol/L, 4.94±0.08 mmol/L, 4.87±0.08 mmol/L, 5.27±0.09 mmol/L; female participants: 5.12±0.07 mmol/L, 5.21±0.07 mmol/L, 5.28±0.05 mmol/L, 5.28±0.07 mmol/L). Furthermore, urinary caffeine and theophylline were positively associated with high-density lipoprotein cholesterol in SKIPOGH male participants. CONCLUSION: Plasma and urinary caffeine, paraxanthine, and theophylline were positively associated with plasma lipids, whereas the associations involving theobromine were less clear. We postulate that the positive association between caffeine intake and plasma lipids may be related to the sympathomimetic function of methylxanthines, mitigating the overall health-beneficial effect of caffeine intake.

Microbial and metabolomic profiles in correlation with depression and anxiety co-morbidities in diarrhoea-predominant IBS patients.

BACKGROUND: Psychological co-morbidities in irritable bowel syndrome (IBS) have been widely recognized, whereas less is known regarding the role of gut microbial and host metabolic changes in clinical and psychological symptoms in IBS. RESULTS: A total of 70 diarrhoea-predominant IBS (IBS-D) patients and 46 healthy controls were enrolled in this study. Stool and urine samples were collected from both groups for 16S rRNA gene sequencing and metabolomic analysis. The results showed that fecal microbiota in IBS-D featured depleted Faecalibacterium (adjusted P = 0.034), Eubacterium rectale group (adjusted P = 0.048), Subdoligranulum (adjusted P = 0.041) and increased Prevotella (adjusted P = 0.041). O-ureido-L-serine, 3,4-dihydroxybenzenesulfonic acid and (R)-2-Hydroxyglutarate demonstrated lower urinary concentrations in IBS-D patients. We further built correlation matrices between gut microbe abundance, differentiated metabolite quantities and clinical parameters. Dialister manifested negative association with IBS severity (r = - 0.285, P = 0.017), anxiety (r = - 0.347, P = 0.003) and depression level (r = - 0.308, P = 0.010). Roseburia was negatively associated with IBS severity (r = - 0.298, P = 0.012). Twenty metabolites correlated with anxiety or depression levels, including 3,4-dihydroxymandelaldehyde with SAS (r = - 0.383, P = 0.001), 1-methylxanthine with SDS (r = - 0.347, P = 0.004) and 1D-chiro-inositol with SAS (r = - 0.336, P = 0.005). In analysis of microbe-metabolite relationship, 3,4-dihydroxymandelaldehyde and 1-methylxanthine were negatively correlated with relative abundance of Clostridiumsensu stricto. CONCLUSIONS: Our findings demonstrated altered microbial and metabolomic profiles associated with clinically and psychological symptoms in IBS-D patients, which may provide insights for further investigations.

Alterations of Proximal Tubular Secretion in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND AND OBJECTIVES: In autosomal dominant polycystic kidney disease (ADPKD), the GFR often remains normal despite significant nephron loss. Proximal tubular secretory clearance may be reduced in ADPKD before detectable changes in GFR. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We used targeted mass spectrometry to quantify secretory solutes from blood and urine samples from 31 patients with ADPKD and preserved GFR (mean eGFR =111±11 ml/min per 1.73 m2) and 25 healthy control individuals as well as from 95 patients with ADPKD and reduced GFR (mean eGFR =53±21 ml/min per 1.73 m2) and 92 individuals with non-ADPKD CKD. We used linear regression to compare the fractional excretion of each solute between ADPKD and control groups. Among 112 patients with ADPKD, we used linear regression to determine associations of solute fractional excretion with height-adjusted total kidney volume. RESULTS: After adjusting for demographics, clinical characteristics, and kidney function measures, the fractional excretions of three secretory solutes were lower in patients with ADPKD and preserved GFR compared with healthy individuals: 52% lower cinnamoylglycine excretion (95% confidence interval, 24% to 70%), 53% lower tiglylglycine excretion (95% confidence interval, 23% to 71%), and 91% lower xanthosine excretion (95% confidence interval, 83% to 95%). In addition to lower excretions of tiglylglycine and xanthosine, patients with ADPKD and reduced GFR also demonstrated 37% lower dimethyluric acid excretion (95% confidence interval, 21% to 50%), 58% lower hippurate excretion (95% confidence interval, 48% to 66%), 48% lower isovalerylglycine excretion (95% confidence interval, 37% to 56%), and 31% lower pyridoxic acid excretion (95% confidence interval, 16% to 42%) compared with patients with non-ADPKD CKD and comparable eGFR. Among patients with ADPKD, solute fractional excretions were not associated with differences in kidney volume. CONCLUSIONS: Patients with ADPKD and preserved and reduced GFR demonstrate lower tubular secretory solute excretion compared with healthy controls and patients with non-ADPKD CKD. Our results suggest that tubular secretion is impaired in ADPKD independent of GFR.

N-Acetyltransferase-2 (NAT2) phenotype is influenced by genotype-environment interaction in Ethiopians.

BACKGROUND AND OBJECTIVES: N-acetyltransferase 2 (NAT2) metabolize several drugs including isoniazid. We investigated the effect of genotype, geographical difference, and smoking habit on NAT2 phenotype in Ethiopians. METHODS: Genotyping for NAT2 191G > A, 341 T > C, 590G > A, and 857G > A was performed in 163 unrelated healthy Ethiopians (85 living in Ethiopia and 78 living in Sweden). The NAT2 phenotype was determined using caffeine as a probe and log AFMU/(AFMU + 1X + 1 U) urinary metabolic ratio (MR) as an index. RESULTS: The frequencies of NAT2*4, *5, *6, *7, and *14 haplotypes were 14.1, 48.5, 30.1, 5.5, and 1.8%, respectively. The frequencies of rapid (NAT2*4/*4), intermediate (heterozygous *4), and slow (no *4 allele) acetylator genotypes were 1.8, 24.6, and 73.6%, respectively. The distribution NAT2 MR was bimodal with 70% being phenotypically slow acetylators. NAT2 genotype (p < 0.0001) and country of residence (p = 0.004) independently predicted NAT2 phenotype. Controlling for the effect of genotype, ethnic Ethiopians living in Ethiopia had significantly higher NAT2 MR than those living in Sweden (p = 0.006). NAT2 genotype-phenotype concordance rate was 75%. Distinct country-of-residence-based genotype-phenotype discordance was observed. The proportion of phenotypically determined rapid acetylators was significantly higher and slow acetylators was lower in Ethiopians living in Ethiopia (39% rapid, 61% slow) than in Sweden (20% rapid, 80% slow). Sex and smoking had no significant effect on NAT2 MR. CONCLUSIONS: We report a high prevalence of NAT 2 slow acetylators in Ethiopians and a conditional NAT2 genotype-phenotype discordance implicating a partial phenotype conversion and metabolic adaptation. Gene-environment interactions regulate NAT2 phenotype.

Quantification of xanthine- and uric acid-related compounds in urine using a "dilute-and-shoot" technique coupling ultra-high-performance liquid chromatography and high-resolution Orbitrap mass spectrometry.

Quantitative analysis of relevant metabolites in biofluids such as urine is often a tedious procedure, since it usually requires extraction, purification or preconcentration. For instance, in the analysis of methylxanthines in urine, a solid-phase extraction is often required. In the current work, a rapid and highly sensitive "dilute-and-shoot" method combining ultra-high-performance liquid chromatography and high-resolution mass spectrometry (UHPLC/HRMS) was validated for urinary determination of twelve analytes: uric acid, hypoxanthine, xanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, theophylline, theobromine, paraxanthine and caffeine. These analytes are the major physiological metabolites of caffeine, theobromine or theophylline, or final products of purine catabolism. The separation was carried out on a core-shell Kinetek EVO C18 column coupled to a Q Exactive Orbitrap high-resolution mass spectrometer equipped with a heated electrospray ionization (HESI) probe, that operated both in positive and negative ionization modes. The twelve analytes eluted from between 1.5 and 10.5min. The lower limit of quantification (LLOQ) values ranged from 0.25 to 2.5ng/mL, and the calibration curves were linear from the LLOQ to 100ng/mL. The only pretreatment needed was to dilute each urine sample (typically to 1/500) with 0.1% formic acid solution, and then filter the diluted sample before injecting it into the UHPLC system. With this high dilution, there were no significant matrix effects, and the intra- and inter-day precision and accuracy values were acceptable (coefficients of variance and relative errors below 15%, except for the LLOQ, for which they were below 20%). Furthermore, the analysis of spiked urine samples with 25ng/mL of the target analytes showed excellent recoveries and precision levels for the twelve analytes. To our knowledge, there is no other published method that allows for the simultaneous determination of the concentrations of these twelve compounds, nor has a previously reported method been indicated to show such low LLOQ values as we have for the majority of the analytes. We expect our protocol to be useful for nutritional assessments, interventional studies, kidney stone research, and purine metabolism studies.

Prediction of serum theophylline concentrations and cytochrome P450 1A2 activity by analyzing urinary metabolites in preterm infants.

AIMS: The purpose of this study was to explore clinical markers reflecting developmental changes in drug clearance by preterm infants. METHODS: Preterm infants administered aminophylline or theophylline to treat apnoea of prematurity were enrolled in this study. Trough and one of 2 h, 4 h or 6 h post-dose blood samples and urine samples were collected during steady state, to determine the concentrations of theophylline and its targeted metabolites. CYP1A2*1C and CYP1A2*1F genotypes were analyzed. Total, renal and nonrenal clearances of theophylline were calculated, and cytochrome P450 1A2 (CYP1A2) activity was obtained from the ratio of 1-methyluric acid and 3-methylxanthine to theophylline in urine. Multiple linear regression analysis was performed to evaluate the relationships between theophylline clearance and the clinical characteristics of preterm infants. RESULTS: A total of 152 samples from 104 preterm infants were analyzed. A strong association between the serum trough and urine theophylline concentrations was found (P < 0.001). Total, renal and nonrenal clearances of theophylline were 0.50 ± 0.29 ml kg-1  min-1 , 0.16 ± 0.06 ml kg-1  min-1 and 0.34 ± 0.28 ml kg-1  min-1 , respectively. CYP1A2 activity correlated positively with the postnatal age and postmenstrual age. However, CYP1A2 genotype was not associated with CYP1A2 activity, which was significantly associated with nonrenal clearance. CYP1A2 activity, postnatal age , weight and 24-h urine output were significantly associated with total theophylline clearance. CONCLUSIONS: CYP1A2 activity can be monitored using noninvasive random urine samples, and it can be used to assess developmental changes in theophylline clearance by preterm infants.

Cytochrome P450 2A6 Phenotyping Using Dietary Caffeine Salivary Metabolite Ratios and Genotyping Using Blood on Storage Cards in Non-smoking Japanese Volunteers.

BACKGROUND: A simple method of genotyping and phenotyping cytochrome P450 2A6 (CYP2A6) was previously reported using individual blood samples and urinary caffeine metabolite ratios of 1,7-dimethyluric acid (17U) to 1-methylxanthine (1X). OBJECTIVE: Blood spotted onto storage cards and salivary caffeine metabolites were analyzed in 27 healthy non-smoking Japanese volunteers with no prior abstention from dietary caffeine intake. METHODS: 1,7-Dimethylxanthine (17X), 17U, 1X, and caffeine levels in spot saliva samples were determined in Japanese non-smokers by high-performance liquid chromatography under normal dietary caffeine consumption. RESULTS: 17U/17X ratios in saliva were almost constant over time, but those of 17U/1X were variable in two subjects tested before and 1-2.5 h after caffeine treatment (a cup of black tea). In seven subjects, 17U/17X ratios in saliva were highly correlated with those in plasma (r = 0.98, p < 0.01) and well correlated with those in urine samples (r = 0.78, p < 0.05). The average 17U/17X ratios, but not 17U/1X ratios, in saliva under dietary caffeine consumption obtained from subjects with CYP2A6*1/*4 (n=11) and CYP2A6*4/*4 (whole-gene deletion, n=2) genotypes were significantly lower than those from subjects with wild-type CYP2A6*1/*1 (n=14). Genotyping was done by a multiplex real-time polymerase chain reaction method using blood spotted onto storage cards. CONCLUSION: The present results suggest that the decreased CYP2A6 function associated with the whole-gene deletion genotype (determined using blood samples) could be detected using 17U/17X ratios, but not 17U/1X ratios, in spot saliva samples under normal dietary caffeine consumption in Japanese non-smokers, just as it could be detected using urinary 17U/1X ratios.

Urine excretion of caffeine and select caffeine metabolites is common in the U.S. population and associated with caffeine intake.

BACKGROUND: Caffeine is a widely consumed psychoactive stimulant and is of epidemiologic interest. Major sources of caffeine are challenging to standardize, and the use of biomarkers is proposed as an alternative means of assessing intake. OBJECTIVE: We described urine caffeine and caffeine metabolite concentrations (n = 2466) and excretion rates (n = 2261) in the US population ≥6 y by age, sex, race-ethnicity, and caffeine intake (from foods, beverages, and dietary supplements). METHODS: We measured caffeine and 14 of its metabolites in spot urine samples from the cross-sectional NHANES 2009-2010 by use of LC-tandem mass spectrometry. RESULTS: Caffeine and its metabolites were detectable in the urine of most persons, generally at concentrations ≥1 µmol/L. Median concentrations (95% CI) ranged from 0.560 (0.497, 0.620) µmol/L to 58.6 (48.6, 67.2) µmol/L; median excretion rates from 0.423 (0.385, 0.468) nmol/min to 46.0 (40.7, 50.2) nmol/min. Urine concentrations and excretion rates for 9 analytes (caffeine, theophylline, paraxanthine, 1-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, 1,3,7-trimethyluric acid, and 5-acetylamino-6-amino-3-methyluracil) had moderate correlations with caffeine intake (Spearman ρ = 0.55-0.68, P < 0.0001); the remaining analytes had low correlations (ρ = 0.15-0.33, P < 0.0001). We observed larger differences in geometric mean concentrations and excretion rates between the highest vs. lowest quartiles of caffeine intake for these 9 compounds than the rest. Consistent with dietary caffeine intake, we observed that urine concentrations and excretion rates for most compounds were significantly (P < 0.05) higher in men than women, non-Hispanic whites than Hispanics and non-Hispanic blacks, and highest in persons aged 40-59 y. CONCLUSION: Excretion of caffeine and its metabolites in urine is common in the US population. According to the observed associations between spot urine concentrations or excretion rates with caffeine intake, several of these compounds show promise as potential biomarkers of caffeine intake.

Levels of caffeine and its metabolites among U.S. smokers and nonsmokers.

Data from National Health and Nutrition Examination Survey for the years 2009-2010 were used to estimate the levels of caffeine and 14 of its metabolite among U.S. smokers and nonsmokers after adjustments were made for other factors that affect observed caffeine levels. In this study, when adjusted for daily caffeine intake, adjusted levels (AGM) of caffeine and its metabolites were not found to be statistically significantly different between smokers and nonsmokers. AGMs for caffeine and all of its metabolites were found to be statistically significantly higher (p < 0.01) among females aged ≥ 12 years than males. For caffeine, 1,3-dimethylxanthine, and 1,7-dimethylxanthine, those aged ≥ 20 years had statistically significantly higher (p < 0.01) AGM than those aged 12-19 years but the reverse was true for 7-methylxanthine and 3,7-dimethylxanthine (p ≤ 0.02). The order of the AGMs by race/ethnicity was non-Hispanic whites > Hispanics > non-Hispanic blacks and most of the differences were statistically significant, at least between non-Hispanic whites and non-Hispanic blacks (p < 0.01). In general, there was a statistically significant positive association between the levels of caffeine and its metabolites and body mass index as well as daily caffeine intake. However, the levels of 7-methylxanthine were negatively associated with body mass index.

[Quantitative analysis of theophylline and its metabolites in urine of Chinese healthy subjects after oral administration of theophylline sustained-release tablets].

To study the metabolite excretion of theophylline, a rapid and specific method by liquid chromatography with heated electrospray ionization tandem mass spectrometry (LC-HESI/MS/MS) method for simultaneous determination of theophylline, 1, 3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX) and 1-methyluric acid (1-MU) in human urine was developed using theophylline-d6 and 5-fluorouracil as internal standards. Selected reaction monitoring (SRM) with heated electrospray ionization (HESI) was used in the negative mode for mass spectrometric detection. After diluted with methanol and centrifuged, the analytes and ISs were separated on a XDB-Phenyl (150 mm x 4.6 mm, 5 microm) column with a mixture of water-methanol-formic acid (30 : 70 : 0.15) as mobile phase at a flow rate of 0.6 mL x min(-1). The linear calibration curves for theophylline, 1, 3-DMU, 3-MX and 1-MU were obtained in the concentration range of 1.0-250 microg x mL(-1), separately. The method herein described is effective and convenient, and can be used for determination of theophylline and its three metabolites. The results showed that urinary excretion ratio of theophylline, 1,3-DMU, 3-MX and 1-MU is approximately 1 : 3 : 1 : 2 in Chinese subjects, which is similar to the reported excretion pattern in Caucasian.

[Effect of Tibetan medicine zuotai on the activity, protein and mRNA expression of CYP1A2 and NAT2].

To study the effect of Tibetan medicine Zuotai on the activity, protein and mRNA expression of CYP1A2 and NAT2, three different doses (1.2, 3.8 and 12 mg x kg(-1)) of Zuotai were administrated orally to rats once a day or once daily for twelve days, separately. Rats were administrated orally caffeine (CF) on the second day after Zuotai administration, and the urine concentration of CF metabolite 5-acetylamino-6-formylamino-3-methyl-uracil (AFMU), 1-methyluric acid (1U), 1-methylxanthine (1X), 1, 7-dimethylxanthine (17U) at 5 h after study drug administration was determined by RP-HPLC. The activity of CYP1A2 and NAT2 was evaluated by the ratio of metabolites (AFMU+1X+1U)/17U and the ratio of AFMU/(AFMU+1X+1U), respectively. The protein and mRNA expression of CYP1A2 and NAT2 were determined by ELISA and RT-PCR method, respectively. After single administration of Zuotai 3.8 mg x kg(-1) and repeated administration of Zuotai 3.8 and 12 mg x kg(-1), the activity of CYP1A2 and NAT2 decreased significantly compared with control group and there was no significant difference between other dose group and control group. The protein expression of CYP1A2 was significant lower than that in control group after repeated administration of Zuotai 12 mg x kg(-1), and the mRNA expression of CYP1A2 decreased significantly compared with that of control group after single administration of Zuotai 3.8 mg x kg(-1) and repeated admistration of Zuotai 12 mg x kg(-1), separately. The protein expression of NAT2 decreased significantly compared with that of control group after single and repeated administration of Zuotai 3.8 mg x kg(-1), respectively, and the mRNA expression of CYP1A2 decreased significantly compared with control group after single administration of Zuotai 3.8 mg x kg(-1). This study found that Tibetan medicine Zuotai had significant effect on the activity, protein and mRNA expression of CYP1A2 and NAT2.

New biomarkers of coffee consumption identified by the non-targeted metabolomic profiling of cohort study subjects.

Coffee contains various bioactives implicated with human health and disease risk. To accurately assess the effects of overall consumption upon health and disease, individual intake must be measured in large epidemiological studies. Metabolomics has emerged as a powerful approach to discover biomarkers of intake for a large range of foods. Here we report the profiling of the urinary metabolome of cohort study subjects to search for new biomarkers of coffee intake. Using repeated 24-hour dietary records and a food frequency questionnaire, 20 high coffee consumers (183-540 mL/d) and 19 low consumers were selected from the French SU.VI.MAX2 cohort. Morning spot urine samples from each subject were profiled by high-resolution mass spectrometry. Partial least-square discriminant analysis of multidimensional liquid chromatography-mass spectrometry data clearly distinguished high consumers from low via 132 significant (p-value<0.05) discriminating features. Ion clusters whose intensities were most elevated in the high consumers were annotated using online and in-house databases and their identities checked using commercial standards and MS-MS fragmentation. The best discriminants, and thus potential markers of coffee consumption, were the glucuronide of the diterpenoid atractyligenin, the diketopiperazine cyclo(isoleucyl-prolyl), and the alkaloid trigonelline. Some caffeine metabolites, such as 1-methylxanthine, were also among the discriminants, however caffeine may be consumed from other sources and its metabolism is subject to inter-individual variation. Receiver operating characteristics curve analysis showed that the biomarkers identified could be used effectively in combination for increased sensitivity and specificity. Once validated in other cohorts or intervention studies, these specific single or combined biomarkers will become a valuable alternative to assessment of coffee intake by dietary survey and finally lead to a better understanding of the health implications of coffee consumption.

Bioappearance and pharmacokinetics of bioactives upon coffee consumption.

Habitual consumption of medium amounts of coffee over the whole life-span is hypothesized to reduce the risk to develop diabetes type 2 (DM2) and Alzheimer's disease (AD). To identify putative bioactive coffee-derived metabolites, first, pooled urine from coffee drinkers and non-coffee drinkers were screened by UPLC-HDMS. After statistical data analysis, trigonelline, dimethylxanthines and monomethylxanthines, and ferulic acid conjugates were identified as the major metabolites found after coffee consumption. For quantitative analysis of these markers in body fluids, targeted methods based on stable-isotope dilution and UPLC-MS/MS were developed and applied to plasma samples from a coffee intervention study (n = 13 volunteers) who consumed a single cup of caffeinated coffee brew after a 10-day washout period. Chlorogenic acid-derived metabolites were found to be separated into two groups showing different pharmacokinetic properties. The first group comprised, e.g., ferulic acid and feruloyl sulfate and showed early appearance in the plasma (~1 h). The second group contained particularly chlorogenic acid metabolites formed by the intestinal microflora, appearing late and persisting in the plasma (>6 h). Trigonelline appeared early but persisted with calculated half-life times ~5 h. The plasma levels of caffeine metabolites significantly and progressively increased 2-4 h after coffee consumption and did not reach c max within the time frame of the study. The pharmacokinetic profiles suggest that particularly trigonelline, caffeine, its metabolites, as well as late appearing dihydroferulic acid, feruloylglycine and dihydroferulic acid sulfate formed from chlorogenic acid by the intestinal microflora accumulate in the plasma due to their long half-life times during habitual consumption of several cups of coffee distributed over the day. Since some of these metabolites have been reported to show antioxidant effects in vivo, antioxidant-response-element activating potential, and neuroprotective properties, respectively, some of these key metabolites might account for the inflammation- and DM2/AD risk reducing effects reported for habitual life time consumption of coffee.

Mechanism of allopurinol induced TPMT inhibition.

Up to 1/5 of patients with wildtype thiopurine-S-methyltransferase (TPMT) activity prescribed azathioprine (AZA) or mercaptopurine (MP) demonstrate a skewed drug metabolism in which MP is preferentially methylated to yield methylmercaptopurine (MeMP). This is known as thiopurine hypermethylation and is associated with drug toxicity and treatment non-response. Co-prescription of allopurinol with low dose AZA/MP (25-33%) circumvents this phenotype and leads to a dramatic reduction in methylated metabolites; however, the biochemical mechanism remains unclear. Using intact and lysate red cell models we propose a novel pathway of allopurinol mediated TPMT inhibition, through the production of thioxanthine (TX, 2-hydroxymercaptopurine). In red blood cells pre-incubated with 250 µM MP for 2h prior to the addition of 250 µM TX or an equivalent volume of Earle's balanced salt solution, there was a significant reduction in the concentration of MeMP detected at 4h and 6h in cells exposed to TX (4 h, 1.68, p=0.0005, t-test). TX acts as a direct TPMT inhibitor with an apparent Ki of 0.329 mM. In addition we have confirmed that the mechanism is relevant to in vivo metabolism by demonstrating raised urinary TX levels in patients receiving combination therapy. We conclude that the formation of TX in patients receiving combination therapy with AZA/MP and allopurinol, likely explains the significant reduction of methylated metabolites due to direct TPMT inhibition.

Cytochrome P450 2A6 phenotyping based on dietary caffeine intake in a Japanese population of non-smokers.

Phenotyping of cytochrome P450 2A6 (CYP2A6) was determined by assessing urinary caffeine metabolites in a Japanese population with a high frequency of CYP2A6 whole-gene deletion (CYP2A6*4). The levels of 1,7-dimethyluric acid (17U), 1-methylxanthine (1X), and 1,7-dimethylxanthine (17X) were measured in non-smokers whose CYP2A6 and NAT2 genotypes had been determined. Low 17U/1X ratios were observed in accumulated overnight urine samples of subjects genotyped as CYP2A6*4/*4 after caffeine treatment. The individual 17U/1X ratios in spot urine samples were almost constant before and 2-8 h after caffeine treatment, with or without prior abstention from dietary caffeine. The average 17U/1X ratios obtained from subjects with CYP2A6 *4/ *4 or CYP2A6 *1/ *4 genotypes were significantly lower than those from subjects with wild-type CYP2A6 *1/ *1 under dietary caffeine consumption. The present results suggest that impaired CYP2A6 function associated with CYP2A6 *4/ *4 could be determined using the 17U/1X ratios in spot urine samples under normal dietary caffeine consumption in Japanese non-smokers, without the need for additional caffeine administration or prior abstention from caffeine.

Comparison of N-acetyltransferase-2 enzyme genotype-phenotype and xanthine oxidase enzyme activity between Swedes and Koreans.

The aim of this study was to compare xanthine oxidase (XO) and N-acetyltransferase-2 (NAT2) genotype and phenotype between Swedes (n = 113) and Koreans (n = 150), as well as to investigate the effect of sex, smoking, age, and oral contraceptive (OC) use on enzyme activities, using caffeine as a probe. XO and NAT2 activities were estimated by 1U/(1U+1X) and AFMU/(AFMU+1X+1U) urinary ratios, respectively. Participants were genotyped for 191G>A, 341T>C, 590G>A, and 857G>A NAT2 polymorphisms. There was no significant difference in XO activity between Swedes and Koreans. In Swedes, higher XO activity was observed in women (P < .003). There were significant differences in NAT2 genotype and phenotype between Swedes and Koreans. Koreans display significantly higher frequency of NAT2 fast acetylator genotype (89%), whereas the slow acetylator genotype is predominant (62%) in Swedes (P < .0001). Significantly higher NAT2 activity was observed in Koreans compared to Swedes (P < .0001). Having the same NAT2 fast acetylator genotype, Koreans display higher enzyme activity than Swedes (P < .004). OC use significantly increased NAT2 activity in Swedish women. In conclusion, Koreans display higher NAT2 activity than Swedes regardless of NAT2 genotype. Ethnicity, OC use, and genotype determine NAT2 activity, whereas sex is the only determinant of XO activity.

N-Acetyltransferase-2 (NAT2) gene polymorphisms and enzyme activity in Serbs: unprecedented high prevalence of rapid acetylators in a White population.

The aim of this study was to investigate N-acetyltransferase 2 (NAT2) genetic polymorphism and enzyme activity in Serbs, and to examine the influence of NAT2 genotype, sex, and smoking on the phenotype. Genotyping for 190C>T, 282C>T, 341T>C, 403C>G, 411T>A, 481C>T, 590G>A, 803A>G, and 857G>A in the NAT2 gene, was performed in 140 healthy Serbs. NAT2 activity was determined as AFMU/ (AFMU + 1X + 1U) urinary ratio in 100 subjects using caffeine as a probe. The most frequent NAT2 haplotypes were NAT2*5B (38.2%), NAT2*6A (26.0%), and NAT2*4 (24.4%). The log-transformed NAT2 activity indices exhibited trimodal distribution with 9%, 36%, and 55% of slow, intermediate, and rapid acetylators, respectively. Significant NAT2 genotype-phenotype correlation was observed (P < .0001). The frequency of NAT1*10 and NAT1*11 were 27.5% and 6.9%, respectively. There was no significant influence of sex or cigarette smoking on NAT2 enzyme activity. Eight subjects displayed rapid NAT2 acetylators phenotype despite being homozygous for NAT2 slow alleles, and NAT1 fast acetylators genotype (NAT1*10 and NAT1*11) had no implication. In contrast to other white populations described hitherto, rapid acetylator is the predominant NAT2 phenotype in Serbs. NAT2 genotype, but not sex and cigarette smoking, influence enzyme activity. NAT1 fast acetylators genotypes do not contribute for NAT2 genotype-phenotype discordance.

Simultaneous determination of epinephrine, uric acid and xanthine in the presence of ascorbic acid using an ultrathin polymer film of 5-amino-1,3,4-thiadiazole-2-thiol modified electrode.

This paper describes the simultaneous determination of epinephrine (EP), uric acid (UA) and xanthine (XN) in the presence of ascorbic acid (AA) using electropolymerized ultrathin film of 5-amino-1,3,4-thiadiazole-2-thiol (p-ATT) modified glassy carbon (GC) electrode in 0.2 M phosphate buffer solution (pH 5). Although bare GC electrode resolves the voltammetric signals of AA and XN, it fails to resolve the voltammetric signals of EP and UA in a mixture. However, the p-ATT modified electrode not only separates the voltammetric signals of AA, EP, UA and XN with potential difference of 150, 120 and 400 mV between AA-EP, EP-UA and UA-XN, respectively but also shows higher oxidation current for these molecules. The p-ATT modified electrode exhibits excellent selectivity towards the oxidation of EP, UA and XN in the presence of 40-fold higher concentration of AA. Further, the p-ATT modified electrode was also used for the selective determination of EP in the presence of 40-fold higher concentrations of AA, UA and XN. Using amperometric method, we achieved the lowest detection of 40 nM EP and 60nM each UA and XN. The amperometric current response was increased linearly with increasing EP concentration in the range of 4.0 x 10(-8) to 4.0 x 10(-5) M and the detection limit was found to be 27 x 10(-11) M (S/N = 3). The practical application of the present modified electrode was demonstrated by determining the concentration of EP in epinephrine tartrate injection and XN in human urine samples.

Phenotyping of N-acetyltransferase type 2 and xanthine oxidase with caffeine: when should urine samples be collected?

OBJECTIVES: Individual activities of N-acetyltransferase 2 (NAT2) and of xanthine oxidase (XO) can be assessed using ratios of urinary caffeine metabolites. We investigated how ratios changed over time and which urine collection interval would be the best for NAT2 and XO activity assessments. METHODS: On two occasions separated by 14 days, 16 healthy male Caucasians collected urine before and 0-2, 2-4, 4-6, 6-8, 8-12, 12-16 and 16-24 h after a dose of 150 mg caffeine given in the framework of a phenotyping cocktail study. The metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U) were quantified with LC-MS/MS. The molar ratio (AFMU + AAMU)/(1X + 1U + AFMU + AAMU) was used as a NAT2 metric, while the ratio 1U/(1X + 1U) served as XO metric. RESULTS: The NAT2 ratios were stable in the intervals 4-24 h after caffeine dosing. Mean intra-individual coefficients of variation were 11-23% starting 4 h post-dose, while inter-individual variability reached 37-75%. The XO ratios increased gradually by 14% from the 2-4 to the 16-24 h interval. The mean intra- and inter-individual coefficients of variation of XO activity were 3-18 and 7-10% respectively. No significant differences between study occasions were observed. CONCLUSIONS: Any sampling interval at least 4 h after caffeine dosing is suitable for NAT2 and XO activity assessments. XO activities can only be compared between volunteers and studies if the same urine collection schedule has been respected. The low intraindividual variability allows for sample sizes of 16 and 6 participants in crossover interaction studies of NAT2 and XO activity respectively.

Possible involvement of organic anion and cation transporters in renal excretion of xanthine derivatives, 3-methylxanthine and enprofylline.

Whether organic anion and cation transporters are involved in the renal excretion of xanthine derivatives, 3-methylxanthie and enprofylline, remains unclear. In this study, we have investigated the effects of typically predominant substrates for organic anion and cation transporters on the tubular secretion of 3-methylxanthine and enprofylline in rats. In the renal clearance experiments using typical substrates for organic anion transporters, probenecid and p-aminohippurate, probenecid (20 mg/kg), but not p-aminohippurate (100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. The typical substrates for organic cation transport systems, tetraethylammonium (30.6 mg/kg) and cimetidine (50 or 100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. These results suggest that the renal secretory transport of 3-methylxanthine and enprofylline are mediated by probenecid-, cimetidine- and tetraethylammonium-sensitive transport systems. Uric acid, an organic anion, significantly inhibited the renal secretion of 3-methylxanthine, but not enprofylline, suggesting that the renal tubular transport of 3-methylxanthine is also mediated via uric acid-sensitive transport system. These findings suggest the possibility that both organic anion and cation transporters are, at least, involved in the renal tubular transport of 3-methylxanthine and enprofylline in rats.