title: In vivo evaluation of CYP1A2, CYP2A6, NAT-2 and xanthine oxidase activities in a Greek population sample by the RP-HPLC monitoring of caffeine metabolic ratios.
abstract: A RP-HPLC method was developed for the assessment of caffeine and its metabolites in urine and was used for the evaluation of the CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyl-transferase-2 (NAT-2) in vivo activities in 44 Greek volunteers (21 men, 23 women). Spot urine samples were analyzed 6 h after 200 mg caffeine consumption, following a 30 h methylxantine-free diet. The major urinary caffeine metabolites are 1-methyluric acid (1U), 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1X), 1,7-dimethyluric acid (17U) and 1,7-dimethylxanthine (17X). CYP1A2, CYP2A6, XO and NAT-2 activities were estimated from the metabolic ratios (AFMU + 1U + 1X)/17U, 17U/17X, 1U/(1X + 1U) and AFMU/(AFMU + 1U + 1X), respectively. Metabolites and internal standard were extracted with chloroform/isopropanol (85:15, v/v) and separated on a C18 column by an isocratic HPLC system using a two-step elution with manual switch from solvent A (0.1% acetic acid-methanol-acetonitrile, 92:4:5 v/v) to solvent B (0.1% acetic acid-methanol, 60:40, v/v), and detected at 280 nm. The method exhibited adequate metabolite separation (resolution factors >1.48), accuracy (94.1-106.3%) and intraday and interday precision <8.02 and <8.78%, respectively (n = 6). Smoking affected only CYP1A2, whereas gender had no effect in any enzyme activity. NAT-2 exhibited bimodal distribution, 63.6% of volunteers being slow acetylators. The developed RP-HPLC method was fully validated and successfully applied for the evaluation of CYP1A2, CYP2A6, XO and NAT-2 activities.
authors.affiliation: Laboratory of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Thessaly, 22 Papakiriazi St, 41222 Larissa, Greece.
authors.forename: E
authors.initials: E
authors.lastname: Begas
authors.forename: E
authors.initials: E
authors.lastname: Kouvaras
authors.forename: A
authors.initials: A
authors.lastname: Tsakalof
authors.forename: S
authors.initials: S
authors.lastname: Papakosta
authors.forename: E K
authors.initials: EK
authors.lastname: Asprodini
mesh_terms: D000328:Adult; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism*; D002110:Caffeine / Q000378:metabolism* / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012907:Smoking / Q000378:metabolism; D013806:Theophylline / Q000652:urine; D014498:Uracil / Q000031:analogs & derivatives / Q000652:urine; D014527:Uric Acid / Q000031:analogs & derivatives / Q000652:urine; D014969:Xanthine Oxidase / Q000378:metabolism*; D014970:Xanthines / Q000652:urine
chemical_list: D014970:Xanthines; D014527:Uric Acid; C030530:1-methyluric acid; D002110:Caffeine; D014498:Uracil; C053085:1-methylxanthine; C036692:5-acetylamino-6-formylamino-3-methyluracil; D013806:Theophylline; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C585710:CYP2A6 protein, human; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D014969:Xanthine Oxidase; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human; C021183:1,7-dimethylxanthine; C053084:1,7-dimethyluric acid