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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
Author(s) -
Begas E.,
Kouvaras E.,
Tsakalof A.,
Papakosta S.,
Asprodini E. K.
Publication year - 2007
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.736
Subject(s) - chemistry , chromatography , cyp1a2 , acetic acid , xanthine oxidase , high performance liquid chromatography , metabolite , caffeine , urine , cyp2a6 , metabolism , biochemistry , enzyme , cytochrome p450 , endocrinology , medicine
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 C 18 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. Copyright © 2007 John Wiley & Sons, Ltd.