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 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

In vivo evaluation of CYP2A6 and xanthine oxidase enzyme activities in the Serbian population

“…Previous studies, using nicotine and cotinine as probes, showed an inhibitory effect of cigarette smoking on CYP2A6 enzyme activity [5,6]. Sex differences have been observed as well [4,[7][8][9][10], but the findings were not always consistent [11,12]. Nevertheless, the major cause for the observed variability in enzyme activity seems to be genetic polymorphisms of the CYP2A6 gene [1].…”

“…Xanthine oxidase (XO) is a multifunctional enzyme responsible for oxidation processes of a wide range of substrates, including a number of endogenous compounds and several clinically important drugs [11,[13][14][15][16][17]. Previous studies, using radiochemical assay [13] or caffeine test [11,14,18,19], showed that XO activity differs among individuals.…”

“…Previous studies, using radiochemical assay [13] or caffeine test [11,14,18,19], showed that XO activity differs among individuals. Known functional genetic variations in the XO gene are rare [20].…”

“…Known functional genetic variations in the XO gene are rare [20]. Although environmental factors seem to be responsible for the described variability [15,17,19], it is generally accepted that cigarette smoking does not affect XO activity [11,19,21]. Sex differences have been extensively studied as well, but with contradictory results [11,13,14,19,21].…”

“…Because of its pharmacokinetics and noninvasive properties, caffeine as a probe drug represents the safest method for evaluation of CYP2A6 and XO in humans [11,18,22,23].…”