A Nyéki | PubMed Health

PubMed2003

NAT2 and CYP1A2 phenotyping with caffeine: head-to-head comparison of AFMU vs. AAMU in the urine metabolite ratios.

AIMS: (i) To compare the phenotyping of healthy subjects for NAT2 and CYP1A2 activities with caffeine, by the simultaneous assay of the urinary metabolites AFMU and AAMU, and (ii) to ascertain whether NAT2 and CYP1A2 phenotyping is influenced by the use of AFMU or AAMU in the metabolite ratio. METHODS: Thirty-five healthy subjects (16 men, 19 women) participated to the study. Caffeine metabolite concentrations were measured in urine collected 8 h after 2.5 mg kg-1 caffeine intake using a new validated h.p.l.c. method. The metabolite ratios AFMU/1X, AFMU/(AFMU+1X+1U), AAMU/1X, AAMU/(AAMU+1X+1 U), and (AFMU+1U+1X)/17U, (AAMU+1U+1X)/17U were determined as indices of NAT2 and CYP1A2 activity, respectively. RESULTS: Slow and rapid acetylators were similarly identified using the four NAT2 metabolite ratios in 139 out of 140 measurements. An appreciable amount of AAMU was present in urine that was immediately acidified and analysed. Consequently, the ratio using AFMU was lower than that using total AAMU following transformation of AFMU in basic conditions. The proportion of AFMU in urine analysed immediately expressed as AFMU/(AFMU+AAMU) ratio did not correlate with urine pH, but was a function of the acetylation phenotype, with a low intergroup variability (64 +/- 3% and 32 +/- 5%, for rapid and slow acetylators, respectively; P < 0.00001, anova). Regarding CYP1A2 activity, a good correlation (r = 0.99) was observed between the metabolite ratios calculated from AFMU and AAMU, although the ratios calculated from AFMU were proportionately and systematically lower P < 0.00001, paired t-test, slope 1.2). CONCLUSIONS: This study demonstrates that both AFMU and AAMU can be used for NAT2 and CYP1A2 metabolite ratio determinations. The reported conversion of AFMU into AAMU is unlikely to explain the large amount of AAMU in urine that was acidified and analysed immediately after voiding. The results suggest that AAMU is formed not solely through a nonenzymatic hydrolysis in urine, but in vivo by a NAT2 phenotype-dependent pathway.

Adult↗

PubMed2001

Extractionless method for the simultaneous high-performance liquid chromatographic determination of urinary caffeine metabolites for N-acetyltransferase 2, cytochrome P450 1A2 and xanthine oxidase activity assessment.

Urinary metabolic ratios of caffeine are used in humans to assess the enzymatic activities of cytochrome P450 isoenzyme 1A2 (CYP1A2), xanthine oxidase (XO) and for phenotyping individuals for the bimodal N-acetyltransferase 2 (NAT2), all of them involved in the activation or detoxification of various xenobiotic compounds. Most reported analytical procedures for the measurement of the urinary metabolites of caffeine include a liquid-liquid extraction of urine samples prior to their analysis by reversed-phase HPLC. At neutral to basic pH however, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), a metabolite of caffeine, spontaneously decomposes to 5-acetylamino-6-amino-3-methyluracil (AAMU). Since AAMU is not extracted in most organic solvents, the extent of AFMU decomposition cannot be precisely assessed. Although the decomposition reaction can be minimized by immediate acidification of the urine, accurate results can only be obtained when both AAMU and AFMU are monitored, or alternatively, if AAMU is measured after complete transformation of AFMU into AAMU in basic conditions. We report a liquid chromatographic method for the simultaneous quantitative analysis of the five urinary metabolites of caffeine used for the CYP1A2, XO and NAT2 phenotyping studies: AAMU, AFMU, 1-methylxanthine, 1-methyluric acid and 1,7-dimethyluric acid. These metabolites are satisfactory separated from all other known caffeine metabolites as well as endogenous urinary constituents. Sample treatment does not require any liquid-liquid extraction procedure. Urine samples are diluted and centrifuged before being injected (10 microl) onto a YMC-Pack Polyamine II (250x4.6 mm) column. A step-wise gradient elution program is applied using acetonitrile-0.75% (v/v) formic acid: (91:9) at 0 min-->(75:25) at 25 min-->(65:35) at 35 min-->(65:35) at 45 min, followed by a re-equilibration step to the initial solvent composition. The flow-rate is 1.0 ml/min and the separations are monitored by UV absorbance at 260 and 280 nm. The procedure described here represents a substantial improvement over previous methods: a single analysis and a minimal urine sample treatment enables the simultaneous quantitation of five caffeine metabolites, notably AFMU and AAMU, used for the determination of CYP450 1A2, XO and NAT2 enzyme activity. Importantly enough, phenotyping individuals for the bimodal NAT2 is made possible without the uncertainty associated with the deformylation of AFMU, which is likely to happen at all steps prior to the analysis, during sample storage and even in the bladder of the subjects.

Arylamine N-Acetyltransferase↗