ANALYSIS OF WITHIN-SUBJECT VARIATION OF CAFFEINE METABOLISM WHEN USEDTO DETERMINE CYTOCHROME P4501A2 AND N-ACETYLTRANSFERASE-2 ACTIVITIES

Cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 (NAT2) are hepat ic enzymes that may activate some procarcinogens. Previous reports hav e determined CYP1A2 and NAT2 phenotypes by quantitating relative amoun ts of urinary caffeine and metabolites. However, a number of experimen tal issues with this approach remain. To address these, we measured ca ffeine and 4 metabolites in urine samples from 20 healthy volunteers o n 3 separate occasions at 7-day intervals. Two additional volunteers w ere studied to measure the pattern of excretion of these analytes in u rine over time. The molar ratio of two compounds (1,7-dimethylxanthine /1,3,7-trimethylxanthine) was used to phenotype CYP1A2, while the mola r ratio of two other compounds ino-6-formylamino-3-methyluracil/1-meth ylxanthine) served to phenotype NAT2. Within-subject variation was les s than 25% for most participants. In instances when within-subject var iation of the metabolic ratio was >25%, metabolite peaks were usually present in one or more control urine samples. Some caffeine metabolite s were observed in urine samples at detectable levels up to 48 h after caffeine ingestion. We conclude that: (a) this assay for determining CYP1A2 and NAT2 activities (phenotyping) has an acceptably low within- subject variation over 3 consecutive weeks for most subjects who were caffeine-free for 36 h prior to study; (b) collecting and analyzing ur ine samples prior to testing can indicate if subjects are excreting ca ffeine metabolites and will aid in locating metabolite peaks on chroma tograms; (c) refraining from caffeine for 48 h before testing is the b est compromise between convenience for the subject and obtaining repro ducible results; (d) determining metabolite molar ratios in urine coll ected 4-5 h after ingesting caffeine provides an acceptable time for t he measurement; and (e) different ratios of metabolites for determinin g CYP1A2 phenotype have different advantages.