title: Analysis of within-subject variation of caffeine metabolism when used to determine cytochrome P4501A2 and N-acetyltransferase-2 activities.
abstract: Cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 (NAT2) are hepatic enzymes that may activate some procarcinogens. Previous reports have determined CYP1A2 and NAT2 phenotypes by quantitating relative amounts of urinary caffeine and metabolites. However, a number of experimental issues with this approach remain. To address these, we measured caffeine and 4 metabolites in urine samples from 20 healthy volunteers on 3 separate occasions at 7-day intervals. Two additional volunteers were studied to measure the pattern of excretion of these analytes in urine over time. The molar ratio of two compounds (1,7-dimethylxanthine/1,3,7-trimethylxanthine) was used to phenotype CYP1A2, while the molar ratio of two other compounds (5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine) served to phenotype NAT2. Within-subject variation was less than 25% for most participants. In instances when within-subject variation of the metabolic ratio was > 25%, metabolite peaks were usually present in one or more control urine samples. Some caffeine metabolites 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 urine samples prior to testing can indicate if subjects are excreting caffeine metabolites and will aid in locating metabolite peaks on chromatograms; (c) refraining from caffeine for 48 h before testing is the best compromise between convenience for the subject and obtaining reproducible results; (d) determining metabolite molar ratios in urine collected 4-5 h after ingesting caffeine provides an acceptable time for the measurement; and (e) different ratios of metabolites for determining CYP1A2 phenotype have different advantages.
authors.affiliation: Department of Pharmacology and Toxicology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: McQuilkin
authors.forename: D W
authors.initials: DW
authors.lastname: Nierenberg
authors.forename: E
authors.initials: E
authors.lastname: Bresnick
mesh_terms: D000328:Adult; D001191:Arylamine N-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D002110:Caffeine / Q000235:genetics / Q000378:metabolism* / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D005260:Female; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D013056:Spectrophotometry, Ultraviolet; D013806:Theophylline / Q000652:urine; D013997:Time Factors; D014498:Uracil / Q000031:analogs & derivatives / Q000652:urine; D014970:Xanthines / Q000652:urine
chemical_list: D014970:Xanthines; D002110:Caffeine; D014498:Uracil; C053085:1-methylxanthine; C036692:5-acetylamino-6-formylamino-3-methyluracil; D003577:Cytochrome P-450 Enzyme System; D013806:Theophylline; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D001191:Arylamine N-Acetyltransferase; C021183:1,7-dimethylxanthine