Validation of urine caffeine metabolite ratios with use of stable isotope-labeled caffeine clearance.

OBJECTIVE: A number of caffeine metabolite ratios have been proposed to measure CYP1A2 activity in vivo. The data to validate these ratios are scanty. The objective of this study was to validate urine caffeine metabolite ratios versus stable isotope-labeled caffeine clearance under different caffeine dosing conditions. STUDY DESIGN: Two experiments, one with nine nonsmoking subjects and the other with 12 cigarette smokers, were performed. We explored the relationship between caffeine clearance, measured by means of intravenous infusions of stable isotope-labeled caffeine, and a number of caffeine metabolite ratios during administration of different single or multiple doses of caffeine to smokers and nonsmokers on three different occasions over a 2-week period, using different durations of urine collections, including spot urines. The stable isotope technique allowed simultaneous oral dosing of caffeine and measurement of caffeine metabolite ratios and caffeine clearance, the latter reflecting CYP1A2 activity. RESULTS: The caffeine metabolite ratio of AAMU + 1U +1X/17U (5-acetylamino-6-amino-3-methyluracil + 1-methyluric acid + 1 methylxanthine/1,7-dimethyluric acid) maintained a significant correlation with caffeine clearance for all the above conditions (gamma2 range, 0.4 to 0.9) except for dose. With high doses of caffeine (12 mg/kg), a significant relationship was not observed. AAMU + 1U + 1X/17U also correlated with the formation clearance of paraxanthine (gamma2 = 0.6, p = 0.002). Other reported caffeine metabolite ratios did not display the same robust correlation with caffeine clearance under all these different conditions. CONCLUSIONS: We conclude that AAMU+1U+1X/17U measured from a single spot urine collection is a valid measure of CYP1A2 activity except at very high levels of caffeine dosing. The validity of the other proposed caffeine metabolite ratios is questionable.

Caffeine, estradiol, and progesterone interact with human CYP1A1 and CYP1A2. Evidence from cDNA-directed expression in Saccharomyces cerevisiae.

Heterologous expression of cytochrome P-450 cDNAs in yeast is a potent instrument for the study of enzyme-specific parameters and can be used to answer questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities. Two cDNAs of human CYP1A1 and CYP1A2 were expressed in yeast Saccharomyces cerevisiae, and microsomes of transformed strains contained substantial amounts of functional heterologous enzymes. Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in KM values of 0.017 and 1.67 microM and Vmax values of 840 and 387 pmol/mg/min for CYP1A1 and CYP1A2, respectively. Both heterologous enzymes showed an overlapping substrate specificity pattern assayed with different phenoxazone ethers and caffeine. Caffeine was shown to be metabolized by CYP1A2 and CYP1A1. Both enzymes formed paraxanthine and minor amounts of theobromine; however, trimethyluric acid was exclusively formed by CYP1A1. The fact that theophylline was not formed by either enzyme anticipates the involvement of additional enzyme(s) in the primary metabolism of caffeine. Inhibition studies with caffeine, phenacetin, 17 beta-estradiol, and progesterone as inhibitors of the CYP1A1 and CYP1A2 catalyzed O-deethylation of 7-ethoxyresorufin suggest all compounds as possible substrates of CYP1A enzymes. 17 beta-estradiol inhibited CYP1A1-catalyzed paraxanthine and trimethyluric acid formation. In contrast 17 beta-estradiol did not inhibit CYP1A2-catalyzed formation of primary caffeine metabolites. These data clearly demonstrate the capacity of human CYP1A1 and CYP1A2 to metabolize caffeine. Furthermore, possible consequences of CYP1A enzyme inhibition by caffeine, phenacetin, 17 beta-estradiol, and progesterone will be discussed.