Abstract

Antipyrine biotransformation has been used extensively in clinical studies as a marker for general hepatic oxidative or cytochrome P450 (P450)-mediated, metabolism. Studies have indicated that more than one P450 is involved in the formation of the three major human metabolites, 4-hydroxyantipyrine, norantipyrine, and 3-hydroxymethylantipyrine. However, the specific P450s involved have not yet been determined. We have developed a sensitive method for the measurement of antipyrine metabolites formed in the in vitro incubations and applied it to determine the P450s participating in the formation of each metabolite in human liver microsomes. The identification of these P450s was accomplished through the use of simple and multivariate regression analysis, selective chemical inhibitors, and microsomes containing cDNA-expressed enzymes. These methods implicated P450s 1A2, 3A, and 2A6 in the formation of 4-hydroxyantipyrine. The predominant form involved in 3-hydroxymethylantipyrine formation was found to be 1A2, although 2C9 and 2E1 seemed to participate to a lesser extent. All forms considered (1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A) seemed to be involved in the formation of norantipyrine, although 1A2 and 2C9 only were accepted in multivariate regression analysis. Thus, antipyrine clearance is indeed a general measure of P450 oxidative capacity, with a slight to moderate weight toward 1A2, depending on the degree of 1A2 expression. Because of the multiplicity of the enzymes involved in the formation of each metabolite, the determination of these individual metabolites would be ineffective as an indicator of the levels of specific P450 forms in human subjects.