Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

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Abstract
To investigate the potential interaction of the various pathways of androgen hydroxylation, we have conducted studies to identify the profile of products formed during the time course of metabolism of androst-4-ene-3,17-dione (AD). Incubates containing AD, NADPH, and liver microsomes (from rats pretreated with phenobarbital) were sampled at times between 0 and 20 min and the metabolites resolved by reverse-phase (C18) high-performance liquid chromatography. By this method, the pattern of formation and of utilization of eight major primary and secondary metabolites of AD was determined. We report here the formaton of two previously unidentified major metabolites of AD: 6.beta.,16.alpha.-dihydroxyandrost-4-ene-3,17-dione and 6.beta.,16.beta.-dihydroxyandrost-4-ene-3,17-dione. We propose that liver microsomal cytochromes P-450 can sequentially hydroxylate a single molecule of AD at multiple sites. These hydroxylase activities are presumably a result of multiple cytochrome P-450 isozymes acting on AD resulting in a transient time course for the appearance of some nonohydroxylated metabolites. In addition, a unidirectional conversion of the metabolite 16.alpha.-hydroxyandrost-4-ene-3,17-dione to 16.beta.-dihydroxyandrost-4-ene-3,17-dione is described. Evidence is provided to support the role of cytochrome P-430 in catalyzing this reaction.