Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Abstract

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16.alpha.-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2.alpha.-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6.alpha.-hydroxyprogesterone and 7.alpha.-hydroxyprogesterone were only formed by P-450a. 6.beta.-Hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15.beta.-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, P-450i. None of the isozymes catalyzed 17.alpha.-hydroxylation of progesterone, and only cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 .mu.M), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7a, and 2.alpha.-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed > 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.