CHARACTERIZATION OF TESTOSTERONE 11β-HYDROXYLATION CATALYZED BY HUMAN LIVER MICROSOMAL CYTOCHROMES P450

Abstract

A combination of accelerator mass spectrometry (AMS) and liquid chromatography-tandem mass spectrometry has been used to clarify some new aspects of testosterone metabolism. The main pathway of testosterone oxidative metabolism by human liver microsomes is the formation of 1beta-, 2alpha-/beta-, 6beta-, 15beta-, and 16beta-hydroxytestosterones, mainly catalyzed by cytochromes P450 2C9, 2C19, and 3A4. We now report the first determination that 11beta-hydroxytestosterone (11beta-OHT) can also be formed by human liver microsomal fractions. The structures of five hydroxylated metabolites of testosterone (2beta-, 6beta-, 11beta-, 15beta-, and 16beta-OHT) and the C-17 oxidative metabolite androstenedione were determined by liquid chromatography with UV detection at 240 nm and liquid chromatography-tandem mass spectrometry. Corresponding results were obtained by high-performance liquid chromatography-AMS analysis of incubations of [4-14C]testosterone with human liver microsomes. 6beta-Hydroxylation was always the dominant metabolic pathway, but 2beta-, 15beta-, and 16beta-OHT, and androstenedione were also formed. The previously undetected hydroxytestosterone, 11beta-OHT, was found to be a minor metabolite formed by human liver microsomal enzymes. It was formed more readily by CYP3A4 than by either CYP2C9 or CYP2C19. 11beta-Hydroxylation was inhibited by ketoconazole (IC50 = 30 nM) at concentrations similar to the IC50 (36 nM) for 6beta-hydroxylation Therefore, CYP3A4 could be mainly responsible for testosterone 11beta-hydroxylation in the human liver. These findings identify human hepatic biotransformation of testosterone to 11beta-OHT as a previously unrecognized extra-adrenal metabolic pathway.