Characterization of testosterone 11β-hydroxylation catalyzed by human liver microsomal cytochromes P450

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 1 beta-, 2 alpha- /beta-, 6 beta-, 15 beta-, and 16 beta-hydroxytestosterones, mainly catalyzed by cytochromes P450 2C9, 2C19, and 3A4. We now report the first determination that 11 beta-hydroxytestosterone ( 11 beta-OHT) can also be formed by human liver microsomal fractions. The structures of five hydroxylated metabolites of testosterone (2 beta-, 6 beta-, 11 beta-, 15 beta-, and 16 beta-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-C-14] testosterone with human liver microsomes. 6 beta-Hydroxylation was always the dominant metabolic pathway, but 2 beta-, 15 beta-, and 16 beta-OHT, and androstenedione were also formed. The previously undetected hydroxytestosterone, 11 beta-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. 11 beta-Hydroxylation was inhibited by ketoconazole (IC50 = 30 nM) at concentrations similar to the IC50 (36 nM) for 6 beta-hydroxylation Therefore, CYP3A4 could be mainly responsible for testosterone 11 beta-hydroxylation in the human liver. These findings identify human hepatic biotransformation of testosterone to 11 beta-OHT as a previously unrecognized extra-adrenal metabolic pathway.