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