Testosterone 1β-hydroxylation by human cytochrome P450 3A4

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6beta-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6beta- and 2beta-hydroxy products, was identified as 1beta-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of approximate to 6 mug of this product. Mass spectrometry established the formula as C19H29O3 (MH+ 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the beta-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1alpha-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.