Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6-Hydroxylation

Testosterone and other steroid hormones have been studied as prototypic examples of endogenous substrates for hepatic cytochrome P450 (P450) enzymes. CYP3A enzymes from various species, including human, metabolize testosterone by a 6β-hydroxylation reaction, which is unique to this P450 subfamily. A thin-layer chromatographic method is described for the determination of 6β-hydroxytestosterone formed enzymatically by incubation of [¹⁴ C]-testosterone with cDNA-expressed CYP3A enzymes or liver microsomes. ¹⁴ C-labeled enzymatic products are applied to silica gel thin-layer plates, which are developed sequentially with methylene chloride:acetone (80:20) followed by chloroform, ethyl acetate, and absolute ethanol (80:20:14). Metabolite quantification is performed by autoradiography and liquid scintillation counting. This method is applicable to enzymatic studies for the determination of CYP3A-dependent testosterone 6β- hydroxylation activity in both human and animal liver microsomes.