title: Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6β-Hydroxylation.
abstract: 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 [14C]-testosterone with cDNA-expressed CYP3A enzymes or liver microsomes.14C-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.
authors.affiliation: Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Waxman
authors.affiliation: Faculty of Pharmaceutical Sciences, University of British Columbia, Vaqncouver, British Columbia, Canada.
authors.forename: Thomas K H
authors.initials: TK
authors.lastname: Chang
keywords: 6β-hydroxytestosterone; CYP3A; CYP3A4; CYP3A43; CYP3A5; CYP3A7; Cytochrome P450; Testosterone; testosterone 6β-hydroxylation