| 16719384 |
Waxman DJ, Chang TK: Thin-layer chromatography analysis of human CYP3A-catalyzed testosterone 6beta-hydroxylation. Methods Mol Biol. 2006;320:133-41.
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 6beta-hydroxylation reaction, which is unique to this P450 subfamily. A thin-layer chromatographic method is described for the determination of 6beta-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 6beta-hydroxylation activity in both human and animal liver microsomes. |
84(1,1,1,4) |