Thin-layer chromatography analysis of human CYP3A-catalyzed testosterone 6beta-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 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.
Waxman
David J
DJ
Department of Biology, Boston University, MA, USA.
Chang
Thomas K H
TK
D001345:Autoradiography; D002855:Chromatography, Thin Layer / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000201:enzymology; D012588:Scintillation Counting; D013739:Testosterone / Q000378:metabolism*
D013739:Testosterone; D051544:Cytochrome P-450 CYP3A