Waxman David J, Chang Thomas K H
Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA, USA.
Faculty of Pharmaceutical Sciences, University of British Columbia, Vaqncouver, British Columbia, Canada.
Methods Mol Biol. 2006;320:133-141. doi: 10.1385/1-59259-998-2:133.
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 [C]-testosterone with cDNA-expressed CYP3A enzymes or liver microsomes. 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.
睾酮及其他类固醇激素已作为肝细胞色素P450（P450）酶的内源性底物的典型例子进行了研究。包括人类在内的各种物种的CYP3A酶通过6β-羟基化反应代谢睾酮，这是该P450亚家族所特有的。本文描述了一种薄层色谱法，用于测定通过将[C] - 睾酮与cDNA表达的CYP3A酶或肝微粒体孵育而酶促形成的6β-羟基睾酮。将C标记的酶促产物应用于硅胶薄层板，先用二氯甲烷：丙酮（80:20）展开，然后依次用氯仿、乙酸乙酯和无水乙醇（80:20:14）展开。代谢物定量通过放射自显影和液体闪烁计数进行。该方法适用于酶促研究，以测定人和动物肝微粒体中CYP3A依赖性睾酮6β-羟基化活性。
