title: Isocratic high-performance liquid chromatographic measurement of optimal 5 alpha-steroid reductase activity in Hep-G2 cells.
abstract: Measurement of 5 alpha-reductase activity usually involves quantitation of the radiolabelled products of [3H]testosterone. Recently, however, it has been claimed that the activity of 5 alpha-reductase is masked by the activities of 17 beta-hydroxysteroid dehydrogenase and 3-ketosteroid reductase. Therefore in determining 5 alpha-reductase activity in Hep-G2 cells, we have monitored the concentration of androstenedione to ensure that the conditions for measurement of optimum enzyme activity are maintained. Using a polar (cyano) bonded-phase column and hexane-isopropanol (9:1, v/v) as eluent, the ratio of relative retention times (methyl lithocholate used as the reference standard) of the closest peaks, dihydrotestosterone and estradiol, was 1.2, whilst the highest inter-assay coefficient of variation was 2.7%. Therefore this technique appears suitable for the evaluation of 5 alpha-reductase in cell and tissue samples.
authors.affiliation: Gastroenterology Unit, U.M.D.S. of Guy's Hospital, London, UK.
authors.forename: A P
authors.initials: AP
authors.lastname: Stubbs
authors.forename: G M
authors.initials: GM
authors.lastname: Murphy
authors.forename: M L
authors.initials: ML
authors.lastname: Wilkinson
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology*; D013739:Testosterone / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D013739:Testosterone; D015067:17-Hydroxysteroid Dehydrogenases; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase