Isocratic high-performance liquid chromatographic measurement of optimal 5 alpha-steroid reductase activity in Hep-G2 cells.
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.
Stubbs
A P
AP
Gastroenterology Unit, U.M.D.S. of Guy's Hospital, London, UK.
Murphy
G M
GM
Wilkinson
M L
ML
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
D013739:Testosterone; D015067:17-Hydroxysteroid Dehydrogenases; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase