Isocratic high-performance liquid chromatographic measurement of optimal 5 alpha-steroid reductase activity in Hep-G2 cells

• Published in: Journal of chromatography Vol. 570; no. 2; p. 293
• Main Authors: Stubbs, A P, Murphy, G M, Wilkinson, M L
• Format: Journal Article
• Language: English
• Published: Netherlands 04-10-1991
• Subjects: 17-Hydroxysteroid Dehydrogenases - metabolism; 3-Oxo-5-alpha-Steroid 4-Dehydrogenase - metabolism; Carcinoma, Hepatocellular - enzymology; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Liver Neoplasms - enzymology; Testosterone - metabolism; Tumor Cells, Cultured
• DOI: 10.1016/0378-4347(91)80532-H

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.