等度高效液相色谱法测定Hep-G2细胞中最佳5α-类固醇还原酶活性

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

作者信息

Stubbs A P, Murphy G M, Wilkinson M L

机构信息

Gastroenterology Unit, U.M.D.S. of Guy's Hospital, London, UK.

出版信息

J Chromatogr. 1991 Oct 4;570(2):293-9. doi: 10.1016/0378-4347(91)80532-h.

DOI:10.1016/0378-4347(91)80532-h

PMID:1665848

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.

摘要

5α-还原酶活性的测定通常涉及对[3H]睾酮放射性标记产物的定量。然而，最近有观点认为，5α-还原酶的活性被17β-羟基类固醇脱氢酶和3-酮类固醇还原酶的活性所掩盖。因此，在测定Hep-G2细胞中的5α-还原酶活性时，我们监测了雄烯二酮的浓度，以确保维持最佳酶活性的测量条件。使用极性（氰基）键合相柱，以己烷-异丙醇（9:1，v/v）作为洗脱剂，最接近的峰——双氢睾酮和雌二醇的相对保留时间之比（以甲基石胆酸作为参考标准）为1.2，而最高的批间变异系数为2.7%。因此，该技术似乎适用于评估细胞和组织样本中的5α-还原酶。