Matsuzaki K, Kimura K, Kurokawa K, Miyazaki T
First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
J Steroid Biochem Mol Biol. 1991 May;38(5):657-62. doi: 10.1016/0960-0760(91)90324-x.
The A6 cell line of the toad kidney is well known to form an Na+ transporting tight epithelium in culture and is often used as an experimental model for Na+ transport systems. Although it has been shown that A6 cells can convert aldosterone to polar metabolites, these metabolites have not been identified. Therefore, in this study, we tried to identify the metabolites of aldosterone formed by A6 cells in culture. A6 cells at confluence were incubated with serum-free culture media containing [3H]aldosterone. When radioactive compounds in incubation media were separated by reversed phase high-pressure liquid chromatography (HPLC), four fractions (fractions A-D) were obtained. Fraction A, a mixture of two components, comprised the majority of metabolites formed. The more polar material (fraction A-1) and the less polar material (fraction A-2) of fraction A contained 47-71 and 9-19% of total radioactivity, respectively. When incubated in cell-free media, fraction A-2 was found to be unstable and partially converted to fraction A-1. Fraction B, 0.7-1.5% of total radioactivity, and fraction C, 8-21% of total radioactivity, cochromatographed with iso-aldosterone and D-aldosterone, respectively. Fraction D, 4-8% of total radioactivity, was a mixture of two components, which cochromatographed with 3 beta,5 beta-tetrahydroaldosterone and 5 alpha-dihydroaldosterone, respectively. In order to identify fraction A-2 material, large-scale cultures were performed and fraction A-2 was separated and purified by reversed phase HPLC. The purified material was analyzed by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. These two procedures unambiguously revealed that this material was 6 beta-hydroxyaldosterone. These results demonstrate that aldosterone can be converted to at least four metabolites by the incubation with A6 cells, and that major metabolites are polar compounds, a portion of which is 6 beta-hydroxyaldosterone.
蟾蜍肾A6细胞系在培养中能形成运输钠离子的紧密上皮，常用于钠离子转运系统的实验模型。虽然已有研究表明A6细胞能将醛固酮转化为极性代谢产物，但这些代谢产物尚未得到鉴定。因此，在本研究中，我们试图鉴定A6细胞在培养中形成的醛固酮代谢产物。将汇合的A6细胞与含有[3H]醛固酮的无血清培养基一起孵育。当通过反相高压液相色谱（HPLC）分离孵育培养基中的放射性化合物时，得到了四个组分（组分A - D）。组分A是两种成分的混合物，构成了形成的代谢产物的大部分。组分A中极性较强的物质（组分A - 1）和极性较弱的物质（组分A - 2）分别含有总放射性的47 - 71%和9 - 19%。当在无细胞培养基中孵育时，发现组分A - 2不稳定，部分转化为组分A - 1。组分B占总放射性的0.7 - 1.5%，组分C占总放射性的8 - 21%，分别与异醛固酮和D - 醛固酮共色谱。组分D占总放射性的4 - 8%，是两种成分的混合物，分别与3β,5β - 四氢醛固酮和5α - 二氢醛固酮共色谱。为了鉴定组分A - 2物质，进行了大规模培养，并通过反相HPLC分离和纯化了组分A - 2。通过快原子轰击质谱和核磁共振光谱对纯化后的物质进行了分析。这两种方法明确显示该物质是6β - 羟基醛固酮。这些结果表明，醛固酮与A6细胞孵育后可转化为至少四种代谢产物，且主要代谢产物是极性化合物，其中一部分是6β - 羟基醛固酮。
