Kudo K, Emmons G T, Casserly E W, Via D P, Smith L C, St Pyrek J, Schroepfer G J
Department of Biochemistry, Rice University, Houston, TX 77251.
J Lipid Res. 1989 Jul;30(7):1097-111.
The separation of the acetate derivatives of a number of oxygenated sterols was achieved by medium pressure liquid chromatography on silica gel columns and by normal and reversed phase high performance liquid chromatography. We have explored the application of these chromatographic systems for the analysis of oxygenated sterols of plasma samples from two normal human subjects. The addition of highly purified [14C]cholesterol to plasma permitted the detection and quantitation of oxygenated sterols formed by autoxidation of cholesterol during processing of the samples. Special attempts to suppress autoxidation of cholesterol included the use of an all-glass closed system for saponification and extraction under argon followed by rapid removal of cholesterol from the polar sterols by reversed phase medium pressure liquid chromatography. Chromatographic analyses of the [3H]acetate derivatives of the polar sterols provided a sensitive approach for the detection and quantitation of the individual oxygenated sterols. Oxygenated sterols detected in plasma included cholest-5-ene-3 beta,26-diol, (24S)-cholest-5-ene-3 beta,24-diol, and cholest-5-ene-3 beta,7 alpha-diol. After correction for their formation by autoxidation of cholesterol during processing of the samples, very little or none of the following sterols were observed: cholest-5-ene-3 beta,7 beta-diol, 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol, 5 beta,6 beta-epoxy-cholestan-3 beta-ol, and cholestane- 3 beta, 5 alpha,6 beta-triol, and the 25-hydroxy, 22R-hydroxy, 21-hydroxy, 20 alpha-hydroxy, and 19-hydroxy derivatives of cholesterol.
通过硅胶柱上的中压液相色谱法以及正相和反相高效液相色谱法实现了多种氧化甾醇乙酸酯衍生物的分离。我们探索了这些色谱系统在分析两名正常人类受试者血浆样品中氧化甾醇方面的应用。向血浆中添加高度纯化的[14C]胆固醇能够检测和定量样品处理过程中胆固醇自氧化形成的氧化甾醇。抑制胆固醇自氧化的特殊尝试包括使用全玻璃封闭系统在氩气下进行皂化和萃取，随后通过反相中压液相色谱法从极性甾醇中快速去除胆固醇。极性甾醇的[3H]乙酸酯衍生物的色谱分析为检测和定量单个氧化甾醇提供了一种灵敏的方法。血浆中检测到的氧化甾醇包括胆甾 - 5 - 烯 - 3β,26 - 二醇、(24S) - 胆甾 - 5 - 烯 - 3β,24 - 二醇和胆甾 - 5 - 烯 - 3β,7α - 二醇。在对样品处理过程中胆固醇自氧化形成的这些氧化甾醇进行校正后，未观察到以下甾醇中的极少部分或未观察到：胆甾 - 5 - 烯 - 3β,7β - 二醇、5α,6α - 环氧胆甾烷 - 3β - 醇、5β,6β - 环氧胆甾烷 - 3β - 醇以及胆甾烷 - 3β,5α,6β - 三醇以及胆固醇的25 - 羟基、22R - 羟基、21 - 羟基、20α - 羟基和19 - 羟基衍生物。
