DETERMINATION OF NEUROSTEROIDS IN HUMAN CEREBROSPINAL FLUID

这项研究的主要目的是确定 乙醇对中枢神经系统中神经类固醇状态的影响。 神经类固醇，如5a-孕烷-3a-醇-20-酮(THP或别孕酮) 和5a-孕烷-3a，21-二醇-20-酮(THDOC)被证明调节 GABA/苯二氮卓类药物结合部位及抗焦虑和催眠作用 效果。因此，分析中枢神经中的神经类固醇 系统可能提供与压力相关的生化理解 在人类中的情况，如在酗酒者身上观察到的 戒烟。它们的大脑浓度的最佳近似值 神经类固醇可以通过测量它们在脑脊液中的水平来制造。 液体(脑脊液)。 作为这个项目的第一步，我们开发了一种敏感的 允许检测脑脊液中神经类固醇的技术，使用 电喷雾和热喷雾液质联用 (LC/MS)。样品经乙酸乙酯提取后直接进样。 没有衍生化的LC/MS系统。各种神经类固醇 包括双氢睾酮(DHT)、睾酮、孕酮、 脱氢表雄酮(DHEA)、双氢孕酮(DHP)、THDOC、THP、 孕烯醇酮和孕烯醇酮在ODS柱上分离， 被检测为分子离子物种。目前对大多数的检测下限 类固醇大约在10-20 pg范围内。响应曲线 通过选择性地监测分子离子而产生的 氚标记内标物在20pg~5 ng范围内线性关系良好 射程。使用LC/MS技术，我们能够识别所有 人类脑脊液样本中含有上述神经类固醇。

The principal objective of this study is to determine the effect of ethanol on the status of neurosteroids in the central nervous system. Neurosteroids such as 5a-pregnane-3a-ol-20-one (THP or Allopregnanolone) and 5a-pregnane-3a,21-diol-20-one (THDOC) have been shown to modulate the GABA/benzodiazepine binding sites and to exert anxiolytic and hypnotic effects. Therefore, analysis of neurosteroids in the central nervous system may provide a biochemical understanding of stress-related situations in humans such as those observed in alcoholics during withdrawal. The best approximation of the brain concentration of these neurosteroids may be made by measuring their levels in the cerebrospinal fluid (CSF). As the first step for this project, we have developed a sensitive technique which allows the detection of neurosteroids in CSF, using electrospray and thermospray liquid chromatography/mass spectrometry (LC/MS). Samples extracted with ethyl acetate were directly introduced to an LC/MS system without derivatization. Various neurosteroids including dihydrotestosterone (DHT), testosterone, progesterone, dehydroepiandrosterone (DHEA), dihydroprogesterone (DHP), THDOC, THP, pregnenolone and pregnanolone were separated on an ODS column and detected as molecular ion species. The present detection limit for most steroids is approximately in the 10-20 pg range. The response curves generated by selectively monitoring molecular ions in the presence of deuterium-labeled internal standards were linear in the 20 pg to 5 ng range. Using the LC/MS technique, we were able to identify all of the neurosteroids listed above in human CSF samples.