DETERMINATION OF NEUROSTEROIDS IN HUMAN CEREBROSPINAL FLUID

人脑脊液中神经甾醇的测定

• 批准号: 5200241
• 负责人: H-Y KIM
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200241
• 关键词: central nervous system; cerebrospinal fluid; chemical binding; dehydroepiandrosterone; deuterium; dihydropyridines; dihydrotestosterone; drug interactions; electrospray ionization mass spectrometry; ethanol; human tissue; mass spectrometry; pregnanolone; pregnenolone; progesterone; steroids; testosterone

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.

本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的作用。 神经甾体，如5a-甾烷-3a-醇-20-酮（THP或别孕烯醇酮） 和5 α-胆甾烷-3 α，21-二醇-20-酮（THDOC）已被证明可以调节 GABA/苯二氮卓类结合位点，并发挥抗焦虑和催眠作用 方面的影响. 因此，分析中枢神经系统中的神经甾体 系统可以提供与压力相关的生物化学理解， 人类的情况，如在酗酒者中观察到的情况， 戒断 这些物质在大脑中浓度的最佳近似值 神经类固醇可以通过测量它们在脑脊髓中的水平来制备 脑脊液（CSF）。 作为这个项目的第一步，我们已经开发了一个敏感的 一种允许检测CSF中神经类固醇的技术，使用 电喷雾和热喷雾液相色谱/质谱 (LC/MS）。 样品经乙酸乙酯萃取后直接进样 至LC/MS系统，无需衍生化。 各种神经类固醇 包括双氢睾酮（DHT）、睾酮、黄体酮， 脱氢表雄酮（DHEA），二氢孕酮（DHP），THDOC，THP， 在ODS柱上分离双烯醇酮和孕烯醇酮， 检测为分子离子种类。 目前的检测限大多数 类固醇大约在10-20 pg范围内。 的响应曲线 通过选择性地监测分子离子， 氘标记的内标物在20 pg至5 ng范围内呈线性 范围 使用LC/MS技术，我们能够识别所有 在人CSF样品中的上述神经类固醇。