DETERMINATION OF NEUROSTEROIDS IN HUMAN CEREBROSPINAL FLUID

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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.