It was demonstrated that the anesthetic steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one mediates the loss of the righting response (LRR) in mice, in contrast to its metabolites, which are formed in vivo. To reach these conclusions, it was necessary to quantitate levels for 3 alpha-hydroxy-5 alpha-pregnan-20-one and its metabolites at the time of LRR. Methods were used that blocked the production of essentially all of the metabolites. Chromatography of brain extracts by thin-layer chromatography and high-performance liquid chromatography after anesthesia with 3 alpha-hydroxy-5 alpha-[3H]pregnan-20-one showed only a single peak that corresponded to 3 alpha-hydroxy-5 alpha-[3H]pregnan-20-one.
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