Studies on neurosteroids

The identification and quantification of tetrahydrocorticosterone isomers (THBs; 3α,5α-, 3β,5α-, 3α,5β- and 3β,5β-THB) in rat brains using liquid chromatography (LC)-mass spectrometry (MS) are described. For the identification, the THBs were converted to the atmospheric pressure chemical ionization (APCI)-active derivatives, i.e., the dinitrobezoyl esters and 2-nitro-4-trifluoromethylphenyl hydrazones, and detected in the negative-ion mode. These derivatives showed 60- and 40-fold higher sensitivities, respectively, than intact steroids measured in the positive-APCI-MS. The derivatized THBs were satisfactorily separated from the others during the reversed-phase LC. The THBs were not detected at all in the brains of the unstressed rats. When the rats were exposed to the immobilization for 20 min, 3α,5α- and 3β,5α-THB were detected as the major metabolites together with small amounts of 3α,5β- and 3β,5β-THB in the male rat brain, while only 3α,5α-THB was detected in the female rats. Thus, the steroid variety found in the brains was different between the sexes. In the next step, 3α,5α-THB, a major metabolite found in the brains of the stressed rats, was quantified as its dinitrobezoyl ester. This method was accurate and reproducible, and the limit of quantitation was 1.0 ng/g tissue when a 50 mg tissue sample was used. There was also a sex difference in the brain 3α,5α-THB level; it was significantly higher in the female rats than in the male rats (P < 0.05), although the brain corticosterone level was not higher in the stressed female rats than in the male rats (no statistical difference).