Effects of ethanol on GABAergic neuroactive steroids in rat and humans measured by highly specific GC/MS assay

Acute ethanol administration increases 3alpha-hydroxy,5alpha-pregnan-20-one (3a,5a-THP) and 3alpha,21-dihydroxy,5alpha-pregnan-20-one (3a,5a-THDOC) in rat plasma and brain with mixed effects in human plasma. To determine the effects of all 3a,5a and 3a,5b -reduced GABAergic neuroactive steroids, we developed a highly specific gas chromatography/mass spectrometry (GC/MS) assay. We used this assay to measure serum levels of the GABAergic 3a,5a- and 3a,5b-reduced metabolites of progesterone, deoxycorticosterone, DHEA and testosterone as well the precursors pregnenolone and DHEA. Male rats were injected i.p. with 2 g/kg ethanol or saline and were sacrificed 60 minutes later (BEC, 170-190 mg/dl). Acute ethanol administration selectively increased serum levels of pregnenolone, 3a,5a-THP, 3a,5a-THDOC and 3a,5b-androsterone (+589, +191, +291%, p<0.0001 and +78%, p<0.05, respectively), compared to saline control levels. In contrast, ethanol did not alter 3a,5a-androsterone, 3a,5a-androstandiol, 3a,5b-THP or DHEA levels. 3a,5b-THDOC and 3a,5b-androstandiol were not detected in serum following vehicle or ethanol. Young healthy human males consumed 0.8 g/kg ethanol over 30 minutes and achieved peak BECs of 0.75±0.1 mg/dl. No changes in pregnenolone, DHEA or any 3a,5a- or 3a,5b-reduced GABAergic neuroactive steroid were detected after 60 or 120 min. Studies are underway with lower does of ethanol in rats to evaluate the role of dose in the species differences in ethanol responses. The highly selective GC/MS assay can be used to investigate the physiological and pathological role of GABAergic neuroactive steroids in health and disease.