Effects of ethanol on serum levels of eight gabaergic neuroactive steroids in rats measured by highly specific GC/MS assay

Acute ethanol administration increases plasma and brain levels of the progesterone and deoxycorticosterone metabolites 3a-hydroxy,5a-pregnan-20-one (3a,5a-THP) and 3a, 21-dihydroxy,5a-pregnan-20-one (3a,5a-THDOC). However, little is known about the effects of ethanol on other GABAergic neuroactive steroids, such as the dehydroepiandrosterone (DHEA) and testosterone metabolites, 3a-hydroxy-5a-androstan-17-one (3a,5a-A) and 3ahydroxy- 5a-androstan-17b-diol (3a,5a-A-Diol). We used a highly sensitive and specific gas chromatography/mass spectrometry (GC/MS) assay to measure serum levels of the 3a,5a- and 3a,5b-reduced GABAergic neuroactive steroids and their precursors pregnenolone and DHEA, following acute ethanol administration. Seven male rats were injected i.p. with 2 g/kg ethanol and were sacrificed 60 minutes later. Nine control rats received an equivalent volume of saline. Ethanol administration selectively increased serum levels of GABAergic neuroactive steroids. Pregnenolone, 3a,5a-THP, 3a,5a-THDOC and 3a,5b-A levels were significantly increased (+589, +191, +291, p<0.0001 and +78, p<0.05, respectively), compared to control levels. In contrast, ethanol did not alter 3a,5a-A and 3a,5a-A-Diol levels. Basal 3a,5b-THP and DHEA were detected only in 4/9 and 3/9 controls, but levels did not change after ethanol administration. 3a,5b-THDOC and 3a,5b-ADiol were not detected in serum from both control and ethanol-treated rats. To better validate the biological relevance of this GC/MS assay, we also investigated the effects of pregnenolone administration (50 mg/kg, i.p.) to male rats (n=8). Pregnenolone significantly increased levels of 3a,5a-THP (+1488%, p=0.0002), 3a,5a-THDOC (+205%, p=0.003), 3a,5a-A (+216, p=0.0005), 3a,5a-A-Diol (+190%, p=0.001). DHEA levels were also elevated (+1549, p=0.04), although basal levels were detected only in 3/9 rats. 3a,5b-THDOC and 3a,5b-A-Diol were increased from undetectable levels to 271 ± 100 and 2.4 ± 0.9, respectively (pg±SEM), but only in 5/8 rats. 3a,5b-THP and 3a,5b-A were not altered by pregnenolone administration. Pregnenolone levels were dramatically increased (greater than 1000 fold) in all rats, but an accurate estimate was not possible using the same GC/MS settings for concurrent analysis. The highly specific GC/MS assay can be used to investigate the physiological and pathological role of neuroactive steroids, develop biomarkers and new therapeutics for neuropsychiatric diseases, including alcoholism.