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.
Effects of ethanol on serum levels of eight gabaergic neuroactive steroids in rats measured by highly specific GC/MS assay
Porcu P;
2008
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
