Studies on neurosteroids XIV. Levels of dehydroepiandrosterone sulfate in rat brain and serum determined with newly developed enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) of dehydroepiandrosterone sulfate (DHEAS), one of the neurosteroids, has been developed for measuring its brain and serum levels in rats without deconjugation. 11 alpha-Hemiglutaryloxy-DHEAS was newly synthesized, conjugated with bovine serum albumin (BSA), and immunized to rabbits for the production of anti-DHEAS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 100 pg. Although the antibody significantly cross-reacted with epiandrosterone sulfate, it easily discriminated the unconjugated steroids and pregnenolone sulfate, which is reported to exist in the brain at a much higher level when compared with DHEAS. The brain homogenate or serum was treated with hexane to remove the lipophilic compounds and purified with an OASIS HLB cartridge. The DHEAS levels were then determined by ELISA. The overall recovery rate through the pretreatment was a satisfactory and constant (81.8 +/- 3.4% for brain, 89.3 +/- 3.0% for serum, mean +/- standard deviation). This ELISA afforded a satisfactory serial dilution study and recovery test. The intra- and inter-assay coefficients of variation were lower than 13%, which showed the precision of the proposed method. The applied method showed that DHEAS was not detected in some brain samples and its levels were much lower than those previously reported and than its serum levels.

Neurosteroid analogues. 6. The synthesis and GABAA receptor pharmacology of enantiomers of dehydroepiandrosterone sulfate, pregnenolone sulfate, and (3alpha,5beta)-3-hydroxypregnan-20-one sulfate.

The unnatural enantiomers of dehydroepiandrosterone sulfate (1), pregnenolone sulfate (2), and (3alpha,5beta)-3-hydroxypregnan-20-one sulfate (3), compounds 4-6, respectively, were prepared by total steroid synthesis. The enantioselectivity of the compounds as negative modulators of the GABAA receptors present in cultured rat hippocampal neurons was examined using electrophysiological methods. Enantioselectivity was found for the inhibitory actions of the dehydroepiandrosterone enantiomers. The IC50s for compounds 1 and 4 were 11 +/- 1 and 80 +/- 14 microM, respectively. Little, if any, enantioselectivity was found for the other two pairs of steroid sulfate inhibitors. The IC50s for compounds 2 and 5 were 82 +/- 12 and 76 +/- 27 microM, respectively. The IC50s for compounds 3 and 6 were 39 +/- 7 and 46 +/- 2 microM, respectively. The results suggest that the sites of action for the androstane and pregnane series of steroid sulfate blockers of GABA-mediated current are different. The observed enantioselectivity for the actions of dehydroepiandrosterone sulfate indicates that its inhibitory actions are mediated via a chiral recognition site and provides new evidence in support of the earlier hypothesis that there is a binding site for this compound on GABAA receptors. Conversely, the failure to observe enantioselectivity for the actions of pregnenolone sulfate and steroid sulfate 3 indicates that a chiral recognition site for these steroids does not exist on GABAA receptors and suggests that the effects of these compounds on this receptor's function may arise indirectly as a consequence of steroid-induced membrane perturbation.

Synthesis of sodium androst-5-ene-17-one-3 beta-methylene sulfonate.

The synthesis of sodium androst-5-ene-17-one-3 beta-methylene sulfonate 2, a stable analog of memory-enhancing neurosteroid dehydroepiandrosterone sulfate, is described. The synthesis of compound 2 is carried out in six steps from dehydroepiandrosterone.