Abstract
A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.
Highlights
A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain
Unconjugated and lipoidal PREG and DHEA were detected in selected ion monitoring (SIM) mode, and PREG-HFB and DHEA-HFB were identified by their GC retention times and the ratio between two specific diagnostic ions
The concentration of DHEA-S in brain exceeded that of free DHEA in brain and DHEA-S in plasma and persisted in brain after adrenalectomy and gonadectomy
Summary
The radioactive steroids [3H]PREG ([7-3H]PREG; 25 Ci/mmol), [3H]DHEA-S ([1,2,6,7-3H]DHEA-S; 92.5 Ci/mmol), and [3H]PREG palmitate ([7-3H]PREG; 25 Ci/mmol) were supplied by New England Nuclear (Boston, MA), and [3H]PREG-S ([7-3H]PREG-S; 25 Ci/mmol) was prepared in our laboratory from [3H]PREG using pyridine sulfur trioxide. Steroids were extracted from tissues by adding 10 volumes (w/v) of methanol (MeOH), and the samples were sonicated and left overnight at room temperature and centrifuged at 3,000 g for 5 min. Two nanograms of [2H4]PREG-S in MeOH was added as an internal standard to aliquots of brain and plasma and to the total extracts of hippocampus, liver, and adrenals for quantification of PREG-S and DHEA-S. The extracts were dried, taken up in 1 ml of MeOH, and sonicated This protocol was applied in the analysis of PREG and DHEA conjugates in 150 mg of whole brain from 2 month old Swiss male mice. Analogous experiments were carried out by adding tritiated PREG and PREG-S to a methanolic extract of rat brain for 0 and 24 h
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