Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain

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 ( Ͻ 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 ( ‫ف‬ 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. Four years ago, we developed and validated an analytical procedure for measuring trace amounts of neurosteroids in brain tissue by GC-MS (1). This method, which includes solid-phase extraction (SPE) and HPLC as fractionation and purification steps, is very suitable for quantifying simultaneously numerous neurosteroids in small individual regions of the central nervous system (CNS) (2) or peripheral nervous system (3) with high sensitivity and accuracy. Interest was primarily focused on pregnenolone (PREG), dehydroepiandrosterone (DHEA), and their sulfated conjugates (PREG-S and DHEA-S, respectively) and on progesterone (PROG) and allopregnanolone (3 ␣ -hydroxy-5 ␣ -pregnan-20-one).Neurosteroids are synthesized by glial cells and neurons from cholesterol or from blood-borne precursors (4). In particular, PREG-S and DHEA-S are known to be neuroactive, and their major effects are the modulation of several neuronal membrane receptors, such as ␥ -aminobutyric acid (5, 6), N -methyl-d -aspartate (7), and σ receptors, by affecting neuronal excitability and behavior (8, 9). Notably, a physiologic function of PREG-S was suggested by the positive correlation between PREG-S levels in the hippocampus of aged rats and their spatial memory performance (10). However, some studies do not favor the concept that PREG-S and DHEA-S are actually neurosteroids. Indeed, contradictory results have been obtained concerning the presence and activity of the hydroxysteroid sulfotransferase, implied in the sulfation of free steroid.

Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods

“…In the method developed in this study, addition of 30% MeOH and decreasing the pH to 5 in the intestinal fluid before SPE was sufficient to improve the sorption of TAGs, FFAs, and PLs. This is also in accordance with previous studies (54)(55)(56)(57)(58). More than 30% MeOH in the intestinal fluid resulted in a breakthrough of conjugated BAs in this study.…”

Differential hypothalamic–pituitary–adrenal activation of the neuroactive steroids pregnenolone sulfate and deoxycorticosterone in healthy controls and alcohol-dependent subjects

“…Stress is known to interfere with HPA axis functioning and we cannot rule out the possibility that the alterations in PREG-S and deoxycorticosterone responses to HPA axis challenge seen in these patients are strictly related to the alcohol-dependence. Also, recent findings using highly sensitive mass spectrometry techniques suggest that PREG-S is not present in rat brain and plasma (Liu et al, 2003;Liere et al, 2004). However, these authors were able to measure PREG-S levels in human plasma and values are comparable to the ones we obtained using a radioimmunoassay with a commercially available antibody.…”

Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat

“…The glucuronide forms of the adrenal steroids CCS and 11-DCC were detected in the blood and whole organs at high concentrations (Table III), suggesting that these glucuronides were delivered to the target cells that express a glucuronide transporter and that the steroid can perform its respective function after deconjugation. Neurosteroid sulphates have not been previously found in the rodent brain (35,36), however, sulphate conjugates of PGT, 11-DCC, CCS and TS were detected in the brain at very low levels (1020 pmol/g) (Table III) in this and earlier studies (37,38). Because the glucuronides and sulphates of major steroids were detected in the blood and kidney at similar levels, these conjugates were excreted into the urine via the kidneys.…”