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• W2121753150 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 (∼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. 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 (∼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 (1Liere P. Akwa Y. Weill-Engerer S. Eychenne B. Pianos A. Robel P. Sjovall J. Schumacher M. Baulieu E.E. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 2000; 739: 301-312Crossref PubMed Scopus (148) Google Scholar). 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) (2Weill-Engerer S. David J.P. Sazdovitch V. Liere P. Eychenne B. Pianos A. Schumacher M. Delacourte A. Baulieu E.E. Akwa Y. Neurosteroid quantification in human brain regions: comparison between Alzheimer's and nondemented patients.J. Clin. Endocrinol. Metab. 2002; 87: 5138-5143Crossref PubMed Scopus (287) Google Scholar) or peripheral nervous system (3Ferzaz B. Brault E. Bourliaud G. Robert J.P. Poughon G. Claustre Y. Marguet F. Liere P. Schumacher M. Nowicki J.P. Fournier J. Marabout B. Sevrin M. George P. Soubrie P. Benavides J. Scatton B. SSR180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair.J. Pharmacol. Exp. Ther. 2002; 301: 1067-1078Crossref PubMed Scopus (116) Google Scholar) 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 (3α-hydroxy-5α-pregnan-20-one). Neurosteroids are synthesized by glial cells and neurons from cholesterol or from blood-borne precursors (4Baulieu E.E. Robel P. Schumacher M. Neurosteroids: beginning of the story.Int. Rev. Neurobiol. 2001; 46: 1-32Crossref PubMed Scopus (21) Google Scholar). 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 γ-aminobutyric acid (5Majewska M.D. Schwartz R.D. Pregnenolone-sulfate: an endogenous antagonist of the gamma-aminobutyric acid receptor complex in brain?.Brain Res. 1987; 404: 355-360Crossref PubMed Scopus (305) Google Scholar, 6Majewska M.D. Demirgoren S. Spivak C.E. London E.D. The neurosteroid dehydroepiandrosterone sulfate is an allosteric antagonist of the GABAA receptor.Brain Res. 1990; 526: 143-146Crossref PubMed Scopus (427) Google Scholar), N-methyl-d-aspartate (7Wu F.S. Gibbs T.T. Farb D.H. Pregnenolone sulfate: a positive allosteric modulator at the N-methyl-D-aspartate receptor.Mol. Pharmacol. 1991; 40: 333-336PubMed Google Scholar), and σ receptors, by affecting neuronal excitability and behavior (8Paul S.M. Purdy R.H. Neuroactive steroids.FASEB J. 1992; 6: 2311-2322Crossref PubMed Scopus (1470) Google Scholar, 9Rupprecht R. Holsboer F. Neuroactive steroids: mechanisms of action and neuropsychopharmacological perspectives.Trends Neurosci. 1999; 22: 410-416Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). 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 (10Vallée M. Mayo W. Darnaudery M. Corpechot C. Young J. Koehl M. Moal M. Le Baulieu E.E. Robel P. Simon H. Neurosteroids: deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus.Proc. Natl. Acad. Sci. USA. 1997; 94: 14865-14870Crossref PubMed Scopus (290) Google Scholar). 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. In some studies, the enzyme was not detected in human (11Meloche C.A. Falany C.N. Expression and characterization of the human 3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b).J. Steroid Biochem. Mol. Biol. 2001; 77: 261-269Crossref PubMed Scopus (86) Google Scholar, 12Sharp S. Barker E.V. Coughtrie M.W. Lowenstein P.R. Hume R. Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans.Eur. J. Biochem. 1993; 211: 539-548Crossref PubMed Scopus (60) Google Scholar), mouse (13Shimizu C. Fuda H. Yanai H. Strott C.A. Conservation of the hydroxysteroid sulfotransferase SULT2B1 gene structure in the mouse: pre- and postnatal expression, kinetic analysis of isoforms, and comparison with prototypical SULT2A1.Endocrinology. 2003; 144: 1186-1193Crossref PubMed Scopus (53) Google Scholar), or rat (12Sharp S. Barker E.V. Coughtrie M.W. Lowenstein P.R. Hume R. Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans.Eur. J. Biochem. 1993; 211: 539-548Crossref PubMed Scopus (60) Google Scholar) brain, although some groups have found the enzyme in rat (14Shibuya K. Takata N. Hojo Y. Furukawa A. Yasumatsu N. Kimoto T. Enami T. Suzuki K. Tanabe N. Ishii H. Mukai H. Takahashi T. Hattori T.A. Kawato S. Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction.Biochim. Biophys. Acta. 2003; 1619: 301-316Crossref PubMed Scopus (120) Google Scholar, 15Shimada M. Yoshinari K. Tanabe E. Shimakawa E. Kobashi M. Nagata K. Yamazoe Y. Identification of ST2A1 as a rat brain neurosteroid sulfotransferase mRNA.Brain Res. 2001; 920: 222-225Crossref PubMed Scopus (32) Google Scholar) and in frog (16Beaujean D. Mensah-Nyagan A.G. Do-Rego J.L. Luu-The V. Pelletier G. Vaudry H. Immunocytochemical localization and biological activity of hydroxysteroid sulfotransferase in the frog brain.J. Neurochem. 1999; 72: 848-857Crossref PubMed Scopus (61) Google Scholar) brain. Other studies have reported a very low hydroxysteroid sulfotransferase activity in rat (17Rajkowski K.M. Robel P. Baulieu E.E. Hydroxysteroid sulfotransferase activity in the rat brain and liver as a function of age and sex.Steroids. 1997; 62: 427-436Crossref PubMed Scopus (68) Google Scholar) and human (18Knapstein P. David A. Wu C.H. Archer D.F. Flickinger G.L. Touchstone J.C. Metabolism of free and sulfoconjugated DHEA in brain tissue in vivo and in vitro.Steroids. 1968; 11: 885-896Crossref PubMed Scopus (80) Google Scholar) brain. These results are in apparent contradiction to the reported high levels of PREG-S and DHEA-S measured in mammalian brain by RIA (19Lanthier A. Patwardhan V.V. Sex steroids and 5-en-3 beta-hydroxysteroids in specific regions of the human brain and cranial nerves.J. Steroid Biochem. 1986; 25: 445-449Crossref PubMed Scopus (169) Google Scholar, 20Corpéchot C. Robel P. Axelson M. Sjovall J. Baulieu E.E. Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.Proc. Natl. Acad. Sci. USA. 1981; 78: 4704-4707Crossref PubMed Scopus (601) Google Scholar, 21Corpéchot C. Synguelakis M. Talha S. Axelson M. Sjovall J. Vihko R. Baulieu E.E. Robel P. Pregnenolone and its sulfate ester in the rat brain.Brain Res. 1983; 270: 119-125Crossref PubMed Scopus (354) Google Scholar, 22Young J. Corpéchot C. Perché F. Haug M. Baulieu E.E. Robel P. Neurosteroids: pharmacological effects of a 3beta-hydroxy-steroid dehydrogenase inhibitor.Endocrine. 1994; 2: 505-509Google Scholar) and the higher concentrations of PREG-S and DHEA-S in rodent brain than in blood (20Corpéchot C. Robel P. Axelson M. Sjovall J. Baulieu E.E. Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.Proc. Natl. Acad. Sci. USA. 1981; 78: 4704-4707Crossref PubMed Scopus (601) Google Scholar, 21Corpéchot C. Synguelakis M. Talha S. Axelson M. Sjovall J. Vihko R. Baulieu E.E. Robel P. Pregnenolone and its sulfate ester in the rat brain.Brain Res. 1983; 270: 119-125Crossref PubMed Scopus (354) Google Scholar, 22Young J. Corpéchot C. Perché F. Haug M. Baulieu E.E. Robel P. Neurosteroids: pharmacological effects of a 3beta-hydroxy-steroid dehydrogenase inhibitor.Endocrine. 1994; 2: 505-509Google Scholar). Moreover, PREG-S and DHEA-S were found to be still present in the brains of castrated and adrenalectomized male rats, whereas they disappeared from plasma. These latter results, together with the low permeability of the blood-brain barrier for sulfated steroids (18Knapstein P. David A. Wu C.H. Archer D.F. Flickinger G.L. Touchstone J.C. Metabolism of free and sulfoconjugated DHEA in brain tissue in vivo and in vitro.Steroids. 1968; 11: 885-896Crossref PubMed Scopus (80) Google Scholar, 23Kishimoto Y. Hoshi M. Dehydroepiandrosterone sulphate in rat brain: incorporation from blood and metabolism in vivo.J. Neurochem. 1972; 19: 2207-2215Crossref PubMed Scopus (47) Google Scholar), are consistent with their in situ synthesis. In agreement with the concentration range for PREG-S and DHEA-S in these reports, their average levels in rat brain measured by GC-MS were 8.3 ± 0.80 ng/g and 2.5 ± 0.27 ng/g, respectively, and were higher than the levels of the unconjugated steroids (1Liere P. Akwa Y. Weill-Engerer S. Eychenne B. Pianos A. Robel P. Sjovall J. Schumacher M. Baulieu E.E. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 2000; 739: 301-312Crossref PubMed Scopus (148) Google Scholar). However, several experimental oddities in terms of reproducibility and linearity were observed for PREG-S and DHEA-S measurements. We also became aware of the fact that the chemical identity of the sulfated steroids had never been established. The analytical protocol was thus modified to improve the reliability of PREG-S and DHEA-S quantification. Our results demonstrate that the sulfated forms of PREG and DHEA are not present in rat and mouse brain. The previous detection of steroid sulfates resulted from the indirect method of analysis used in earlier studies that was based on the measure of unconjugated PREG and DHEA after hydrolysis of fractions supposed to contain the endogenous sulfated conjugates. We show here that large amounts of PREG and DHEA are derivatized from lipoidal complexes by acylation derivatization reaction in the presence of triethylamine (TEA). The presence of these lipoidal derivatives of PREG and DHEA in rodent brain could explain the variations observed for PREG-S and DHEA-S measurements with direct (24Higashi T. Daifu Y. Shimada K. Studies on neurosteroids. XIV. Levels of dehydroepiandrosterone sulfate in rat brain and serum determined with newly developed enzyme-linked immunosorbent assay.Steroids. 2001; 66: 865-874Crossref PubMed Scopus (19) Google Scholar, 25Higashi T. Daifu Y. Ikeshima T. Yagi T. Shimada K. Studies on neurosteroids. XV. Development of enzyme-linked immunosorbent assay for examining whether pregnenolone sulfate is a veritable neurosteroid.J. Pharm. Biomed. Anal. 2003; 30: 1907-1917Crossref PubMed Scopus (32) Google Scholar, 26Higashi T. Sugitani H. Yagi T. Shimada K. Studies on neurosteroids. XVI. Levels of pregnenolone sulfate in rat brains determined by enzyme-linked immunosorbent assay not requiring solvolysis.Biol. Pharm. Bull. 2003; 26: 709-711Crossref PubMed Scopus (39) Google Scholar, 27Liu S. Sjovall J. Griffiths W.J. Neurosteroids in rat brain: extraction, isolation, and analysis by nanoscale liquid chromatography-electrospray mass spectrometry.Anal. Chem. 2003; 75: 5835-5846Crossref PubMed Scopus (124) Google Scholar) and indirect (1Liere P. Akwa Y. Weill-Engerer S. Eychenne B. Pianos A. Robel P. Sjovall J. Schumacher M. Baulieu E.E. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 2000; 739: 301-312Crossref PubMed Scopus (148) Google Scholar, 19Lanthier A. Patwardhan V.V. Sex steroids and 5-en-3 beta-hydroxysteroids in specific regions of the human brain and cranial nerves.J. Steroid Biochem. 1986; 25: 445-449Crossref PubMed Scopus (169) Google Scholar, 20Corpéchot C. Robel P. Axelson M. Sjovall J. Baulieu E.E. Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.Proc. Natl. Acad. Sci. USA. 1981; 78: 4704-4707Crossref PubMed Scopus (601) Google Scholar, 21Corpéchot C. Synguelakis M. Talha S. Axelson M. Sjovall J. Vihko R. Baulieu E.E. Robel P. Pregnenolone and its sulfate ester in the rat brain.Brain Res. 1983; 270: 119-125Crossref PubMed Scopus (354) Google Scholar, 22Young J. Corpéchot C. Perché F. Haug M. Baulieu E.E. Robel P. Neurosteroids: pharmacological effects of a 3beta-hydroxy-steroid dehydrogenase inhibitor.Endocrine. 1994; 2: 505-509Google Scholar) methods of analysis. The chemical identities of these lipoidal derivatives still need to be elucidated. 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. PREG, DHEA, PREG-S (potassium salt), and DHEA-S (sodium salt) were obtained from Roussel-Uclaf (Romainville, France), and tetradeuterated [2H4]PREG-S ([17,21,21,21-2H4]PREG-S; trimethylammonium salt) was synthesized by Dr. R. Purdy (The Scripps Research Institute, La Jolla, CA). The nonpolar steroids PREG palmitate, DHEA stearate, and DHEA acetate were purchased from Sigma (Saint-Louis, MO), and PREG tosylate was supplied by Steraloids (Newport, RI). The derivatization reagents heptafluorobutyric anhydride (HFBA), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), and methoxyamine hydrochloride were provided by Pierce (Rockford, IL), and TEA was supplied by Sigma. All other reagents and solvents were of analytical grade. The extraction procedure is roughly similar to that reported in our previous paper (1Liere P. Akwa Y. Weill-Engerer S. Eychenne B. Pianos A. Robel P. Sjovall J. Schumacher M. Baulieu E.E. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 2000; 739: 301-312Crossref PubMed Scopus (148) Google Scholar). The entire brains (∼2 g) of 2 month old male Sprague-Dawley rats [Centre d'Elevage R. Janvier, (CERJ), Le Genest St-Isle] were removed and weighed. Rat blood was sampled in heparinized polypropylene Falcon tubes through a funnel containing a heparin-treated gauze filter. Blood was centrifuged at 3,000 g for 10 min at 4°C, and the plasma was collected. 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. The supernatants were collected, and the residues were extracted with 10 volumes (w/v) of MeOH/CHCl3 (1:1, v/v) and centrifuged again. The proportions of solvent in all solvent mixtures are given as volume proportions throughout the article. The supernatants were combined, and aliquots corresponding to 100 mg of rat brain, unless otherwise stated, were analyzed. The same protocol was used for analysis of 1 ml of plasma. One hundred milligram aliquots of the anterior brain and the entire hippocampus (144.5 ± 13.0 mg; n = 5), liver (340.8 ± 77.4 mg; n = 5), and adrenal glands (38.9 ± 6.2 mg; n = 5) were also used in this study. 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 also applied in the analysis of PREG and DHEA conjugates in 150 mg of whole brain from 2 month old Swiss male mice. One hundred milligram aliquots from the cerebellum of a 91 year old man and from the frontal cortex of an 88 year old woman were also analyzed. These patients were hospitalized and died in a geriatric unit (Service de Gérontologie, 3 Hôpital Emile Roux, Limeil-Brévannes, France), and the postmortem delay before dissection was less than 24 h. These brain tissues were obtained from a previous study concerning neurosteroid quantification in human brain in Alzheimer's and nondemented patients (2Weill-Engerer S. David J.P. Sazdovitch V. Liere P. Eychenne B. Pianos A. Schumacher M. Delacourte A. Baulieu E.E. Akwa Y. Neurosteroid quantification in human brain regions: comparison between Alzheimer's and nondemented patients.J. Clin. Endocrinol. Metab. 2002; 87: 5138-5143Crossref PubMed Scopus (287) Google Scholar). These investigations were in agreement with French legislation (the French bioethic Huriet law from 1994) requiring the explicit consent of patients or the family for medical research purposes. A blood sample from a healthy adult man (50 years old) was analyzed as a positive control for validation of the steroid sulfate methodology. A protocol derived from the techniques used in our laboratory for RIA analysis in the period 1980–1995 (21Corpéchot C. Synguelakis M. Talha S. Axelson M. Sjovall J. Vihko R. Baulieu E.E. Robel P. Pregnenolone and its sulfate ester in the rat brain.Brain Res. 1983; 270: 119-125Crossref PubMed Scopus (354) Google Scholar, 22Young J. Corpéchot C. Perché F. Haug M. Baulieu E.E. Robel P. Neurosteroids: pharmacological effects of a 3beta-hydroxy-steroid dehydrogenase inhibitor.Endocrine. 1994; 2: 505-509Google Scholar, 28Corpechot C. Young J. Calvel M. Wehrey C. Veltz J.N. Touyer G. Mouren M. Prasad V.V.K. Banner C. Sjövall J. Baulieu E.E. Robel P. Neurosteroids: 3 alpha-hydroxy-5 alpha-pregnan-20-one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats.Endocrinology. 1993; 133: 1003-1009Crossref PubMed Scopus (0) Google Scholar, 29Young J. Corpechot C. Haug M. Gobaille S. Baulieu E.E. Robel P. Suppressive effects of dehydroepiandrosterone and 3 beta-methyl-androst-5-en-17-one on attack towards lactating female intruders by castrated male mice. II. Brain neurosteroids.Biochem. Biophys. Res. Commun. 1991; 174: 892-897Crossref PubMed Scopus (65) Google Scholar) was used for comparison with the standard method with regard to the detection of PREG-S and DHEA-S in rat brain. The halves of rat anterior brain were dissected and weighed (∼650 mg). One-half was extracted by the standard method, as described above, and the other was homogenized in 1 ml of PBS buffer (10 mM, pH 7.4) containing 0.8% NaCl in a Teflon/glass homogenizer at 4°C. [2H4]PREG-S and 2 volumes of ethyl acetate-isooctane (1:1) were added. The mixture was left overnight at room temperature and then centrifuged at 3,000 g for 5 min. The organic phase was removed and the extraction was repeated twice. The aqueous phase, containing steroid sulfates, was taken to dryness, and the residue was dissolved in 1 ml of MeOH. These experiments were performed with a homogenate of rat brain in a Tris hypotonic buffer (pH 7.4). Aliquots of ∼300 mg of homogenate were incubated with [3H]PREG for 1, 4, and 48 h at 37°C. Control experiments were carried out by incubating [3H]PREG with a boiled homogenate for 4 h and in the absence of rat brain homogenate. Analogous experiments were carried out by adding tritiated PREG and PREG-S to a methanolic extract of rat brain for 0 and 24 h. The first purification and fractionation step was achieved by SPE on C18 columns (500 mg, 6 ml; International Sorbent Technology, Mid Glamorgan, UK). The protocol has been changed relative to that described in our previous paper (1Liere P. Akwa Y. Weill-Engerer S. Eychenne B. Pianos A. Robel P. Sjovall J. Schumacher M. Baulieu E.E. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 2000; 739: 301-312Crossref PubMed Scopus (148) Google Scholar) for several reasons explained below. This procedure was the same as that described in our recent paper (1Liere P. Akwa Y. Weill-Engerer S. Eychenne B. Pianos A. Robel P. Sjovall J. Schumacher M. Baulieu E.E. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 2000; 739: 301-312Crossref PubMed Scopus (148) Google Scholar) except that a washing step with 5 ml of water was introduced before the elution of sulfated and free steroids with 5 ml each of MeOH/water (1:1) and MeOH/water (9:1), respectively. In this system, the C18 cartridge was conditioned with 5 ml of MeOH, 5 ml of water, and 5 ml of MeOH. The brain extract in MeOH was applied to the cartridge, which was then washed with 5 ml of MeOH. The flow-through and the washing were combined and diluted with water to MeOH/water (8:2) and again applied to the same cartridge. Then, the flow-through was diluted to MeOH/water (6:4) and reapplied to the cartridge, and the same process was repeated with MeOH/water (4:6), MeOH/water (2:8), and finally water. This sequence of adsorptions was defined as a sample recycling by SPE. The second part of the procedure was the stepwise recovery of steroids by eluting the C18 cartridge with increasing concentrations of MeOH in water from 0% to 100% in 5% increments. A simplified recycling/elution SPE protocol was established for quantifying sulfated, unconjugated, and lipoidal PREG and DHEA (Fig. 1). Samples were dissolved in 1 ml of MeOH, applied to the cartridge, and 5 ml of MeOH/water (85:15) was added. The flow-through was collected and dried. The column was reconditioned with 5 ml of water, and samples were dissolved in MeOH/water (2:8) and applied to the cartridge. After a preliminary wash with 5 ml of water, sulfated, free, and lipoidal steroids were eluted with 5 ml of MeOH/water (1:1), 5 ml of MeOH/water (9:1), and 5 ml of MeOH/CHCl3 (1:1), respectively. In this paper, lipoidal PREG and DHEA (L-PREG and L-DHEA, respectively) designate derivatives of unknown structure eluted in the MeOH/CHCl3 (1:1) fraction. Fatty acid esters of PREG and DHEA were also eluted in the same fraction. The recovery of L-PREG and L-DHEA was increased by additional elution with 5 ml of hexane. Another SPE procedure was used for the purification of steroid esters as proposed by Aguilera et al. (30Aguilera R. Catlin D.H. Becchi M. Phillips A. Wang C. Swerdloff R.S. Pope H.G. Hatton C.K. Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols.J. Chromatogr. B Biomed. Sci. Appl. 1999; 727: 95-105Crossref PubMed Scopus (95) Google Scholar). The heptafluorobutyrate derivatives prepared from the free and lipoidal fractions were dissolved in 1 ml of CH3CN/water (1:1). This solution was applied to a C18 cartridge (International Sorbent Technology; 500 mg) previously conditioned with 5 ml of CH3CN, 5 ml of water, and 5 ml of CH3CN/water (1:1), and PREG and DHEA derivatives were eluted with 6 ml of CH3CN after a washing step with 4 ml of CH3CN/water (1:1). For this purpose, the lipoidal fraction obtained after SPE fractionation was dissolved in different solvents for 30 min or 24 h at different temperatures and in the presence or absence of light. Then, the samples were dried and taken up in 1 ml of MeOH for a second C18 SPE fractionation. The sulfated, unconjugated, and lipoidal fractions were collected as described above. As shown in Fig. 1, sulfated, unconjugated, and lipoidal steroids were derivatized separately. Simultaneous hydrolysis/derivatization of PREG-S and DHEA-S with HFBA was investigated. Touchstone and Dobbins (31Touchstone J.C. Dobbins M.F. Direct determination of steroidal sulfates.J. Steroid Biochem. 1975; 6: 1389-1392Crossref PubMed Scopus (16) Google Scholar) reported that some sulfated steroids can react with HFBA for their conversion into heptafluorobutyrates suitable for GC-MS analysis, and Murray and Baillie (32Murray S. Baillie T.A. Direct derivatization of sulphate esters for analysis by gas chromatography mass spectrometry.Biomed. Mass Spectrom. 1979; 6: 82-89Crossref Scopus (28) Google Scholar) showed that this single-step reaction was quantitative only in the case of aromatic sulfated steroids and steroids with a 3β-sulfoxy-Δ5 structure, such as PREG-S and DHEA-S. The potential usefulness of this reaction was of interest because of its simplicity and the selectivity of the hydrolysis/acylation reaction. The reaction mechanism proposed by Murray and Baillie (32Murray S. Baillie T.A. Direct derivatization of sulphate esters for analysis by gas chromatography mass spectrometry.Biomed. Mass Spectrom. 1979; 6: 82-89Crossref Scopus (28) Google Scholar) is schematized in Fig. 2. The MeOH/water (1:1) and MeOH/water (9:1) fractions from the SPE containing sulfated and free steroids, respectively, were dissolved in 100 μl of acetone. Twenty microliters of HFBA was added, and the samples were allowed to stand at room temperature for 30 min. A subsequent C18 SPE purification step with the CH3CN/water (1:1) system was used only for unconjugated steroids. The MeOH/CHCl3 (1:1) fraction from the SPE containing lipoidal steroids was derivatized with HFBA or TEA/HFBA. The reaction with HFBA was performed as described above for sulfated and free steroids except that the temperature was kept at 70°C. Concerning the reaction with TEA/HFBA, the residue was dissolved in 100 μl of acetone, 20 μl of TEA and 20 μl of HFBA were added, and the mixture was heated for 30 min at 70°C. This reaction has been optimized according to several parameters, such as the relative amount of reagent, reaction duration, and temperature. A liquid-liquid partition step between water and hexane was performed. The organic phase was removed, and the extraction was repeated. The combined organic extracts were dried under nitrogen at 70°C. The heptafluorobutyrates obtained in both the HFBA and TEA/HFBA reactions were purified by C18 SPE with the CH3CN/water (1:1) system described above. Two other derivatization reagents were used for analyzing L-PREG and L-DHEA by GC-MS. Fifty microliters of MSTFA or 100 μl of methoxyamine hydrochloride (2% in pyridine) was added to the lipoidal fraction at 70°C for 30 min. The reaction product with MSTFA [i.e., trimethylsilyl ether of PREG (PREG-TMS)] was purified with SPE using the CH3CN/water (1:1) system, whereas the PREG-20-methoxime (PREG-20-MO) synthesized with methoxyamine hydrochloride was submitted to SPE with simplified recycling and fractionation. In the latter case, MeOH/water (9:1) and MeOH/CHCl3 (1:1) fractions were collected, and 20 μl of HFBA and 100 μl of acetone were added to these fractions for 30 min at 70°C to form the 3-heptafluorobutyrate-20-methoxime of PREG (PREG-3-HFB-20-MO). In some experiments, PREG palmitate, DHEA stearate (10 ng each), and 100 ng of PREG tosylate were added" @default.
• W2121753150 created "2016-06-24" @default.
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• W2121753150 date "2004-12-01" @default.
• W2121753150 modified "2023-10-18" @default.
• W2121753150 title "Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain" @default.
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• W2121753150 doi "https://doi.org/10.1194/jlr.m400244-jlr200" @default.
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