Identification of Neuroactive Steroids and Their Precursors and Metabolites in Adult Male Rat Brain
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《内分泌学杂志》
Department of Physiology (M.J.E., D.I.C., H.H., J.P.F.), University College London, London WC1E 6BT, United Kingdom
SAS Laboratory (J.W.H.), Clinical Biochemistry, University College London Hospitals, London W1T 4EU, United Kingdom
Abstract
Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3,5-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3,5-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5-dihydroprogesterone. Furthermore, the presence of 3-, 11-, 17-, and 20-hydroxylated metabolites of 3,5-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20-reduced metabolites could also be found for pregnenolone, progesterone, and 5-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3,5-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.
Introduction
STEROID HORMONES HAVE long been known to enter the mammalian nervous system to influence its development and function. More recently, some steroids have also been shown to be present in brain tissue independently of peripheral sources (1, 2, 3), the so-called neurosteroids (4). In support of this concept, mRNA and protein have been detected in rodent brain for the steroidogenic cholesterol P450 side chain cleavage (P450scc or CYP11A1) enzyme and for certain other steroid-metabolizing enzymes (see Refs.5 and 6 and Discussion). However, there is a paucity of information on the actual steroid content of mammalian nervous tissue. Such information is essential for a proper understanding of the regulation and function of steroids in the brain.
The first compounds to be defined as neurosteroids were pregnenolone and dehydroepiandrosterone (DHEA), which had been detected in adult male rat brain as both the free steroids and their 3-sulfate esters (1, 2). Both the free and sulfated forms of these steroids persisted at normal or only slightly reduced concentrations after adrenalectomy and gonadectomy, suggesting synthesis within the brain. Comparable evidence has since been provided for progesterone and its reduced metabolites 5-dihydroprogesterone and 3,5-tetrahydroprogesterone (allopregnanolone), although there appears to be more of a contribution from the gonads in the female rat (3, 7). Other steroids measured in adult mammalian brain include estradiol and testosterone (8, 9), aldosterone, corticosterone, and 3,5-tetrahydrodeoxycorticosterone (10, 11, 12). In the male rat brain, testosterone, corticosterone, aldosterone, and 3,5-tetrahydrodeoxycorticosterone appear to arise from endocrine sources (1, 11, 12).
Steroids in the brain are of profound physiological significance. There are well-established sites of action for estradiol, testosterone, progesterone, corticosterone, and aldosterone through transcription factors in the mammalian brain (13, 14). More rapid, nongenomic effects of steroids include those of the 3,5-reduced metabolites of progesterone and deoxycorticosterone, which are well known to modulate synaptic and extrasynaptic inhibition by acting at nanomolar concentrations as enhancers of -aminobutyric acid (GABA) at GABAA receptors (15, 16). By contrast and at lower (micromolar) potencies, pregnenolone sulfate and DHEA sulfate negatively modulate GABAA receptor function, and pregnenolone sulfate also appears to enhance excitatory glutamate actions at the N-methyl-D-aspartate receptor. Other rapid and relatively low-potency effects of brain steroids on neuronal membrane receptors include negative modulation by progesterone of the nicotinic acetylcholine receptor (17) and the strychnine-sensitive glycine receptor and positive modulation of -receptors by DHEA and DHEA sulfate and of voltage-gated K+ and Ca2+ channels by pregnenolone sulfate (18, 19). Moreover, there is increasing evidence for interactions between transcription factor-mediated and nongenomic actions of steroids on the brain (see Refs.19, 20, 21, 22, 23).
Despite the longstanding interest in steroid effects on the mammalian brain, there has to our knowledge been no detailed profiling of the steroid metabolites present in this tissue. This information could provide a foundation for identifying not only the origins of neuroactive steroids but also pathways for their inactivation in the brain. To a large extent, the lack of this information reflects technical difficulties. Concentrations of the above steroids have mostly been measured in brain tissue by immunoassay, which is generally accepted as the most sensitive method for the estimation of these compounds. However, problems with cross-reactivity of antibodies, especially at the low concentrations of steroid present and with interference from the lipidic, cholesterol-rich tissue matrix, mean that brain extracts require fractionation by solvent partitioning and/or liquid chromatography before any confidence can be placed on the steroid measurements (24, 25). In some cases, additional evidence of brain steroid identity has been obtained by gas capillary chromatography-mass spectrometry (GC-MS). With this technique, as for immunoassay, the steroids in brain extracts require fractionation before analysis and in addition need to be stabilized by derivatization before injection onto the GC. However, GC-MS has the advantage that steroid derivatives can chemically be identified, both by their retention time on the chromatograph and also by their ion fragments in the mass spectrometer. Thus, pregnenolone, DHEA, testosterone, progesterone, 5-dihydroprogesterone, and 3,5- and 3,5-tetrahydroprogesterone have all been characterized in rat brain extracts by the appearance of at least two and sometimes more, diagnostic ions at the appropriate retention time on GC-MS (7, 8, 26, 27, 28, 29, 30). The sulfate esters of pregnenolone and DHEA were also identified by GC-MS as their corresponding free steroids after solvolysis of brain extracts (1, 2, 26).
Whether estimating brain steroids by immunoassay or by GC-MS, the above studies have each only focused on a few compounds at a time and revealed several discrepancies. Moreover, studies of steroidogenic enzyme expression suggest that additional neuroactive metabolites could be produced in rat brain and/or enter from peripheral sources (see Discussion). To provide a framework in which the physiological actions of neurosteroids can be understood, and to facilitate additional investigations on the regulation of steroid metabolism in the brain and the ways in which these pathways might interact with peripheral sources of steroid, we now report a detailed analysis of steroids in adult male rat brain, using GC-MS and applying the identification criteria of retention time and diagnostic ion ratios at a high level of stringency. The steroids to be screened were chosen from products of enzymes known to be expressed in mammalian central nervous system and peripheral endocrine sources.
Materials and Methods
Chemicals
Reference steroids were obtained from Sigma-Aldrich (Dorset, UK) or Steraloids, Inc. (Newport, RI). [3H]Progesterone (5106.0 GBq/mmol) was from PerkinElmer LAS Ltd. (Buckinghamshire, UK). All chemicals were analytical grade from VWR International (Leicestershire, UK) or Sigma-Aldrich, unless stated otherwise, and the solvents were redistilled before use. Water was double glass-distilled, and silanized glassware was used throughout. Of the derivatization reagents for GC-MS, methoxyamine (MO) hydrochloride and heptafluorobutyric acid anhydride (HFBA) were from Sigma-Aldrich, whereas trimethylsilylimidazole (TMSI) and hexamethyldisilazane (HMDS) came from Pierce (Perbio Science Ltd., Cheshire, UK). Lipidex 5000 gel (PerkinElmer) was washed before use with cyclohexane and then stored in cyclohexane/HMDS/pyridine 98:1:1 (vol/vol/vol). Oasis hydrophilic-lipophilic balance (HLB) (5 ml, 200 mg) and mixed-mode anion exchange (3 ml, 60 mg) solid-phase extraction cartridges were obtained from Waters Corp. (Milford, MA). Ecoscint H (National Diagnostics, Yorkshire, UK) was used for scintillation counting.
Brain samples
Adult male Sprague Dawley rats (250–450 g) from the breeding colony at Biological Services, University College London, were kept in a 12-h lighting regimen (lights on at 0800 h) and fed rat chow and water ad libitum. For experiments to test the extraction of endogenous [3H]progesterone and its metabolites from brain tissue, rats were killed by CO2 inhalation. All other rats were killed between 1100 and 1500 h by cervical dislocation. The whole brain (including cerebellum but excluding olfactory bulbs) was rapidly removed and stripped of meninges then stored at –70 C until extraction. All animal procedures were given local ethical committee approval and performed under license from the UK Government Home Office.
Tissue extraction and fractionation
Pooled tissue samples from two to four rats were homogenized in 5 vol ice-cold potassium phosphate buffer (5 mM, pH 7) with a Polytron blender. A 0.5-ml portion of the homogenate was removed to determine blood contamination of the brain samples as described below and stored at –20 C. The remaining homogenate was added dropwise to 20 vol acetic acid (3%, vol/vol) in 96% ethanol in polypropylene tubes in an ultrasonicating bath. After additional sonication for 10 min followed by incubation at –20 C overnight, the extracts were sonicated on ice with an MSE Soniprep probe. After another 30 min on ice, extracts were centrifuged at 28,000 x g for 30 min at 25 C. Lipid was removed from the supernatants by partitioning three times against 10 vol isooctane (previously saturated with 77% ethanol in potassium phosphate buffer (5 mM, pH 7) plus acetic acid (2.4%, vol/vol). After drying down under vacuum, the extracts were resuspended in 4 vol of 60% ethanol (in H2O, vol/vol) and then centrifuged at 1000 x g for 12 min, and the supernatant (corresponding to up to 8 g of original tissue) was passed through a 200-mg reverse-phase Oasis HLB cartridge for the additional removal of lipidic material. An additional 4.4 vol of 60% ethanol in potassium phosphate buffer (5 mM, pH 7, vol/vol) were passed through the cartridge to ensure elution of steroids, and the combined eluate was dried down under vacuum. For separation of free and conjugated steroids, the extracts were then redissolved in 3.75 vol of 20% ethanol in potassium phosphate buffer and passed through 60-mg reverse-phase Oasis mixed-mode anion exchange cartridges. After a wash with 5 ml of 20% ethanol in ammonium acetate buffer (20 mM, pH 7, vol/vol), free steroids were eluted in 4 ml ethyl acetate. Any steroid glucuronides present could then be eluted with 20 ml of 60% ethanol in formate/pyridine buffer (20 mM, pH 3, vol/vol). Finally, after a wash with 2 ml ethyl acetate (dried over Na2SO4), the steroid sulfates were eluted in 15 ml of 50 mM benzene sulfonic acid in ethyl acetate saturated with 2 M H2SO4. The free steroid eluates were dried under nitrogen and derivatized as described below. Steroid sulfate fractions were solvolysed before derivatization by incubation at 40 C in the acidified ethyl acetate eluant for 16 h in the presence of Na2SO4. After neutralization with pyridine, ethyl acetate was evaporated from these solvolysed fractions and the residue extracted three times with 2 ml ether. The combined ether phase was dried down for derivatization by MO-TMSI or HFBA. If the latter, the ether phase was washed beforehand three times with 1 ml H2O. Reagent blank samples were carried alongside each tissue sample throughout the above procedures.
Extraction efficiency of ethanol/acetic acid for brain steroids was estimated after ip injection of two rats with 0.7 MBq/kg [3H]progesterone in PBS (5 ml/kg). These rats were killed after 2.5–3 h by CO2 inhalation and their brains quickly removed before storage at –70 C. As described above, brains were homogenized in 5 vol ice-cold potassium phosphate buffer (5 mM, pH 7). The total brain radioactivity was then estimated by removing portions of homogenate for solubilization in 3 vol Soluene 350. These solubilized samples were then bleached with hydrogen peroxide (final concentration of 1%, wt/vol) for 2 h at 50 C before counting in Ecoscint H containing Triton X-100 (6%, vol/vol), glacial acetic acid (0.6%, vol/vol), and butylated hydroxytoluene (2%, wt/vol). The remaining homogenate was extracted into ethanol/acetic acid as already described. Radioactivity contained in these brain extracts was found to be 88.2% of total brain radioactivity.
Blood contamination of the brain samples was estimated by the spectrophotometric determination of hemoglobin. Portions (0.5 ml) of brain homogenates that had been withheld from steroid extraction and stored at –20 C were thawed and centrifuged (28,000 x g for 30 min at 4 C). Hemoglobin was then measured in these supernatants by the change in absorbance between 560 and 578 nm upon reduction by the addition of sodium dithionite (to a final concentration of 10 mM). Changes in absorbance were calibrated with respect to hemoglobin standards (Sigma-Aldrich) and the blood content of the original brain samples then calculated to be 1.26 ± 0.10% (vol/vol; mean ± SEM, n = 12), assuming a hemoglobin concentration in rat blood of 157 g/liter (31).
Sample derivatization
For samples to be derivatized with MO and TMSI, the internal standards 16-dehydropregnenolone, 6-methyl-17-hydroxyprogesterone, and prednisolone were added beforehand at 50–100 ng. These samples were then dried under nitrogen and redissolved in 200 μl MO in pyridine (2%, wt/vol). In the case of the steroid sulfate fractions, improved yields were obtained if samples were reduced in volume to approximately 50 μl after dissolving in MO/pyridine (32). Both steroid sulfates and free steroids were incubated with MO/pyridine at 60 C for 1 h. The TMSI was then added at 100 μl and the mixture heated another 3 h at 100 C. At the end of this derivatization, pyridine was evaporated under nitrogen and the residue dissolved in cyclohexane/HMDS/pyridine 98:1:1 (vol/vol/vol) and passed through a Lipidex 5000 gel column (0.5 cm diameter and 8 cm high). After additional elution with 2 ml of the same solvent, the pooled eluates were dried under nitrogen and dissolved in cyclohexane for injection onto the GC.
For samples to be derivatized with HFBA, the internal standard 16-dehydropregnenolone was added at 50 ng. After drying under nitrogen, these samples were then redissolved in 30 μl benzene and 30 μl HFBA for incubation at 60 C for 30 min. After this incubation, samples were dried under nitrogen and the residue redissolved in 1 ml cyclohexane/pyridine 98:2 (vol/vol) for passage through Lipidex 5000 gel columns. Columns were eluted with another 2 ml of the same solvent and to the pooled eluates the internal standards tetracosane and octacosane were then added at 25 ng each. Finally, the samples were dried under nitrogen and dissolved in cyclohexane for injection into the GC.
GC-MS analysis
All analyses were carried out on a Shimadzu 17A GC coupled to a QP 5050A MS (Shimadzu, Milton Keynes, UK), equipped with autosampler AOC-20s. The system was controlled and data processed by the Shimadzu Class 5000 software. A 30-m-long Zebron ZB1 wall coated open tubular column (Phenomenex, Macclesfield, UK) with 0.25 mm inner diameter and 0.25 μm film thickness was used for GC with helium as the carrier gas at a constant flow rate of 0.7 ml/min. All analyses were performed in the splitless mode, and the injector purge valve was opened after 4 or 2 min for analysis of MO-TMS or HFB derivatives, respectively. For the analysis of MO-TMS derivatives, the injector temperature was kept constant at 280 C and the pressure at 400 kPa for 5 min. Thereafter the pressure was decreased to 34.2 kPa, followed by a rise of 6.5 kPa/min to 81 kPa. After 0.33 min at this pressure, the gradient was set at 1.6 kPa/min to a final pressure of 111.7 kPa, which was held for 4 min. The oven temperature was at 70 C for 5 min and then rose at 20 C/min to 220 C. After 0.33 min, the gradient was 5 C/min to 315 C, which was held for 4 min. The interface temperature was constant at 315 C. For analysis of HFB derivatives, the injector temperature was constant at 250 C and the pressure at 400 kPa for 2 min. Thereafter, the pressure was decreased to 34.2 kPa followed by a rise of 5.9 kPa/min to 79 kPa. After 0.33 min at this pressure, the gradient was set at 1.5 kPa/min to 98 kPa, which was held for 5 min. Oven temperature was at 70 C for 2 min followed by a rise of 20 C/min to 220 C and then, after 0.33 min, a rise of 5 C/min to 285 C, which was held for 5 min. The interface temperature was constant at 285 C. Samples were ionized by electron impact ionization with an energy of 70 eV. The detector voltage was at 1.7 kV. For initial characterization, retention indices after Kovats (33) were determined for derivatized reference steroids according to the following equation:
where N is the number of carbon atoms in the alkane eluting before the compound of interest, n the increment in number of carbon atoms from this alkane eluting to the one eluting after the compound of interest, t(A) the retention time (min) of the compound of interest, t(N) the retention time of the alkane eluting before the compound of interest and t(N + n) the retention time of the alkane eluting after the compound of interest. For highest possible sensitivity, the MS was run in selected ion monitoring (SIM) mode. In two-ion SIM of MO-TMS derivatives, pairs of target and qualifier ions for each steroid were monitored in groups of four to six ions at a time such that potentially overlapping pairs did not coincide. The ions were monitored in four different injections with the detector settings changed according to retention index as indicated in Table 1.
For confirmation of compound identities, three ions were monitored for MO-TMS derivatives or two ions for HFB derivatives in groups of three to six ions. These ions are shown in Table 2.
Analysis of results
Peaks were integrated manually, and retention time and integration data were additionally processed in Microsoft Excel. For identification, qualifier to target ion ratios (Q/T) were calculated from their areas. Compounds had to meet target values of Q/T of reference compounds run alongside tissue samples within 0.67 and 1.5 (±20% of relative abundance of target and qualifier ions). Further identification was obtained from relative retention times (RRT). These RRTs were calculated as the ratio of retention times of analyte and the closest of one of three internal standards. For positive identification in SIM, RRTs of analytes had to lie within ±0.5% of the RRTs of reference compounds run alongside tissue samples.
For calibration, increasing amounts of reference steroids (0.5–10 ng) were run together with fixed amounts (25–100 ng) of internal standards. The ratios of reference to standard target ion areas could then be plotted against amount ratios. Regression lines were fitted to these plots, which were linear up to 10 ng. Endogenous brain steroids could then be quantified by using the area ratio of their target ions to those of the closest internal standard.
Accuracy of this quantitation was examined by assaying mixtures of standards at 0.5, 1.6, 4.0, and 10.0 ng injected mass per steroid (results shown only for 0.5 ng). The amount of each steroid in each sample was determined and the percent accuracy calculated as the amount measured over the calculated amount. The intra- and interassay reproducibilities were calculated from the assays carried out for determination of accuracy. The percent coefficients of variation (CV) were calculated from the SDs and means within and between assays (results shown only for 0.5 ng).
Detection limits of the overall extraction and assay procedure were determined for brain steroids from area ratios of peaks in reagent blanks extracted and assayed alongside the brain samples. For detection, area ratios had to be three times those of peaks at the same RRT in the corresponding extraction blank. The concentrations at those three-times blank values were calculated using the calibration curves. Detection limits shown in Table 3 were the minimal values found from all extracts.
Results
Two-ion SIM of steroids in rat brain
The first stage of the present investigation was to screen for a wide range of compounds in both the free steroid and sulfate conjugate fractions of rat brain extracts. For this purpose, we used gas capillary chromatography-electron impact mass spectrometry (GC-EIMS) in two-ion SIM mode after derivatization of these fractions with MO and TMSI. The steroids for which we screened are listed in Table 1. Pure standards of these steroids were first used to adjust sample injection, temperature, and pressure conditions of the gas chromatograph for their optimum resolution as MO-TMS derivatives (see Materials and Methods). The MO-TMS derivatives of each steroid were then individually analyzed for their mass spectrometric behavior. An example of a mass spectrum is shown in Fig. 1 for MO-TMS-pregnenolone. Diagnostic ions for each compound were chosen from their mass spectra according to high relative abundance and selectivity, excluding those that might originate from overlapping elutions of steroid derivatives. Choice of these diagnostic ions then allowed SIM for endogenous brain steroids at increased sensitivity. Usually the ion of higher relative abundance was chosen as the target (T) ion for quantitation and the second ion as a qualifier (Q) to aid in identification. These ions are also listed for the nonbiological steroids that were used in the present study as internal standards for quantitation. To maintain high analytical sensitivity during SIM, the T and Q ions were monitored in groups of no more than six and changed at set times during the elution. To monitor all T and Q ions of the MO-TMS derivatives of the compounds listed in Table 1, four separate injections were performed for each sample and altogether four different brain extracts screened in this way. This initial screen tentatively identified 32 free steroids and 23 steroid sulfates in the rat brain extracts, as indicated in Table 1. Also shown are the accuracy and intra- and interassay reproducibilities determined at the 0.5-ng level for all compounds in this screening procedure.
Confirmation and quantitation of brain steroids
Confirmation of the identities of the compounds revealed in the above two-ion SIM screening was attempted using three-ion SIM of their MO-TMS derivatives and/or two-ion SIM of their HFB derivatives. The additional ions for the former derivatives were chosen following the same principles as for the two-ion SIM methods. Likewise, specific ions were identified for two-ion SIM of the HFB derivatives. Figures 2 and 3 illustrate typical chromatograms obtained from three-ion SIM of MO-TMS derivatives and from two-ion SIM of HFB derivatives, respectively. In both Figs. 2 and 3, the trace from a brain extract is shown below that of the appropriate reference steroid. Examples are shown not only for identified brain steroids but also for some of those steroids that were not confirmed as present in the brain extracts.
Table 2 lists all 17 free steroids together with one steroid sulfate, DHEA sulfate, that met the criteria for unequivocal identification in the present study. Table 2 also shows the diagnostic ions for three-ion SIM of MO-TMS derivatives and for two-ion SIM of HFB derivatives for each compound and allows comparison of the RRTs and Q/T ratios of brain analytes with those of standard steroids. Overall definitive identification of an endogenous brain steroid is achieved if coelution of three ions of the compound derivatized with MO and TMSI or of two ions from both the MO-TMS and HFB derivatives occurred within the RRT and Q/T limits of the standard. For such confirmation, the RRTs and Q/T ratios of brain analytes had to be within ±0.5% and 0.67 and 1.5, respectively, of those of standards analyzed on the same day (see Discussion). Confidence limits at the 99.9% level for RRTs and Q/T ratios were also calculated from series of standard samples and found to coincide well with the above pragmatic limits (not shown).
Identified rat brain steroids were quantified using two- or three-ion SIM, and their concentrations are shown in Table 3. Also shown in Table 3 are the recoveries for all of the steroids screened by two-ion SIM in the present study after their addition as pure standards to rat brain homogenates and subsequent extraction, fractionation, and derivatization with MO-TMSI. In addition, for each steroid, we give the minimal detection limits of the overall extraction and fractionation procedure, as determined from reagent blank samples run alongside the tissue samples.
Discussion
The present study has employed ethanolic extraction of steroids from adult male rat brain followed by mixed-mode hydrophobic interaction and anion exchange chromatography to completely separate free steroids and steroid sulfates, before their identification and assay by GC-EIMS. Others have also used methanol or ethanol to extract free and sulfated steroids from brain tissue (12, 30, 34, 35). With the addition of acetic acid as a denaturant, Liere et al. (28) obtained comparable apparent extraction efficiencies for pregnenolone to those reported here for progesterone. As for previous studies, we were unable to estimate extraction efficiencies for steroid sulfates because these undergo desulfation after systemic injection and do not remain in the brain in significant quantities as the original ester (7, 36). However, the polar steroid sulfates are soluble in aqueous ethanol and so should extract more efficiently than the free steroids. Lipoidal steroid conjugates (26) were not analyzed in the present study. These would not extract well into ethanol and in any case should have been removed during the initial clean-up of the extracts by isooctane partitioning and then passage through the HLB cartridges.
After extraction and fractionation, the free steroids and steroid sulfates from rat brain were derivatized for identification and assay by GC-EIMS. Identification was based on comparison with steroid standards and relied on two criteria: RRT (with respect to the nearest internal standard) on the GC and diagnostic ions from the MS. For the former, we adopted the European Commission recommendation (37) of accepting only RRTs that fell within ±0.5% of a reference standard analyzed under the same conditions. As for diagnostic ions in the MS, it was not possible to obtain complete spectra for endogenous brain steroids, and SIM had to be employed to increase sensitivity. An initial screen of rat brain extracts monitoring only two diagnostic ions for each compound indicated the possible presence of 32 free steroids and 23 steroid sulfates. A more rigorous analysis then confirmed 17 free steroids plus one steroid sulfate, DHEA sulfate, as present in adult male rat brain. To be considered as identified, the brain steroid derivatives had to meet not only the ±0.5% RRT criteria but also to have relative ion abundance for three diagnostic ions within ±20% of the standard. Previous evaluations of mass spectral databases have shown such limits on the three-ion criterion alone to provide an unambiguous identification of target compounds (38, 39).
With reference to previous analyses of steroids in nervous tissue (see Introduction), the present results confirm the identification of pregnenolone, progesterone, 5-dihydroprogesterone, 3,5- and 3,5-tetrahydroprogesterone, DHEA, DHEA sulfate, and testosterone in adult male rat brain extracts. The GC-MS analysis reported here has also provided chemical identification for the 3,5-tetrahydrodeoxycorticosterone and corticosterone previously measured by immunoassay in rat brain. Whole-brain concentrations of the above steroids were found to be in the same ranges as given in the previous reports cited here apart from one study (26), which found approximately 10- to 40-fold higher concentrations of pregnenolone, 3,5- and 3,5-tetrahydroprogesterone, and DHEA. In addition to the above steroids, we have also identified 20- and 20-dihydropregnenolone, 20-dihydroprogesterone, 5,20-tetrahydroprogesterone, 5-pregnane-3,20-diol, 5-pregnane-3,17-diol-20-one, and 5-pregnane-3,11-diol-20-one as present in adult male rat brain. None of the steroids identified here has been reported to occur in plasma at concentrations that would contribute significantly to the amounts measured in the present brain extracts, and contamination with blood can be excluded as a source.
In contrast to one previous report that employed two-ion SIM on GC-MS (26), epiandrosterone was not identified in either the free or the sulfated steroid fractions of male rat brain. This steroid was detected in two-ion SIM of the sulfate fraction in the present study but not confirmed with three-ion SIM. The present results also differ from several previous reports (see, for example, Refs.2 , 26 , and 28) in failing to detect pregnenolone sulfate in rat brain. These previous studies have employed solvent phase partitioning and/or hydrophobic interaction chromatography to separate free and sulfated steroids and relied on indirect measurements of pregnenolone sulfate in which the ester is solvolysed to yield pregnenolone for identification and assay. In retrospect, these earlier measurements of pregnenolone sulfate seem likely to have assayed contaminating free steroid or another solvolysable, possibly lipoidal, conjugate from the original extract (40). Direct immunoassay of pregnenolone sulfate in adult male rat brain reported this conjugate at less than 0.4 ng/g adult male rat brain (41) and direct liquid chromatography-MS failed to detect it at a limit of 0.3 ng/g tissue (35). The present study relied on the solvolysis of sulfate esters but employed previous anion exchange chromatography for the complete separation of free steroids from steroid sulfates and failed to detect pregnenolone sulfate at a limit of 0.05 ng/g brain (uncorrected for procedural losses). This is consistent with recent functional studies in rat hippocampus that suggest a role for pregnenolone sulfate as a retrograde synaptic messenger, but only during development and not older than postnatal d 5 (42). Nevertheless and in contrast to the lack of pregnenolone sulfate in adult male rat brain, we were able to detect DHEA sulfate in this tissue. The latter was found at concentrations close to those given in the initial characterization of this rat brain steroid (1).
Steroids identified as present in adult male rat brain by the present study are shown in Fig. 4 in relation to the possible metabolic conversions that could give rise to these compounds. The rats used in our study had not been adrenalectomized or gonadectomized, and so endocrine sources cannot be excluded for some of these brain steroids. Indeed, 3,5-tetrahydrodeoxycorticosterone, corticosterone, and testosterone were found in the absence of detectable concentrations of their respective precursors, 5-dihydrodeoxycorticosterone, 11-deoxycorticosterone, and androstenedione, suggesting endocrine origins. Previous studies in adrenalectomized and/or castrated rats have suggested endocrine sources for tetrahydrodeoxycorticosterone, corticosterone, and testosterone in male rat brain (1, 11, 12). For corticosterone, the 11-keto metabolite could not be detected, confirming that the 11-hydroxysteroid dehydrogenase in brain works predominantly as a reductase and so would not attenuate the glucocorticoid receptor actions of this steroid (43). Figure 4 illustrates that DHEA was also found in the present survey in the absence of detectable concentrations of its known precursor in endocrine tissues. In these tissues, 17-hydroxypregnenolone is an intermediate produced by the enzyme CYP17, which catalyzes the conversion of pregnenolone to DHEA and possesses both steroid 17-hydroxylase and 17,20-lyase activities (44). The absence of 17-hydroxypregnenolone in the present extracts is consistent with previous investigations that have failed to detect activity of the CYP17 enzyme in adult mammalian brain (5, 6). Nevertheless and unlike the above corticosteroids and testosterone, DHEA persists in male rat brain after adrenalectomy and castration (1) and is therefore thought to arise within this tissue.
For the remaining steroids identified as present in adult male rat brain and shown in black in Fig. 4, enzymes previously identified in terms of mRNA, protein, and/or activity (5, 6) can account for their presence. Thus, pregnenolone can be converted to 20-dihydropregnenolone or progesterone by the brain enzymes 20-hydroxysteroid dehydrogenase or 3-hydroxysteroid dehydrogenase/isomerase, respectively. It is unclear at present whether the 20-reduced metabolites are formed by a specific 20-hydroxysteroid oxidoreductase or by one of the 3-hydroxysteroid dehydrogenases (6, 43). The latter can also give rise to 20-reduced metabolites, which would account for the presence of 20-dihydropregnenolone, although we were unable to detect 20-dihydroprogesterone. By contrast, the 20-reduced metabolite of progesterone was detected, although only a small proportion of progesterone appears to undergo this conversion in rat brain homogenates in vitro (45).
For progesterone, the major metabolic route in rat brain appears to be 5-reduction to yield 5-dihydroprogesterone, on the pathway to the GABAA receptor-enhancing metabolite 3,5-tetrahydroprogesterone (see Introduction). Consistent with the 5-reductase enzyme being the highest-affinity, rate-limiting step on this pathway (46), we could not detect 3- or 3-dihydroprogesterone. The present study also failed to detect 5-reduced metabolites of progesterone, in agreement with the reported lack of 5-reductase activity in mammalian brain tissue. However, we did find 3,5-tetrahydroprogesterone. Unlike their respective 3,5-reduced compounds, the 3,5-reduced steroids are not active at the GABAA receptor (47). Nevertheless, they would be substrates for the 3-diol hydroxylase enzyme, which hydroxylates at positions 6 or 7 and is found throughout the brain. Competitive inhibition of 3-diol hydroxylase increases the duration of anesthesia induced by 3,5-tetrahydroprogesterone in the rat, and this enzyme has therefore been suggested to provide an inactivation pathway for 3,5-reduced pregnanes and androstanes in the central nervous system (48). The 3,5-tetrahydroprogesterone found in the present study could have arisen through epimerization of 3,5-tetrahydroprogesterone (49) or direct from 5-dihydroprogesterone. The latter would serve as a diversion of substrate from the formation of neuroactive 3,5-tetrahydroprogesterone.
The present results also identify 11-, 17-, and 20-hydroxylation as possible routes for the inactivation of 3,5-tetrahydroprogesterone. The first two metabolites appear to be inactive at the GABAA receptor, whereas the latter has partial agonist properties at the 3,5-tetrahydroprogesterone-modulated site (47, 50). An 11-hydroxylase (CYP11B) is widely expressed in brain and usually denoted as the final step in the production of corticosterone from deoxycorticosterone. However, the lack of evidence in this and previous studies for 21-hydroxylase (CYP21) activity and thus deoxycorticosterone formation in brain suggests that the 11-hydroxylase may actually be more important in this tissue for the inactivation of 3,5-tetrahydroprogesterone. Formation of 5-pregnane-3,17-diol-20-one is harder to explain in view of the apparent lack of CYP17 activity in brain (see above), although 17-hydroxylation could result from variable site specificity of one of the 3-hydroxysteroid dehydrogenases (43) or another as yet unknown mechanism. As mentioned above, 20-dihydroprogesterone was also detected in the present survey. This steroid is known to be a better substrate than progesterone for rat brain 5-reductase (51), which would explain the presence of 5,20-tetrahydroprogesterone. Further reduction to 5-pregnane-3,20-diol may therefore indicate an alternative metabolic route rather than a true inactivation pathway for 3,5-tetrahydroprogesterone. Likewise, the formation of 20-dihydropregnenolone may serve to divert pregnenolone from the formation of neuroactive progesterone and its 3,5-reduced metabolites.
To conclude, the present study has chemically identified several neuroactive free steroids in adult male rat brain. Some of these compounds were found together with precursors, suggesting they could be formed within this tissue, whereas others appear to enter from peripheral sources, although future studies will be necessary to confirm such origins. For 3,5-tetrahydroprogesterone, which is known to enhance the interaction of GABA with the GABAA receptor, 11-, 17-, and 20-hydroxylated metabolites were identified, thereby implicating inactivation pathways for this potent neuromodulatory compound. Our results also confirm the suggestion that epimerization could serve as a first step on the inactivation of 3,5-reduced steroids in brain. In addition, the presence of 20- and 20-reduced pregnenolone indicates a possible regulation on the production of progesterone and 3,5-tetrahydroprogesterone. For the steroid sulfates, only DHEA sulfate was confirmed, and pregnenolone sulfate was not detected at limits below those quoted by previous authors. Despite its reported effects on GABAA and N-methyl-D-aspartate receptors, pregnenolone sulfate is therefore of doubtful physiological significance in the adult central nervous system. Additional studies are now needed to elucidate the regulation of steroid production within the brain and to evaluate how these processes interact with peripheral sources of steroid.
Acknowledgments
We are grateful to E. Conway and R. Hodkinson for help with the GC-MS analyses, to M. Mojet for the hemoglobin measurements, and to M. Farrant for helpful comments on the manuscript.
Footnotes
This study was supported by the European Commission Marie Curie Fellowship Programe, M.J.E. as an individual Training and Mobility of Researchers Fellow and both D.C. and H.H. as Visiting Host Fellows at a UCL Training Site.
Present address for M.J.E.: Procognia Limited, Unit 4, The Switchback, Gardner Road, Maidenhead, Berkshire SL6 7RJ, United Kingdom
Present address for D.C.: Crop Performance Improvement Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom.
Present address for H.H.: Institute of Endocrinology, Národní 8, 116 94, Prague 1, Czech Republic.
First Published Online October 13, 2005
Abbreviations: CV, Coefficient of variation; DHEA, dehydroepiandrosterone; GABA, -aminobutyric acid; GC-EIMS, gas capillary chromatography-electron impact mass spectrometry; GC-MS, GC-mass spectrometry; HFBA, heptafluorobutyric acid anhydride; HLB, hydrophilic-lipophilic balance; HMDS, hexamethyldisilazane; MO, methoxyamine; Q, qualifier; RRT, relative retention time; SIM, selected ion monitoring; T, target; TMSI, trimethylsilylimidazole.
Accepted for publication October 4, 2005.
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SAS Laboratory (J.W.H.), Clinical Biochemistry, University College London Hospitals, London W1T 4EU, United Kingdom
Abstract
Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3,5-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3,5-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5-dihydroprogesterone. Furthermore, the presence of 3-, 11-, 17-, and 20-hydroxylated metabolites of 3,5-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20-reduced metabolites could also be found for pregnenolone, progesterone, and 5-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3,5-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.
Introduction
STEROID HORMONES HAVE long been known to enter the mammalian nervous system to influence its development and function. More recently, some steroids have also been shown to be present in brain tissue independently of peripheral sources (1, 2, 3), the so-called neurosteroids (4). In support of this concept, mRNA and protein have been detected in rodent brain for the steroidogenic cholesterol P450 side chain cleavage (P450scc or CYP11A1) enzyme and for certain other steroid-metabolizing enzymes (see Refs.5 and 6 and Discussion). However, there is a paucity of information on the actual steroid content of mammalian nervous tissue. Such information is essential for a proper understanding of the regulation and function of steroids in the brain.
The first compounds to be defined as neurosteroids were pregnenolone and dehydroepiandrosterone (DHEA), which had been detected in adult male rat brain as both the free steroids and their 3-sulfate esters (1, 2). Both the free and sulfated forms of these steroids persisted at normal or only slightly reduced concentrations after adrenalectomy and gonadectomy, suggesting synthesis within the brain. Comparable evidence has since been provided for progesterone and its reduced metabolites 5-dihydroprogesterone and 3,5-tetrahydroprogesterone (allopregnanolone), although there appears to be more of a contribution from the gonads in the female rat (3, 7). Other steroids measured in adult mammalian brain include estradiol and testosterone (8, 9), aldosterone, corticosterone, and 3,5-tetrahydrodeoxycorticosterone (10, 11, 12). In the male rat brain, testosterone, corticosterone, aldosterone, and 3,5-tetrahydrodeoxycorticosterone appear to arise from endocrine sources (1, 11, 12).
Steroids in the brain are of profound physiological significance. There are well-established sites of action for estradiol, testosterone, progesterone, corticosterone, and aldosterone through transcription factors in the mammalian brain (13, 14). More rapid, nongenomic effects of steroids include those of the 3,5-reduced metabolites of progesterone and deoxycorticosterone, which are well known to modulate synaptic and extrasynaptic inhibition by acting at nanomolar concentrations as enhancers of -aminobutyric acid (GABA) at GABAA receptors (15, 16). By contrast and at lower (micromolar) potencies, pregnenolone sulfate and DHEA sulfate negatively modulate GABAA receptor function, and pregnenolone sulfate also appears to enhance excitatory glutamate actions at the N-methyl-D-aspartate receptor. Other rapid and relatively low-potency effects of brain steroids on neuronal membrane receptors include negative modulation by progesterone of the nicotinic acetylcholine receptor (17) and the strychnine-sensitive glycine receptor and positive modulation of -receptors by DHEA and DHEA sulfate and of voltage-gated K+ and Ca2+ channels by pregnenolone sulfate (18, 19). Moreover, there is increasing evidence for interactions between transcription factor-mediated and nongenomic actions of steroids on the brain (see Refs.19, 20, 21, 22, 23).
Despite the longstanding interest in steroid effects on the mammalian brain, there has to our knowledge been no detailed profiling of the steroid metabolites present in this tissue. This information could provide a foundation for identifying not only the origins of neuroactive steroids but also pathways for their inactivation in the brain. To a large extent, the lack of this information reflects technical difficulties. Concentrations of the above steroids have mostly been measured in brain tissue by immunoassay, which is generally accepted as the most sensitive method for the estimation of these compounds. However, problems with cross-reactivity of antibodies, especially at the low concentrations of steroid present and with interference from the lipidic, cholesterol-rich tissue matrix, mean that brain extracts require fractionation by solvent partitioning and/or liquid chromatography before any confidence can be placed on the steroid measurements (24, 25). In some cases, additional evidence of brain steroid identity has been obtained by gas capillary chromatography-mass spectrometry (GC-MS). With this technique, as for immunoassay, the steroids in brain extracts require fractionation before analysis and in addition need to be stabilized by derivatization before injection onto the GC. However, GC-MS has the advantage that steroid derivatives can chemically be identified, both by their retention time on the chromatograph and also by their ion fragments in the mass spectrometer. Thus, pregnenolone, DHEA, testosterone, progesterone, 5-dihydroprogesterone, and 3,5- and 3,5-tetrahydroprogesterone have all been characterized in rat brain extracts by the appearance of at least two and sometimes more, diagnostic ions at the appropriate retention time on GC-MS (7, 8, 26, 27, 28, 29, 30). The sulfate esters of pregnenolone and DHEA were also identified by GC-MS as their corresponding free steroids after solvolysis of brain extracts (1, 2, 26).
Whether estimating brain steroids by immunoassay or by GC-MS, the above studies have each only focused on a few compounds at a time and revealed several discrepancies. Moreover, studies of steroidogenic enzyme expression suggest that additional neuroactive metabolites could be produced in rat brain and/or enter from peripheral sources (see Discussion). To provide a framework in which the physiological actions of neurosteroids can be understood, and to facilitate additional investigations on the regulation of steroid metabolism in the brain and the ways in which these pathways might interact with peripheral sources of steroid, we now report a detailed analysis of steroids in adult male rat brain, using GC-MS and applying the identification criteria of retention time and diagnostic ion ratios at a high level of stringency. The steroids to be screened were chosen from products of enzymes known to be expressed in mammalian central nervous system and peripheral endocrine sources.
Materials and Methods
Chemicals
Reference steroids were obtained from Sigma-Aldrich (Dorset, UK) or Steraloids, Inc. (Newport, RI). [3H]Progesterone (5106.0 GBq/mmol) was from PerkinElmer LAS Ltd. (Buckinghamshire, UK). All chemicals were analytical grade from VWR International (Leicestershire, UK) or Sigma-Aldrich, unless stated otherwise, and the solvents were redistilled before use. Water was double glass-distilled, and silanized glassware was used throughout. Of the derivatization reagents for GC-MS, methoxyamine (MO) hydrochloride and heptafluorobutyric acid anhydride (HFBA) were from Sigma-Aldrich, whereas trimethylsilylimidazole (TMSI) and hexamethyldisilazane (HMDS) came from Pierce (Perbio Science Ltd., Cheshire, UK). Lipidex 5000 gel (PerkinElmer) was washed before use with cyclohexane and then stored in cyclohexane/HMDS/pyridine 98:1:1 (vol/vol/vol). Oasis hydrophilic-lipophilic balance (HLB) (5 ml, 200 mg) and mixed-mode anion exchange (3 ml, 60 mg) solid-phase extraction cartridges were obtained from Waters Corp. (Milford, MA). Ecoscint H (National Diagnostics, Yorkshire, UK) was used for scintillation counting.
Brain samples
Adult male Sprague Dawley rats (250–450 g) from the breeding colony at Biological Services, University College London, were kept in a 12-h lighting regimen (lights on at 0800 h) and fed rat chow and water ad libitum. For experiments to test the extraction of endogenous [3H]progesterone and its metabolites from brain tissue, rats were killed by CO2 inhalation. All other rats were killed between 1100 and 1500 h by cervical dislocation. The whole brain (including cerebellum but excluding olfactory bulbs) was rapidly removed and stripped of meninges then stored at –70 C until extraction. All animal procedures were given local ethical committee approval and performed under license from the UK Government Home Office.
Tissue extraction and fractionation
Pooled tissue samples from two to four rats were homogenized in 5 vol ice-cold potassium phosphate buffer (5 mM, pH 7) with a Polytron blender. A 0.5-ml portion of the homogenate was removed to determine blood contamination of the brain samples as described below and stored at –20 C. The remaining homogenate was added dropwise to 20 vol acetic acid (3%, vol/vol) in 96% ethanol in polypropylene tubes in an ultrasonicating bath. After additional sonication for 10 min followed by incubation at –20 C overnight, the extracts were sonicated on ice with an MSE Soniprep probe. After another 30 min on ice, extracts were centrifuged at 28,000 x g for 30 min at 25 C. Lipid was removed from the supernatants by partitioning three times against 10 vol isooctane (previously saturated with 77% ethanol in potassium phosphate buffer (5 mM, pH 7) plus acetic acid (2.4%, vol/vol). After drying down under vacuum, the extracts were resuspended in 4 vol of 60% ethanol (in H2O, vol/vol) and then centrifuged at 1000 x g for 12 min, and the supernatant (corresponding to up to 8 g of original tissue) was passed through a 200-mg reverse-phase Oasis HLB cartridge for the additional removal of lipidic material. An additional 4.4 vol of 60% ethanol in potassium phosphate buffer (5 mM, pH 7, vol/vol) were passed through the cartridge to ensure elution of steroids, and the combined eluate was dried down under vacuum. For separation of free and conjugated steroids, the extracts were then redissolved in 3.75 vol of 20% ethanol in potassium phosphate buffer and passed through 60-mg reverse-phase Oasis mixed-mode anion exchange cartridges. After a wash with 5 ml of 20% ethanol in ammonium acetate buffer (20 mM, pH 7, vol/vol), free steroids were eluted in 4 ml ethyl acetate. Any steroid glucuronides present could then be eluted with 20 ml of 60% ethanol in formate/pyridine buffer (20 mM, pH 3, vol/vol). Finally, after a wash with 2 ml ethyl acetate (dried over Na2SO4), the steroid sulfates were eluted in 15 ml of 50 mM benzene sulfonic acid in ethyl acetate saturated with 2 M H2SO4. The free steroid eluates were dried under nitrogen and derivatized as described below. Steroid sulfate fractions were solvolysed before derivatization by incubation at 40 C in the acidified ethyl acetate eluant for 16 h in the presence of Na2SO4. After neutralization with pyridine, ethyl acetate was evaporated from these solvolysed fractions and the residue extracted three times with 2 ml ether. The combined ether phase was dried down for derivatization by MO-TMSI or HFBA. If the latter, the ether phase was washed beforehand three times with 1 ml H2O. Reagent blank samples were carried alongside each tissue sample throughout the above procedures.
Extraction efficiency of ethanol/acetic acid for brain steroids was estimated after ip injection of two rats with 0.7 MBq/kg [3H]progesterone in PBS (5 ml/kg). These rats were killed after 2.5–3 h by CO2 inhalation and their brains quickly removed before storage at –70 C. As described above, brains were homogenized in 5 vol ice-cold potassium phosphate buffer (5 mM, pH 7). The total brain radioactivity was then estimated by removing portions of homogenate for solubilization in 3 vol Soluene 350. These solubilized samples were then bleached with hydrogen peroxide (final concentration of 1%, wt/vol) for 2 h at 50 C before counting in Ecoscint H containing Triton X-100 (6%, vol/vol), glacial acetic acid (0.6%, vol/vol), and butylated hydroxytoluene (2%, wt/vol). The remaining homogenate was extracted into ethanol/acetic acid as already described. Radioactivity contained in these brain extracts was found to be 88.2% of total brain radioactivity.
Blood contamination of the brain samples was estimated by the spectrophotometric determination of hemoglobin. Portions (0.5 ml) of brain homogenates that had been withheld from steroid extraction and stored at –20 C were thawed and centrifuged (28,000 x g for 30 min at 4 C). Hemoglobin was then measured in these supernatants by the change in absorbance between 560 and 578 nm upon reduction by the addition of sodium dithionite (to a final concentration of 10 mM). Changes in absorbance were calibrated with respect to hemoglobin standards (Sigma-Aldrich) and the blood content of the original brain samples then calculated to be 1.26 ± 0.10% (vol/vol; mean ± SEM, n = 12), assuming a hemoglobin concentration in rat blood of 157 g/liter (31).
Sample derivatization
For samples to be derivatized with MO and TMSI, the internal standards 16-dehydropregnenolone, 6-methyl-17-hydroxyprogesterone, and prednisolone were added beforehand at 50–100 ng. These samples were then dried under nitrogen and redissolved in 200 μl MO in pyridine (2%, wt/vol). In the case of the steroid sulfate fractions, improved yields were obtained if samples were reduced in volume to approximately 50 μl after dissolving in MO/pyridine (32). Both steroid sulfates and free steroids were incubated with MO/pyridine at 60 C for 1 h. The TMSI was then added at 100 μl and the mixture heated another 3 h at 100 C. At the end of this derivatization, pyridine was evaporated under nitrogen and the residue dissolved in cyclohexane/HMDS/pyridine 98:1:1 (vol/vol/vol) and passed through a Lipidex 5000 gel column (0.5 cm diameter and 8 cm high). After additional elution with 2 ml of the same solvent, the pooled eluates were dried under nitrogen and dissolved in cyclohexane for injection onto the GC.
For samples to be derivatized with HFBA, the internal standard 16-dehydropregnenolone was added at 50 ng. After drying under nitrogen, these samples were then redissolved in 30 μl benzene and 30 μl HFBA for incubation at 60 C for 30 min. After this incubation, samples were dried under nitrogen and the residue redissolved in 1 ml cyclohexane/pyridine 98:2 (vol/vol) for passage through Lipidex 5000 gel columns. Columns were eluted with another 2 ml of the same solvent and to the pooled eluates the internal standards tetracosane and octacosane were then added at 25 ng each. Finally, the samples were dried under nitrogen and dissolved in cyclohexane for injection into the GC.
GC-MS analysis
All analyses were carried out on a Shimadzu 17A GC coupled to a QP 5050A MS (Shimadzu, Milton Keynes, UK), equipped with autosampler AOC-20s. The system was controlled and data processed by the Shimadzu Class 5000 software. A 30-m-long Zebron ZB1 wall coated open tubular column (Phenomenex, Macclesfield, UK) with 0.25 mm inner diameter and 0.25 μm film thickness was used for GC with helium as the carrier gas at a constant flow rate of 0.7 ml/min. All analyses were performed in the splitless mode, and the injector purge valve was opened after 4 or 2 min for analysis of MO-TMS or HFB derivatives, respectively. For the analysis of MO-TMS derivatives, the injector temperature was kept constant at 280 C and the pressure at 400 kPa for 5 min. Thereafter the pressure was decreased to 34.2 kPa, followed by a rise of 6.5 kPa/min to 81 kPa. After 0.33 min at this pressure, the gradient was set at 1.6 kPa/min to a final pressure of 111.7 kPa, which was held for 4 min. The oven temperature was at 70 C for 5 min and then rose at 20 C/min to 220 C. After 0.33 min, the gradient was 5 C/min to 315 C, which was held for 4 min. The interface temperature was constant at 315 C. For analysis of HFB derivatives, the injector temperature was constant at 250 C and the pressure at 400 kPa for 2 min. Thereafter, the pressure was decreased to 34.2 kPa followed by a rise of 5.9 kPa/min to 79 kPa. After 0.33 min at this pressure, the gradient was set at 1.5 kPa/min to 98 kPa, which was held for 5 min. Oven temperature was at 70 C for 2 min followed by a rise of 20 C/min to 220 C and then, after 0.33 min, a rise of 5 C/min to 285 C, which was held for 5 min. The interface temperature was constant at 285 C. Samples were ionized by electron impact ionization with an energy of 70 eV. The detector voltage was at 1.7 kV. For initial characterization, retention indices after Kovats (33) were determined for derivatized reference steroids according to the following equation:
where N is the number of carbon atoms in the alkane eluting before the compound of interest, n the increment in number of carbon atoms from this alkane eluting to the one eluting after the compound of interest, t(A) the retention time (min) of the compound of interest, t(N) the retention time of the alkane eluting before the compound of interest and t(N + n) the retention time of the alkane eluting after the compound of interest. For highest possible sensitivity, the MS was run in selected ion monitoring (SIM) mode. In two-ion SIM of MO-TMS derivatives, pairs of target and qualifier ions for each steroid were monitored in groups of four to six ions at a time such that potentially overlapping pairs did not coincide. The ions were monitored in four different injections with the detector settings changed according to retention index as indicated in Table 1.
For confirmation of compound identities, three ions were monitored for MO-TMS derivatives or two ions for HFB derivatives in groups of three to six ions. These ions are shown in Table 2.
Analysis of results
Peaks were integrated manually, and retention time and integration data were additionally processed in Microsoft Excel. For identification, qualifier to target ion ratios (Q/T) were calculated from their areas. Compounds had to meet target values of Q/T of reference compounds run alongside tissue samples within 0.67 and 1.5 (±20% of relative abundance of target and qualifier ions). Further identification was obtained from relative retention times (RRT). These RRTs were calculated as the ratio of retention times of analyte and the closest of one of three internal standards. For positive identification in SIM, RRTs of analytes had to lie within ±0.5% of the RRTs of reference compounds run alongside tissue samples.
For calibration, increasing amounts of reference steroids (0.5–10 ng) were run together with fixed amounts (25–100 ng) of internal standards. The ratios of reference to standard target ion areas could then be plotted against amount ratios. Regression lines were fitted to these plots, which were linear up to 10 ng. Endogenous brain steroids could then be quantified by using the area ratio of their target ions to those of the closest internal standard.
Accuracy of this quantitation was examined by assaying mixtures of standards at 0.5, 1.6, 4.0, and 10.0 ng injected mass per steroid (results shown only for 0.5 ng). The amount of each steroid in each sample was determined and the percent accuracy calculated as the amount measured over the calculated amount. The intra- and interassay reproducibilities were calculated from the assays carried out for determination of accuracy. The percent coefficients of variation (CV) were calculated from the SDs and means within and between assays (results shown only for 0.5 ng).
Detection limits of the overall extraction and assay procedure were determined for brain steroids from area ratios of peaks in reagent blanks extracted and assayed alongside the brain samples. For detection, area ratios had to be three times those of peaks at the same RRT in the corresponding extraction blank. The concentrations at those three-times blank values were calculated using the calibration curves. Detection limits shown in Table 3 were the minimal values found from all extracts.
Results
Two-ion SIM of steroids in rat brain
The first stage of the present investigation was to screen for a wide range of compounds in both the free steroid and sulfate conjugate fractions of rat brain extracts. For this purpose, we used gas capillary chromatography-electron impact mass spectrometry (GC-EIMS) in two-ion SIM mode after derivatization of these fractions with MO and TMSI. The steroids for which we screened are listed in Table 1. Pure standards of these steroids were first used to adjust sample injection, temperature, and pressure conditions of the gas chromatograph for their optimum resolution as MO-TMS derivatives (see Materials and Methods). The MO-TMS derivatives of each steroid were then individually analyzed for their mass spectrometric behavior. An example of a mass spectrum is shown in Fig. 1 for MO-TMS-pregnenolone. Diagnostic ions for each compound were chosen from their mass spectra according to high relative abundance and selectivity, excluding those that might originate from overlapping elutions of steroid derivatives. Choice of these diagnostic ions then allowed SIM for endogenous brain steroids at increased sensitivity. Usually the ion of higher relative abundance was chosen as the target (T) ion for quantitation and the second ion as a qualifier (Q) to aid in identification. These ions are also listed for the nonbiological steroids that were used in the present study as internal standards for quantitation. To maintain high analytical sensitivity during SIM, the T and Q ions were monitored in groups of no more than six and changed at set times during the elution. To monitor all T and Q ions of the MO-TMS derivatives of the compounds listed in Table 1, four separate injections were performed for each sample and altogether four different brain extracts screened in this way. This initial screen tentatively identified 32 free steroids and 23 steroid sulfates in the rat brain extracts, as indicated in Table 1. Also shown are the accuracy and intra- and interassay reproducibilities determined at the 0.5-ng level for all compounds in this screening procedure.
Confirmation and quantitation of brain steroids
Confirmation of the identities of the compounds revealed in the above two-ion SIM screening was attempted using three-ion SIM of their MO-TMS derivatives and/or two-ion SIM of their HFB derivatives. The additional ions for the former derivatives were chosen following the same principles as for the two-ion SIM methods. Likewise, specific ions were identified for two-ion SIM of the HFB derivatives. Figures 2 and 3 illustrate typical chromatograms obtained from three-ion SIM of MO-TMS derivatives and from two-ion SIM of HFB derivatives, respectively. In both Figs. 2 and 3, the trace from a brain extract is shown below that of the appropriate reference steroid. Examples are shown not only for identified brain steroids but also for some of those steroids that were not confirmed as present in the brain extracts.
Table 2 lists all 17 free steroids together with one steroid sulfate, DHEA sulfate, that met the criteria for unequivocal identification in the present study. Table 2 also shows the diagnostic ions for three-ion SIM of MO-TMS derivatives and for two-ion SIM of HFB derivatives for each compound and allows comparison of the RRTs and Q/T ratios of brain analytes with those of standard steroids. Overall definitive identification of an endogenous brain steroid is achieved if coelution of three ions of the compound derivatized with MO and TMSI or of two ions from both the MO-TMS and HFB derivatives occurred within the RRT and Q/T limits of the standard. For such confirmation, the RRTs and Q/T ratios of brain analytes had to be within ±0.5% and 0.67 and 1.5, respectively, of those of standards analyzed on the same day (see Discussion). Confidence limits at the 99.9% level for RRTs and Q/T ratios were also calculated from series of standard samples and found to coincide well with the above pragmatic limits (not shown).
Identified rat brain steroids were quantified using two- or three-ion SIM, and their concentrations are shown in Table 3. Also shown in Table 3 are the recoveries for all of the steroids screened by two-ion SIM in the present study after their addition as pure standards to rat brain homogenates and subsequent extraction, fractionation, and derivatization with MO-TMSI. In addition, for each steroid, we give the minimal detection limits of the overall extraction and fractionation procedure, as determined from reagent blank samples run alongside the tissue samples.
Discussion
The present study has employed ethanolic extraction of steroids from adult male rat brain followed by mixed-mode hydrophobic interaction and anion exchange chromatography to completely separate free steroids and steroid sulfates, before their identification and assay by GC-EIMS. Others have also used methanol or ethanol to extract free and sulfated steroids from brain tissue (12, 30, 34, 35). With the addition of acetic acid as a denaturant, Liere et al. (28) obtained comparable apparent extraction efficiencies for pregnenolone to those reported here for progesterone. As for previous studies, we were unable to estimate extraction efficiencies for steroid sulfates because these undergo desulfation after systemic injection and do not remain in the brain in significant quantities as the original ester (7, 36). However, the polar steroid sulfates are soluble in aqueous ethanol and so should extract more efficiently than the free steroids. Lipoidal steroid conjugates (26) were not analyzed in the present study. These would not extract well into ethanol and in any case should have been removed during the initial clean-up of the extracts by isooctane partitioning and then passage through the HLB cartridges.
After extraction and fractionation, the free steroids and steroid sulfates from rat brain were derivatized for identification and assay by GC-EIMS. Identification was based on comparison with steroid standards and relied on two criteria: RRT (with respect to the nearest internal standard) on the GC and diagnostic ions from the MS. For the former, we adopted the European Commission recommendation (37) of accepting only RRTs that fell within ±0.5% of a reference standard analyzed under the same conditions. As for diagnostic ions in the MS, it was not possible to obtain complete spectra for endogenous brain steroids, and SIM had to be employed to increase sensitivity. An initial screen of rat brain extracts monitoring only two diagnostic ions for each compound indicated the possible presence of 32 free steroids and 23 steroid sulfates. A more rigorous analysis then confirmed 17 free steroids plus one steroid sulfate, DHEA sulfate, as present in adult male rat brain. To be considered as identified, the brain steroid derivatives had to meet not only the ±0.5% RRT criteria but also to have relative ion abundance for three diagnostic ions within ±20% of the standard. Previous evaluations of mass spectral databases have shown such limits on the three-ion criterion alone to provide an unambiguous identification of target compounds (38, 39).
With reference to previous analyses of steroids in nervous tissue (see Introduction), the present results confirm the identification of pregnenolone, progesterone, 5-dihydroprogesterone, 3,5- and 3,5-tetrahydroprogesterone, DHEA, DHEA sulfate, and testosterone in adult male rat brain extracts. The GC-MS analysis reported here has also provided chemical identification for the 3,5-tetrahydrodeoxycorticosterone and corticosterone previously measured by immunoassay in rat brain. Whole-brain concentrations of the above steroids were found to be in the same ranges as given in the previous reports cited here apart from one study (26), which found approximately 10- to 40-fold higher concentrations of pregnenolone, 3,5- and 3,5-tetrahydroprogesterone, and DHEA. In addition to the above steroids, we have also identified 20- and 20-dihydropregnenolone, 20-dihydroprogesterone, 5,20-tetrahydroprogesterone, 5-pregnane-3,20-diol, 5-pregnane-3,17-diol-20-one, and 5-pregnane-3,11-diol-20-one as present in adult male rat brain. None of the steroids identified here has been reported to occur in plasma at concentrations that would contribute significantly to the amounts measured in the present brain extracts, and contamination with blood can be excluded as a source.
In contrast to one previous report that employed two-ion SIM on GC-MS (26), epiandrosterone was not identified in either the free or the sulfated steroid fractions of male rat brain. This steroid was detected in two-ion SIM of the sulfate fraction in the present study but not confirmed with three-ion SIM. The present results also differ from several previous reports (see, for example, Refs.2 , 26 , and 28) in failing to detect pregnenolone sulfate in rat brain. These previous studies have employed solvent phase partitioning and/or hydrophobic interaction chromatography to separate free and sulfated steroids and relied on indirect measurements of pregnenolone sulfate in which the ester is solvolysed to yield pregnenolone for identification and assay. In retrospect, these earlier measurements of pregnenolone sulfate seem likely to have assayed contaminating free steroid or another solvolysable, possibly lipoidal, conjugate from the original extract (40). Direct immunoassay of pregnenolone sulfate in adult male rat brain reported this conjugate at less than 0.4 ng/g adult male rat brain (41) and direct liquid chromatography-MS failed to detect it at a limit of 0.3 ng/g tissue (35). The present study relied on the solvolysis of sulfate esters but employed previous anion exchange chromatography for the complete separation of free steroids from steroid sulfates and failed to detect pregnenolone sulfate at a limit of 0.05 ng/g brain (uncorrected for procedural losses). This is consistent with recent functional studies in rat hippocampus that suggest a role for pregnenolone sulfate as a retrograde synaptic messenger, but only during development and not older than postnatal d 5 (42). Nevertheless and in contrast to the lack of pregnenolone sulfate in adult male rat brain, we were able to detect DHEA sulfate in this tissue. The latter was found at concentrations close to those given in the initial characterization of this rat brain steroid (1).
Steroids identified as present in adult male rat brain by the present study are shown in Fig. 4 in relation to the possible metabolic conversions that could give rise to these compounds. The rats used in our study had not been adrenalectomized or gonadectomized, and so endocrine sources cannot be excluded for some of these brain steroids. Indeed, 3,5-tetrahydrodeoxycorticosterone, corticosterone, and testosterone were found in the absence of detectable concentrations of their respective precursors, 5-dihydrodeoxycorticosterone, 11-deoxycorticosterone, and androstenedione, suggesting endocrine origins. Previous studies in adrenalectomized and/or castrated rats have suggested endocrine sources for tetrahydrodeoxycorticosterone, corticosterone, and testosterone in male rat brain (1, 11, 12). For corticosterone, the 11-keto metabolite could not be detected, confirming that the 11-hydroxysteroid dehydrogenase in brain works predominantly as a reductase and so would not attenuate the glucocorticoid receptor actions of this steroid (43). Figure 4 illustrates that DHEA was also found in the present survey in the absence of detectable concentrations of its known precursor in endocrine tissues. In these tissues, 17-hydroxypregnenolone is an intermediate produced by the enzyme CYP17, which catalyzes the conversion of pregnenolone to DHEA and possesses both steroid 17-hydroxylase and 17,20-lyase activities (44). The absence of 17-hydroxypregnenolone in the present extracts is consistent with previous investigations that have failed to detect activity of the CYP17 enzyme in adult mammalian brain (5, 6). Nevertheless and unlike the above corticosteroids and testosterone, DHEA persists in male rat brain after adrenalectomy and castration (1) and is therefore thought to arise within this tissue.
For the remaining steroids identified as present in adult male rat brain and shown in black in Fig. 4, enzymes previously identified in terms of mRNA, protein, and/or activity (5, 6) can account for their presence. Thus, pregnenolone can be converted to 20-dihydropregnenolone or progesterone by the brain enzymes 20-hydroxysteroid dehydrogenase or 3-hydroxysteroid dehydrogenase/isomerase, respectively. It is unclear at present whether the 20-reduced metabolites are formed by a specific 20-hydroxysteroid oxidoreductase or by one of the 3-hydroxysteroid dehydrogenases (6, 43). The latter can also give rise to 20-reduced metabolites, which would account for the presence of 20-dihydropregnenolone, although we were unable to detect 20-dihydroprogesterone. By contrast, the 20-reduced metabolite of progesterone was detected, although only a small proportion of progesterone appears to undergo this conversion in rat brain homogenates in vitro (45).
For progesterone, the major metabolic route in rat brain appears to be 5-reduction to yield 5-dihydroprogesterone, on the pathway to the GABAA receptor-enhancing metabolite 3,5-tetrahydroprogesterone (see Introduction). Consistent with the 5-reductase enzyme being the highest-affinity, rate-limiting step on this pathway (46), we could not detect 3- or 3-dihydroprogesterone. The present study also failed to detect 5-reduced metabolites of progesterone, in agreement with the reported lack of 5-reductase activity in mammalian brain tissue. However, we did find 3,5-tetrahydroprogesterone. Unlike their respective 3,5-reduced compounds, the 3,5-reduced steroids are not active at the GABAA receptor (47). Nevertheless, they would be substrates for the 3-diol hydroxylase enzyme, which hydroxylates at positions 6 or 7 and is found throughout the brain. Competitive inhibition of 3-diol hydroxylase increases the duration of anesthesia induced by 3,5-tetrahydroprogesterone in the rat, and this enzyme has therefore been suggested to provide an inactivation pathway for 3,5-reduced pregnanes and androstanes in the central nervous system (48). The 3,5-tetrahydroprogesterone found in the present study could have arisen through epimerization of 3,5-tetrahydroprogesterone (49) or direct from 5-dihydroprogesterone. The latter would serve as a diversion of substrate from the formation of neuroactive 3,5-tetrahydroprogesterone.
The present results also identify 11-, 17-, and 20-hydroxylation as possible routes for the inactivation of 3,5-tetrahydroprogesterone. The first two metabolites appear to be inactive at the GABAA receptor, whereas the latter has partial agonist properties at the 3,5-tetrahydroprogesterone-modulated site (47, 50). An 11-hydroxylase (CYP11B) is widely expressed in brain and usually denoted as the final step in the production of corticosterone from deoxycorticosterone. However, the lack of evidence in this and previous studies for 21-hydroxylase (CYP21) activity and thus deoxycorticosterone formation in brain suggests that the 11-hydroxylase may actually be more important in this tissue for the inactivation of 3,5-tetrahydroprogesterone. Formation of 5-pregnane-3,17-diol-20-one is harder to explain in view of the apparent lack of CYP17 activity in brain (see above), although 17-hydroxylation could result from variable site specificity of one of the 3-hydroxysteroid dehydrogenases (43) or another as yet unknown mechanism. As mentioned above, 20-dihydroprogesterone was also detected in the present survey. This steroid is known to be a better substrate than progesterone for rat brain 5-reductase (51), which would explain the presence of 5,20-tetrahydroprogesterone. Further reduction to 5-pregnane-3,20-diol may therefore indicate an alternative metabolic route rather than a true inactivation pathway for 3,5-tetrahydroprogesterone. Likewise, the formation of 20-dihydropregnenolone may serve to divert pregnenolone from the formation of neuroactive progesterone and its 3,5-reduced metabolites.
To conclude, the present study has chemically identified several neuroactive free steroids in adult male rat brain. Some of these compounds were found together with precursors, suggesting they could be formed within this tissue, whereas others appear to enter from peripheral sources, although future studies will be necessary to confirm such origins. For 3,5-tetrahydroprogesterone, which is known to enhance the interaction of GABA with the GABAA receptor, 11-, 17-, and 20-hydroxylated metabolites were identified, thereby implicating inactivation pathways for this potent neuromodulatory compound. Our results also confirm the suggestion that epimerization could serve as a first step on the inactivation of 3,5-reduced steroids in brain. In addition, the presence of 20- and 20-reduced pregnenolone indicates a possible regulation on the production of progesterone and 3,5-tetrahydroprogesterone. For the steroid sulfates, only DHEA sulfate was confirmed, and pregnenolone sulfate was not detected at limits below those quoted by previous authors. Despite its reported effects on GABAA and N-methyl-D-aspartate receptors, pregnenolone sulfate is therefore of doubtful physiological significance in the adult central nervous system. Additional studies are now needed to elucidate the regulation of steroid production within the brain and to evaluate how these processes interact with peripheral sources of steroid.
Acknowledgments
We are grateful to E. Conway and R. Hodkinson for help with the GC-MS analyses, to M. Mojet for the hemoglobin measurements, and to M. Farrant for helpful comments on the manuscript.
Footnotes
This study was supported by the European Commission Marie Curie Fellowship Programe, M.J.E. as an individual Training and Mobility of Researchers Fellow and both D.C. and H.H. as Visiting Host Fellows at a UCL Training Site.
Present address for M.J.E.: Procognia Limited, Unit 4, The Switchback, Gardner Road, Maidenhead, Berkshire SL6 7RJ, United Kingdom
Present address for D.C.: Crop Performance Improvement Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom.
Present address for H.H.: Institute of Endocrinology, Národní 8, 116 94, Prague 1, Czech Republic.
First Published Online October 13, 2005
Abbreviations: CV, Coefficient of variation; DHEA, dehydroepiandrosterone; GABA, -aminobutyric acid; GC-EIMS, gas capillary chromatography-electron impact mass spectrometry; GC-MS, GC-mass spectrometry; HFBA, heptafluorobutyric acid anhydride; HLB, hydrophilic-lipophilic balance; HMDS, hexamethyldisilazane; MO, methoxyamine; Q, qualifier; RRT, relative retention time; SIM, selected ion monitoring; T, target; TMSI, trimethylsilylimidazole.
Accepted for publication October 4, 2005.
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