Simultaneous determination of dehydroepiandrosterone, its 7-hydroxylated metabolites, and their sulfates in rat brain tissues

Steroids 69 (2004) 667–674

Simultaneous determination of dehydroepiandrosterone, its 7-hydroxylated metabolites, and their sulfates in rat brain tissues Hana Kazihnitkov´aa , Hana Tejkalov´ab , Olga Beneˇsov´ab , Marie Biˇc´ıkov´aa , Martin Hilla , Richard Hampla,∗ a

Institute of Endocrinology, N´arodn´ı 8, 11694 Praha 1, Czech Republic b Prague Psychiatric Centre, Czech Republic

Received 10 February 2004; received in revised form 7 June 2004; accepted 10 June 2004 Available online 18 September 2004

Abstract A method is described for simultaneous assessment of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and their 7-hydroxylated metabolites in cortex and subcortex of the rat brain. The procedure for determination of unconjugated steroids and DHEAS involved diethyl ether extraction of the homogenized tissue, solvent partition of the dry extract, and final quantification by specific radioimmunoassays. In addition, determination of 7-hydroxy-dehydroepiandrosterone sulfates required solvolysis, followed by high-performance liquid chromatography for separation of 7-hydroxylated metabolites from their precursor. The losses during this process were monitored by measurement of spiked radioactivity of [3 H]testosterone or [3 H]dehydroepiandrosterone sulfate. The content of dehydroepiandrosterone sulfate in both brain tissues was of the order of ten(s) nmol/g tissue irrespective its type (cortex or subcortex), while concentrations of other steroids were about 10 times lower in both tissues. In contrast to the ratio of sulfated/unconjugated DHEA, the levels of unconjugated 7-hydroxylated metabolites and their sulfates were close to each other. The reproducibility of the method with respect to coefficients of variation varied from 12 to 25%. An age-related decrease of sulfated dehydroepiandrosterone in the cortex of animals was also observed. © 2004 Elsevier Inc. All rights reserved. Keywords: Dehydroepiandrosterone; 7-hydroxydehydroepiandrosterone; Determination; Rat brain

1. Introduction Dehydroepiandrosterone (DHEA), as a sulfate (DHEAS), is the most abundant circulating steroid in humans and is believed to possess many, mostly beneficial, effects. Among other actions, it acts in brain by allosteric modulation of the ␥-aminobutyric acid receptor, type A (GABAA -R). Since the latter is linked to the chloride channel, it may regulate chloride influx and thus, increase chloride conductance, resulting in hyperpolarization of neuronal membrane. In this way, brain function is influenced [1]. In addition, both DHEA and DHEAS have been shown to positively modulate the N-methyl-d-aspartate (NMDA)-type of glutamate receptor either directly or indirectly through an interaction with the ∗

Corresponding author. Tel.: +420 224 905 289; fax: +420 224 905 325. E-mail address: [email protected] (R. Hampl).

0039-128X/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2004.06.002

sigma receptor; for the literature, see [2] and other papers in this volume. The main source of circulating DHEA/S in human beings are adrenals, but a certain portion of these steroids originates from brain. For such steroids, the term neurosteroids was introduced by Baulieu, as steroids “synthesized in central and peripheral nervous cells (particularly, but not exclusively in myelinating glial cells) from cholesterol and precursors from peripheral sources” [3,4]. In rodents, in contrast to primates, concentrations of circulating DHEA/S are very low because the rodent adrenal does not express P450c17 and therefore does not synthesize these steroids [5]. In spite of that, remarkable concentrations of both steroids are found in rat brain tissues [6], indicating that rat brain cells are capable of producing them in situ. Initial experiments revealed that these steroids remained in the brain after removal of potential glandular sources, that is, after

668

H. Kazihnitkov´a et al. / Steroids 69 (2004) 667–674

adrenalectomy and gonadectomy [3]. The local production of DHEA/S in rat brain is in concordance with the known fact that steroid conjugates can pass only with difficulty across the blood–brain barrier [7,8]. The steroid modulation of GABAA R is bimodal: DHEA/S or pregnenolone sulfate interact with the receptor as antagonists, while some unconjugated steroids as 5␣-saturated metabolites of progesterone act as agonists [9]. Generally, the modulatory effects of sulfated and non-sulfated steroids on neurotransmitter receptors may differ, emphasizing thus the importance of local activities of steroid sulfotransferase and sulfatase, both enzymes found in rat brain tissues [10,11]. Many, but not all beneficial actions of DHEA are ascribed to its immunomodulatory and immunoprotective effects, including moderation of the excessive effects of glucocorticoids [12]. In addition, DHEA/S was repeatedly reported to possess various neuroprotective effects (for reviews, see [1,13]). Recent reports indicate that not only DHEA/S but some of its metabolites also are the local agents, responsible for the above-mentioned immunoprotective and neuroprotective effects. This concerns particularly the 7␣- and 7␤hydroxylated metabolites of DHEA and other 17-oxosteroids [14–16]. Indeed, 7␣-hydroxylation in murine brain structures was repeatedly demonstrated [14,17], the responsible enzymes of the cytochrome P450 family were characterized [18], and the major gene encoding these enzymes was identified as CYP7B1 species [19,20]. Taken together, rat brain is the site of intensive DHEA/S metabolism leading to bioactive steroids influencing various brain functions via their targets. Levels of these steroids in blood need not reflect their concentration at the site of action, and therefore, knowledge of their actual concentrations in the brain tissue is needed. A method, based on extraction, chromatographic separation, and final determination by gas chromatography–mass spectrometry have been reported for DHEA/S in rat brain [21,22], but a method for simultaneous estimation of DHEA/S along with their 7-hydroxylated metabolites is lacking. Here, such a method is described, taking advantage of specific radioimmunoassays for both 7-OH-DHEA isomers, recently developed in the author’s laboratory [23,24]. The method is now being used to follow respective steroid concentrations in rat brain tissues in studies of the effect of DHEA and another 7-oxygenated metabolite, 7-oxo-DHEA, on various physiological parameters and behavior.

2. Materials and methods 2.1. Animals and tissues Wistar/Han male rats (BioTest Kon´arovice, Czech Republic) of three age groups (A: 6 months, B: 15.5 months, C: 20.5 months, weighing A: 449.4 ± 6.4, B: 547.3 ± 10.4, C: 637.3 ± 17.2 g, respectively were used for the experiments. All animals were housed in a temperature and humidity-controlled

room (22 ± 2 ◦ C; humidity 30–70%) under a 12-h light/dark cycle in accordance with regulations of the National Committee for the Care and Use of Laboratory Animals. Animals were killed by cervical dislocation. After decapitation, brains were immediately removed, put on an ice-cooled plate, and dissected to frontal cortex and subcortex. The latter consisted of basal ganglia and striatum, but without hippocampus and hypothalamus. The wet tissues were weighed and then frozen at −70 ◦ C until processed (separately, cortex and subcortex). The study was approved by the Institution’s Animal Care Committee and admitted by the Czech Ministry of Health under no. 2/2000 and 14/2001, in agreement with the Czech Animal Protection Law no. 249/1992 and the later Regulation no. 311/1997. 2.2. Materials: reagents, chemicals and instruments [1,2,6,7-3 H]Testosterone and [1,2,6,7-3 H(n)] dehydroepiandrosterone sulfate, sodium salt, specific radioactivities 3.1 and 2.9 TBq/mmol, respectively, were purchased from Radiochemical Center, Amersham (UK). Radioactive steroids were purified by thin-layer chromatography on silica gel (Kieselgel 60 F254 , Merck, Darmstadt, FRG) with dichloromethane–methanol 95:5, and ethyl acetate–methanol–ammonium hydroxide (0.1%) 75:25:2, respectively. Purified tracers were dissolved in ethanol to a final concentration 330 Bq (20,000 dpm) in 10 ␮l. Buffers are A: 50 mmol/l citrate–phosphate, pH 8.6, containing estrone sulfamate (sulfatase inhibitor, 1 ␮g/10 ml, Sigma-Aldrich, Czech Division, Praha, Czech Rep.); B: 20 mmol/sodium phosphate containing 0.1 g/100 ml BSA. C: 50 mmol/l sodium-acetate, pH 6.0. Dextran-coated charcoal: 0.025 g Dextran T-70 (Pharmacia, Uppsala, Sweden) and 0.25 g Norit A (Merck) in 100 ml buffer B. All solvents, i.e., diethyl ether, light petroleum ether, methanol, and ethanol were of analytical grade from Merck. The GILSON HPLC system (Villiers le Bel, France) consisted of a programmable pump 305 with a manometric module 805, a slave pump 306, dynamic mixer 811C, autoinjector 234, and a programmable sample collector FC 203B. The UV detector LCD 2082 and a column thermostat LCO 100 were from ECOM (Praha, Czech Republic). Reverse phase C18 column ET 250/4 NUCLEOSIL® 100-5 was from ¨ MACHEREY-NAGEL (D¨uren, FRG). Radioimmunoassay (RIA) kits for determination of unconjugated DHEA and DHEAS were from Immunotech (Marseille, France). The origin and preparation of the reagents used for determination of unconjugated 7␣- and 7␤OH-DHEA (radioligands, antisera, standards) are described in detail in the referenced publications [23,24]. A Stratec automatic analyzer (Immunotech) was used for radioimmunoassays. 3 H radioactivity was measured on a liquid scintillation counter LS 6500 (Beckman Instruments, Irwine, CA, USA) using scintillation cocktail OptiPhase HiSafe 2 (Wallac Oy, Turku, Finland). 125 I radioactivity was measured on a 12channel gamma counter Berthold LB 2104 (Wien, Austria).

H. Kazihnitkov´a et al. / Steroids 69 (2004) 667–674

2.3. Determination of unconjugated steroids: DHEA, 7␣-, 7␤-OH-DHEA, and DHEAS 2.3.1. Homogenization and extraction Approximately 200 mg of tissue was weighed and put into a stoppered glass homogenization tube. Buffer A (600 ␮l) and 10 ␮l of an ethanolic solution of [3 H]testosterone of known radioactivity (approximately 330 Bq), were added, and the mixture was homogenized using a small electric hand-held homogenizer with a Teflon piston. The homogenate was then extracted with diethyl ether (3 ml) for 1 min using a vortex mixer. The lower water phase was freezed in a solid carbon dioxide bath, and the upper ether phase was decanted into another glass tube without a stopper. Following thawing of the homogenate, it was re-extracted with ether (2 ml), which after freezing of the homogenate was added to the main portion. The combined extracts were then evaporated to dryness in a speed-vac centrifuge. The dry residues were used for determination of unconjugated steroids: DHEA, 7␣- and 7␤OH-DHEA. The remaining homogenate was put aside in a deep freezer, and after thawing, it was used for determination of DHEAS. 2.3.2. Solvent partition of the dry residue from the extract and its further processing In order to separate fatty components which may interfere with the assay, the combined ether extracts from the previous paragraph containing unconjugated steroids were evaporated, and processed as follows: petroleum ether and the mixture water–methanol 1:4 (1 ml each) were added to the dry residue in a glass tube. The mixture was vortexed briefly (30 s), and after partition, the upper, petroleum ether phase was carefully removed with a Pasteur pipette and discarded. The lower, aqueous-methanolic phase containing the most of unconjugated steroids was evaporated again to dryness in a speed-vac centrifuge. The dry residue was dissolved in RIA buffer (B, 400 ␮l). The following analyses had to be performed within 2–3 days because buffer solutions of steroids are unstable. The first portion (100 ␮l) was used for determination of unconjugated DHEA (see below). The next two 100 ␮l portions were used for determination of both 7-OHDHEA isomers as described below. The remaining 100 ␮l (all the rest in the tube) served for assessment of the losses during processing. The tubes were washed twice with 0.5 ml

669

of a mixture of water–methanol 1:4, which was transferred into scintillation vials containing liquid scintillator (4 ml). 3 H Radioactivity was then measured by liquid scintillation counting. 2.3.3. Radioimmunoassay of unconjugated DHEA The reagents: antibody-coated tubes, standard solutions (0, 50, 100, 300, and 3000 pg), and radioligand were supplied in the RIA kit for determination of unconjugated DHEA. The cross-reactions of selected steroids with the monoclonal antibody provided by the manufacturer (in percent) were as follows: DHEAS, 106; 3␤-hydroxy-5␣-androstan-17-one (epiandrosterone), 25.3; 3␣-hydroxy-5␣-androstan-17-one (androsterone), 7.1; those with other DHEA metabolites and major hormonal steroids were lower than 0.2%. The crossreactions of both 7-OH-DHEA isomers with the antibody, as measured in our laboratory, were lower than 0.1% each. The detection limit reported by the manufacturer for blood serum was 104 pmol (300 pg/sample), the mean intra- and inter-assay coefficients of variation were 7.3 and 11.1%, respectively. The latter values differed from those obtained for tissue samples (see Table 1). Buffered sample solution (100 ␮l) prepared as described in the Section 2.3.2 or of standards (100 ␮l) were pipetted into antibody-coated tubes. The tracer (500 ␮l) was then added, and the tubes were put into the analyzer. The analyzer performed incubation, removing of the solution, and measurement of the radioactivity remaining in the tube. The values provided by the analyzer in pg/tube were corrected to losses during processing and recalculated to nmol/g tissue. 2.4. Radioimmunoassay of unconjugated 7␣- and 7␤-OH-DHEA Both 7-OH-DHEA isomers were analyzed in the same way. Two 100-␮l portions of buffered solutions prepared as described above (see Section 2.3.2) were taken for analysis of 7␣- and 7␤-OH-DHEA. Rabbit polyclonal antisera against 19-O-(carboxymethyl) oxime: BSA conjugate and tracers (homologous 19-[125 I]iodo-tyrosine methyl esters of both 7OH-DHEA isomers) were prepared as described elsewhere [21,22]. The 7␤-OH-DHEA antibody cross-reacted with 7␣OH-DHEA by 0.28, with DHEA by 0.18%, while 7␣-OHDHEA antibody cross-reacted with 7␤-OH-DHEA by 1.16,

Table 1 Reproducibilty of steroid determination in two rat brain tissues. The remains of brain tissues from ten animals, four from the A group (6 months old animals), and three from each of the other groups (15.5 and 20.5 months) were pooled and minced. Six aliquots (approx. 200 mg) were weighed and processed separately for analyses of DHEA, DHEAS, 7-OH isomers, and their sulfates. The data (means ± SD) are given in pmol/g.tissue DHEA

DHEAS

7␣-OH-DHEA

7␤-OH-DHEA

7␣-OH-DHEAS

7␤-OH-DHEAS

Cortex Mean ± SD C.V. (%)

0.83 ± 0.12 14.5

23.8 ± 4.62 19.4

0.64 ± 0.11 17.4

0.70 ± 0.15 21.6

0.42 ± 0.08 19.6

0.59 ± 0.10 16.3

Subcortex Mean ± SD C.V. (%)

0.87 ± 0.13 14.9

16.9 ± 3.23 19.1

0.44 ± 0.09 20.5

0.56 ± 0.10 18.2

0.67 ± 0.08 11.6

0.47 ± 0.12 25.3

670

H. Kazihnitkov´a et al. / Steroids 69 (2004) 667–674

and with DHEA by 1.95%, respectively. Cross-reactivities of both antibodies with other unconjugated DHEA metabolites and major hormonal steroids were lower than 0.1%. Standard solutions contained 50, 25, 12.5, 6.25, and 3.125 pg/100 ␮l of each isomer (six points). The 7th standard was the zero point (buffer, 100 ␮l). Assay characteristics: 7␤-OH-DHEA, intra-assay c.v. 6.7%, inter-assay c.v. 10.0%, detection limit 3.6 fmol (1.1 pg); 7␣-OH-DHEA intra-assay c.v. 7.1%, interassay c.v. 10.6%, detection limit 3.1 fmol (0.95 pg). The buffered solutions of the antisera (working dilution 1:80,000 for 7␣-OH-DHEA and 1: 40,000 for 7␤-OHDHEA.) and tracers, approximately 250 Bq (15,000 cpm), 100 ␮l each, were added to the polystyrene tubes containing either standards or sample solutions. Only the radioligand and buffer were pipetted into the tubes for determination of the non-specific binding and total radioactivity. The contents of the tubes were vortexed briefly, and samples were incubated overnight at 2–4 ◦ C. Stirred, dextran-coated charcoal suspension (500 ␮l) was added to all tubes, with the exception of the “total”. The contents were mixed again, and after 10 min at 2–4 ◦ C, the tubes were centrifuged at 4 ◦ C for 10 min. The supernatant was decanted into another set of polystyrene tubes in which 125 I radioactivity was measured. The results in pg/tube were recalculated to final S.I. units as described above, taking into account the efficacy of sample processing, the weight of the tissue, and the molecular weight of 7-OH-DHEA (304.4 Da) 2.5. Determination of DHEAS The homogenate remaining after ether extraction (see Section 2.3.1) was used. The rest of the ether was removed by a stream of nitrogen. The tube walls were washed with water (400 ␮l), and the suspension was centrifuged at 4 ◦ C and 2500–3000 rpm for 15 min. The supernatant, after removal of debris and undissolved particles, was transferred carefully to another small glass tube using a Pasteur pipette. The sediment in the extraction tube was suspended again in water (200 ␮l), the centrifugation was repeated, and the supernatant was added to the main portion. If the combined supernatant was still turbid, it was re-centrifuged and decanted into another tube. 2.5.1. RIA The reagents (radioiodinated tracer and standards) and antibody-coated tubes from the kit for DHEAS RIA (Immunotech) were used as follows. Six tubes were needed for the standard curve (including zero) and one tube was used for each sample. For the standard curve, 25 ␮l of each standard and 600 ␮l of water were pipetted into the tubes. To the other tubes combined supernatant from the homogenate was transferred quantitatively using a Pasteur pipette. The glass tubes were washed with an additional 100 ␮l of water, which was added to the main portion. The radioligand (500 ␮l) was then pipetted into all tubes, which were put into the STRATEC analyzer. The analyzer incubated the samples,

removed the incubation mixture, washed the tubes, measured the radioactivity, and calculated the results. The results (in ng/tube) were multiplied by the value 1000/(weight of the processed tissue in mg) and recalculated to S.I. units. No correction for losses was made since all the homogenate was analyzed. 2.6. Determination of 7␣- and 7␤-OH-DHEA sulfates Since DHEAS may interfere considerably with the assay of 7-OH-DHEA sulfates, the analyses were carried out separately from the determination of unconjugated steroids and DHEAS. The whole procedure included homogenization, removal of unconjugated steroids, solvolysis, extraction of freed steroids, their separation by HPLC, and final RIA, as described below. 2.7. Homogenization and removal of unconjugated steroids Approximately 100 mg of tissue was cut off, weighed, and put into stoppered glass homogenization tube. Ethanolic solution of [3 H]DHEAS (10 ␮l) of known radioactivity (approximately 330 Bq) and acetate buffer (C, 500 ␮l) were added, and the mixture was homogenized using a small, electric hand-held homogenizer with a Teflon piston. The homogenate was extracted with a mixture of diethyl ether–light petroleum ether 1:1 (2 ml) for 1 min using a vortex mixer. The homogenate in water phase was frozen in the carbon dioxide bath, and the organic phase was transferred into a small glass tube in which it was evaporated to dryness with a speedvac centrifuge. The dry residue was dissolved in a mixture of methanol–water (1 ml) and transferred into a scintillation vial. The tube was washed with another 0.5 ml of the solvent mixture, which was added to the main portion. 3 H radioactivity was then measured for determination of the losses during homogenization and extraction (the value b); see paragraph “Calculation of the results” below. 2.8. Solvolysis After thawing the homogenate, the rest of the extraction mixture was removed by a stream of nitrogen. The homogenate was then centrifuged at 6000 rpm for 5 min. The supernatant containing the analyzed steroid sulfates was carefully transferred into another glass tube with a Pasteur pipette. The sediment was stirred again with water (1 ml) using a vortex mixer, the centrifugation was repeated, and the supernatant was added to the first portion. The sediment was then suspended in water (0.5 ml), transferred into a scintillation vial, and the tube was washed with another 0.5 ml of water, which was added to the scintillation vial. The washing was repeated, and the remaining 3 H radioactivity in the sediment was then measured for determination of the losses during this step (value c). Concentrated sulfuric acid (20 ␮l) and a small amount (approximately 50 mg) of

H. Kazihnitkov´a et al. / Steroids 69 (2004) 667–674

671

Fig. 1. Flow-chart diagram of determination of DHEA/S, its 7-hydroxylated metabolites, and their sulfates in rat brain tissues. The brains after removal were dissected to frontal cortex and subcortex. The latter consisted of basal ganglia and striatum, but without hippocampus and hypothalamus. Both tissues were processed in the same way.

672

H. Kazihnitkov´a et al. / Steroids 69 (2004) 667–674

sodium sulfate were added to the supernatant, the mixture was mixed briefly, and ethyl acetate (3 ml) was added. The mixture was mixed again and left at room temperature for 30 min. The water phase was then frozen in a carbon dioxide bath, and the organic layer was decanted into another glass tube. Extraction of the water phase was repeated twice with ethyl acetate (1 ml each extraction). The ethyl acetate extracts were combined and incubated overnight at 37 ◦ C. The extract was then evaporated to dryness in a speed-vac centrifuge. 2.9. High-performance liquid chromatography (HPLC) The dry residue was prepared as described in the previous paragraph, and the solvolyzed steroid sulfates were dissolved in methanol (70 ␮l) and transferred to stoppered HPLC vials. An aliquot (25 ␮l) was then injected into the chromatographic system. Elution was carried out in a binary gradient system. The mobile phase A consisted of acetonitrile (15%) and water (85%) with 100 mg/1 of ammonium bicarbonate. Mobile phase B: methanol. The gradient was as follows: from the start to the 1st min, 0% of phase B, then, a linear gradient from 45 to 55% B until the 7th min. Steep gradient followed to 100% B in the 8th min. A plateau was then maintained between the 8th and 12th min with 100% B, which was replaced by another plateau using 0% B, lasting until the 18th min. The fractions corresponding to authentic 7␤and 7␣-OH-DHEA (Rt = 9.77 and 10.87 min, respectively) were collected into small glass tubes. The Rt of DHEA was 16.86 min (not collected). The eluent was evaporated to dryness in a speed-vac centrifuge, and the dry residues were used for RIA. 2.10. Radioimmunoassay Both 7-OH-DHEA isomers were determined by radioimmunoassay in the dry residues from the fractions collected by HPLC separation (see Section 2.9). The RIA was described above (see Section 2.4).

2.11. Calculation of 7␣ and 7␤-OH-DHEAS concentrations The concentrations of 7-OH-DHEAS isomers in pmol/g tissue were calculated according to the formula: 9.19Ta w(T − b − c) where T is the mean total radioactivity of spiked [3 H]DHEAS (dpm); b the losses during extraction with petroleum etherdiethyl ether (dpm; see Section 2.7); c the losses during separation of the homogenate pellet (dpm) (see Section 2.8); w wet weight of the tissue taken for analysis (mg); a the value of 7-OH-DHEA concentration determined by RIA (in pg/tube). The flow-chart of the whole procedure is shown on Fig. 1. 3. Results and discussion A method is described and evaluated for simultaneous estimation of DHEA, its sulfate, and their 7␣- and 7␤hydroxylated metabolites in rat brain tissues. While DHEA/S in rat brain have been already measured by others [6,21,22], our method enabling simultaneous assessment of 7OHDHEA isomers and their sulfates is described for the first time. The reproducibility of the method was assessed by a parallel determination (n = 6) of all six steroids in pooled samples of cortex and subcortex from 10 rat brains. The results with the respective coefficients of variation are shown in Table 1. The data from three individual experiments with groups of animals differing in age are summarized in Table 2. The aim of this work was to create a suitable method for assessing DHEA and its 7-hydroxylated metabolites in rat brain tissue to be used in further experiments. Although we were not specifically studying the effect of age on DHEA/S, there was a distinct decline of sulfated DHEA content with age and a shift to 7␤-hydroxylated metabolites in the oldest animals. This indicates an age dependence of the responsible enzymatic activities.

Table 2 Concentrations of DHEA, 7-OH DHEA isomers, and their sulfates in rat brain tissues. Summarized data from three independent experiments with rat groups differing in age. The data are given in pmol/g.tissue Group

Cortex 7␣

Subcortex

Age (months)

DHEA

DHEAS

7␤

7␣S

7␤S

DHEA

DHEAS

7␣

7␤

7␣S

7␤S

A 6

Mean SD Median n

1.24 1.14 0.50 8

31.67 29.98 14.28 8

0.63 0.50 0.48 8

0.48 0.25 0.48 8

0.57 0.36 0.50 8

0.48 0.16 0.61 8

1.49 1.55 0.70 8

14.33 11.49 8.60 8

0.71 0.58 0.50 8

0.38 0.29 0.24 8

0.43 0.35 0.20 8

0.21 0.23 0.08 8

B 15.5

Mean SD Median n

1.22 1.09 0.53 14

29.43 30.29 7.93 14

0.50 0.37 0.38 14

0.29 0.19 0.30 14

0.41 0.41 0.16 14

0.31 0.20 0.33 14

1.44 1.17 0.77 14

17.67 16.10 12.40 14

0.45 0.38 0.21 14

0.27 0.21 0.19 14

0.43 0.41 0.17 14

0.22 0.21 0.08 14

C 20.5

Mean SD Median n

3.61 1.94 3.34 13

9.59 5.77 7.55 11

0.53 0.51 0.50 13

1.02 0.42 0.80 13

1.35 0.67 1.45 13

2.53 0.60 2.60 13

2.36 1.31 2.18 12

13.90 9.03 17.55 10

1.72 2.51 0.72 13

1.48 0.90 1.24 13

0.42 0.40 0.26 13

2.62 1.75 2.00 13

H. Kazihnitkov´a et al. / Steroids 69 (2004) 667–674

673

Table 3 Comparison of our results of DHEA/S content in rat brain tissue with those of other authors. The values in paraentheses mean number of experiments Author (year)

Reference

Brain tissue

DHEA (pmol/g)

DHEAS (pmol/g)

Robel and Baulieu (1995)

[19]

Anterior Posterior

1.50 ± 0.3 (4) 0.38 ± 0 (4)

4.10 ± 0.51 (10) 12.6 ± 2.82 (11)

Liere et al. (2000)

[20]

Whole brain

1.56 (10)

6.33 (10)

Cortex Subcortex

2.11 ± 1.42 (35) 1.78 ± 1.31 (34)

23.4 ± 22.0 (33) 15.6 ± 12.7 (32)

This work (2003)

The content of DHEA was close to that reported by others [21,22]; while that of DHEAS was two to three-fold higher. It should be emphasized, however, that much older rats were investigated here than in cited studies, and in addition, there was a great inter-individual variation. A comparison of our results with those of the cited authors is shown in Table 3. While Liere et al. [22], using gas chromatography–mass spectrometry as an end-point measurement technique, determined the steroids in small amounts of the whole brain tissue, Robel and Baulieu [21] used RIA for quantification and took anterior and posterior brain samples for analysis. We measured all the steroids separately in the cortex and subcortex because we wanted to correlate the data with cognitive and motor parameters. The amount of both 7-OH-DHEA isomers in both brain tissues was of the same order of magnitude as unconjugated DHEA in agreement with the finding of intensive 7-hydroxylation in brain cells [13,14]. With respect to the neuroprotective effects of 7-hydroxylated C19 steroids [13,14,16], determination of 7-OH-DHEA and other 7hydroxylated steroids such as 7␤-hydroxy-epiandrosterone [16] in situ, opens the possibility of further studies on the role of these steroids in brain. The levels of 7-OH-DHEA sulfates in both brain tissues were close to or lower than those of unconjugated steroids; the ratio of sulfated/unconjugated steroids was close to 1. It is in sharp contrast to the ratio of DHEAS/DHEA with an order of magnitude higher than concentration of the sulfate. This finding supports the idea that unconjugated DHEA is preferred as a substrate for 7-hydroxylation to DHEAS, similarly as shown by others in rat liver as early as in 1967, where the rate of formation of 7␣-hydroxylated compounds was four to five times greater with free DHEA as the substrate than with DHEAS [25].

Acknowledgements The work was supported by the Research Intention no. 23761-5 from the Czech Ministry of Health.

References [1] Wolf OT, Kirschbaum C. Actions of dehydroepiandrosterone and its sulfate in the central nervous system: effects on cognition and emotion in animals and humans. Brain Res Rev 1999;30:264–88.

[2] Barbaccia ML, Sidiropoulou T. The brain, a putative source and target of DHEA. In: Morfin R, editor. DHEA and the brain. London: Taylor and Francis; 2002. p. 1–23. [3] Baulieu EE. Neurosteroids: of the nervous system, by the nervous system, for the nervous system. Rec Prog Horm Res 1996;52:1–32. [4] Baulieu EE, Robel P. Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as neuroactive steroids. Proc Natl Acad Sci USA 1998;95:4089–91. [5] Brock BJ, Waterman MR. Biochemical differences between rat and human cytochrome P450c17 support the different steroidogenic needs of these two species. Biochemistry 1999;38:1598–606. [6] Corp´echot C, Robel P, Axelson M, Sjovall J, Baulieu EE. Characterization and measurement of dehydroepiandrosterone sulfate in rat brain. Proc Natl Acad Sci USA 1981;78:4704–7. [7] Guazzo EP, Kirkpatrick PJ, Goodyer IM, Shiers HM, Herbert J. Cortisol, dehydroepiandrosterone (DHEA), and DHEA sulfate in the cerebrospinal fluid of man: relation to blood levels and the effect of age. J Clin Endocrinol Metab 1996;81:3951–60. [8] Asaba H, Hosoya K, Takanaga H, Ohtsuki S, Tamura E, Takizawa T, et al. Blood–brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2. J Neurochem 2000;75:1907–16. [9] Majewska MD. DHEA: hormone of youth and resilience: still a maverick. In: Morfin R, editor. DHEA and the brain. London: Taylor and Francis; 2002. p. 65–80. [10] Suitters AJ, Shaw S, Wales MR, Porter JR, Leonard J, Woodger R, et al. Immune enhancing effects of dehydroepiandrosterone and dehydroepiandrosterone sulphate and the role of steroid sulphatase. Immunology 1997;91:314–21. [11] Johnson DA, Tzu-Hua WU, Pui-Kai LI, Maher TJ. The effect of steroid sulfatase inhibition on learning and spatial memory. Brain Res 2000;865:286–90. [12] Kalimi M, Regelson W., editors. Dehydroepiandrosterone (DHEA): biochemical, physiological and clinical aspects. Berlin: Walter de Gruyter; 2000. [13] Ngai LY, Herbert J. Postulated roles of DHEA in the decline of neural function with age. In: Morfin R, editor. DHEA and the brain. London: Taylor and Francis; 2002. p. 40–64. [14] Trincal M, Loeper D, Pompon D, Hauw JJ, Morfin R. DHEA metabolism in the brain production and effects of the 7␣hydroxylated derivative. In: Morfin R, editor. DHEA and the brain. London: Taylor and Francis; 2002. p. 117–28. [15] Morfin R. Involvement of steroids and cytochromes P450 species in the triggering of immune defense (review). J Steroid Biochem Mol Biol 2002;80:273–90. [16] Pringle AK, Schmidt W, Deans JK, Wulfert E, Reymann KG, Sundstron LE. 7-Hydroxylated epiandrosterone (7-OH-EPIA) reduces ischaemia-induced neuronal damage both in vivo and in vitro. Eur J Neurosci 2003;18:117–24. [17] Attal-Kh´emis S, Dalmeyda V, Morfin R. Change of 7␣-hydroxydehydroepiandrosterone levels in serum of mice treated by cytochrome P450-modifying agents. Life Sciences 1998;63:1543–53. [18] Doostzadeh J, Cotillon AC, Morfin R. Dehydroepiandrosterone 7alpha- and 7-beta-hydroxylation in mouse brain microsomes Effects of cytochrome P450 inhibitors and structure-specific inhibition by steroid hormones. J Neuroendocrinol 1997;9:923–8.

674

H. Kazihnitkov´a et al. / Steroids 69 (2004) 667–674

[19] Stapleton G, Steel M, Richardson M, Mason JO, Rose KA, Morris RG, et al. A novel cytochrome P450 expressed primarily in brain. J Biol Chem 1995;270:29739–45. [20] Rose K, Allan A, Gauldie S, Stapleton G, Dobbie L, Dott K, et al. Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation. J Biol Chem 2001;276:29937–44. [21] Robel P, Baulieu EE. Dehydroepiandrosterone is a neuroactive steroid. Ann NY Acad Sci USA 1995;774:82–110. [22] Liere P, Akwa Y, Weill-Engerer S, Eychenne B, Pianos A, Robel P, et al. Validation of an analytical procedure to measure trace amounts

of neurosteroids in brain tissue by gas chromatography–mass spectrometry. J Chromat B 2000;739:301–12. [23] Lapcik O, Hampl R, Hill M, Starka L. Immunoassay of 7-hydroxysteroids: 2 Radioimmunoassay of 7-alpha-hydroxy-dehydroepiandrosterone. J Steroid Biochem Mol Biol 1999;71:231–7. [24] Lapcik O, Hampl R, Hill M, Bicikova M, Starka L. Immunoassay of 7-hydroxysteroids: 1. Radioimmunoassay of 7beta-hydroxy-dehydroepiandrosterone. J Steroid Biochem Mol Biol 1998;67:439–45. [25] St´arka L, D¨ollefeld E, Breuer H. Biogenese von freiem und sulfatiertem 7␣-hydroxy-androstenolon in Zellfraktionen der Rattenleber. Hoppe-Seyler’s Z Physiol Chem 1967;348:293–302.