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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 11 5199-5206
Copyright © 2003 by The Endocrine Society

Neurosteroids: Cerebrospinal Fluid Levels for Alzheimer’s Disease and Vascular Dementia Diagnostics

Sae-Bom Kim, Martin Hill, Yong-Tae Kwak, Richard Hampl, Do-Hyun Jo and Robert Morfin

Biotechnologie, Conservatoire National des Arts et Métiers (S.-B.K., R.M.), 75003 Paris, France; Institute of Endocrinology (M.H., R.H.), 11694 Prague, Czech Republic; Yongin Hyoja Hospital (Y.-T.K.), Yongin-City, Kyonggi-do, 449-910, Korea; and Biotechnology, Ajou University (D.-H.J.), Suwon, 441-749, Paldal, Korea

Address all correspondence and requests for reprints to: R. Morfin, D.Sc., Biotechnologie, Conservatoire National des Arts et Métiers, 2 rue Conté, 75003 Paris, France. E-mail: morfin{at}cnam.fr.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
A neurodegenerative disease such as Alzheimer’s disease (AD) is associated with significantly higher dehydroepiandrosterone (DHEA) levels in cerebrospinal fluid (CSF). Because the human brain is known to transform DHEA into DHEA sulfate (DHEAS), 7{alpha}-hydroxy-DHEA, 7ß-hydroxy-DHEA, and 16{alpha}-hydroxy-DHEA, it is possible that DHEA accumulation in the brain results from a decreased production of such metabolites. To test this hypothesis, we have measured and compared CSF levels of DHEA, DHEAS, 7{alpha}-hydroxy-DHEA, 7ß-hydroxy-DHEA, and 16{alpha}-hydroxy-DHEA in 14 patients with AD, 12 controls, and eight patients with another common dementia, vascular dementia (VD). Results indicated that DHEAS CSF levels were significantly decreased in AD and VD (P < 0.007), whereas other metabolite levels were not significantly changed. Use of steroid level ratios, such as DHEA/(7{alpha}-hydroxy-DHEA + 7ß-hydroxy-DHEA), 7ß-hydroxy-DHEA/DHEA, and DHEAS/DHEA ratios, resulted in significant differences between diseased and control patients (P < 0.0003, P < 0.002, and P < 0.002, respectively). In addition, the 7{alpha}-hydroxy-DHEA/7ß-hydroxy-DHEA ratio was significantly different between AD and VD (P < 0.0001) and could be used for differentiating AD from VD. These results indicate that, in AD and VD, increased DHEA levels are not neuroprotective and are neither better sulfated nor better hydroxylated at the 7{alpha}, 7ß, and 16{alpha} positions than in controls. The results also suggest that, in AD and VD brains, the sulfotransferase and the cytochromes P450 responsible for the 7{alpha}-, 7ß-, and 16{alpha}-hydroxylations of DHEA are either present at lower levels or transformed through natural polymorphism into less-efficient enzymes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE NEUROSTEROID dehydroepiandrosterone (DHEA) is produced in the brain (1) and is further hydroxylated by the cytochrome P450 (P450) species, which leads to 7{alpha}-hydroxy-DHEA and 7ß-hydroxy-DHEA production (2, 3, 4). New findings in humans showed that only neurons contained the P45017{alpha} necessary for the oxidative cleavage of the pregnenolone (PREG) side chain and subsequent DHEA production (5). In contrast, glial cells use a putative alternate pathway leading to DHEA production through Fe2+- catalyzed oxidation of PREG (6, 7). Such DHEA production increases considerably in the presence of the ß-amyloid protein (5, 6) and leads to DHEA levels up to 40 times higher in the cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD) than in age-matched controls (7, 8). On the other hand, the human P4507B1, responsible for the 7{alpha}- hydroxylation of DHEA, is located mainly in the hippocampus (4) where neurons are the only cells expressing this P450 (9). Because of these findings, one may hypothesize that an overproduction of DHEA results both from the alternate pathway and from deficient DHEA sulfatation and hydroxylations in AD brains.

In contrast to plasma, CSF steroid levels reflect neurosteroid production and metabolism in the brain. Thus, CSF from patients of both sexes with dementia pathologies, such as AD and vascular dementia (VD), and CSF from nondemented control subjects were used for the measurement and comparison of neurosteroid levels. The neurosteroid levels examined were those of the DHEA precursors, PREG and PREG sulfate (PREGS), and those of DHEA, DHEA sulfate (DHEAS), 7{alpha}-hydroxy-DHEA, 7ß-hydroxy-DHEA, and 16{alpha}-hydroxy-DHEA. Our results show that levels of these neurosteroids in CSF may contribute to the diagnosis of neurodegenerative diseases and may be used to differentiate AD from VD.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Clinical cases and controls

This study was carried out with 34 subjects of both sexes (Table 1Go) and included 14 patients with probable AD [age: mean ± SD, 75.14 ± 9.88 yr; range, 53–88 yr; Mini-Mental State Evaluation (10): mean ± SD, 8.21 ± 7.29; range, 2–16; Clinical Dementia Rating (CDR) (11): mean ± SD, 1.69 ± 0.68; range, 1–5] and eight patients with VD (age: mean ± SD, 78.50 ± 4.82 yr; range, 71–87 yr; MMSE: mean ± SD, 15.63 ± 4.17; range, 11–21; CDR: mean ± SD, 1.94 ± 0.68; range, 0.5–3). All AD and VD patients were in an advanced stage of illness. To exclude secondary forms of dementia, cerebral tomographies were carried out for each patient before diagnosis. A pool of 12 nondemented patients were selected as controls who did not qualify for MMSE and CDR testing (Table 1Go). Clinical findings could emulate some sort of dementia in patients with hydrocephalus (typical clinical trials, gait disturbance, incontinence, and dementia). For each patient with hydrocephalus, the cause of the condition was identified as the disturbance of CSF resorption, which was proven both by lumbar infusion test and by positive effect of the therapy (implantation of ventriculo-peritoneal shunt). A sample of CSF (1 ml) was obtained from each patient and was frozen and lyophilized for transportation. Informed consent was obtained from all patients and all procedures were approved by the ethical committees of the Institute of Endocrinology of Prague and of the Yongin Hyoja Hospital of Yongin-City. The performances of each group of patients were compared with those obtained from the CSF of nondemented control patients.


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  		TABLE 1. Clinical cases involved in the present study

 
Neurosteroid measurements

DHEA and 16{alpha}-hydroxy-DHEA were from Steraloids (Newport, RI). 7{alpha}-Hydroxy-DHEA and 7ß-hydroxy-DHEA were prepared using the original procedure of Stárka (12), and 16{alpha}-hydroxy-DHEA was from Sigma-Aldrich (St. Louis, MO).

The lyophilized CSF samples were reconstituted in water (1 ml) for extraction with diethyl ether (3 ml) in a stoppered glass tube. The content of each tube was frozen, and the ether phase was transferred and dried in vacuum centrifuge (Heto, Melsungen, Germany) and further partitioned between 1 ml of methanol-water (8:1, vol/vol) and light petroleum ether. The nonpolar phase was removed, and 0.8 ml of the water-methanol phase was transferred into a glass tube and evaporated.

The HPLC system was from Gilson (Villiers Le Bel, France) and fitted with a ET 250/4 Nucleosil 100-5 C18 column from Macherey-Nägel (Düren, Germany). The data obtained were treated using Chromatography Station for Windows software from DataApex (Prague, Czech Republic).

HPLC separations used mobile phase A: 15% acetonitrile in water with 100 mg/liter of ammonium bicarbonate; mobile phase B: methanol; flow rate: 1 ml/min; and gradient: start, 1 min, 0% B; 1–11 min, linear gradient from 55–65% B; 11–14 min, 100% B; 14–20 min, 0% B; overall time, 20 min. Reference 7ß-hydroxy-DHEA, 7{alpha}-hydroxy-DHEA, 16{alpha}- hydroxy-DHEA, DHEA, and PREG were detected by UV at 205 nm [retention time (Rt) in min/collection window/dead time]: 7ß-hydroxy-DHEA (9.8/9.5–10.3/0.4); 7{alpha}-hydroxy-DHEA (11.6/11.1–11.9/+0.4); 16{alpha}-hydroxy-DHEA (12.9/12.7–13.5/0.4); DHEA (15.6/15.4–16.2/+0.4), and PREG (16.5/1.2–17.0/0.4). Based on these Rts, fractions from injected samples were collected automatically overnight in glass tubes. The dry residues were dissolved in methanol (100 µl) before further analysis.

RIA was used for the measurements of 7ß- and 7{alpha}-hydroxy-DHEA from HPLC fractions (13, 14) and for the measurements of DHEA and PREG with the commercial kit from Immunotech (Marseille, France). Both DHEAS and PREGS were recovered by ether extraction of the water residues. DHEAS levels were determined using the commercial kit from Immunotech, and PREGS levels were measured as previously described (15), except that 100 µl of the sample, instead of 10 µl, was taken out for analysis.

The gas chromatography (GC)/mass spectrometry (MS) system was supplied by SHIMADZU (Kyoto, Japan) and consisted of a GC 17A gas chromatograph equipped with automatic flow control, AOC-20 autosampler, and, for the MS, QP 5050A quadrupole electron-impact detector with a fixed electron voltage of 70 eV.

The HPLC individual fractions containing 16{alpha}-hydroxy-DHEA were derivatized by using a mixture of bis-(trimethylsilyl)trifluoroacetamide, trimethylchlorosilane (99:1, 10 µl), and pyridine (30 µl, 80 C, 45 min). Analysis by GC/MS used a medium-polarity column (ZEBRON ZB-50, 15 m x 0.25 mm, 0.15-µm film thickness, catalog no. 7EG-G004-05) (Phenomenex, Torrance, CA) and a simple quadrupole electron-impact MS detector in single-ion monitoring (SIM) mode. The standard mixtures used for calibration contained 17{alpha}-methylandrostenediol (1000 pg/µl) as an internal standard and one of the following: 1000 pg/µl, 100 pg/µl, or 10 pg/µl of 16{alpha}-hydroxy-DHEA, 7{alpha}-hydroxy-DHEA, or 7ß-hydroxy-DHEA. Standard mixtures and samples were derivatized and analyzed with the same procedure. SIM mode was at mass-to-charge ratio (m/z) 253, m/z 268 for internal standard (Rt = 3.25 min), m/z 266, 356, and 446 for 16{alpha}-hydroxy-DHEA (Rt = 4.19 min), and at m/z 343, 358, and 359 for 7{alpha}-hydroxy-DHEA (Rt = 13.6 min) and for 7ß-hydroxy-DHEA (Rt = 10.6 min).

The extraction yields for each steroid were determined after HPLC separation of the standard mixtures processed exactly as the samples. Recovery of steroids that underwent all of the separation steps was 64.7. The sensitivity of the analysis was 0.63 pg. The interassay coefficient of variation was 5.8%, and the response was linear within the range of physiological values of the steroids.

Statistical methods

For evaluation of the relationships between the steroids, a correlation analysis was applied and followed by principal component analysis (PCA). Special care was focused on the data pretreatment regarding non-Gaussian data distribution, heteroscedasicity (nonconstant variance), and nonhomogeneity. The data were transformed by power transformation to minimum skewness in individual dimensions. To eliminate the influence of univariate outliers, only the data with absolute studentized values less than 2 were considered. The multivariate outliers were searched using F-distributed Mahalanobis distance (statistical software NCSS 2000, NCSS, Kaysville, UT) after transformation. Adjustments of data for age and sex were performed using the analysis of covariance model in statistical software Statgraphics Plus 3.1. (Rockville, MD). Once treated, the data underwent correlation analysis followed by PCA (statistical software Statgraphics Plus 3.1) on the complete data matrix. The VARIMAX algorithm rotation was then performed for simplification and interpretation of results. The covariates of age and sex were transformed to minimum skewness to eliminate the occurrence of influential points, because the analysis of covariance model is a mixture of ANOVA and regression.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Based on the separation of steroid standards by HPLC (Fig. 1AGo), steroids from extracts were recovered at proper Rts and used for measurements by RIA procedures or GC/MS analysis. All steroids were measured by RIA, except for 16{alpha}-hydroxy-DHEA. GC/MS measurements were used for 16{alpha}-hydroxy-DHEA and were also used to confirm the identity of both 7{alpha}-hydroxy-DHEA and 7ß-hydroxy-DHEA. Thus, based on the characteristic fragment ions in mass spectra of authentic 7{alpha}-hydroxy-DHEA (343, 358, and 359; Fig. 1BGo), 7ß-hydroxy-DHEA (343, 358, and 359; Fig. 1CGo), and 16{alpha}-hydroxy-DHEA (266, 356, and 466; Fig. 1DGo), each steroid recovered after HPLC separation of CSF extract was derivatized and analyzed by SIM. Results provided 16{alpha}-hydroxy-DHEA levels in CSF and confirmation of the 7{alpha}-hydroxy-DHEA and 7ß-hydroxy-DHEA levels obtained by RIA.



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  		FIG. 1. HPLC separation of steroid standards detected by UV absorbance at 205 nm (A); mass spectrum of the trimethyl silyl ether (TMS) derivatives of authentic 7{alpha}-hydroxy-DHEA (B) and 7ß-hydroxy-DHEA (C) showing characteristic ion fragments at m/z 343, 358, and 359; and mass spectrum of the TMS derivatives of authentic 16{alpha}-hydroxy-DHEA (D) showing characteristic ion fragments at m/z 266, 356, and 446.

 
The raw data obtained in measurements of DHEAS, DHEA, 7{alpha}-hydroxy-DHEA, 7ß-hydroxy-DHEA, 16{alpha}-hydroxy- DHEA, PREG, and PREGS were adjusted for age differences and were used for the computation of ratios. Comparisons through statistical analysis showed that CSF levels of DHEA were lower in controls than in AD and VD patients (P < 0.02) and that CSF levels of DHEAS were higher in controls than in AD and VD patients (P < 0.007; Fig. 2Go, A and B). Taken together, these differences could help discrimination between controls and AD or VD patients.



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  		FIG. 2. Neurosteroid levels in the CSF of 14 patients with AD, eight patients with VD, and 12 nondemented control subjects (C). Significance of difference in levels between relevant paired groups is indicated. Relationships with age were analyzed using age as a covariate. 7{alpha}-OH-DHEA, 7{alpha}-hydroxy-DHEA; 7ß-OH-DHEA, 7ß-hydroxy-DHEA; 16{alpha}-OH-DHEA, 16{alpha}-hydroxy-DHEA; NS, not significant.

 
No significant difference was found between controls and AD and VD patients for CSF levels of PREG, PREGS, 7{alpha}-hydroxy-DHEA, 7ß-hydroxy-DHEA, and 16{alpha}-hydroxy-DHEA (Fig. 2Go, C–G).

In contrast, the DHEA/(7{alpha}-hydroxy-DHEA + 7ß-hydroxy- DHEA) ratio was significantly lower in controls than in AD and VD patients (P < 0.0003; Fig. 3AGo), and the 7ß-hydroxy-DHEA/DHEA ratio was significantly higher in controls than in AD and VD patients (P < 0.002; Fig. 3BGo). Specific differences between AD and VD were found; the 7{alpha}-hydroxy-DHEA/7ß-hydroxy-DHEA ratio was significantly lower in VD patients than in AD patients and controls (P < 0.0001; Fig. 3CGo), whereas the 7{alpha}-hydroxy-DHEA/DHEA ratio was found to be significantly higher in AD patients than in VD patients and controls (P < 0.02; Fig. 3DGo). Taken together, these discrepancies in CSF steroid ratios could help discriminate between controls and AD or VD.



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  		FIG. 3. Ratios of neurosteroid levels in the CSF of 14 patients with AD, eight patients with VD, and 12 control subjects (C). Significance of difference in ratio levels between relevant paired groups is indicated. Relationships with age were analyzed using age as a covariate. 7{alpha}-OH-DHEA, 7{alpha}-hydroxy-DHEA; 7ß-OH-DHEA, 7ß- hydroxy-DHEA; 16{alpha}-OH-DHEA, 16{alpha}- hydroxy-DHEA;NS, not significant.

 
Other CSF steroid ratios could be of help for differentiating AD and VD from controls. Thus, significant differences were found with the 7{alpha}-hydroxy-DHEA/DHEA ratio, with the DHEAS/DHEA ratio, and with the 16{alpha}-hydroxy-DHEA/DHEA ratio, which were significantly larger in controls than in AD and VD patients (P < 0.02, P < 0.002, and P < 0.005, respectively; Fig. 3Go, D–F).

Analysis with the covariate of sex provided no significant correlation with all steroid-dependent variables. Analysis with the covariate of age showed that it was positively correlated with the dependent variable 7ß-hydroxy-DHEA (Fig. 2FGo) and the DHEA/PREGS ratio (not shown) with statistical significance (P < 0.008).

A summarized presentation of the results obtained is shown in Fig. 4Go, in which known and unknown steroid metabolisms in the brain are related with the steroid CSF levels in controls and AD and VD patients.



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  		FIG. 4. DHEA metabolism in the brain of control subjects and of patients with AD or VD. The steroid levels shown in proportional font size were measured in the CSF. Italicized putative metabolites were not measured. Dashed arrows with question marks indicate enzymatic pathways that were not established in human brain. 7{alpha}-OH-DHEA, 7{alpha}-hydroxy-DHEA; 7ß-OH-DHEA, 7ß-hydroxy-DHEA; 16{alpha}-OH-DHEA, 16{alpha}-hydroxy-DHEA.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We found steroid levels in human CSF to be consistently different from those reported in serum and in brain. Thus, DHEA and DHEAS levels were highly correlated with serum and CSF, with much higher levels in serum than in CSF (16, 17). The levels of PREG and PREGS in CSF did not differ significantly from those reported in human serum (18, 19). These findings differ from those reported for human brain steroid levels, where DHEA, DHEAS, PREG, and PREGS were found in much higher levels than in serum and CSF (20, 21, 22). Thus, overall levels of DHEA, DHEAS, PREG, and PREGS reported in different-aged human brain regions were 2 orders of magnitude higher than the levels found in CSF (22). Therefore, it is possible that levels in CSF reflect only a portion of such steroids that are produced and differentially retained in brain.

Our findings show that, in the CSF of patients with AD and VD, DHEA and DHEAS levels are significantly increased and decreased, respectively, when compared with controls. These findings support those reported on CSF DHEA levels (6, 8) and those on DHEAS levels after hippocampal perfusion in AD (23). In AD, the occurrence of a ß-amyloid-magnified process involving reactive oxygen species and Fe2+ and catalyzed by an unknown protein may contribute to the increase in the DHEA levels (8). It is possible that such a process exists in VD because of the CSF DHEA levels similar to those of AD. Thus, AD and VD cannot be differentiated on the sole basis of CSF DHEA and DHEAS levels.

We report here for the first time CSF levels in 7{alpha}-hydroxy-DHEA, 7ß-hydroxy-DHEA, and 16{alpha}-hydroxy-DHEA. These steroids are derived from DHEA metabolism in human brain, where 7{alpha}-hydroxylation and 16{alpha}-hydroxylation are catalyzed by CYP7B1 and CYP3A7, respectively (7, 24, 25). Because knockout mice for P4507b1 produce neither 7{alpha}- hydroxy-DHEA nor 7ß-hydroxy-DHEA (26), 7ß-hydroxy-DHEA may derive from 7{alpha}-hydroxy-DHEA. The processes leading to 7ß-hydroxylation of DHEA in brain lead to questions and debates. Incubation of AD and control brain homogenates provided evidence for 7{alpha}-hydroxylation of DHEA but not for 7ß-hydroxy-DHEA production (27). Our experience with human P4507B1 expressed in yeast microsomes and incubated with reduced nicotinamide adenine dinucleotide phosphate showed that DHEA was 7{alpha}- and 7ß-hydroxylated in major and minor quantities, respectively (28). In any case, the P4507B1-catalyzed production yields of 7ß-hydroxy-DHEA are not sufficient for justifying the 7ß-hydroxy-DHEA levels found in CSF. Other 7ß-hydroxylation mechanisms in brain are at stake and may be based upon the facts that the brain mitochondria are a major source for 7{alpha}-hydroxylation and were not extensively studied (2) and that the 7ß-hydroxy-DHEA may arise from allylic interactions of its precursor with Fe2+-dependent noncytochromic brain peroxidases (29, 30). We found that the CSF levels of 7{alpha}-hydroxy-DHEA, 7ß-hydroxy-DHEA, and 16{alpha}-hydroxy-DHEA were not significantly different in AD and VD patients when compared with controls. Due to the high levels of DHEA, the ratios of 7{alpha}-hydroxy-DHEA/DHEA levels, 7ß-hydroxy-DHEA/DHEA levels, and 16{alpha}-hydroxy-DHEA/DHEA levels were significantly larger in controls than in AD and VD patients. We found a more significant difference between AD and VD patients and controls with examination of the DHEA/(7{alpha}-hydroxy-DHEA + 7ß-hydroxy-DHEA) ratio. This finding implies a decreased production of 7- hydroxylated metabolites in brain and is supported by the recent report of decreased 7{alpha}-hydroxylation in homogenates of AD brain when compared with controls (27). According to Michaelis enzyme kinetics, it is well known that velocities in product formation are augmented with increased substrate concentrations. A lesser augmentation is observed either when lower quantities of enzyme are present or when the Michaelis-Menten constant (Km) of the enzyme is increased. Increase in Km may be observed with amino acid changes in the enzyme sequence, as observed in many cases of P450 polymorphism. Thus, in AD brain, P4507B1 may either be present in lower quantities than in controls or be a polymorphic form resulting in a higher Km. Among precedents for human P450 polymorphism association with AD, one concerns CYP46 (31, 32) for which an intronic polymorphism was associated with AD through increased brain ß-amyloid load and CSF levels in ß-amyloid peptide and phosphorylated {tau} (33). Because excess cholesterol in hippocampal neurons promotes the cleavage of the amyloid precursor protein into amyloidogenic components with the consequence of the acceleration of neuronal degeneration (34), the P45046-mediated 24S-hydroxylation of cholesterol is the major pathway for the elimination of brain cholesterol and the maintenance of brain cholesterol homeostasis (35). The described CYP46 polymorphism leading to increased 24S-hydroxycholesterol/cholesterol ratio in the brain (31, 32, 33) may predispose to AD and is a risk factor for late-onset sporadic AD (33). Such a risk could be increased if a natural polymorphism of human P4507B1 exists and could result in a decrease in the neuroprotective function suggested by several authors for 7-hydroxylated DHEA metabolites (24, 26, 36, 37).

Yet, even though we obtained highly significant differences with these DHEA-depending ratios, they did not permit differentiation between AD and VD. In contrast, the ratio of 7{alpha}-hydroxy-DHEA/7ß-hydroxy-DHEA levels was very significantly decreased in VD patients when compared with controls and AD patients. This implies that in VD brain, 7ß-hydroxy-DHEA levels are higher than 7{alpha}-hydroxy-DHEA levels and that 7ß-hydroxy-DHEA may be more neuroprotective than its 7{alpha}-hydroxy-DHEA precursor. Therefore, we can propose that the 7{alpha}-hydroxy-DHEA/7ß-hydroxy-DHEA ratio could be of help in discriminating AD from VD.

Our results infer that the DHEA produced in brain and found in CSF does not help to protect brain from neurodegenerative disease onset and progression. Involvement of DHEAS in neuroprotection has been suggested through memory consolidation tests (38, 39) and from comparison of DHEAS levels in brain regions of AD patients and controls (22) where DHEAS levels were negatively correlated with those of ß-amyloid protein. We found that DHEAS levels were significantly decreased in the CSF of AD and VD patients. Examination of the DHEAS/DHEA ratio provided an even more clear-cut difference between AD and VD patients and controls. The fact that human brain contains a specific sulfotransferase (40) and may produce DHEAS from DHEA is documented (41, 42). It is presently unknown whether the 7{alpha}- and 7ß-hydroxylated metabolites of DHEA produced in human brain are substrates for the brain sulfotransferase and whether DHEAS is a substrate for 7-hydroxylation (Fig. 3Go). Putative productions of 7{alpha}- and 7ß-hydroxy-DHEAS are supported by reports of such a transformation in rat liver (43) and by the isolation and identification of 7{alpha}-hydroxy-DHEAS in human urine (44) and its measurement in human serum (45).

Reports discussing the 7-hydroxylated derivatives of DHEA neuroprotective potencies are available (24, 26, 36). Recent findings in the neuroprotection exerted by 5{alpha}- reduced 7-hydroxylated derivatives are also available (37). Neuroprotection may result either from 7{alpha}-hydroxy-DHEA and 7ß-hydroxy-DHEA or from their unknown sulfated metabolites. Nevertheless, putative neuroprotection by unknown 7{alpha}-hydroxy-DHEA- and 7ß-hydroxy-DHEA-sulfated metabolites cannot be ascertained yet. The finding that the 7{alpha}-hydroxy-DHEA/7ß-hydroxy-DHEA ratio is significantly lower in VD patients than in AD patients implies that CSF from VD patients contained relatively more 7ß-hydroxy-DHEA than CSF from AD patients. MMSE and CDR gave better scores for VD patients than for AD patients, and the 7ß-hydroxy-DHEA levels in VD patients may be related to the better scores and to a better neuroprotection. In addition, evidence exists for a better neuroprotection by a 7ß-hydroxysteroid than by a 7{alpha}-hydroxysteroid (37). Taken together, these findings suggest that 7ß-hydroxylated steroids are more potent than their 7{alpha} epimers for neuroprotection.

Our findings of 16{alpha}-hydroxy-DHEA in CSF imply that this steroid may be produced in brain after 16{alpha}-hydroxylation of DHEA by CYP3A7. Major amounts of CYP3A7 are found in human endometrium, placenta, and fetal liver (46). Other embryonic tissues contain negligible amounts of CYP3A7 (25). Even though adult human liver carries out 16{alpha}- hydroxylation of DHEA (47), no direct evidence is available for this transformation in brain. We found no significant difference in 16{alpha}-hydroxy-DHEA levels in CSF between controls and AD and VD patients. However, when compared with DHEA levels through the 16{alpha}-hydroxy-DHEA/DHEA ratio, significantly larger values were found in controls than in AD and VD patients. This finding demonstrates that in AD and VD, the large amounts of DHEA are not better 16{alpha}-hydroxylated than the smaller DHEA amounts in controls, and the putative transformation in brain of 16{alpha}-hydroxy-DHEA into its sulfated derivative is presently unknown.

In conclusion, significantly greater amounts of DHEA and lesser amounts of DHEAS are found in the CSF of patients with neurodegenerative diseases such as AD and VD. The well-studied procedure involving Fe2+ and reactive oxygen species in brain for DHEA production from PREG (5, 6, 8, 29, 30) may be the key for such a production. In AD and CD, the increased DHEA levels do not result in increased sulfatation or increased hydroxylation at the 7{alpha}, 7ß, and 16{alpha} positions. These findings suggest that, in AD and VD brain, the sulfotransferase and the P450s responsible for 7{alpha}- and 16{alpha}-hydroxylations of DHEA are either present in lower levels or transformed through natural polymorphism into less- efficient enzymes.


   Acknowledgments
 
We thank Dr. Milan Mohalp (Department of Neurosugery, Central Military Hospital, Prague, Czech Republic) for his help in the selection of control patients.


   Footnotes
 
This work was supported in part by Grant NB 6691-3 from Internal Grant Agency of the Czech Republic, by International Project Grant from Korean Science and Engineering Foundation (KOSEF), by a grant from Hunter-Fleming Ltd., and by travel grants from Association for Research with Industrial and Educational Links/KOSEF and from the French North Atlantic Treaty Organization fellowship program. S.-B.K. was a recipient of a French government fellowship for her doctorate in France.

Abbreviations: AD, Alzheimer’s disease; CDR, Clinical Dementia Rating; CSF, cerebrospinal fluid; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; GC, gas chromatography; Km, Michaelis- Menten constant; MMSE, Mini-Mental State Evaluation; MS, mass spectrometry; P450, cytochrome P450; PREG, pregnenolone; PREGS, PREG sulfate; Rt, retention time; SIM, single-ion monitoring; VD, vascular dementia.

Received April 15, 2003.

Accepted July 21, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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