• liquid chromatography-tandem mass spectrometry;
  • neurosteroids;
  • pregnenolone;
  • progesterone metabolites;
  • relapsing-remitting multiple sclerosis;
  • testosterone metabolites


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
Thumbnail image of graphical abstract

Neuroactive steroid family includes molecules synthesized in peripheral glands (i.e., hormonal steroids) and directly in the nervous system (i.e., neurosteroids) which are key regulators of the nervous function. As already reported in clinical and experimental studies, neurodegenerative diseases affect the levels of neuroactive steroids. However, a careful analysis comparing the levels of these molecules in cerebrospinal fluid (CSF) and in plasma of multiple sclerosis (MS) patients is still missing. To this aim, the levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in CSF and plasma of male adults affected by Relapsing-Remitting MS and compared with those collected in control patients. An increase in pregnenolone and isopregnanolone levels associated with a decrease in progesterone metabolites, dihydroprogesterone, and tetrahydroprogesterone was observed in CSF of MS patients. Moreover, an increase of 5α-androstane-3α,17β-diol and of 17β-estradiol levels associated with a decrease of dihydrotestosterone also occurred. In plasma, an increase in pregnenolone, progesterone, and dihydrotestosterone and a decrease in dihydroprogesterone and tetrahydroprogesterone levels were reported. This study shows for the first time that the levels of several neuroactive steroids, and particularly those of progesterone and testosterone metabolites, are deeply affected in CSF of relapsing-remitting MS male patients.

We here demonstrated that, the cerebrospinal fluid and plasma levels of several neuroactive steroids are modified in relapsing remitting multiple sclerosis male patients. Interestingly, we reported for the first time that, the levels of progesterone and testosterone metabolites are deeply affected in cerebrospinal fluid. These findings may have an important relevance in therapeutic and/or diagnostic field of multiple sclerosis.

Abbreviations used





blood–brain barrier


central nervous system


cerebrospinal fluid








liquid chromatography-tandem mass spectrometry


multiple sclerosis









As demonstrated in humans (Weill-Engerer et al. 2002; Luchetti et al. 2011; Noorbakhsh et al. 2011; Melcangi et al. 2013) and rodents (Labombarda et al. 2006; Meffre et al. 2007; Caruso et al. 2010, 2013a; Giatti et al. 2010; Pesaresi et al. 2010; Noorbakhsh et al. 2011; Melcangi et al. 2014), neurodegenerative diseases alter the levels of key regulators of the nervous system, such as the neuroactive steroids (Melcangi et al. 2011; Panzica et al. 2012). Therefore, changes in neuroactive steroid levels, that can reliably be measured in plasma or in cerebrospinal fluid (CSF), might represent possible biomarkers of pathological alterations in the nervous system. Incidence, progression, and severity of multiple sclerosis (MS), an inflammatory, demyelinating disease of the central nervous system (CNS) with relevant neurodegenerative aspects have well-recognized sex-dependent features. The incidence of MS is higher in female with approximately a 2 : 1 ratio (Melcangi and Garcia-Segura 2010) and the disease mainly affects young and post-pubertal women with a relapsing-remitting course. However, women experience a more benign course (Melcangi and Garcia-Segura 2010), and men have a worse long-term prognosis in terms of risk of reaching severe disability (Confavreux et al. 2003). In agreement with these gender-related features of MS, physiological situations characterized by changes in sex steroid plasma levels, such as menstrual cycle, menopause, or pregnancy affect the disease course (Vukusic and Confavreux 2006; Melcangi and Garcia-Segura 2010). Finally, MS may also affect sex steroid plasma levels by itself (Foster et al. 2003; Safarinejad 2008), suggesting that these molecules might be somewhat relevant in MS pathogenesis. Additional support to a role of neuroactive steroids in MS is provided by our recent observations. In experimental autoimmune encephalomyelitis (EAE), we have shown that the levels of neuroactive steroids are modified in the CNS in a sex-dimorphic way, depending on the acute and chronic phases of the disease (Caruso et al. 2010; Giatti et al. 2010).

A careful analysis comparing neuroactive steroid levels in CSF and in matched plasma samples in male MS patients is still missing. In this study, the levels of several neuroactive steroids, such as pregnenolone (PREG), progesterone (PROG) and its derivatives, dihydroprogesterone (DHP), tetrahydroprogesterone (THP also known as allopregnanolone), and isopregnanolone, dehydroepiandrosterone (DHEA), testosterone (T) and its derivatives dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α-diol), and 17β-estradiol (17β-E) were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described (Caruso et al. 2013b) in plasma and CSF of male adult MS patients and compared to those present in male control patients.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

PREG, PROG, DHP, THP, isopregnanolone, T, DHT, 3α-DIOL, DHEA, and 17β-E were purchased from Sigma-Aldrich, Milano, Italy. 17,21,21,21-D4-PREG (D4-PREG) was kindly synthesized by Dr P. Ferraboschi (Dept. of Med. Biotech. & Translational Medicine, University of Milano, Italy); 2,2,4,6,6-17α,21,21,21-D9-PROG (D9-PROG) was obtained from Medical Isotopes, (Pelham, NH, USA); 2,3,4-13C3-17β-estradiol (13C3-17β-E) was obtained from Sigma-Aldrich. SPE cartridges (Discovery DS-C18 500 mg) were from Supelco, Milano, Italy. All solvents and reagents were HPLC grade (Sigma-Aldrich).

Study design and sample preparation

The aim of this study was to analyze neuroactive steroid levels in plasma and CSF samples from male adult MS patients, comparing these results with the analysis of plasma and CSF samples collected from male controls. CSF was collected from 26 male MS patients who underwent lumbar puncture at the Multiple Sclerosis Centre of Cagliari for diagnostic reasons (i.e., diagnosis of MS) between January 2011 and March 2013. The procedure was approved by the Ethics Committee of the University of Cagliari. At the moment of lumbar puncture, all MS patients were affected by relapsing-remitting MS (Polman et al. 2011). Moreover, CSF and plasma were collected from 12 subjects who underwent spinal anesthesia for orthopedic surgery at the San Gerardo Hospital of Monza. These subjects were carefully screened for the absence of any neurological disorder in their personal or family history and gave their written informed consent to the use for scientific purpose of the aliquot (approx 100–200 μL) of CFS drawn to verify the correct position of the spinal needle, according to the procedure approved by the Ethics Committee of the S. Gerardo Hospital in Monza.

Blood–brain barrier damage examination and isoelectrofocusing analysis

Paired CSF and plasma samples were examined for blood–brain barrier damage (i.e., Reiber's quotient diagram); isoelectrofocusing analysis was performed and classified according to Consensus Statement (Freedman et al. 2005).

Quantitative analysis of neuroactive steroids by LC-MS/MS

Extraction and purification of the samples were performed as previously described (Caruso et al. 2013b). Briefly, samples were spiked with 13C3-17β-E (1 ng/sample), D9-PROG (0.2 ng/sample), and D4-PREG (5 ng/sample), as internal standards (IS) and homogenized in MeOH/acetic acid (99:1 v/v) using a tissue lyser (Qiagen, Jesi, Italy). After an overnight extraction at 4°C, samples were centrifuged at 15 300 g for 5 min and the pellet was extracted twice with 1 mL of MeOH/acetic acid (99:1 v/v). The organic residues were resuspended with 3 mL of MeOH/H20 (10:90 v/v) and passed through a SPE cartridge, the steroids were eluted in MeOH, concentrated and transferred in autosampler vials before the LC-MS/MS analysis.

Calibration curves

Quantitative analysis was performed on the basis of calibration curves daily prepared and analyzed as described previously (Caruso et al. 2013b). Linear least-square regression analysis was performed and, in addition, a blank (non-spiked sample) and a zero sample (only spiked with IS) were run to demonstrate the absence of interferences at the retention times and m/z corresponding to all the analytes. Moreover, the precision of the assay, inter-assay accuracy, precision, and reproducibility are calculated as described in (Caruso et al. 2013b) and are within tolerance range for all the neuroactive steroids.

Instrumental conditions

Positive atmospheric pressure chemical ionization (APCI+) experiments were performed with a linear ion trap – mass spectrometer (LTQ, ThermoElectron Co, San Jose, CA, USA) using nitrogen as sheath, auxiliary, and sweep gas. The instrument was equipped with a Surveyor liquid chromatography (LC) Pump Plus and a Surveyor Autosampler Plus (ThermoElectron Co). The mass spectrometer (MS) was employed in tandem mode (MS/MS) using helium as collision gas.

The LC mobile phases were previously described (Caruso et al. 2013b). The Hypersil Gold column (100 × 3 mm, 3 μm; ThermoElectron Co) was maintained at 40°C. Peaks of the LC-MS/MS were evaluated using a Dell workstation by means of the software Excalibur® release 2.0 SR2 (ThermoElectron Co). Samples were analyzed using the transitions previously reported (Caruso et al. 2013b).

Statistical analysis

The linearity of the standard curve (r2) and all the validation parameters of the method were judged by GraphPad4 PRISM (version 5). Student's t-tests were used to compare control subjects and MS patients. A p-value of < 0.05 was considered significant.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

Levels of neuroactive steroids were assessed by LC-MS/MS in paired CSF and plasma samples obtained from 26 male patients affected by relapsing-remitting MS and compared to the levels observed in 12 age-matched male controls. As reported in Table 1, mean age of MS patients was 34 years, expanded disability status scale median was 2.0 and mean time from onset of MS was 1.96 years (Min 0, Max 11). Blood–brain barrier damage examination showed low or moderate damage in 12 (46%) patients. Isoelectrofocusing analysis was performed in all patients: 24 (92.4%) patients had type 2 Oligoclonal Bands (OB), 1 (3.8%) patient had type 1 OB, and 1 (3.8%) patient had type 3 OB. Mean age of the control subjects was 29 years (p = 0.145 vs. MS patients).

Table 1. Demographic, clinic, and cerebrospinal fluid characteristics of multiple sclerosis patients
Number of patients26
Age (mean)34.5 years old
EDSS (median)2.0 (Min 0–Max 3.5)
 Features at lumbar puncture timeSteroid therapy timing
  Ongoing RelapseRemittedStabilized   
 Relapsing Remitting-relapse occurred within 30 days- -relapse occurred before 30 days and within 60 days--relapse occurred before 60 days-within 30 days30 days-6 months>6 months
 BBB damageOligoclonal bands NOligoclonal bands Type
 NOLowModerate< 33–6> 6Type1Type2Type3Type4

As reported in Fig. 1, the levels of PREG (i.e., the first steroid formed from cholesterol by the action of the enzyme P450 side chain cleavage) were significantly higher in CSF and plasma of MS patients in comparison to those observed in controls. PROG, which is synthesized from PREG by the enzyme 3β-hydroxysteroid dehydrogenase, was significantly higher in the plasma, but not in CSF of MS patients. PROG is then converted by the action of the enzyme 5α-reductase into DHP and this is then further converted by the enzyme 3α-hydroxysteroid oxido-reductase into THP; the levels of these two PROG metabolites showed a significant decrease both in CSF and plasma of MS patients. On the contrary, the levels of another PROG metabolite, isopregnanolone, which is formed from DHP by the action of the enzyme 3β-hydroxysteroid oxido-reductase, were increased in CSF, but not in plasma.


Figure 1. Pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), and isopregnanolone levels in CSF and in plasma of control (CTRL) and multiple sclerosis (MS) patients. Data (n = 12 for CTRL and 26 for MS patients) are expressed as pg/μL ± SEM. **p < 0.01; ***p < 0.001; the detection limit for THP and isopregnanolone is < 0.1 pg/μL.

Download figure to PowerPoint

Figure 2 reports the levels of DHEA and of its metabolites. Data obtained indicate that DHEA and its metabolite, T, were unchanged both in CSF and in plasma of MS patients. On the contrary, DHT, which is synthesized from T by the action of the enzyme 5α-reductase, was significantly decreased in CSF but significantly increased in plasma. DHT is then further metabolized by the enzyme 3α-hydroxysteroid oxido-reductase into 3α-diol; this steroid was significantly increased in CSF and resulted under detection limit in plasma of both controls and MS patients. Levels of 17β-E, which is formed from T by the action of the enzyme aromatase, were significantly decreased in CSF, while in plasma were under detection limit both in MS patients and controls.


Figure 2. Dehydroepiandrosterone (DHEA), testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α, 17β-diol (3α-diol), and 17β-estradiol (17β-E) levels in CSF and in plasma of control (CTRL) and multiple sclerosis (MS) patients. Data (n = 12 for CTRL and 26 for MS patients) are expressed as pg/μL ± SEM. **p < 0.01; ***p < 0.001; the detection limit for 3α-diol and 17β-E is < 0.05 and < 0.02 pg/μL, respectively.

Download figure to PowerPoint

Neuroactive steroid levels were also analyzed in correlation with the timing of methylprednisolone administration (i.e., within 30 days, 30 days–6 months and >6 months). No statistically significant difference among these three groups was observed (data not shown).


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

Data reported here indicate an increase in the PREG, PROG, and DHT, associated with a decrease in DHP and THP levels, in the plasma of male patients affected by relapsing-remitting MS. Similar to what has been observed in acute and chronic phases of EAE model (Caruso et al. 2010; Giatti et al. 2010), a decrease in DHP levels was detected in the plasma of MS patients. However, here, for the first time, we have also measured the levels of neuroactive steroids in CSF of male MS patients. The changes in CSF we observed were only partially reproduced in plasma. For instance, while increase of PREG and decrease of DHP and THP occurred in both plasma and CSF, PROG levels were not modified in CSF, and levels of DHT were significantly increased in plasma, but decreased in CSF. Moreover, neuroactive steroids that were unmodified in plasma were affected in CSF. Indeed, we observed an increase in isopregnanolone and 3α-diol, associated with a decrease in 17β-E. In this context, it is interesting to note that similar changes have been reported in the chronic phase (i.e., a phase more similar to relapsing-remitting MS) of EAE model in Dark Agouti male rats (Caruso et al. 2010). In these animals, a decrease of DHP and DHT in spinal cord and an increase of isopregnanolone in cerebral cortex were demonstrated (Caruso et al. 2010). Altogether, these observations indicate that an increase of PREG was linked to changes in the levels of PROG (i.e., DHP, THP, and isopregnanolone) and T (DHT and 3α-diol) metabolites as well as of 17β-E. Our observations also indicate that the plasma levels of T were not significantly modified in relapsing-remitting MS patients. It is interesting to note that the situation seems to be different in progressive MS. Indeed, as reported by others (Wei and Lightman 1997; Safarinejad 2008), the plasma levels of T are significantly decreased in progressive MS in comparison to what has been observed in relapsing-remitting MS patients. This finding might suggest a different impairment depending on the forms and/or phases of MS. However, it should be noted that differences in age of the patients (i.e., relapsing-remitting MS patients are generally younger in comparison to progressive MS patients) and in the analytical methodology applied (mass spectrometrometry vs. radioimmunoassay) can help explain the differences existing among our current and previous observations (Wei and Lightman 1997; Safarinejad 2008).

It is interesting to note that not only the pathology affect the levels of neuroactive steroids present in plasma and CSF differently but also that the neuroactive steroid profile present in plasma and CSF of control subjects is quite different. We reported similar findings in rodents, where the plasma levels of these molecules are not fully correlated with their levels in CSF (Caruso et al. 2013b). This seems to be particularly evident in the case of PROG and T metabolites. Indeed, DHP, THP, DHT, and 17β-E levels in CSF are significantly higher than those in plasma (p < 0.001 in case of DHP and 17β-E; p < 0.05 in case of THP and DHT). In this context, it is important to highlight that intensive PROG and T metabolism occur in the nervous system (Melcangi et al. 2008). On this basis, a different contribution to the levels of CSF and plasma might be hypothesized.

The observed changes in neuroactive steroid levels may be relevant not only to better understand MS pathophysiology but also to design new possible therapeutic strategies. Indeed, neuroactive steroids exert important protective effects. For instance, 17β-E regulates reactive microglia activation and the release of proinflammatory cytokines (Kipp and Beyer 2009; Cerciat et al. 2010), while estriol promotes release of anti-inflammatory cytokines, like TGF-β and IL-10 (Papenfuss et al. 2011). PROG decreases the proliferation of microglia and reactive astrocytes and microglia activation (Garcia-Segura and Melcangi 2006). Moreover, it increases the proliferation and cellular branching of oligodendrocyte precursors and the rate of myelination in vitro (Garcia-Segura and Melcangi 2006). Furthermore, PROG increases myelin basic protein expression in explant cultures of cerebellar slices (Garcia-Segura and Melcangi 2006) and in the lesioned rat spinal cord experimental model (Labombarda et al. 2009). Finally, it induces a small but significant enhancement in remyelination of toxin-induced demyelination in aged male rats (Ibanez et al. 2004). On this basis, promising effects of estrogens, PROG, or T administration have already been ascertained in EAE model and in clinical study (Voskuhl and Palaszynski 2001; El-Etr et al. 2005; Nicot 2009; Spence and Voskuhl 2012; Khalaj et al. 2013; Melcangi et al. 2014).

However, few observations have been obtained about the possible effects of PROG and T metabolites, although some of these molecules, such as DHP and THP, have well-established neuroprotective properties and may mediate the neuroprotective actions exerted by PROG and decrease microglia proinflammatory responses (Garcia-Segura and Melcangi 2006). In EAE model, DHT reduces pro-inflammatory IFN-γ, whereas it increases anti-inflammatory IL-10 levels (Dalal et al. 1997), and THP treatment reduces the immunoreactivity of Iba1, the monocytoid cell marker, and CD3ε, a lymphocytic marker, in lumbar spinal cord (Noorbakhsh et al. 2011). In particular, THP diminishes phorbol ester myristate-induced expression of pro-inflammatory genes in primary monocyte-derived macrophage cultures, while it does not affect antigen-specific proliferation of CD4+ T-cells, as well as the expression of IFN-γ and IL-17 in vitro in the presence of myelin oligodendrocyte glycoprotein (Noorbakhsh et al. 2011). These results, together with the absence of monocytoid and lymphocytic markers in the spinal cord, suggest that THP specifically inhibits the activation of both macrophages and microglia as well as the penetration of circulating lymphocytes and macrophages toward the CNS, thus preventing the exacerbation of the immune response. Consistent with the role of a deregulated inflammatory response in the progression of CNS diseases, THP treatment diminishes EAE severity (Noorbakhsh et al. 2011).

Based on these experimental observations, PROG or T metabolites, or synthetic ligands capable of interacting with their receptors, might be considered as an interesting approach to MS treatment. In this context, it is important to highlight that DHP and DHT, metabolites of PROG and T, respectively, still maintain the capacity to interact with their classical steroid receptors (i.e., PR and AR), but the subsequent metabolism of DHP and DHT results in the biosynthesis of neuroactive steroids that are able to bind to non-classical steroid receptors such as the GABA-A receptor (Melcangi et al. 2008). Thus, THP (i.e., the metabolite of DHP) and 3α-diol (i.e., metabolite of DHT) activate GABA-A receptor, while isopregnanolone (i.e., a further metabolite of DHP), which does not bind directly to the GABA-A receptor, can antagonize the effect of THP on the GABA-A receptor (Melcangi et al. 2008).

The present results also suggest the possible importance of these metabolites as relevant biomarkers, since it has been proposed that the pathology of the normal appearing white matter in MS, which is characterized by structurally altered and less stable myelin, as well as the axonal loss, failure of remyelination and the development of focal lesions might all be related to a dysregulation of neuroactive steroid synthesis (Leitner 2010).

In conclusion, the present observations demonstrate for the first time that the CSF levels of several neuroactive steroids, and in particular the metabolites of PROG and T, are deeply affected by relapsing-remitting MS in male patients. These findings may have an important relevance in therapeutic and/or diagnostic field of MS.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

We wish to thank Bayer S.p.A. for the financial support. The authors declare no conflict of interest.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
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