Methods in endogenous steroid profiling - A comparison of gas chromatography mass spectrometry (GC-MS) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS).

In various fields of endocrinology, the determination of steroid hormones synthesised by the human body plays an important role. Research on central neurosteroids has been intensified within the last years, as they are discussed as biomarkers for various cognitive disorders. Their concentrations in cerebrospinal fluid (CSF) are considered to be regulated independently from peripheral fluids. For that reason, the challenging matrix CSF becomes a very interesting specimen for analysis. Concentrations are expected to be very low and available amount of CSF is limited. Thus, a comprehensive method for very sensitive quantification of a set of analytes as large as possible in one analytical aliquot is desired. However, high structural similarities of the selected panel of 51 steroids and steroid sulfates, including numerous isomers, challenges achievement of chromatographic selectivity. Since decades the analysis of endogenous steroids in various body fluids is mainly performed by gas chromatography (GC) coupled to (tandem) mass spectrometry (MS(/MS)). Due to the structure of the steroids of interest, derivatisation is performed to meet the analytical requirements for GC-MS(/MS). Most of the laboratories use a two-step derivatisation in multi-analyte assays that was already published in the 1980s. However, for some steroids this elaborate procedure yields multiple isomeric derivatives. Thus, some laboratories utilize (ultra) high performance liquid chromatography ((U)HPLC)-MS/MS as alternative but, even UHPLC is not able to separate some of the isomeric pairs. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to GC and (U)HPLC may help to overcome these issues. Within this project the two most promising methods for endogenous steroid profiling were investigated and compared: the "gold standard" GC-MS and the orthogonal separation technique SFC-MS/MS. Different derivatisation procedures for gas chromatographic detection were explored and the formation of multiple derivatives described and confirmed. Taken together, none of the investigated derivatisation procedures provided acceptable results for further method development to meet the requirements of this project. SFC with its unique selectivity was able to overcome these issues and to distinguish all selected steroids, including (pro-)gestagens, androgens, corticoids, estrogens, and steroid sulfates with appropriate selectivity. Valued especially in the separation of enantiomeric analytes, SFC has shown its potential as alternative to GC. The successful separation of 51 steroids and steroid sulfates on different columns is presented to demonstrate the potential of SFC in endogenous steroid profiling.

New steps forward in the neuroactive steroid field.

Evidence accumulated in recent years suggests that the systemic treatment with neuroactive steroids, or the pharmacological modulation of its production by brain cells, represent therapeutic options to promote neuroprotection. However, new findings, which are reviewed in this paper, suggest that the factors to be considered for the design of possible therapies based on neuroactive steroids are more complex than previously thought. Thus, although as recently reported, the nervous system regulates neuroactive steroid synthesis and metabolism in adaptation to modifications in peripheral steroidogenesis, the neuroactive steroid levels in the brain do not fully reflect its levels in plasma. Even, in some cases, neuroactive steroid level modifications occurring in the nervous tissues, under physiological and pathological conditions, are in the opposite direction than in the periphery. This suggests that the systemic treatment with these molecules may have unexpected outcomes on neural steroid levels. In addition, the multiple metabolic pathways and signaling mechanisms of neuroactive steroids, which may change from one brain region to another, together with the existence of regional and sex differences in its neural levels are additional sources of complexity that should be clarified. This complexity in the levels and actions of these molecules may explain why in some cases these molecules have detrimental rather than beneficial actions for the nervous system. This article is part of a Special Issue entitled 'Steroid Perspectives'.

Patients treated for male pattern hair with finasteride show, after discontinuation of the drug, altered levels of neuroactive steroids in cerebrospinal fluid and plasma.

Observations performed in a subset of patients treated for male pattern hair loss indicate that persistent sexual side effects as well as anxious/depressive symptomatology have been reported even after discontinuation of finasteride treatment. Due to the capability of finasteride to block the metabolism of progesterone (PROG) and/or testosterone (T) we have evaluated, by liquid chromatography-tandem mass spectrometry, the levels of several neuroactive steroids in paired plasma and cerebrospinal fluid (CSF) samples obtained from post-finasteride patients and in healthy controls. At the examination, post-finasteride patients reported muscular stiffness, cramps, tremors and chronic fatigue in the absence of clinical evidence of any muscular disorder or strength reduction. Although severity of the anxious/depressive symptoms was quite variable in their frequency, overall all the subjects had a fairly complex and constant neuropsychiatric pattern. Assessment of neuroactive steroid levels in CSF showed a decrease of PROG and its metabolites, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), associated with an increase of its precursor pregnenolone (PREG). Altered levels were also observed for T and its metabolites. Thus, a significant decrease of dihydrotestosterone (DHT) associated with an increase of T as well as of 3α-diol was detected. Changes in neuroactive steroid levels also occurred in plasma. An increase of PREG, T, 3α-diol, 3ß-diol and 17ß-estradiol was associated with decreased levels of DHP and THP. The present observations show that altered levels of neuroactive steroids, associated with depression symptoms, are present in androgenic alopecia patients even after discontinuation of the finasteride treatment. This article is part of a Special Issue entitled 'Sex steroids and brain disorders'.

Neuroactive steroid levels in plasma and cerebrospinal fluid of male multiple sclerosis patients.

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,17ß-diol and of 17ß-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.

Neuroactive steroid levels are modified in cerebrospinal fluid and plasma of post-finasteride patients showing persistent sexual side effects and anxious/depressive symptomatology.

INTRODUCTION: Observations performed in a subset of subjects treated with finasteride (an inhibitor of the enzyme 5α-reductase) for male pattern hair loss seem to indicate that sexual dysfunction as well as anxious/depressive symptomatology may occur at the end of the treatment and continue after discontinuation. AIM: A possible hypothesis to explain depression symptoms after finasteride treatment might be impairment in the levels of neuroactive steroids. Therefore, neuroactive steroid levels were evaluated in paired plasma and cerebrospinal fluid samples obtained from male patients who received finasteride for the treatment of androgenic alopecia and who, after drug discontinuation, still show long-term sexual side effects as well as anxious/depressive symptomatology. METHODS: The levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in three postfinasteride patients and compared to those of five healthy controls. MAIN OUTCOME MEASURES: Neuroactive steroid levels in plasma and cerebrospinal fluid of postfinasteride patients and healthy controls. RESULTS: At the examination, the three postfinasteride patients reported muscular stiffness, cramps, tremors, and chronic fatigue in the absence of clinical evidence of any muscular disorder or strength reduction. Severity and frequency of the anxious/depressive symptoms were quite variable; overall, all the subjects had a fairly complex and constant neuropsychiatric pattern. Assessment of neuroactive steroid levels in patients showed some interindividual differences. However, the most important finding was the comparison of their neuroactive steroid levels with those of healthy controls. Indeed, decreased levels of tetrahydroprogesterone, isopregnanolone and dihydrotestosterone and increased levels of testosterone and 17ß-estradiol were reported in cerebrospinal fluid of postfinasteride patients. Moreover, decreased levels of dihydroprogesterone and increased levels of 5α-androstane-3α,17ß-diol and 17ß-estradiol were observed in plasma. CONCLUSION: The present observations confirm that an impairment of neuroactive steroid levels, associated with depression symptoms, is still present in androgenic alopecia patients treated with finasteride despite the discontinuation of the treatment.

Peripheral neuroactive steroids may be as good as the steroids in the cerebrospinal fluid for the diagnostics of CNS disturbances.

To compare the predictivity of the neuroactive steroids in the cerebrospinal fluid and peripheral blood for the diagnostics of CNS disturbances, eighteen unconjugated steroids were quantified in the cerebrospinal fluid (CSF) from the 3rd ventricle and 18 unconjugated steroids and 7 steroid polar conjugates were measured in the serum using GC-MS and RIA. Eight postmenopausal women (56-78 years of age) and 7 men (22-88 years of age) with hydrocephalus were enrolled in the study. The sensitivity of the method ranged from low femtogram to low picogram levels depending on the steroid fragmentation pattern. Using multivariate regression, a model for simultaneous prediction of the CSF steroids from the serum steroids was completed. Then, the penetrability of the individual steroids across the blood-brain-barrier was evaluated and the sources of various brain steroids were estimated. Our data show that a part of the steroids may be synthesized de novo in the CNS. However, substantial part of the steroid metabolites may be synthesized in the CNS from the steroid precursors or directly transported from the periphery. The CNS in situ synthesis and transport from periphery might be complementary in some cases, i.e. brain synthesis might provide minimum level of steroids, which are indispensable for the CNS functions.

The importance of steroidomics in the study of neurodegenerative disease and ageing.

In this mini review, the importance of experimental steroidomics in the study of neurodegenerative disease and aging is discussed. Attention is focused on just one class of lipid which is based on the cyclopentanoperhydro-phenanthrene ring system. Experimental methods for steroidomic analysis are reviewed, and the potential to use these methods to diagnose disease and to gain a better understanding of neurodegenerative disorders is examined.

Decreased plasma and cerebrospinal fluid content of neuroactive steroids in Parkinson's disease.

The levels of the neuroactive steroids allopregnanolone (THP) and 5alpha-dihydroprogesterone (DHP) were quantified in the plasma of 11 (group 1) and in the liquor of 12 (group 2) Parkinson's disease (PD) patients using a gas-chromatographic/mass-spectrometric method. When compared with controls, both groups showed a significant decrease in DHP and THP concentrations. These decreases could be a useful marker of PD. Moreover, in view of the importance of GABA-ergic transmission to substantia nigra (SN) neurons and GABA-ergic modulation exerted by the two neuroactive steroids, our data indicate a global dysregulation of the SN GABA-ergic system in PD patients. Moreover, a lack of neuroprotective factors (i. e., GDNF, BDNF), promoted by DHP, may contribute to dopaminergic cell death.

Central nervous system monoamine correlates of social dominance in cynomolgus monkeys (Macaca fascicularis).

Social dominance is a fundamental component of both human and nonhuman primate sociality. However, its neurobiological correlates remain incompletely understood. We evaluated the association between dominance status and monoamine metabolite concentrations in cisternal cerebrospinal fluid (CSF) in adult male (n = 25) and female (n = 21) cynomolgus macaques (Macaca fascicularis) housed in unisexual social groups. Concentrations of the metabolites of dopamine (homovanillic acid [HVA]), norepinephrine (3-methoxy-4-hydroxyphenylglycol [MHPG]) and serotonin (5-hydroxyindoleacetic acid [5-HIAA]) were assayed. Dominant monkeys, both males and females, had significantly higher CSF HVA concentrations than did subordinates (p values <.05). Among males, but not females, dominants also had lower CSF 5-HIAA than subordinates (p <.05). The Dominance-HVA association observed here is consistent with recent speculation that social extraversion, a dominance-related personality trait in humans, may also reflect heightened central nervous system dopaminergic activity.

Profiling neurosteroids in cerebrospinal fluids and plasma by gas chromatography/electron capture negative chemical ionization mass spectrometry.

A quantitative method for the determination of allopregnanolone (5alpha,3alpha-THP) and related neurosteroids in CSF and plasma was established using gas chromatography/electron capture negative chemical ionization mass spectrometry (GC/ECNCI/MS). Neurosteroids were converted to carboxymethoxime, pentafluorobenzyl and trimethylsilyl derivatives and detected as intense (M-181)(-) fragment ions generated under the negative ion chemical ionization process. The response curves constructed using d(4)-dihydrotestosterone (DHT) and d(4)-5alpha,3alpha-THP as internal standards showed linearity in the concentration range of 10-1000 pg/ml. The variation of response ratios determined against internal standards over a 2-month period was less than 10%. Instrumental detection limits for most neurosteroids were in the low picogram range with the exception of progesterone and dihydroprogesterone (DHP) which were detected with approximately 10 times less sensitivity in comparison to other steroids. In conjunction with solid-phase extraction, this method allowed the quantification of at least four neurosteroids, including androsterone, testosterone, 5alpha,3alpha-THP, and pregnenolone in 1-2 ml of human cerebrospinal fluid (CSF). While the level of 5alpha, 3alpha-THP in human CSF was comparable to that in the human plasma, other steroid levels were significantly lower. Although individual CSF and plasma samples showed widely varying neurosteroid levels, species specificity appeared to exist. The levels of 5alpha, 3alpha-THP and pregnenolone in human CSF were higher than those of monkey CSF where these steroids were often not detected with our current detection limit. In comparison to human plasma, rat plasma samples contained considerably lower levels of androsterone and pregnenolone. Among THP stereoisomers, 5beta,3alpha-THP and 5alpha, 3beta-THP were observed only in human plasma, while 5beta,3beta-THP was detected only in rat plasma.

Nanoelectrospray mass spectrometry and precursor ion monitoring for quantitative steroid analysis and attomole sensitivity.

Nanoelectrospray ionization (nanoESI) mass spectrometry was performed on naturally occurring steroid sulfates and unconjugated steroids derivatized to their sulfate esters using precursor ion monitoring. Initially, an extraction method was developed based on a combinatorial approach employed to obtain the most efficient liquid/liquid extraction protocol. The new method allowed unconjugated steroids and their sulfated analogues to be isolated separately in a two-step procedure using diethyl ether/hexane (90:10, v/v) in the first step to extract the unconjugated steroids and chloroform/2-butanol (50:50, v/v) in the second step to extract steroid sulfates. Precursor ion scanning performed with a triple-quadrupole mass spectrometer was used to examine quantitatively the extracted unconjugated and sulfated steroids, where the recovery efficiency averaged 70 and 87%, respectively. In addition, some steroids could be structurally elucidated by employing tandem mass spectrometry. The limit of detection for steroid sulfates from the biological matrix was 200 amol/microL (approximately 80 fg/microL) with only 1 microL of sample being injected. Endogenous levels of the unconjugated and sulfated steroids were detected and quantified from physiological samples including urine and blood. Internal standards, pregnenolone-d4 sulfate and dehydroepiandrosterone-d2 (DHEA), were used for quantitation. Extraction and nanoESI analyses were also performed on cerebrospinal fluid where the neurosteroid DHEA sulfate was detected. The small amount of material consumed (typically less than 20% of the injection volume) suggests that nanoESI has even greater potential for high sensitivity when combined with nanoLC approaches, especially for monitoring reproductive and adrenal steroids, as well as for the analysis of the less abundant neurosteroids.

Steroid hormones and steroid hormone binding globulins in cerebrospinal fluid studied in individuals with intact and with disturbed blood-cerebrospinal fluid barrier.

We measured in simultaneously withdrawn cerebrospinal fluid (CSF) and serum samples from 56 endocrinologically grossly normal patients the concentrations of several lipophilic unconjugated steroids [i.e. dehydroepiandrosterone (DHEA), androstenedione, cortisol, progesterone, testosterone] and their hydrophilic counterparts, i.e. DHEA-sulfate, or hydrophilic binding proteins, i.e. albumin, sex hormone-binding globulin (SHBG) and corticosterone-binding globulin (CBG). CSF levels of total (i.e. free plus protein-bound) DHEA, androstenedione, cortisol, progesterone, and testosterone were found to be in the 0.02-2 nM range and only cortisol reached levels approximately 20 nM. These values were of the same order of magnitude as the reported and calculated free serum levels of these steroids. In patients with disturbed (abnormally leaky) blood-CSF barrier (BCB) function, CSF levels of these steroids were not different from those with intact BCB, in contrast to DHEA-sulfate, CBG and SHBG whose CSF levels were significantly elevated, that is similar (i.e. 2-5) fold as those of albumin. In vitro demonstrated low affinity (micromolar) interactions of steroids with neuronal membrane-bound neurotransmitter receptors should be considered in perspective to the here reported finding that steroids occur in vivo at best in nanomolar concentrations in the CSF. Whether in other extracellular fluid compartments of the brain higher levels of steroids than in CSF can accumulate is as yet not clear. Very probably, pathological production or excessive dosage of steroids that are negligibly bound to SHBG or CBG will produce CSF and brain levels in the near micromolar range.

Demonstration of sex hormone binding globulin in human cerebrospinal fluid.

This study demonstrates the existence of sex hormone binding globulin (SHBG) in human cerebrospinal fluid (CSF) by means of a highly sensitive radioligand saturation assay that has been recently described by us for measurement of SHBG in human plasma. The molecular similarity of this 5 alpha-dihydrotestosterone binding protein with plasma-SHBG was substantiated by a number of experiments in which the CSF-protein displayed the same properties as plasma-SHBG with respect to thermolability, affinity, specificity and sedimentation rate. SHBG levels in the CSF of normal women were found to be 0.139 +/- 0.04 nmol/l (mean +/- standard deviation), and in normal men to be 0.083 +/- 0.03 nmol/l respectively. CSF-SHBG in patients with a variety of neurological diseases associated with different degrees of a blood-CSF barrier disturbance, showed a good correlation with commonly determined parameters such as CSF-albumin and CSF-IgG that are known to be of plasma origin. The concept of CSF-SHBG originating from plasma by restricted diffusion is strongly supported by the finding that the CSF/plasma ratio of SHBG is independent of the plasma-SHBG concentration in the entire physiological range. Possible diagnostic and pathophysiological implications of this so far undetected CSF-constituent are discussed with regard to neurological and endocrine abnormalities.

Physiological neuroendocrinology of peptides, steroids and other hormones in cerebrospinal fluid.

Cerebrospinal fluid acts as a conduit in neuroendocrine regulation. Valid assessment of normal cerebrospinal fluid levels of peptides, steroids and other hormones requires clarification of reference concentrations in control patients and normal volunteers. Awareness of factors which may alter neuronal activity and, in turn, the relative composition of cerebrospinal fluid constituents is essential to the accurate sampling and hormonal analysis of cerebrospinal fluid.

Neuroendocrinology of cerebrospinal fluid: peptides, steroids, and other hormones.

Cerebrospinal fluid (CSF) has been implicated as a conduit in neuroendocrine integration. Evidence suggests that the ventricular CSF may promote the central distribution, enable the dilutional inactivation (sink effect), and facilitate the peripheral delivery of neurally secreted hormones. This discussion of the sites of origin and concentration gradients of CSF hormones and of both physiological and pharmacological variations in the hormonal content of the CSF provides insight into the putative role of CSF in neuroendocrine regulation. Normal or control concentrations of peptides, steroids, and other hormones present in human lumbar CSF are listed to provide a physiological base line to which the CSF hormonal profile of patients may be compared. The individual, somatotopic, chronological, endocrinological, pharmacological, and possible artifactual variations in CSF hormonal composition are presented to facilitate the formulation of clinical protocols and to eliminate possible sources of error.