Simultaneous Determination of Selected Steroids with Neuroactive Effects in Human Serum by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Neuroactive steroids are a group of steroid molecules that are involved in the regulation of functions of the nervous system. The nervous system is not only the site of their action, but their biosynthesis can also occur there. Neuroactive steroid levels depend not only on the physiological state of an individual (person's sex, age, diurnal variation, etc.), but they are also affected by various pathological processes in the nervous system (some neurological and psychiatric diseases or injuries), and new knowledge can be gained by monitoring these processes. The aim of our research was to develop and validate a comprehensive method for the simultaneous determination of selected steroids with neuroactive effects in human serum. The developed method enables high throughput and a sensitive quantitative analysis of nine neuroactive steroid substances (pregnenolone, progesterone, 5α-dihydroprogesterone, allopregnanolone, testosterone, 5α-dihydrotestosterone, androstenedione, dehydroepiandrosterone, and epiandrosterone) in 150 µL of human serum by ultrahigh-performance liquid chromatography with tandem mass spectrometry. The correlation coefficients above 0.999 indicated that the developed analytical procedure was linear in the range of 0.90 nmol/L to 28.46 µmol/L in human serum. The accuracy and precision of the method for all analytes ranged from 83 to 118% and from 0.9 to 14.1%, respectively. This described method could contribute to a deeper understanding of the pathophysiology of various diseases. Similarly, it can also be helpful in the search for new biomarkers and diagnostic options or therapeutic approaches.

Sex chromosome complement interacts with gonadal hormones in determining regional-specific neuroactive steroid levels in plasma, hippocampus, and hypothalamus. A study using the four core genotype mouse model.

An important aspect of the neuromodulatory and neuroprotective actions exerted by neuroactive steroids is that they are sex-specific, as determined by the sexually dimorphic levels of these molecules in plasma and the nervous tissue. Thus, the identification of the factors that generate the sex-dimorphic levels of neuroactive steroids may be crucial from a neuroprotectant perspective. The main driver for sex determination in mammals is the SRY gene and the subsequent presence of a specific gonad: testes for males and ovaries for females, thus producing hormonal compounds, primarily androgens and estrogens, respectively. Nowadays, it is well established that despite the relevance of gonads, other factors control sexual features, and, among them, sex chromosome complement is highly relevant. In this study, neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in the hypothalamus, the hippocampus, and plasma of the four core genotype mouse model, to determine the relative contribution of sex chromosome complement and gonads in determining their sex dimorphic levels. The data obtained reveal that although gonads are the main contributing factor for sex differences in neuroactive steroid levels, the levels of some neuroactive steroids, including testosterone, are also influenced in brain and plasma by tissue-specific actions of sex chromosomes. The data presented here adds a new piece to the puzzle of steroid level regulation, which may be useful in designing sex-specific neuroprotective approaches to pathological conditions affecting the nervous system.

Exploring the Impact of the Microbiome on Neuroactive Steroid Levels in Germ-Free Animals.

Steroid hormones are essential biomolecules for human physiology as they modulate the endocrine system, nervous function and behaviour. Recent studies have shown that the gut microbiota is directly involved in the production and metabolism of steroid hormones in the periphery. However, the influence of the gut microbiota on levels of steroids acting and present in the brain (i.e., neuroactive steroids) is not fully understood. Therefore, using liquid chromatography-tandem mass spectrometry, we assessed the levels of several neuroactive steroids in various brain areas and the plasma of germ-free (GF) male mice and conventionally colonized controls. The data obtained indicate an increase in allopregnanolone levels associated with a decrease in those of 5α-androstane-3α, 17ß-diol (3α-diol) in the plasma of GF mice. Moreover, an increase of dihydroprogesterone and isoallopregnanolone in the hippocampus, cerebellum, and cerebral cortex was also reported. Changes in dihydrotestosterone and 3α-diol levels were also observed in the hippocampus of GF mice. In addition, an increase in dehydroepiandrosterone was associated with a decrease in testosterone levels in the hypothalamus of GF mice. Our findings suggest that the absence of microbes affects the neuroactive steroids in the periphery and the brain, supporting the evidence of a microbiota-mediated modulation of neuroendocrine pathways involved in preserving host brain functioning.

Prenatal Exposure to Bisphenols and Phthalates and Postpartum Depression: The Role of Neurosteroid Hormone Disruption.

CONTEXT: Postpartum depression (PPD) is a serious psychiatric disorder. While causes remain poorly understood, perinatal sex hormone fluctuations are an important factor, and allopregnanolone in particular has emerged as a key determinant. Although synthetic environmental chemicals such as bisphenols and phthalates are known to affect sex hormones, no studies have measured allopregnanolone and the consequences of these hormonal changes on PPD have not been interrogated. OBJECTIVE: To investigate associations of repeated measures of urinary bisphenols and phthalates in early and midpregnancy with serum pregnenolone, progesterone, allopregnanolone, and pregnanolone concentrations in midpregnancy and PPD symptoms at 4 months postpartum. METHODS: Prospective cohort study of 139 pregnant women recruited between 2016 and 2018. Bisphenols and phthalates were measured in early and midpregnancy urine samples. Serum sex steroid hormone concentrations were measured in midpregnancy. PPD was assessed at 4 months postpartum using the Edinburgh Postnatal Depression Scale (EPDS). Multiple informant models were fit using generalized estimating equations. Serum levels of allopregnanolone, progesterone, pregnanolone, and pregnenolone were examined as log-transformed continuous variables. PPD symptoms were examined as continuous EPDS scores and dichotomously with scores ≥10 defined as PPD. RESULTS: Di-n-octyl phthalate (DnOP) and diisononyl phthalate (DiNP) metabolites were associated with reduced progesterone concentrations. Log-unit increases in ∑DnOP and ∑DiNP predicted 8.1% (95% CI -15.2%, -0.4%) and 7.7% (95% CI -13.3%, -1.7%) lower progesterone, respectively. ∑DnOP was associated with increased odds of PPD (odds ratio 1.48; 95% CI 1.04, 2.11). CONCLUSION: Endocrine disrupting chemicals may influence hormonal shifts during pregnancy as well as contribute to PPD.

Analytical Methods for the Determination of Neuroactive Steroids.

Neuroactive steroids are a family of all steroid-based compounds, of both natural and synthetic origin, which can affect the nervous system functions. Their biosynthesis occurs directly in the nervous system (so-called neurosteroids) or in peripheral endocrine tissues (hormonal steroids). Steroid hormone levels may fluctuate due to physiological changes during life and various pathological conditions affecting individuals. A deeper understanding of neuroactive steroids' production, in addition to reliable monitoring of their levels in various biological matrices, may be useful in the prevention, diagnosis, monitoring, and treatment of some neurodegenerative and psychiatric diseases. The aim of this review is to highlight the most relevant methods currently available for analysis of neuroactive steroids, with an emphasis on immunoanalytical methods and gas, or liquid chromatography combined with mass spectrometry.

Neurosteroids and Neurotrophic Factors: What Is Their Promise as Biomarkers for Major Depression and PTSD?

Even though major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) are among the most prevalent and incapacitating mental illnesses in the world, their diagnosis still relies solely on the characterization of subjective symptoms (many of which are shared by multiple disorders) self-reported by patients. Thus, the need for objective measures that aid in the detection of and differentiation between psychiatric disorders becomes urgent. In this paper, we explore the potential of neurosteroids and neurotrophic proteins as biomarkers for MDD and PTSD. Circulating levels of the GABAergic neuroactive steroid, allopregnanolone, are diminished in MDD and PTSD patients, which corroborates the finding of depleted neurosteroid levels observed in animal models of these disorders. The neurotrophic protein, brain-derived neurotropic factor (BDNF), is also reduced in the periphery and in the brain of MDD patients and depressed-like animals that express lower neurosteroid levels. Although the role of BDNF in PTSD psychopathology seems less clear and merits more research, we propose a causal link between allopregnanolone levels and BDNF expression that could function as a biomarker axis for the diagnosis of both MDD and PTSD.

Effects of prenatal stress on neuroactive steroid responses to acute stress in adult male and female rats.

Acute swim stress results in the robust production of several neuroactive steroids, which act as mediators of the stress response. These steroids include glucocorticoids, and positive GABAA receptor modulatory steroids such as allopregnanolone and tetrahydrocorticosterone (THDOC), which potentiate inhibitory GABA signalling, thereby playing a role in the negative control of the hypothalamic-pituitary-adrenal (HPA) axis. Prenatally stressed (PNS) offspring exhibit increased vulnerability to stress-related disorders and frequently display exaggerated HPA axis responses to stressors during adulthood, which may be a result of reduced neuroactive steroid production and consequently inhibitory signalling. Here, we investigated whether exposure of rats to prenatal social stress from gestational day 16-20 altered neuroactive steroid production under non-stress conditions and in response to an acute stressor (swim stress) in adulthood. Using liquid chromatography-mass spectrometry, nine neuroactive steroids were quantified (corticosterone, deoxycorticosterone [DOC], dihydrodeoxycorticosterone, THDOC, progesterone, dihydroprogesterone, allopregnanolone, pregnenolone, testosterone) in plasma and in five brain regions (frontal cortex, hypothalamus, amygdala, hippocampus, brainstem) of male and female control and PNS rats. There was no difference in the neuroactive steroid profile between control and PNS rats under basal conditions. The increase in circulating corticosterone induced by acute swim stress was similar in control and PNS offspring. However, greater stress-induced corticosterone and DOC concentrations were observed in the brainstem of male PNS offspring, whereas DOC concentrations were lower in the hippocampus of PNS females compared to controls, following acute stress. Although PNS rats did not show deficits in allopregnanolone responses to acute stress, there were modest deficits in the production of THDOC in the brainstem, amygdala, and frontal cortex of PNS males and in the frontal cortex of PNS females. The data suggest that neuroactive steroid modulation of GABAergic signalling following stress exposure may be affected in a sex- and region-specific manner in PNS offspring.

Low Neuroactive Steroids Identifies a Biological Subtype of Depression in Adults with Human Immunodeficiency Virus on Suppressive Antiretroviral Therapy.

BACKGROUND: The prevalence and mortality risk of depression in people with human immunodeficiency virus (HIV) infection receiving antiretroviral therapy (ART) is higher than in the general population, yet biomarkers for therapeutic targeting are unknown. In the current study, we aimed to identify plasma metabolites associated with depressive symptoms in people with HIV receiving ART. METHODS: This is a prospective study of ART-treated HIV-infected adults with or without depressive symptoms assessed using longitudinal Beck Depression Inventory scores. Plasma metabolite profiling was performed in 2 independent cohorts (total n = 99) using liquid and gas chromatography and tandem mass spectrometry. RESULTS: Participants with depressive symptoms had lower neuroactive steroids (dehydroepiandrosterone sulfate [DHEA-S], androstenediols, and pregnenolone sulfate) compared with those without depressive symptoms. The cortisol/DHEA-S ratio, an indicator of hypothalamic-pituitary-adrenal axis imbalance, was associated with depressive symptoms (P < .01) because of low DHEA-S levels, whereas cortisol was similar between groups. The odds of having depressive symptoms increased with higher cortisol/DHEA-S ratios (adjusted odds ratio, 2.5 per 1-unit increase in z score; 95% confidence interval, 1.3-4.7), independent of age and sex. The kynurenine-to-tryptophan ratio showed no significant associations. CONCLUSIONS: These findings suggest that altered neuroactive steroid metabolism may contribute to the pathophysiological mechanisms of depression in ART-treated HIV-infected adults, representing a potential biological pathway for therapeutic targeting.

Steroid precursors, steroids, neuroactive steroids, and neurosteroids concentrations in serum and saliva of healthy neonatal heifer Holstein calves.

BACKGROUND: Persistence of high neurosteroid concentrations in blood is associated with neonatal encephalopathy and septicemia in foals. This has not been investigated in calves. OBJECTIVES: To determine concentrations of steroid compounds in serum and saliva within the first 48 hours after birth in healthy neonatal calves, identify potential markers for disease, and investigate the association between serum steroid compounds concentrations in calves and their respective dams within 2 hours after birth. ANIMALS: Twelve healthy neonatal heifer Holstein calves and their dams. METHODS: Prospective study. Serum and saliva were collected from calves at 2, 6, 24, and 48 hours after birth. Steroid compounds were analyzed using liquid chromatography-mass spectrometry. A nonlinear regression model was used to determine half-lives of the neurosteroids. Serum concentrations of neurosteroids between the cows and calves were compared using the Wilcoxon signed rank test. RESULTS: Half-lives (95% confidence intervals) of dehydroepiandrosterone (DHEA) and 17α,20α-dihydroxyprogesterone in calf serum were 2.9 (2.1, 4.3), and 2.1 (1.3, 3.0) hours, respectively. Pregnanediol in saliva had a half-life (95% confidence interval) of 24.5 (14.2, 66.5) hours. Serum DHEA (1718.7 ± 2313 vs 57.7 ± 44) and 17α,20α-dihydroxyprogesterone (207.8 ± 198.2 vs 43.5 ± 33.5) concentrations respectively were higher (P < .05) in calves compared to cows. CONCLUSIONS AND CLINICAL IMPORTANCE: Dehydroepiandrosterone, 17α,20α-dihydroxyprogesterone, and pregnanediol could be potential markers of disease in neonatal heifer calves with unexplained failure to thrive or encephalopathy. However, because of the wide 95% confidence interval of the half-life, pregnanediol in saliva might not be a potential marker.

Longitudinal proneuroactive and neuroactive steroid profiles in medication-free women with, without and at-risk for perinatal depression: A liquid chromatography-tandem mass spectrometry analysis.

BACKGROUND: Neuroactive steroids (NAS) are derivatives of cholesterol or steroidal precursors made in the gonads, adrenal gland, placenta and brain. We characterized longitudinal plasma proneuroactive and NAS in healthy perinatal comparison women (HPCW), women at-risk for perinatal depression (AR-PND), and women with PND with/without comorbid anxiety. We hypothesized that AR-PND women who either did or did not go on to develop PND would have elevated NAS concentrations as compared to HPCW and that NAS would be correlated to depressive and anxiety symptoms. METHODS: A prospective cohort study evaluated 75 medication-free perinatal women (HPCW, n = 30; AR-PND, n = 19; PND, n = 26). Standardized depression and anxiety assessments and blood samples were completed across 5 visits. Structured Clinical Interviews for DSM-IV TR Disorders were administered at study entry and exit. Plasma pregnenolone, progesterone, 5α- and 5ß-dihydroprogesterone, pregnanolone, allopregnanolone, deoxycorticosterone and tetrahydrodeoxycorticosterone were quantified by liquid chromatography-tandem mass spectrometry. Longitudinal relationships between risk-group, depression and anxiety symptoms, and NAS concentrations were analyzed using generalized estimating equations to control for repeated measures correlations. RESULTS: Perinatal 5α-dihydroprogesterone, 5ß-dihydroprogesterone, allopregnanolone, deoxycorticosterone, and tetrahydrodeoxycorticosterone concentrations were higher in AR-PND and PND women compared to HPCW (ß = 3.57 ± 1.40 and ß = 2.11 ± 1.12, p = 0.03; ß = 0.18 ± 0.06 and ß = 0.03 ± 0.05, p = 0.02; ß = 1.06 ± 0.42 and ß = 1.19 ± 0.47, p = 0.01; ß = 0.17 ± 0.07 and ß = 0.11 ± 0.06, p = 0.05; ß = 0.03 ± 0.01 and ß = 0.03 ± 0.01, p = 0.05, respectively). Perinatal allopregnanolone, 5α-dihydroprogesterone and tetrahydrodeoxycorticosterone were positively associated with HAM-D17 (all p < 0.02). HAM-A was positively associated with 5α- and 5ß-dihydroprogesterone, pregnanolone, allopregnanolone, deoxycorticosterone and tetrahydrodeoxycorticosterone (all p < 0.05). A history of depression was associated with increased 5α-dihydroprogesterone (2.20 ± 1.09, p = 0.05), deoxycorticosterone (0.13 ± 0.06, p = 0.03) and tetrahydrodeoxycorticosterone (0.03 ± 0.01, p = 0.02). CONCLUSION: To our knowledge, this study represents the largest prospective study of 5-α and 5-ß reductase products of progesterone and deoxycorticosterone in HPCW and women AR-PND. Data suggest that PND is associated with both a reduction of progesterone to 5ß-dihydroprogesterone, 5α-dihydroprogesterone, and allopregnanolone, and the 21-hydroxylation to deoxycorticosterone and tetrahydrodeoxycorticosterone. The shift towards 5α-dihydroprogesterone, deoxycorticosterone and tetrahydrodeoxycorticosterone was associated with a history of depression, a significant risk factor for PND.

Sexually divergent changes in select brain proteins and neurosteroid levels after a history of ethanol drinking and intermittent PTSD-like stress exposure in adult C57BL/6J mice.

Human studies reported that the number of past-year stressors was positively related to current drinking patterns, including binge drinking. In animal models, exposure to predator odor stress (PS), considered a model of traumatic stress, consistently increased ethanol intake. Recently, we reported that repeated PS significantly increased ethanol intake and had a synergistic interaction with prior binge drinking (binge group) in male but not in female C57BL/6J mice, when compared to mice without prior binge exposure (control group). The current studies utilized plasma and dissected prefrontal cortex (PFC) and hippocampal tissue from these animals and from age-matched naïve mice (naïve group). Western blots assessed relative protein levels of P450scc (an enzyme involved in the first step of steroidogenesis), of GABAA receptor α2 and α4 subunits, and of two proteins involved in synaptic plasticity - ARC (activity-regulated cytoskeletal protein) and synaptophysin. Gas chromatography-mass spectrometry simultaneously quantified 10 neurosteroid levels in plasma. A history of ethanol drinking and PS exposure produced brain regional and sex differences in the changes in proteins examined as well as in the pattern of neurosteroid levels versus (vs.) values in naïve mice. For instance, P450scc levels were significantly increased only in binge and control female PFC and hippocampus vs. naïve mice. Some neurosteroid levels were significantly altered by binge treatment in both males and females, whereas others were only significantly altered in males. These sexually divergent changes in neurosteroid and protein levels add to evidence for sex differences in the neurochemical systems influenced by traumatic stress and a history of ethanol drinking.

The anxiolytic-like effects of puerarin on an animal model of PTSD.

Post traumatic stress disorder (PTSD) is a mental illness that affected numerous people. The anti-PTSD-like effects of puerarin is unknown, although the antidepressant- and anxiolytic- like effects of puerarin have been reported. The PTSD behavioral deficits in rats were induced by single prolonged stress (SPS), mainly including the reduced time/entries in the open arms and the elevated time/entries in the closed arms in elevated plus maze test, increased freezing duration in contextual fear paradigm and lowered time/entries in the central zone in open field test. However, the behavioral deficits were attenuated by puerarin (50 and 100 mg/kg) without affecting the locomotor activity. For the evaluation of mechanism, the decreased levels of progesterone, allopregnanolone, and the increased levels of corticosterone, corticotropin releasing hormone, and adrenocorticotropic hormone in the brain or serum were induced by SPS, which is blocked by puerarin. In summary, the anti-PTSD-like effects of puerarin were associated with biosynthesis of neurosteroids and normalized levels of stress hormones in HPA axis.

Neuroactive steroid levels and cocaine use chronicity in men and women with cocaine use disorder receiving progesterone or placebo.

BACKGROUND AND OBJECTIVES: Neuroactive steroids (NAS) may play a role in addiction, with observed increases in response to acute stress and drug use, but decreases with chronic substance use, suggesting that NAS neuroadaptations may occur with chronic substance use. However, levels of NAS in addicted individuals have not been systematically examined. Here, we evaluated a panel of NAS in men and women with cocaine use disorder (CUD) who participated in a clinical laboratory study of progesterone. METHODS: Forty six CUD individuals were enrolled in a randomized placebo-controlled laboratory study to evaluate progesterone effects on levels of various NAS. On day 5 of a 7-day inpatient treatment regimen of 400 mg/day progesterone (15M/8F) or placebo (14M/9F), plasma levels of NAS known to be downstream of progesterone (allopregnanolone, pregnanolone), and NAS not in the progesterone synthesis pathway (androstanediol, testosterone, dehydroepiandrosterone [DHEA] and the NAS precursor, pregnenolone) were analyzed using highly sensitive gas chromatography/mass spectrometry (GC/MS). The relationship between each of the NAS and chronicity of cocaine use was also assessed. RESULTS: Progesterone versus placebo significantly increased the GABAergic NAS allopregnanolone and pregnanolone in both CUD men and women. Levels of pregnenolone, testosterone, its GABAergic metabolite androstanediol, and the non-GABAergic DHEA were unaffected by progesterone treatment, and testosterone and androstanediol levels were significantly higher in men than women. Importantly, lower pregnenolone and androstanediol levels were associated with greater years of cocaine use. SCIENTIFIC SIGNIFICANCE: GABAergic NAS that are upstream from the progesterone synthesis pathway appear susceptible to chronic effects of cocaine use. (Am J Addict 2019;28:16-21).

Resting-state functional connectivity, cortical GABA, and neuroactive steroids in peripartum and peripartum depressed women: a functional magnetic resonance imaging and spectroscopy study.

Postpartum depression (PPD) is associated with abnormalities in resting-state functional connectivity (RSFC) but the underlying neurochemistry is unclear. We hypothesized that peripartum GABAergic neuroactive steroids (NAS) are related to cortical GABA concentrations and RSFC in PPD as compared to healthy comparison women (HCW). To test this, we measured RSFC with fMRI and GABA+/Creatine (Cr) concentrations with proton magnetic resonance spectroscopy (1H MRS) in the pregenual anterior cingulate (pgACC) and occipital cortices (OCC) and quantified peripartum plasma NAS. We examined between-group differences in RSFC and the relationship between cortical GABA+/Cr concentrations with RSFC. We investigated the relationship between NAS, RSFC and cortical GABA+/Cr concentrations. Within the default mode network (DMN) an area of the dorsomedial prefrontal cortex (DMPFC) had greater connectivity with the rest of the DMN in PPD (peak voxel: MNI coordinates (2, 58, 32), p = 0.002) and was correlated to depression scores (peak HAM-D17 voxel: MNI coordinates (0, 60, 34), p = 0.008). pgACC GABA+/Cr correlated positively with DMPFC RSFC in a region spanning the right anterior/posterior insula and right temporal pole (r = +0.661, p = 0.000). OCC GABA+/Cr correlated positively with regions spanning both amygdalae (right amygdala: r = +0.522, p = 0.000; left amygdala: r = +0.651, p = 0.000) as well as superior parietal areas. Plasma allopregnanolone was higher in PPD (p = 0.03) and positively correlated with intra DMPFC connectivity (r = +0.548, p = 0.000) but not GABA+/Cr. These results provide initial evidence that PPD is associated with altered DMN connectivity; cortical GABA+/Cr concentrations are associated with postpartum RSFC and allopregnanolone is associated with postpartum intra-DMPFC connectivity.