The Role of Steroidomics in the Diagnosis of Alzheimer's Disease and Type 2 Diabetes Mellitus.

Epidemiological studies suggest an association between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). This study aimed to investigate the pathophysiological markers of AD vs. T2DM for each sex separately and propose models that would distinguish control, AD, T2DM, and AD-T2DM comorbidity groups. AD and T2DM differed in levels of some circulating steroids (measured mostly by GC-MS) and in other observed characteristics, such as markers of obesity, glucose metabolism, and liver function tests. Regarding steroid metabolism, AD patients (both sexes) had significantly higher sex hormone binding globulin (SHBG), cortisol, and 17-hydroxy progesterone, and lower estradiol and 5α-androstane-3α,17ß-diol, compared to T2DM patients. However, compared to healthy controls, changes in the steroid spectrum (especially increases in levels of steroids from the C21 group, including their 5α/ß-reduced forms, androstenedione, etc.) were similar in patients with AD and patients with T2DM, though more expressed in diabetics. It can be assumed that many of these steroids are involved in counter-regulatory protective mechanisms that mitigate the development and progression of AD and T2DM. In conclusion, our results demonstrated the ability to effectively differentiate AD, T2DM, and controls in both men and women, distinguish the two pathologies from each other, and differentiate patients with AD and T2DM comorbidities.

Altered Steroidome in Women with Gestational Diabetes Mellitus: Focus on Neuroactive and Immunomodulatory Steroids from the 24th Week of Pregnancy to Labor.

Gestational diabetes mellitus (GDM) is a complication in pregnancy, but studies focused on the steroidome in patients with GDM are not available in the public domain. This article evaluates the steroidome in GDM+ and GDM- women and its changes from 24 weeks (± of gestation) to labor. The study included GDM+ (n = 44) and GDM- women (n = 33), in weeks 24-28, 30-36 of gestation and at labor and mixed umbilical blood after delivery. Steroidomic data (101 steroids quantified by GC-MS/MS) support the concept that the increasing diabetogenic effects with the approaching term are associated with mounting progesterone levels. The GDM+ group showed lower levels of testosterone (due to reduced AKR1C3 activity), estradiol (due to a shift from the HSD17B1 towards HSD17B2 activity), 7-oxygenated androgens (competing with cortisone for HSD11B1 and shifting the balance from diabetogenic cortisol towards the inactive cortisone), reduced activities of SRD5As, and CYP17A1 in the hydroxylase but higher CYP17A1 activity in the lyase step. With the approaching term, the authors found rising activities of CYP3A7, AKR1C1, CYP17A1 in its hydroxylase step, but a decline in its lyase step, rising conjugation of neuroinhibitory and pregnancy-stabilizing steroids and weakening AKR1D1 activity.

Changes of BMI, steroid metabolome and psychopathology in patients with anorexia nervosa during hospitalization.

Anorexia nervosa (AN) is associated with various alterations including the dysfunction of the HPA axis and consequently the hypercortisolemia and deficit in sex hormones but the comprehensive evaluation of changes in circulating steroids during the hospitalization of AN patients is lacking. We investigated the effect of realimentation of women with AN during hospitalization on 45 circulating steroids, the relationships between BMI, its change during hospitalization and physical activity, on one side and initial levels and their changes for two adipokines, circulating steroids, anorexia-specific (hunger, appetite and satiety), and anorexia non-specific symptoms (anxiety, depression fatigue, sleep, and body pain) on the other side. We included 33 women with anorexia who were hospitalized for 38(35, 44) days (median with quartiles). The increase of BMI from the initial value 15.2 (13.2, 16.6) kg/m2 was 1.69 (1.37, 2.66) kg/m2. The patients with more severe anorexia showed higher activity in 7ß-, and 16α-hydroxylation of androgen precursors, which declined during hospitalization. Otherwise, the 7α-hydroxylation activity is higher in AN patients with less severe malnutrition and the ratio of 5-androstene-3ß,7α,17ß-triol to 5-androstene-3ß,7ß,17ß-triol increased during the realimentation. Our data allow to speculate that the intensive 7ß-, and 16α- and possibly also the 7α-hydroxylation of C19 Δ5 steroids participate in the pathophysiology of anorexia by additional catabolism of substrates available for synthesis of active androgens and estrogens. However, the question remains whether the synthetic analogues of 7α/ß- and 16α-hydroxy-steroids prevent the catabolism of the sex steroid precursors, or further activate the "energy wasting" mitochondrial thermogenic metabolism.

Activation of Adrenal Steroidogenesis and an Improvement of Mood Balance in Postmenopausal Females after Spa Treatment Based on Physical Activity.

Spa treatment can effectively reestablish mood balance in patients with psychiatric disorders. In light of the adrenal gland's role as a crossroad of psychosomatic medicine, this study evaluated changes in 88 circulating steroids and their relationships with a consolidation of somatic, psychosomatic and psychiatric components from a modified N-5 neurotic questionnaire in 46 postmenopausal 50+ women with anxiety-depressive complaints. The patients underwent a standardized one-month intervention therapy with physical activity and an optimized daily regimen in a spa in the Czech Republic. All participants were on medication with selective serotonin reuptake inhibitors. An increase of adrenal steroidogenesis after intervention indicated a reinstatement of the hypothalamic-pituitary-adrenal axis. The increases of many of these steroids were likely beneficial to patients, including immunoprotective adrenal androgens and their metabolites, neuroactive steroids that stimulate mental activity but protect from excitotoxicity, steroids that suppress pain perception and fear, steroids that consolidate insulin secretion, and steroids that improve xenobiotic clearance. The positive associations between the initial values of neurotic symptoms and their declines after the intervention, as well as between initial adrenal activity and the decline of neurotic symptoms, indicate that neurotic impairment may be alleviated by such therapy provided that the initial adrenal activity is not seriously disrupted.

A Comprehensive Evaluation of Steroid Metabolism in Women with Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a common liver disorder, mostly occurring in the third trimester. ICP is defined as an elevation of serum bile acids, typically accompanied by pruritus and elevated activities of liver aminotransferases. ICP is caused by impaired biliary lipid secretion, in which endogenous steroids may play a key role. Although ICP is benign for the pregnant woman, it may be harmful for the fetus. We evaluated the differences between maternal circulating steroids measured by RIA (17-hydroxypregnenolone and its sulfate, 17-hydroxyprogesterone, and cortisol) and GC-MS (additional steroids), hepatic aminotransferases and bilirubin in women with ICP (n = 15, total bile acids (TBA) >8 µM) and corresponding controls (n = 17). An age-adjusted linear model, receiver-operating characteristics (ROC), and multivariate regression (a method of orthogonal projections to latent structure, OPLS) were used for data evaluation. While aminotransferases, conjugates of pregnanediols, 17-hydroxypregnenolone and 5ß-androstane-3α,17ß-diol were higher in ICP patients, 20α-dihydropregnenolone, 16α-hydroxy-steroids, sulfated 17-oxo-C19-steroids, and 5ß-reduced steroids were lower. The OPLS model including steroids measured by GC-MS and RIA showed 93.3% sensitivity and 100% specificity, while the model including steroids measured by GC-MS in a single sample aliquot showed 93.3% sensitivity and 94.1% specificity. A composite index including ratios of sulfated 3α/ß-hydroxy-5α/ß-androstane-17-ones to conjugated 5α/ß-pregnane-3α/ß, 20α-diols discriminated with 93.3% specificity and 81.3% sensitivity (ROC analysis). These new data demonstrating altered steroidogenesis in ICP patients offer more detailed pathophysiological insights into the role of steroids in the development of ICP.

Circulating C19 steroids and progesterone metabolites in women with acute depression and anxiety disorders.

Depression and anxiety disorders are highly prevalent in women. Although several studies have reported altered circulating steroids accompanying various mental disturbances, knowledge about alterations in the peripheral steroid pattern in such pathologies is incomplete. Therefore, we attempted to add to this knowledge using the simultaneous quantification of circulating steroids by gas chromatography mass spectrometry (GC-MS) in groups of premenopausal women in the follicular phase of the menstrual cycle (22 women with depression, 17 with anxiety disorders, 17 healthy controls). In addition to age-adjusted analysis of covariance (ANCOVA) followed by multiple comparisons, we developed models to successfully discriminate these groups from each other on the basis of steroid levels. Women with depression showed a reduced sulfoconjugation of steroids as well as lower levels of 7α-, 7ß- and 16α-hydroxy-metabolites of C19 Δ5 steroids. Women with depression have significantly lower circulating levels of 5α/ß-reduced pregnane steroids (with exception of free isopregnanolone) than women with anxiety or controls. Finally, our data indicate higher levels of estrogens in women with anxiety disorders when compared to women with depression.

Preliminary evidence of altered steroidogenesis in women with Alzheimer's disease: Have the patients "OLDER" adrenal zona reticularis?

Alzheimer's disease (AD) represents more than half of total dementias. Various factors including altered steroid biosynthesis may participate in its pathophysiology. We investigated how the circulating steroids (measured by GC-MS and RIA) may be altered in the presence of AD. Sixteen women with AD and 22 age- and BMI-corresponding controls aged over 65 years were enrolled in the study. The steroid levels (47 steroids and steroid polar conjugates) and their ratios in AD female patients indicated increased CYP11A1 activity, weakened activity of the CYP17A1C17,20 lyase metabolic step and attenuated sulfotransferase SULT2A1 activity at higher activity of the CYP17A1 17-hydroxylase step. The patients showed diminished HSD3B2 activity for C21 steroids, abated conversion of 17-hydroxyprogesterone to cortisol, and significantly elevated cortisol. The women with AD had also attenuated steroid 7α-hydroxylation forming immunoprotective Δ(5)-C19 steroids, attenuated aromatase activity forming estradiol that induces autoimmunity and a shift from the 3ß-hydroxy-5α/ß-reduced C19 steroids to their neuroinhibitory and antiinflammatory GABAergic 3α-hydroxy- counterparts and showed higher levels of the 3α-hydroxy-5α/ß-reduced C21 steroids and pregnenolone sulfate (improves cognitive abilities but may be both protective and excitotoxic). Our preliminary data indicated functioning of alternative "backdoor" pathway in women with AD showing higher levels of both 5α/ß-reduced C21 steroids but reduced levels of both 5α/ß-reduced C21 steroids, which implied that the alternative "backdoor" pathway might include both 5α- and 5ß-reduced steroids. Our study suggested relationships between AD status in women based on the age of subjects and levels of 10 steroids measured by GC-MS.

Steroid profiling in pregnancy: a focus on the human fetus.

In this review we focused on steroid metabolomics in human fetuses and newborns and its role in the physiology and pathophysiology of human pregnancy and subsequent stages of human life, and on the physiological relevance of steroids influencing the nervous systems with regards to their concentrations in the fetus. Steroid profiling provides valuable data for the diagnostics of diseases related to altered steroidogenesis in the fetal and maternal compartments and placenta. We outlined a potential use of steroid metabolomics for the prediction of reproductive disorders, misbalance of hypothalamic-pituitary-adrenal axis, and impaired insulin sensitivity in subsequent stages of human life. A possible role of steroids exhibiting a non-genomic effect in the development of gestational diabetes and in the neuroprotection via negative modulation of AMPA/kainate receptors was also indicated. Increasing progesterone synthesis and catabolism, declining production of tocolytic 5ß-pregnane steroids, and rising activities of steroid sulfotransferases with the approaching term may be of importance in sustaining pregnancy. An increasing trend was demonstrated with advancing gestation toward the production of ketones (and 3ß-hydroxyl groups in the case of 3α-hydroxy-steroids) was demonstrated in the fetus on the expense of 3α-hydroxy-, 17ß-hydroxy-, and 20α-hydroxy-groups weakening in the sequence C17, C3, and C20. There was higher production of active progestogen but lower production of active estrogen and GABAergic steroids with the approaching term. Rising activities of placental CYP19A1 and oxidative isoforms of HSD17B, and of fetal CYP3A7 with advancing gestation may protect the fetus from hyperestrogenization. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

The steroid metabolome in lamotrigine-treated women with epilepsy.

BACKGROUND: Epilepsy in women may be associated with reproductive disorders and alterations in serum steroid levels. Some steroids can be induced by epilepsy and/or treatment with antiepileptic drugs; however, there are still limited data available concerning this effect on the levels of other neuroactive steroid metabolites such as 3a-hydroxy-5a/b-reduced androstanes. AIM: To evaluate steroid alterations in women with epilepsy (WWE) on lamotrigine monotherapy. SUBJECTS AND METHODS: Eleven WWE and 11 age-matched healthy women underwent blood sampling in both phases of their menstrual cycles (MCs). The steroid metabolome, which included 30 unconjugated steroids, 17 steroid polar conjugates, gonadotropins, and sex hormone-binding globulin (SHBG), was measured using gas chromatography-mass spectrometry (GC-MS) and radioimmunoassay (RIA). RESULTS: WWE had lower cortisol levels (status p<0.001), but elevated levels of unconjugated 17-hydroxypregnenolone (status p<0.001). Progesterone was higher in the follicular menstrual phase (FP) in WWE than in the controls (status×menstrual phase p<0.05, Bonferroni multiple comparisons p<0.05), whereas 17-hydroxyprogesterone was higher in WWE in both menstrual phases (status p<0.001). The steroid conjugates were mostly elevated in WWE. The levels of 5α/ß-reduced androstanes in WWE that were significantly higher than the controls were etiocholanolone (status p<0.001), 5α-androstane-3α,17ß-diol (status p<0.001), and the 5α/ß-reduced androstane polar conjugates (status p<0.001). CONCLUSIONS: WWE showed a trend toward higher circulating 3α-hydroxy-5α/ß-reduced androstanes, increased activity of 17α-hydroxylase/17,20 lyase in the Δ(5)-steroid metabolic pathway, and increased levels of the steroid polar conjugates.

Cigarette smoking and progesterone and androgen metabolites in premenopausal women.

BACKGROUND: Smoking represents the most widespread substance dependence in the world. Several studies show nicotine's ability to alter women hormonal homeostasis. Women smokers have higher testosterone and lower estradiol levels throughout life compared to women non-smokers. This negatively affects women's reproductive function. Furthermore, alteration of neuroactive and neuroprotective steroids occurs in women smokers, and this plays an important role in the activity of the central nervous system, cognition, mental condition, and degree of substance dependence. METHODS: We monitored the effect of smoking discontinuation on steroid spectrum in 40 premenopausal women heavy smokers. These women were examined before they began to discontinue smoking, and after 6, 12, 24 and 48 weeks of abstinence. In each examination, blood was collected to determine steroid spectrum, LH, FSH, and SHBG; basic anthropometric data were also measured using GC-MS or immunoanalysis. Repeated-measures analysis of variance (ANOVA) model was used for evaluation of the data. RESULTS: Given the small number of women who persisted in not smoking, only the data after 6 weeks could be analyzed. No changes were found in C21 steroids, and a slight increase in androgens occurred after the discontinuation of smoking. CONCLUSION: Chronic smoking causes hyperandrogenism in fertile women; after smoking discontinuation, it increases further. Longer-term monitoring is necessary to show the effect of smoking discontinuation on steroid spectrum.

Effects of valproate and carbamazepine monotherapy on neuroactive steroids, their precursors and metabolites in adult men with epilepsy.

Only limited data is available concerning the role of unconjugated Δ(5) C19-steroids and almost no data exists regarding the neuroactive C21 and C19 3α-hydroxy-5α/ß-metabolites in men with epilepsy. To evaluate the alterations in serum neuroactive steroids and related substances in adult men with epilepsy on valproate and carbamazepine monotherapy, we have measured 26 unconjugated steroids, 18 steroid polar conjugates, gonadotropins and sex hormone binding globulin (SHBG) in 6 and 11 patients on valproate and carbamazepine monotherapy, respectively, and in 19 healthy adult men, using the GC-MS and immunoassays. Decreased testosterone, free androgen index, free testosterone, androstenediol, 5α-androstane-3α,17ß-diol (androstanediol), androsterone, epiandrosterone, DHEA, 7ß-hydroxy-DHEA, and DHEAS levels were associated with epilepsy per se. Valproate (VPA) therapy increased 5α-dihydrotestosterone, androsterone, epiandrosterone, DHEA, DHEAS, and 7ß-hydroxy-DHEA levels. Decrease in pregnenolone and 17-hydroxypregnenolone were associated with epilepsy with no effect of antiepileptic drugs (AEDs). Alternatively, the increase in progesterone levels was linked to epilepsy and VPA further increased progesterone levels. Reduced steroid 20α-hydroxy-metabolites and cortisol were connected with epilepsy without an effect of AEDs. Carbamazepine induced only slight decrease in isopregnanolone, 5α,20α-tetrahydroprogesterone, and androstanediol levels.

Steroid metabolome in fetal and maternal body fluids in human late pregnancy.

Despite the extensive research during the last six decades the fundamental questions concerning the role of steroids in the initiation of human parturition and origin and function of some steroids in pregnancy were not definitely answered. Based on steroid metabolomic data found in the literature and our so far unpublished results, we attempted to bring new insights concerning the role of steroids in the sustaining and termination of human pregnancy, and predictive value of these substances for estimation of term. We also aimed to explain enigmas concerning the biosynthesis of progesterone and its bioactive catabolites considering the conjunctions between placental production of CRH, synthesis of bioactive steroids produced by fetal adrenal, localization of placental oxidoreductases and sustaining of human pregnancy. Evaluation of data available in the literature, including our recent findings as well as our new unpublished data indicates increasing progesterone synthesis and its concurrently increasing catabolism with approaching parturition, confirms declining production of pregnancy sustaining 5ß-pregnane steroids providing uterine quiescence in late pregnancy, increased sulfation of further neuroinhibiting and pregnancy sustaining steroids. In contrast to the established concept considering LDL cholesterol as the primary substrate for progesterone synthesis in pregnancy, our data demonstrates the functioning of alternative mechanism for progesterone synthesis, which is based on the utilization of fetal pregnenolone sulfate for progesterone production in placenta. Close relationships were found between localization of placental oxidoreductases and consistently higher levels of sex hormones, neuroactive steroids and their metabolites in the oxidized form in the fetus and in the reduced form in the maternal compartment.

Steroid metabolome in plasma from the umbilical artery, umbilical vein, maternal cubital vein and in amniotic fluid in normal and preterm labor.

The boost in placental production of CRH in late pregnancy is specific for human. CRH receptors are expressed in the fetal zone of the fetal adrenal (FZFA). Hence, we evaluated the associations between the steroid metabolome and gestational age (GA). The levels of 69 steroids and steroid polar conjugates such as 3beta-hydroxy-5-ene steroids (3betaOH5S), 3-oxo-4-ene steroids (3O4S), progesterone 5alpha/beta-reduced metabolites, 20alpha-hydroxy-metabolites of C21 steroids, C19 5alpha/beta-reduced metabolites, 7alpha/beta-hydroxy-metabolites of 3betaOH5S, estrogens and 16alpha-hydroxy-metabolites of 3betaOH5S and 3O4S, were measured by GC-MS in plasma from the umbilical artery (UA), umbilical vein (UV), and maternal cubital vein (MV) and in amniotic fluid (AF) in 12 women at normal labor and 38 women at preterm labor due to pathologies unrelated to steroid status. Using multivariate regression, prediction models for GA were completed for the individual body fluids. The conjugated 3betaOH5S (the key products of the FZFA), estrogens, some polar conjugates of progesterone 5alpha/beta-reduced metabolites and some steroid 7alpha/beta- and 16alpha-hydroxy-metabolites showed strong positive correlations with the GA. The predictivity decreased in the following sequence UV (R=0.950), UA (R=0.945), MV (R=0.895), and AF (R=0.891). Although the predictivity of steroids in maternal blood was slightly less effective when compared with the UV and UA, it was the best solution for further practice.

The distribution of placental oxidoreductase isoforms provides different milieus of steroids influencing pregnancy in the maternal and fetal compartment.

Using information based on the steroid metabolome in maternal and fetal body fluids, we attempted to ascertain whether there is a common mechanism, which is based on the placental distribution of various isoforms of 17ß-hydroxysteroid dehydrogenases and aldo-keto reductases. This system simultaneously provides a higher proportion of active progestogens in fetal circulation and a higher proportion of active estrogens and GABAergic steroids in the maternal compartment. The data obtained using gas chromatography-mass spectrometry completely support the aforementioned hypothesis. We confirmed a common trend to higher ratios of steroids with hydroxy-groups in the 3α-, 17ß-, and 20α-positions to the corresponding 3-oxo-, 17-oxo-, and 20-oxo-metabolites, respectively, in the maternal blood when compared with the fetal circulation, and the same tendency was obvious in the 3α-hydroxy/3ß-hydroxy steroid ratios. A decreasing trend was observed in the ratios of active estrogens and neuro-inhibitory steroids to their inactive counterparts in fetal and maternal body fluids. This was probably associated with a limited capacity of placental oxidoreductases in the converting of estrone to estradiol during the transplacental passage. Although we observed a decreasing trend in pregnancy-sustaining steroids with increasing gestational age, we recorded rising levels of estradiol and particularly of estriol, regardless of the limited capacity of placental oxidoreductases. Besides the estradiol, which is generally known as an active estrogen, estriol may be of importance for the termination of pregnancy with respect to its excessive concentrations near term which allows its binding to estrogen receptors.

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.