Lyudmila Kancheva

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 Based on steroid metabolomic data found in the literature and our so far unpublished results, we attempted to bring new insights concerning the role of steroids in the sustaining and termination of human pregnancy, and predictive value of these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 Evaluation of data available in the literature, including our recent findings as well as our new unpublished data indicates increasing progesterone synthesis and its concurrently increasing catabolism with approaching parturition, confirms declining production of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 5β-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.

Circulating levels of pregnanolone isomers during the third trimester of human pregnancy

This study addresses the question of whether changes in the biosynthesis and metabolism of neuroactive pregnanolone isomers (PIs) might participate in the timing of parturition in humans. The time profiles of unconjugated allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one, P3alpha5alpha), pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one, P3alpha5beta), isopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one, P3beta5alpha) and epipregnanolone (3beta-hydroxy-5beta-pregnan-20-one, P3beta5beta), pregnenolone, their polar conjugates, progesterone, 5alpha-dihydroprogesterone (P5alpha), and 5beta-dihydroprogesterone (P5beta) were monitored in the plasma of 30 healthy women during the third trimester of pregnancy, at 1-week intervals from the 30th week of gestation using GC-MS. Changes in the steroid levels were evaluated by two-way ANOVA with gestational age and subject as independent factors. The mean concentrations of free PIs ranged from 2 to 50 nmol/L, while the mean levels of their polar conjugates were 40-100 x higher. The ratio of 5alpha-PIs to progesterone significantly but inconspicuously culminated in the 35th week. The decelerating biosynthesis of free 5beta-PIs from the 31st week and their escalating sulfation was found from the 30th week. The changes were particularly evident in the second most abundant PI pregnanolone that may, like the allopregnanolone, sustain the pregnancy via attenuation of hypothalamic GABA(A)-receptors and prevent uterine contractility via binding to nuclear pregnane X receptor.

Altered profiles of serum neuroactive steroids in premenopausal women treated for alcohol addiction

Long-term alcohol consumption results in menstrual irregularities due to the inhibition of progesterone secretion. Some progesterone metabolites, including three pregnanolone isomers (PI), abate, while pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) increase, alcohol tolerance. The rationale of this study was to evaluate how the neuroactive steroids reflect the impaired progesterone formation in premenopausal women treated for alcohol addiction, and whether detoxification therapy could restore female reproductive functions and psychosomatic stability by reinstatement of the steroid biosynthesis. Accordingly, serum allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one (P3alpha5alpha)), pregnanolone (P3alpha5beta), isopregnanolone (P3beta5alpha) and epipregnanolone (P3beta5beta), progesterone, PregS, pregnenolone, 17alpha-hydroxy-pregnenolone (Preg17), 17alpha-hydroxy-progesterone (Prog17), DHEA, DHEAS, cortisol and estradiol were measured in 20 women during the therapy (start, 3 days, 14 days, 1 month, 4 months), and in 17 controls, using GC-MS or RIA and evaluated by age-adjusted ANCOVA with status and phase of the menstrual cycle (PMC) as factors, and status-PMC interaction. The patients exhibited depressed progesterone, Prog17, PI, and estradiol, a decreased progesterone/pregnenolone ratio, a decreased ratio of neuroinhibiting P3alpha5alpha to neuroactivating PregS, and an elevated PregS and PregS/pregnenolone ratio. The treatment mostly restored the indices. The reduction of neuroinhibiting pregnanolone isomers in the patients is primarily associated with the impairment in ovarian steroid biosynthesis. Nevertheless, changes in enzyme activities connected with the formation of PI and the influence of altered physiological requirements on the balance between endogenous neuroinhibiting and neuroactivating steroids are also likely. The reinstatement of serum estradiol, progesterone, and PI during the therapy demonstrates its favorable effect on both reproductive functions and the psychosomatic stability of the patients.

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 identification and simultaneous quantification of 7-hydroxylated metabolites of pregnenolone, dehydroepiandrosterone, 3β,17β-androstenediol, and testosterone in human serum using gas chromatography–mass spectrometry

7-Hydroxy-metabolites of dehydroepiandrosterone (DHEA) and 3beta,17beta-androstenediol (AD) possess immunomodulatory and neuroprotective properties; therefore, the measurement of these steroids in patients with autoimmune diseases or disturbances in the CNS may be of interest. A gas chromatography-mass spectrometry (GC-MS) method for the determination of 7-hydroxy-metabolites of pregnenolone, DHEA, AD, and testosterone including the parent steroids was applied to serum samples from 12 adult men (27-66 years), 13 male adolescents (13-20 years), 5 boys (6-10 years), 15 women in the follicular phase of the menstrual cycle (22-45 years), 17 women in the luteal phase (22-45 years), and 4 girls (6-10 years). The steroids were age and sex dependent, but independent of the menstrual cycle. The ratio of the 7alpha-hydroxy-metabolites to their parent steroids were age dependent, exhibiting an increasing trend (p < 0.0001, ANOVA) from pregnenolone (5%) to AD (20%). The ratio of 7beta- to 7alpha-metabolites ranged from 0.6 to 1. These results are consistent with models suggesting 7alpha-hydroxylation of the parent steroid, conversion to a 7-oxo-steroid and finally to the 7beta-hydroxylated-metabolite. Partial correlations suggested that 7-hydroxylation might reduce the concentration of circulating androgens. Despite the three times lower concentration of AD-metabolites, their antiglucocorticoid, immunomodulatory, and neuroprotective effects may be comparable to that of DHEA based on their reported greater biological activity.

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/β-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,17β-diol (androstanediol), androsterone, epiandrosterone, DHEA, 7β-hydroxy-DHEA, and DHEAS levels were associated with epilepsy per se. Valproate (VPA) therapy increased 5α-dihydrotestosterone, androsterone, epiandrosterone, DHEA, DHEAS, and 7β-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.

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/β-reduced androstanes in WWE that were significantly higher than the controls were etiocholanolone (status p<0.001), 5α-androstane-3α,17β-diol (status p<0.001), and the 5α/β-reduced androstane polar conjugates (status p<0.001). CONCLUSIONS WWE showed a trend toward higher circulating 3α-hydroxy-5α/β-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.

The identification and simultaneous quantification of neuroactive androstane steroids and their polar conjugates in the serum of adult men, using gas chromatography-mass spectrometry.

Certain androstane steroids (AS) modulate ionotropic receptors, as do the pregnane steroids. Whereas women produce significant amounts of neuroactive progesterone metabolites, the steroid neuromodulators in men originate mainly from the 3-oxo-4-ene C(19)-steroids, which are converted to their 3alpha- and 3beta-hydroxy-5alpha/5beta-reduced metabolites. The neuromodulating effects of AS prompted us to monitor circulating levels of the steroids to estimate metabolic pathways in the periphery that may influence brain concentrations of AS. Hence, the serum levels of 20 steroids and 16 steroid polar conjugates including 17-oxo- and 17beta-hydroxy-derivatives of 5alpha/beta-androstane-3alpha/beta-hydroxy-androstane steroids were quantified in 15 men (16-62 years of age) using GC-MS. The conjugated AS for the most part reached micromolar concentrations, these being two or three orders of magnitude higher than those of the free steroids. The ratios of conjugates to free steroids were one to two orders of magnitude higher than the values for the corresponding pregnane steroids. This data suggested that conjugation may considerably restrain the transport of free AS from the periphery into the central nervous system.

Reinstatement of serum pregnanolone isomers and progesterone during alcohol detoxification therapy in premenopausal women

BACKGROUND Alcohol abuse is associated with menstrual irregularities related to the inhibition of progesterone secretion involved in regulation of the menstrual cycle. Reduced progesterone metabolites, including pregnanolone isomers (PIs), are efficient neuromodulators. The authors attempted to evaluate whether levels of PIs reflect impairment in progesterone biosynthesis in premenopausal women treated for alcohol addiction and whether alcohol detoxification therapy contributes to the restoration of their reproductive functions and psychosomatic stability by influencing steroid biosynthesis. METHODS Serum allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one; P3alpha5alpha), pregnanolone (P3alpha5beta), isopregnanolone (P3beta5alpha), epipregnanolone (P3beta5beta), progesterone, pregnanolone sulfate (PregS), pregnanolone, and estradiol were measured in 20 women during therapy (at start, three days, 14 days, one month, and four months) by gas chromatography-mass spectrometry or radioimmunoassay. The results were evaluated by a linear mixed model for longitudinal data, with stage of the treatment and subject as categorical factors, phase of the menstrual cycle as a time-varying covariate, and age of the subject as a covariate and by regression in individual stages of the menstrual cycle. RESULTS During detoxification treatment, progesterone increased in the luteal phase. P3alpha5alpha, P3beta5alpha, and P3beta5beta rose in both phases of the menstrual cycle. DISCUSSION Given the similar mechanism in the effects of alcohol and steroids in activating gamma-aminobutyric acid A receptors, the restoration of progesterone and PIs during therapy could be explained by an adaptation to increasing requests for gamma-aminobutyric acid A-receptor activating substances owing to the cessation of alcohol intake or by the regeneration of progesterone formation. In conclusion, the reinstatement of progesterone, P3alpha5alpha, and P3beta5beta serum levels demonstrates the favorable effect of detoxification therapy on both reproductive functions and the psychosomatic stability of premenopausal women treated for alcohol addiction.

Profiling of neuroactive steroids, their precursors and metabolites in patients suffering from multiple sclerosis

We have followed the neuroprotective, GABAergic glycinergic, glutamatergic acetylcholinergic and purinergic NAS, their precursors and metabolites and their conjugates (64 steroids) in the circulation and cerebrospinal fluid of 13 female patients36 years old median age and 8 sex age matched healthy controls (both in the follicular menstrual phase) with the use of gas chromatography – mass spectrometry. Conclusion