Luboslav Stárka

Neurosteroid 7-hydroxylation products in the brain

The neurosteroids pregnenolone (PREG) and dehydroepiandrosterone (DHEA) are precursors for both oxidized and hydroxylated metabolites in the brain. Thus, brain production of 7-hydroxylated derivatives is second to that in the liver, and P4507B1-containing hippocampus is the major site for 7 alpha-hydroxylation. Other P450s and/or oxido-reductive mechanisms may be responsible for 7 beta-hydroxylation. In addition to regulating neurosteroid brain levels, when produced, the 7-hydroxylated derivatives of PREG and DHEA were investigated for antiglucocorticoid-mediated neuroprotective potencies, and both 7 alpha- and 7 beta-hydroxy-DHEA were efficient in preventing the nuclear uptake of [3H]dexamethasone-activated glucocorticoid receptor in brain cells. Activation of 7 alpha-hydroxylation by increased close contacts of astrocytes and after glucocorticoid treatment suggested that the regulated production of 7 alpha-hydroxysteroids was a key event for the neuroprotection conferred by neurosteroids.

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.

Dehydroepiandrosterone: A neuroactive steroid

Dehydroepiandrosterone (DHEA) and its sulfate bound form (DHEAS) are important steroids of mainly adrenal origin. They are produced also in gonads and in the brain. Dehydroepiandrosterone easily crosses the brain-blood barrier and in part is also produced locally in the brain tissue. In the brain, DHEA exerts its effects after conversion to either testosterone and dihydrotestosterone or estradiol via androgen and estrogen receptors present in the most parts of the human brain, through mainly non-genomic mechanisms, or eventually indirectly via the effects of its metabolites formed locally in the brain. As a neuroactive hormone, DHEA in co-operation with other hormones and transmitters significantly affects some aspects of human mood, and modifies some features of human emotions and behavior. It has been reported that its administration can increase feelings of well-being and is useful in ameliorating atypical depressive disorders. It has neuroprotective and antiglucocorticoid activity and modifies immune reactions, and some authors have also reported its role in degenerative brain diseases. Here we present a short overview of the possible actions of dehydroepiandrosterone and its sulfate in the brain, calling attention to various mechanisms of their action as neurosteroids and to prospects for the knowledge of their role in brain disorders.

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.

Immunoassay of 7-hydroxysteroids : 2. Radioimmunoassay of 7α-hydroxy-dehydroepiandrosterone

High sensitivity radioimmunoassay of 3beta, 7alpha-dihydroxy-5-androsten-17-one (7alpha-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [(125)I]iodotyrosine methyl ester as a tracer. Sensitivity of the assay amounted to 3.12 fmol (0.95 pg)/tube, precision as a mean intra- and interassay coefficient of variation was 7.1 and 10.6%, respectively, and the average recovery of the analyte added to steroid-free serum was 110%. Out of the steroids occurring in human serum which may interfere with the assay, the only important cross-reactants were dehydroepiandrosterone and 3beta, 7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) with cross-reactivities of 1.95 and 1.16%, respectively. The levels of free (unconjugated) 7alpha-OH-DHEA have been determined in 29 sera from healthy volunteers (23 females and 6 males), and from 48 patients (43 females and 5 males) in which dehydroepiandrosterone and its sulfate (DHEA/S) had been measured for various endocrinopathies. The levels in healthy subjects ranged from 0.21 to 6.57 (mean 2.33+/-1.50) nM, while those of the patients from 0 to 5. 99 (mean 1.46+/-1.52) nM. The levels of 7alpha-OH-DHEA in patients significantly correlated with those of DHEA and its sulfate.

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.

A novel radioimmunoassay of allopregnanolone

A radioimmunoassay for determination of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in serum or plasma has been developed and evaluated. The method employs rabbit antiserum to 3 alpha-hydroxy-5 alpha-pregnane-11,20-dione-11-O-carboxymethyloxime bovine serum-albumin conjugate and tritiated radioligand. The main cross-reactant interfering in the assay, progesterone, is eliminated by permanganate oxidation. Two assay variants were compared, with and without a micro-column chromatography. The simplified variant appeared to be reliable enough for determination of allopregnanolone in normally menstruating women at luteal phase, whereas the column-chromatography step is necessary when analyzing samples of expected low analyte concentration as in women in follicular phase, postmenopausal women, or in men. The levels of allopregnanolone in healthy women correlated excellently with progesterone in agreement with previous findings.

Aqueous humour and plasma cortisol levels in glaucoma and cataract patients

By means of radioimmunoassay, the content of endogenous cortisol in the aqueous humour and plasma of 35 patients suffering from various types of glaucomas and 35 cataract patients was determined, and the ratio of the plasma to the aqueous humour cortisol levels was calculated. The highest cortisol level in both plasma and aqueous humour was found to occur in patients with open-angle glaucoma suffering at the same time from systemic hypertension. The lowest plasma to aqueous humour cortisol ratio was found in patients with secondary glaucoma treated with steroids. In woman suffering from open-angle glaucoma, the rising cortisol level in plasma with age contrasted to the decreasing cortisol level in aqueous humour. The authors suggest that there is an active interference with homeostatic mechanisms responsible for ensuring the stability of the eye inner milieu and a certain protection of the trabecular meshwork of the angle of the anterior eye chamber against noxious effects of the endogenous cortisol.

Neuroactive steroids, their precursors, and polar conjugates during parturition and postpartum in maternal and umbilical blood: 1. Identification and simultaneous determination of pregnanolone isomers.

A rapid method for the identification and measurement of four pregnanolone isomers and their polar conjugates in human plasma was developed using a simple quadrupole GC/MS system with electron impact ionization. Steroid levels were measured in the plasma of 13 and three women at delivery with subarachnoidal and epidural analgesia, respectively, and in corresponding samples of umbilical plasma. A good correlation (r=0.94, P<0.001, n=8) was found between the allopregnanolone in maternal plasma determined by GC/MS and that measured by RIA. Epipregnanolone (3beta-hydroxy-5beta-pregnan-20-one) was identified and measured for the first time in human plasma; its concentration in both maternal and umbilical plasma was much lower than that of other pregnanolone isomers. The levels of 3beta-hydroxy-pregnanolone isomers were significantly higher in the umbilical plasma than in the maternal plasma, while the differences in 3alpha-hydroxy-isomers were insignificant. The differences in conjugates were insignificant except in the case of allopregnanolone, the levels of which were lower in umbilical plasma. In all of the pregnanolone isomers, a significantly lower conjugated/unconjugated steroid ratio was found in the umbilical plasma than in the maternal plasma. The possible role of the sulfatation of pregnanolone isomers around parturition is discussed.