Richard Hampl

Serum Concentrations of Some Neuroactive Steroids in Women Suffering from Mixed Anxiety-Depressive Disorder

Concentrations of neurosteroids may be influenced by some physiological or pathological factors. We investigated neuroactive steroids in the serum of women suffering from anxiety-depressive disorder treated with fluoxetine and in a control group, in both the follicular and the luteal phases of the menstrual cycle. Two groups of neuroactive steroids were measured by radioimmunoassays: 1) the positive allosteric modulator of GABAA receptors, allopregnanolone with its precursor progesterone and 2) pregnenolone sulfate and dehydroepiandrosterone sulfate (DHEAS) acting on GABAA receptors by an opposite mechanism. Significantly higher levels of pregnenolone sulfate (p < 0.0001) were found in patients in both phases of the menstrual cycle. Significantly higher values were recorded in pregnenolone (p < 0.001) and 17α-hydroxy-pregnenolone (p < 0.01) levels in the patients group in the follicular phase. Our results indicate that imbalance in neuroactive steroids may play a negative role in origin and course of psychiatric and neurological disorders.

Division of Lymphatic Vessels at Varicocelectomy Leads to Testicular Oedema and Decline in Testicular Function According to the LH-RH Analogue Stimulation Test

OBJECTIVES To study the andrological outcome of the division of testicular lymphatic vessels at varicocelectomy in children and adolescents. METHODS Testicular size and basal and stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) values were determined prospectively in 86 patients with left unilateral varicocele grades II-III. 22 patients underwent lymphatic non-sparing surgery (LNS group), 10 of them with artery sparing (LNS/AS) and 12 without artery sparing (LNS/ANS), 23 patients underwent lymphatic sparing repair (LS group) and 41 patients were treated conservatively (NT group). RESULTS The LNS group demonstrated significantly greater left testicular enlargement at six weeks and one year following repair, left testicular hypertrophy developed in 31.8% and hydrocele in 22.7% of patients. Marked oedema of intertubular tissue and a varying degree of tubular injury was observed in boys surgically treated for hydrocele. In the LS group, neither hypertrophy nor hydrocele developed postoperatively, the LH stimulated values were lower than in LNS/ANS group (p<0.05) and the NT group (p<0.04), the FSH stimulated values were lower than in the LNS/ANS group (p<0.001). CONCLUSIONS Division of lymphatic vessels at varicocelectomy is associated with an excessive increase in testicular volume due to oedema, and with a reduced testicular function according to higher LH and FSH stimulated values. Preservation of lymphatics is strongly advised in varicocelectomy in adolescents to ensure better andrological outcome.

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.

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.

Immunoassay of 7-hydroxysteroids : 1. Radioimmunoassay of 7β-hydroxy dehydroepiandrosterone

High sensitivity radioimmunoassay of 3beta,7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against its 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [125I]iodotyrosine methyl ester as a tracer. Alternatively, [3H]tracer has been prepared, which was recognized by the antiserum as well, but the assay sensitivity was lower. The identity of measured immunoreactive material was confirmed by high performance liquid chromatography which separated 7beta-OH-DHEA from its 7alpha-isomer. Using radioiodinated tracer, the sensitivity of the method was 3.48 fmol (1.06 pg) per tube, the mean recovery of standard added to steroid-free serum was 98.5%. Free (unconjugated and not-esterified) 7beta-OH-DHEA amounted in average 5.8% of the total 7beta-OH-DHEA present in human serum. It was measured in 42 normal subjects (28 females and 14 males) and in 92 randomly selected patients with various endocrinopathies. The mean values +/- SD in normals were 2.05 +/- 1.02 nmol l(-1), the broad range of values from undetectable levels to 10.3 nmol l(-1) was found in the patients. Serum levels of free 7beta-OH-DHEA in the patients significantly correlated with DHEA and its sulfate.

Development and validation of LC-MS/MS method for quantification of bisphenol A and estrogens in human plasma and seminal fluid

Bisphenol A (BPA) is a widely known endocrine disruptor with estrogenic, antiestrogenic or antiandrogenic properties. BPA could interfere with estrogen metabolism as well with receptor-mediated estrogen actions. Both environmental BPA and estrogens may be traced in body fluids, of which, besides the blood plasma, the seminal fluid is of particular interest regarding their possible interactions in the testis. The method for simultaneously determining BPA and estrogens is then needed, taking into account that their concentrations in these body fluid may differ. Here the method was developed and validated for measurements of BPA, estrone (E1), estradiol (E2) and estriol (E3) in blood plasma and seminal plasma using liquid chromatography-tandem mass spectrometry. Due to the phenolic moiety of all compounds, dansyl chloride derivatization could be used. The analytical criteria of the method with respect to expected concentration of the analytes were satisfactory. The lower limits of quantifications (LLOQ) amounted to 43.5, 4.0, 12.7, 6.7 pg/mL for plasma BPA, E1, E2 and E3, and 28.9, 4.9, 4.5, 3.4 pg/mL for seminal BPA, E1, E2 and E3, respectively. The concentrations of individual steroids differed between body fluids. To the best of our knowledge, this is the first method that enabled the measurement of estrogens and BPA together in one run. The concentrations of E1, E2 and for the first time also of E3 in seminal plasma in normospermic men are reported.

Two neuroactive steroids in midpregnancy as measured in maternal and fetal sera and in amniotic fluid.

Allopregnanolone and pregnenolone sulfate, which are neuroactive steroids that differentially modulate the sensitivity of GABA(A) and NMDA receptors, were measured simultaneously in maternal and fetal sera and in amniotic fluid for the first time during the second trimester of gravidity. The study included 33 pregnant women, who underwent cordocentesis due to suspicion of fetal abnormality or alloimmunization. Allopregnanolone concentrations in maternal and fetal sera were similar and close to the previously found levels in healthy controls at 20 weeks of gestation The levels of pregnenolone sulfate in maternal serum were 2-3 times higher and in fetal serum, as much as 10-25 times higher than those found by others in the serum of healthy, non-pregnant women. A positive correlation between maternal and fetal allopregnanolone indicate similar 5alpha-reductase activities or the efficient transport of allopregnanolone between the two subjects. No correlation of pregnenolone sulfate levels between mother and fetus was found. This finding suggests the autonomous production of pregnenolone sulfate in mother and fetus.

Determination of steroid metabolome as a possible tool for laboratory diagnosis of schizophrenia.

Metabolomic studies represent a promising tool for early diagnosis of schizophrenia. The aim of this study was to find differences in the steroid spectrum in patients and controls, and to assess the diagnosis of schizophrenia by building a predictive model based on steroid data. Thirty-nine serum steroids (22 neuroactive steroids and their metabolites and 17 polar conjugates) representing steroid metabolome were measured by gas chromatography-mass spectrometry in 22 drug-naive (first episode) schizophrenia patients (13 men and 9 women) before and after six-month treatment with atypical antipsychotics. The results were compared to the data from healthy subjects (22 males, 25 females). In summary the following significant differences were found: (1) In both sexes higher levels of pregnenolone sulfate and sulfated 5α- as well as 5β-saturated metabolites of C21-steroids in progesterone metabolic pathway were found in patients, pointing to decreased activity of sulfatase. (2) In a few instances decreased levels of the respective 5α-metabolites of C21 steroids were found in patients. (3) As C19 steroids concern, in both sexes there were considerably lowered levels of 5β-reduced metabolites in patients. On the other hand, with only a few exceptions, the treatment did not significantly influence most steroid levels. Further, to assess the relationships between schizophrenia status and steroid levels and to build the predictive model of schizophrenia, multivariate regression with reduction of dimensionality (the method of orthogonal projections to latent structures, OPLS) was applied. Irrespective of the small number of patients, use of this model enabled us to state the diagnosis of schizophrenia with almost 100% sensitivity. Our findings suggest that the assessment of steroid levels may become a valid and accurate laboratory test in psychiatry. A limitation of our study is the absence of subjects with a diagnosis other than schizophrenia, so we cannot conclude whether the results are specific for schizophrenia. On the other hand, steroid metabolome model may be used as a diagnostic tool for further studies.