Comparative analysis of steroids in cyclic and pregnant killer whales, beluga whales and bottlenose dolphins by liquid chromatography tandem mass spectrometry.

There exists a surprising diversity in the physiology and endocrinology of pregnancy among mammals in both the source (luteal/placental) and metabolism of progesterone. To evaluate the possible diversity of steroid metabolism within toothed cetaceans, we investigated 5α-reduced progesterone metabolites and androgens in cyclic (luteal phase) and pregnant captive killer whales, belugas and bottlenose dolphins (n = 5/species) bled longitudinally in early, mid- and late pregnancy (0.16, 0.50 and 0.85 fractions of 535, 464 and 380 gestation days, respectively). Mid-luteal samples were also collected. Serum was analyzed by liquid chromatography tandem-mass spectrometry as previously validated for (among others) progesterone, 20αOH-progesterone (20αOHP), 5α-dihydroprogesterone (DHP), several additional 5α-reduced metabolites and androgens (dehydroepiandrosterone, androstenedione and testosterone). The predominant mid-luteal pregnanes were: progesterone, belugas; progesterone and 20αOHP, dolphins; allopregnanolone (3α-DHP) and progesterone, killer whales. Progesterone was 2-4-fold higher in early pregnancy than mid-luteal samples but decreased thereafter. The predominant metabolite, 3ß,20α-dihydroprogesterone (3ß,20α-DHP; 40-80 ng/ml) was higher in mid- and late-than early gestation in all 3 species. Concentrations of 20αOHP and 3ß,20α-DHP were similar at mid-gestation but 20αOHP declined in late-gestation in killer whales, and 20αOHP was lower than 3ß,20α-DHP in belugas and dolphins throughout gestation. Other 5α-reduced metabolites, DHP, 3α-DHP and 20α-DHP, were far lower throughout pregnancy (<10 ng/ml). DHP and 3α-DHP decreased from early to mid-gestation in belugas, but changed little in killer whales and dolphins. These data suggest that progesterone metabolism is relatively conserved among these cetacean species. As in equine pregnancies, 3ß,20α-DHP is the major metabolite, increasing at the expense of progesterone as pregnancy progresses. Androstenedione and testosterone also increased detectably in mid- to late-gestation in these species. The tissue source remains unknown, but progesterone metabolism during gestation in these cetaceans is similar to horses and, together with androgens, may be reliable biomarkers of pregnancy.

Serum and tissue pregnanes and pregnenes after dexamethasone treatment of cows in late gestation.

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3ß-hydroxysteroid dehydrogenase (3ßHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3ßHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3ßHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3ßHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.

Changes in maternal pregnane concentrations in mares with experimentally-induced, ascending placentitis.

The objectives of this study were to compare via liquid chromatography-tandem mass spectrometry (LC-MS/MS) progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, 3ß-hydroxy-5α-pregnan-20-one (3ß5P), 20α-hydroxy-5α-pregnan-3-one (20α5P), 5α-pregnan-3ß,20α-diol (ßα-diol), and 5α-pregnan-3ß,20ß-diol (ßß-diol) concentrations in plasma of mares with experimentally-induced, ascending placentitis compared to gestationally age-matched control mares. Placentitis was induced via intracervical inoculation of Streptococcus equi spp. zooepidemicus between 260 and 280 days of gestation. Placentitis mares were subdivided into those which aborted in less than eight days (n = 6; acute) and those that aborted at ≥ 8 days after inoculation (n = 9; chronic). Ten pregnant mares at similar gestational ages served as healthy controls. Pregnanes were measured for days (-8), -6, -4, -3, -2, -1, and 0 days preceding abortion in the treated mares, and for the matched days of gestation in the control mares by LC-MS/MS and by immunoassay for immunoreactive (ir) P4. In mares with chronic placentitis, concentrations of DHP and its downstream metabolites (allopregnanolone, 3ß5P, 20α5P, ßα-diol) increased at 2-8 days prior to abortion compared to control mares. Of these pregnanes, 20α5P and ßα-diol increased at eight days prior to abortion and demonstrated the largest increase (approximately 3 to 4×) in mares with chronic placentitis compared to control mares. Concentrations of P4 determined by LC-MS/MS were at or below the limit of detection (0.5 ng/mL) for control mares and did not increase significantly in mares with chronic placentitis. Immunoreactive-P4 was increased at two days prior to abortion in mares with chronic placentitis but was not different from controls in mares with acute placentitis. In mares with acute placentitis, concentrations of DHP, allopregnanolone, 3ß5P, 20α5P, and ßα-diol decreased within 0-3 days prior to abortion. In mares with chronic placentitis, the patterns of increased pregnanes metabolized by the placenta was similar to changes in maternal pregnanes noted in normal mares beyond Day 300 of gestation and likely represent the effects of fetal stress and adrenal activation on pregnane metabolism by the fetus and placenta. Decreases in these same pregnanes in mares with acute cases likely reflect extreme fetal or placental compromise.

A comparison of progesterone assays for determination of peripheral pregnane concentrations in the late pregnant mare.

During the latter half of gestation in mares, there is a complex milieu of pregnanes in peripheral blood. Progesterone concentrations are often assessed by immunoassay during late gestation as a measure of pregnancy well-being; however, interpretation of results is complicated by the numerous cross-reacting pregnanes present in high concentrations during late gestation. Further, many mares are supplemented with an exogenous progestin, altrenogest, which may also cross-react with existing assays and further confound interpretation. The objectives of this study were: 1) to compare differences in pregnane concentrations determined with four immunoassays compared to LC-MS/MS and 2) to assess cross-reactivity observed with the same immunoassays, specifically considering pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, and altrenogest. Blood samples from four healthy mares in late gestation were evaluated by immunoassay and by LC-MS/MS. Measured immuno-reactive progesterone (ir-progesterone) concentrations differed (p < 0.0001) between immunoassays, although results were highly correlated (r = 0.85-1.0; p < 0.001). Measured ir-progesterone concentrations by immunoassay were linearly associated (r2 = 0.68-0.76; p < 0.001) with concentrations of P5, P4, DHP, and allopregnanolone determined by LC-MS/MS. There was no detectable cross-reaction of altrenogest in any immunoassay, but varying degrees of cross-reactivity was observed with other pregnanes analyzed. These data confirm ir-progesterone concentrations during late gestation vary depending upon the assay used and the cross-reactivity to other pregnanes present in late gestation, although the synthetic progestin altrenogest did not affect the results of any immunoassay tested.

Simultaneous determination of six C21 steroids of Xiao-Ai-Ping injection in rat plasma by LC-MS/MS.

Xiao-Ai-Ping injection (XAPI) is a traditional Chinese medicine that has been widely used to treat cancer. Modern pharmacological studies have demonstrated that C21 steroids are the main active compounds in XAPI. In this study, a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated the first time for simultanenous determination of three isomeric pregnane genins (17ß-tenacigenin B, tenacigenin B and tenacigenin A) and their corresponding glycosides (tenacigenoside A, tenacissoside F and marsdenoside I) from XAPI in rat plasma. A simple liquid-liquid extraction technique was used after the addition of dexamethasone acetate as internal standard. The chromatography separation of analytes was achieved on an Agilent Zorbax Eclipse XDB-C18 column (3.5 µm, 150 × 3 mm i.d.) using methanol-water as mobile phase in a gradient elution program. Detection was performed in multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method showed satisfactory linearity over a concentration range 5.00-2000.00 ng/mL for tenacigenin B, tenacigenin A, marsdenoside I and tenacissoside F (r(2) > 0.99), 10.00-4000.00 ng/mL for 17ß-tenacigenin B and tenacigenoside A (r(2) > 0.99). Intra- and inter-day precisions (valued as relative standard deviation) were <9.00% and accuracies (as relative error) in the range -6.31 to 7.23%. Finally, this validated method was successfully applied to the pharmacokinetic study of XAPI after intravenous administration to rats.

Effect of insulin on the relationship of estrous behaviors to estradiol and LH surges in intact ewes.

The objective of this study was to determine whether acute stress alters the frequency of spontaneous estrous behaviors and temporal relationships with preovulatory increases in peripheral plasma estradiol and LH. Follicular phases of intact ewes were synchronized with prostaglandin administered at progesterone pessary withdrawal (PW). Twelve ewes served as controls and 12 were acutely stressed (insulin 4 IU/kg given at 30 and 32 h after PW). Ewes being near to ram(s) 21.3+/-1.9h after PW was the first precopulatory behavioral sign and rams nosed the perineal region of ewes after a further 9.0+/-2.0 h (P<0.01), with ewes being nudged and mounted by rams 6.8+/-2.3h later still (P<0.01). Insulin did not affect the frequency or timing (relative to PW) of each behavioral sign of estrus. However, within each animal, estradiol values were more than 2 pg/ml lower (P<0.05) for 6h following insulin, and the onset of the LH surge was delayed in insulin-treated ewes compared to controls (49.5+/-3.3 versus 38.2+/-2.6h; P=0.01). Consequently, the interval between the onset of being mounted and the LH surge was longer in insulin-treated ewes compared to controls (10.4+/-3.0 versus 2.3+/-0.7 h; P<0.01). Maximum LH values were also 15 ng/ml lower after insulin (P<0.01). Thus, acute stress did not alter the timing or frequencies of estrous behaviors but it did reduce estradiol concentrations and delayed the onset and magnitude of the LH surge.

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.

Identification of plasma glucocorticoids in pallid sturgeon in response to stress.

Compared to teleosts, little is known about the stress response in chondrosteans, and the glucocorticoid(s) most responsive to stress have never been definitively determined in sturgeon. In terms of cortisol production, pallid sturgeon (Scaphirhynchus albus) have a low physiological response to stress compared to other sturgeons (Acipenser s.p.). Because of this, our null hypothesis was that cortisol is not the predominant glucocorticoid secreted in response to stress in pallid sturgeon. Our objective was to identify the putative glucocorticoids present in the plasma of pallid sturgeon during the stress response. Pallid sturgeon were subjected to a severe confinement stress (12 h) with an additional handling stressor for the first 6 h. Control fish were not subjected to confinement but were handled only to collect blood. Blood plasma was collected at time 0, 6, and 12 h. Gas chromatography/mass spectrometry was used to screen the plasma for the spectrum of glucocorticoids and determine the putative steroid secreted during the stress response. Cortisol was the primary glucocorticoid detected in stressed pallid sturgeon. In addition, the cortisol metabolites cortisone, alloTHE (5alpha-pregnane-3alpha,17alpha,21-triol-11,20-dione), allo-alpha-cortolone (3alpha,17alpha,20alpha,21-tetrahydro-5alpha-pregnan-11-one), and allo-beta-cortolone (3alpha,17alpha,20beta,21-tetrahydro-5alpha-pregnan-11-one) were detected. Plasma cortisol increased from a resting concentration of 0.67 ng/ml to 10.66 ng/ml at 6h followed by a decrease to 6.78 ng/ml by 12 h. Plasma glucose increased significantly by time 6 and 12 h in both stressed and unstressed groups and remained elevated at time 12h, while resting lactate concentrations were low to non-detectable and did not increase significantly with the stressor over time. Cortisol was the primary glucocorticoid synthesized and secreted in response to a stressor in pallid sturgeon. Though the proportional increase in plasma cortisol in stressed pallid sturgeon was lower than many other species of sturgeon, the concentration was high enough to elicit a secondary stress response as seen by changes in plasma glucose.

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography-tandem mass spectrometry.

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (MGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid-liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE). Analytes were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte. Decision limits (CCalpha) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2 ng mL(-1). Detection capabilities (CCbeta) were estimated using 20 plasma samples fortified at 0.5 ng mL(-1) and were <0.5 ng mL(-1). In the range 0.5-2 ng mL(-1), the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.). Analytes were shown to be stable in fortified plasma samples for >8 months when stored at -20 degrees C.

Studies on neurosteroids XIX. Development and validation of liquid chromatography-tandem mass spectrometric method for determination of 5alpha-reduced pregnane-type neurosteroids in rat brain and serum.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method for the simultaneous determination of 5alpha-reduced pregnan-type neurosteroids, allopregnanolone (AP), epiallopregnanolone and 5alpha-dihydroprogesterone, in rat brain and serum has been developed and validated. The brain and serum steroids were extracted with methanol-acetic acid, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-positive ESI-MS-MS. The limits of quantitation (LOQ) for brain (0.25 ng/g tissue) and serum (0.25 ng/ml) assays using the derivatization-ESI-MS-MS method are 60-150-fold lower than the LOQs for their atmospheric pressure chemical ionization-MS method without derivatization. [17Alpha,21,21,21-2H4]-AP was used as an internal standard. This method allowed the reproducible and accurate quantification of the brain or serum neurosteroids using a 20 mg or 20 microl sample, respectively. That is, the intra- and inter-assay coefficients of variation were below 8.2 and 6.0%, respectively, and the % accuracy values were 98.5-103.0% for all the steroids in both the brain and serum. The application of the developed method to the analysis of changes in the brain and serum neurosteroid levels by immobilization stress and ethanol administration is also presented.

Preliminary evidence of reduced occipital GABA concentrations in puerperal women: a 1H-MRS study.

RATIONALE: Childbirth is associated with rapid neuroendocrine fluctuations, which are thought to contribute to the phatogenesis of postpartum major depression (PPD). OBJECTIVES: The aim of this proton magnetic resonance spectroscopy (1H-MRS) study was two-fold; 1) to examine whether puerperium is associated with alterations in occipital cortex gamma-aminobutyric acid (GABA) concentrations and 2) to determine whether such alterations may be more prominent in women with PPD. MATERIALS AND METHODS: Nine women with PPD, 14 postpartum healthy controls, and ten healthy follicular phase females underwent 1H-MRS at 2.1 Tesla to measure occipital cortex GABA concentrations. Postpartum women were scanned within 6 months of delivery and prior to resumption of menstruation. Healthy non-puerperal controls, drawn from a historical sample, were scanned during the early to mid-follicular phase when ovarian hormone levels would be similar to those found in the puerperium. GABA data were analyzed using analysis of covariance, and regression models were used to explore the relationship between cortical GABA concentrations and blood levels of estradiol, progesterone, and neurosteroids. RESULTS: Cortical GABA and plasma allopregnanolone (ALLO) concentrations were reduced in both groups of postpartum women, regardless of PPD diagnosis, compared to healthy follicular phase women. There was no correlation between cortical GABA concentrations and estradiol, progesterone, ALLO, or pregnenolone (PREG). CONCLUSIONS: This study is the first to describe reductions in occipital cortex GABA levels in the postpartum period, a time of increased vulnerability to mood disturbances in women. The concomitant reduction in peripheral ALLO levels provides further evidence of alterations in the balance between cortical excitation and inhibition during the puerperium. Women with PPD may represent a subgroup of women who fail to adequately adapt to this alteration in the neuroendocrine milieu.

Neuroactive steroids, negative affect, and nicotine dependence severity in male smokers.

RATIONALE: Nicotine administration alters neuroactive steroids in rodent models, and serum levels of the neuroactive steroid DHEAS (dehydroepiandrosterone sulfate) appear to be higher in smokers. These molecules may be relevant to tobacco addiction and affective symptoms. OBJECTIVES: This study aims to investigate DHEAS, allopregnanolone, pregnenolone, and other steroids in male smokers to determine potential associations with nicotine dependence severity and negative affect. MATERIALS AND METHODS: Allopregnanolone and pregnenolone serum levels were determined by gas chromatography/mass spectrometry, while DHEAS and other steroid levels were determined by radioimmunoassay in 28 male smokers. Correlational analyses were performed to determine potential associations with rating measures, including the Fagerstrom Test for Nicotine Dependence (FTND), the addiction subscale of the Ikard Smoking Motivation Questionnaire (ISMQ), the craving item on the Reasons to Smoke (RTS) Questionnaire, and the negative affect and craving subscales of the Shiffman-Jarvik Withdrawal Questionnaire. RESULTS: DHEAS levels were inversely correlated with the negative affect subscale of the Shiffman-Jarvik Withdrawal Questionnaire (r=-0.60, p=0.002) and the RTS craving item (r=-0.43, p=0.03), and tended to be inversely correlated with the FTND scores (r=-0.38, p=0.067) and the ISMQ addiction subscale (r=-0.38, p=0.059), adjusting for age. Allopregnanolone levels were positively correlated with cotinine levels (r=0.57, p=0.006); pregnenolone levels tended to be positively correlated with cotinine levels (r=0.40, p=0.066). CONCLUSIONS: DHEAS levels were inversely correlated with negative affect and craving measures, and may predict nicotine dependence severity. Allopregnanolone levels were positively correlated with cotinine levels, suggesting that this neuroactive steroid may be upregulated in smokers. Neuroactive steroids may represent novel smoking cessation agents.

Observational study on the stability of the psychological status during normal pregnancy and increased blood levels of neuroactive steroids with GABA-A receptor agonist activity.

We investigated whether pregnancy could modify psychological symptoms and whether neuroactive steroids which exert an anti-anxiety effect by acting on the gamma-aminobutyric acid (GABA)-A receptors, are modified during pregnancy in young healthy women. Healthy volunteer women in the Department of Obstetrics and Gynecology at Cagliari University participated in the study. They were divided into women with low (group 1, seven subjects) and high (group 2, seven subjects) psychological score by SCL-90 psychometric scale. Age, body mass index and physiological status of pregnancy did not differ between the groups. The subjects were studied before pregnancy during the follicular phase (FP), and the luteal phase (LP) of the menstrual cycle (MC) and four times during pregnancy (at 14th, 22nd, 30th, and 38th week). SCL-90 psychometric scale, circulating levels of progesterone (P4), 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone, AP), 3alpha,21-dihydroxy-5alpha-pregnan-20-one (allotetrahydrodeoxy-corticosterone, THDOC), cortisol and DHEAS were assayed at each visit. The SCL-90 global score and the intensity of psychological symptoms differ between the groups, but within each group they did not change both during MC and during pregnancy. The DHEAS and cortisol levels did not differ between the groups. DHEAS did not change during the study, whereas cortisol levels increased during pregnancy in both groups. Progesterone, AP, and THDOC levels were higher during LP than during FP and further increased during pregnancy, without any difference between the groups. In conclusion, pregnancy does not seem to interfere with the psychological status of healthy women independently of the psychological basal score. Some neuroactive steroids with anxiolytic activity seem to increase during pregnancy depending on placental function. Their increase could represent some kind of protection against maternal anxiety and stress due to concerns about the pregnancy outcome.

Neuropsychopharmacological properties of neuroactive steroids in depression and anxiety disorders.

Neuroactive steroids modulate neurotransmission through modulation of specific neurotransmitter receptors such as gamma-aminobutyric acid type A (GABAA) receptors. Preclinical studies suggested that neuroactive steroids may modulate anxiety- and depression-related behaviour and may contribute to the therapeutical effects of antidepressant drugs. Attenuations of 3alpha-reduced neuroactive steroids have been observed during major depression. This disequilibrium can be corrected by successful treatment with antidepressant drugs. However, non-pharmacological antidepressant treatment strategies did not affect neuroactive steroid composition independently from the clinical response. Further research is needed to clarify whether enhancement of neuroactive steroid levels might represent a new therapeutical approach in the treatment of affective disorders. Nevertheless, the first studies investigating the therapeutical effects of exogenously administered dehydroepiandosterone revealed promising results in the treatment of major depression. In addition, in various anxiety disorders alterations of neuroactive steroid levels have been observed. In panic disorder, in the absence of panic attacks, neuroactive steroid composition is opposite to that seen in depression, which may represent counter-regulatory mechanisms against the occurrence of spontaneous panic attacks. However, during experimentally induced panic attacks, there was a pronounced decline in GABAergic neuroactive steroids, which might contribute to the pathophysiology of panic attacks. In conclusion, neuroactive steroids contribute to the pathophysiology of affective disorders and the mechanisms of action of antidepressants. They are important endogenous modulators of depression and anxiety and may provide a basis for the development of novel therapeutic agents in the treatment of affective disorders.

Subjective effects and changes in steroid hormone concentrations in humans following acute consumption of alcohol.

BACKGROUND: GABAA receptors are an important site of action of endogenous neurosteroids and an important mediator of several behavioral effects of alcohol. This study examined the effects of alcohol on plasma steroid hormone concentrations on the hypothesis that the endocrine effects mediate some of the subjective effects of alcohol. METHODS: Thirty-two healthy subjects (17 men) with no history of a substance use disorder participated in this human laboratory study. All subjects consumed three standard drinks of grain alcohol. Subjective measures and blood samples for steroid concentrations were collected at baseline and 40 min after alcohol consumption. RESULTS: Alcohol increased self-reported stimulation, alcohol liking, and desire for more alcohol. Alcohol also increased pregnenolone (PREG) and dehydroepiandrosterone (DHEA) concentrations, while it decreased progesterone (PROG) and allopregnanolone (ALLO) concentrations, as well as ALLO/PREG and PROG/PREG ratios. In men, the change in PREG concentration was significantly correlated with alcohol liking, while the alcohol-induced change in ALLO concentration correlated significantly with both alcohol liking and desire for more alcohol. DISCUSSION: These findings provide preliminary support for the hypothesis that endogenous neurosteroids mediate some of the subjective effects of alcohol. Efforts to replicate these findings should aim to specify more clearly the nature and time course of the effects.

Modulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the equine placenta by pregnenolone and progesterone metabolites.

The purpose of this study was to measure 3beta-HSD activity in the equine placenta and to assess the effect of fetal and maternal blood plasma progestagens on 3beta-HSD activity was measured in 8 late gestation (collected by caesarian section at 250 to 320 days) and 7 term (collected by caesarian section at 250 to 320 days) and 7 term (collected at birth) equine placentae using a tritium release assay with [3alpha-3H] pregnenolone as substrate. Mean +/- s.d. Km(app) and Vmax for term placentae were in general higher than for late gestation placentae (0.129 +/- 0.217 micromol/l and 23.85 +/- 9.1 nmol/mg/h respectively vs. 0.016 +/- 0.048 micromol/l and 17.36 +/- 20.9 nmol/mg/h) but there was no statistical difference between them. Inhibition studies were performed on 3 term placentae and 3 late gestation ones. Steroid concentrations used for inhibition studies were close to blood plasma concentrations (0.5 to 2 micromol/l). 3beta-hydroxy compounds (5alpha-pregnene-3beta, 20alpha-diol and 3beta-hydroxy-5alpha-pregnan-20-one) showed noncompetitive or mixed inhibition. Mean Ki(app) of 0.7 micromol/l. Inhibition was competitive with 20alpha-hydroxy-5alpha-pregnan-3-one with a mean Ki(app) of 0.1 micromol/l.

Do neuroactive steroids cause fatigue in pregnancy?

To elucidate factors causing widespread fatigue in pregnancy we studied plasma samples from 23 healthy pregnant volunteers, measuring endogenous neuroactive steroids, endozepines and benzodiazepines by receptor binding assays. The samples were taken at different gestational ages and the women were asked about their feeling of fatigue at the time of sample collection. We found a significant correlation between fatigue in pregnancy and presence of neuroactive steroid-like activity, but could not detect endogenous benzodiazepine-like factors. These results suggest that neuroactive steroids may be involved in fatigue during pregnancy. Their site of synthesis and their biological role remain unclear.

Measurement of steroid hormones in plasma by isocratic high performance liquid chromatography coupled to radioimmunoassay.

The study of steroidal profiles requires simultaneous determinations of various steroid hormones that cannot be appropriately carried out with the conventional routine immunoassays. Moreover, there are several trials for which the assessment of multiple steroids from a single serum sample is mandatory. In this paper we describe a procedure for simultaneously measuring steroid hormones using a unified solid phase extraction which allows the measurement of both unconjugated and conjugated steroids from 1 ml of sample and a combination of HPLC with isocratic elution followed by RIA. The entire procedure was preliminary carried out for the measurement of testosterone, dehydroepiandrosterone and its sulphated conjugate, androstenedione and 17 hydroxyprogesterone. The use of this technique allows precise and accurate measurements of steroid profile with a single serum aliquot and could be helpful in the diagnosis of various form of endocrine disorders.

Determination of a novel steroidal androgen receptor antagonist (Win 49596) in human plasma using solid-phase extraction and high-performance liquid chromatography with ultraviolet detection.

A rapid, sensitive and selective method was developed for the determination of a novel steroidal androgen receptor antagonist (Win 49596, I) in human plasma. The procedure involved extraction from plasma using a solid-phase phenyl support and elution directly onto a reversed-phase C8 column using a mobile phase consisting of 0.2 mol/l sodium acetate buffer at pH 7-acetonitrile (45:55, v/v). Drug was monitored by ultraviolet detection at a wavelength of 238 nm. Linear responses were observed for standards over the range 0.01-5.0 micrograms/ml. The minimum quantifiable level was 0.02 microgram/ml, using a 0.5-ml plasma sample. The precision was 5.5% and the accuracy ranged from -9.4% to 0.23%. The analytical method has been used to quantify I in plasma from dogs and rats and is projected for use with human plasma from clinical trials.