Palmitoylation Controls NMDA Receptor Function and Steroid Sensitivity.

NMDARs are ligand-gated ion channels that cause an influx of Na+ and Ca2+ into postsynaptic neurons. The resulting intracellular Ca2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+ (Ca2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5ß-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5ß-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 µm PAhPim had virtually no effect on NMDAR responses; however, following the Ca2+ challenge, it inhibited the responses by 62%; similarly, the Ca2+ challenge induced a 3.7-fold decrease in the steroid IC50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.

Improved Urinary Cortisol Metabolome in Addison Disease: A Prospective Trial of Dual-Release Hydrocortisone.

CONTEXT: Oral once-daily dual-release hydrocortisone (DR-HC) replacement therapy has demonstrated an improved metabolic profile compared to conventional 3-times-daily (TID-HC) therapy among patients with primary adrenal insufficiency. This effect might be related to a more physiological cortisol profile, but also to a modified pattern of cortisol metabolism. OBJECTIVE: This work aimed to study cortisol metabolism during DR-HC and TID-HC. DESIGN: A randomized, 12-week, crossover study was conducted. INTERVENTION AND PARTICIPANTS: DC-HC and same daily dose of TID-HC were administered to patients with primary adrenal insufficiency (n = 50) vs healthy individuals (n = 124) as controls. MAIN OUTCOME MEASURES: Urinary corticosteroid metabolites were measured by gas chromatography/mass spectrometry at 24-hour urinary collections. RESULTS: Total cortisol metabolites decreased during DR-HC compared to TID-HC (P < .001) and reached control values (P = .089). During DR-HC, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity measured by tetrahydrocortisol + 5α-tetrahydrocortisol/tetrahydrocortisone ratio was reduced compared to TID-HC (P < .05), but remained increased vs controls (P < .001). 11ß-HSD2 activity measured by urinary free cortisone/free cortisol ratio was decreased with TID-HC vs controls (P < .01) but normalized with DR-HC (P = .358). 5α- and 5ß-reduced metabolites were decreased with DR-HC compared to TID-HC. Tetrahydrocortisol/5α-tetrahydrocortisol ratio was increased during both treatments, suggesting increased 5ß-reductase activity. CONCLUSIONS: The urinary cortisol metabolome shows striking abnormalities in patients receiving conventional TID-HC replacement therapy, with increased 11ß-HSD1 activity that may account for the unfavorable metabolic phenotype in primary adrenal insufficiency. Its change toward normalization with DR-HC may mediate beneficial metabolic effects. The urinary cortisol metabolome may serve as a tool to assess optimal cortisol replacement therapy.

A biocatalytic hydroxylation-enabled unified approach to C19-hydroxylated steroids.

Steroidal C19-hydroxylation is pivotal to the synthesis of naturally occurring bioactive C19-OH steroids and 19-norsteroidal pharmaceuticals. However, realizing this transformation is proved to be challenging through either chemical or biological synthesis. Herein, we report a highly efficient method to synthesize 19-OH-cortexolone in 80% efficiency at the multi-gram scale. The obtained C19-OH-cortexolone can be readily transformed to various synthetically useful intermediates including the industrially valuable 19-OH-androstenedione, which can serve as a basis for synthesis of C19-functionalized steroids as well as 19-nor steroidal drugs. Using this biocatalytic C19-hydroxylation method, the unified synthesis of six C19-hydroxylated pregnanes is achieved in just 4 to 9 steps. In addition, the structure of sclerosteroid B is revised on the basis of our synthesis.

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.

Biosynthetic pathway for furanosteroid demethoxyviridin and identification of an unusual pregnane side-chain cleavage.

Furanosteroids, represented by wortmannin, viridin, and demethoxyviridin, are a special group of fungal-derived, highly oxygenated steroids featured by an extra furan ring. They are well-known nanomolar-potency inhibitors of phosphatidylinositol 3-kinase and widely used in biological studies. Despite their importance, the biosyntheses of these molecules are poorly understood. Here, we report the identification of the biosynthetic gene cluster for demethoxyviridin, consisting of 19 genes, and among them 15 biosynthetic genes, including six cytochrome P450 monooxygenase genes, are deleted. As a result, 14 biosynthetic intermediates are isolated, and the biosynthetic pathway for demethoxyviridin is elucidated. Notably, the pregnane side-chain cleavage requires three enzymes: flavin-dependent Baeyer-Villiger monooxygenase, esterase, and dehydrogenase, in sharp contrast to the single cytochrome P450-mediated process in mammalian cells. Structure-activity analyses of these obtained biosynthetic intermediates reveal that the 3-keto group, the C1ß-OH, and the aromatic ring C are important for the inhibition of phosphatidylinositol 3-kinase.

Inhibition of 5α-reductase alters pregnane metabolism in the late pregnant mare.

In the latter half of gestation in the mare, progesterone concentrations decline to near undetectable levels while other 5α-reduced pregnanes are elevated. Of these, 5α-dihydroprogesterone and allopregnanolone have been reported to have important roles in either pregnancy maintenance or fetal quiescence. During this time, the placenta is necessary for pregnane metabolism, with the enzyme 5α-reductase being required for the conversion of progesterone to 5α-dihydroprogesterone. The objectives of this study were to assess the effects of a 5α-reductase inhibitor, dutasteride on pregnane metabolism (pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol and allopregnanolone), to determine circulating dutasteride concentrations and to assess effects of dutasteride treatment on gestational parameters. Pregnant mares (n = 5) received dutasteride (0.01 mg/kg/day, IM) and control mares (n = 4) received vehicle alone from 300 to 320 days of gestation or until parturition. Concentrations of dutasteride, pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol, and allopregnanolone were evaluated via liquid chromatography-tandem mass spectrometry. Samples were analyzed as both days post treatment and as days prepartum. No significant treatment effects were detected in pregnenolone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol or allopregnanolone for either analysis; however, progesterone concentrations were increased (P < 0.05) sixfold in dutasteride-treated mares compared to control mares. Dutasteride concentrations increased in the treated mares, with a significant correlation (P < 0.05) between dutasteride concentrations and pregnenolone or progesterone concentrations. Gestational length and neonatal outcomes were not significantly altered in dutasteride-treated mares. Although 5α-reduced metabolites were unchanged, these data suggest an accumulation of precursor progesterone with inhibition of 5α-reductase, indicating the ability of dutasteride to alter progesterone metabolism.

Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC.

Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the Gloeobacter ligand-gated ion channel (GLIC), a prototypic pLGIC. The neurosteroid-based reagents photolabeled two sites per GLIC subunit, both within the transmembrane domain; one site was an intrasubunit site, and the other was located in the interface between subunits. By using reagents with photoreactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Amino acid substitutions introduced at either site altered neurosteroid modulation of GLIC channel activity, demonstrating the functional role of both sites. These results provide a detailed molecular model of multisite neurosteroid modulation of GLIC, which may be applicable to other mammalian pLGICs.

Classification of Human Pregnane X Receptor (hPXR) Activators and Non-Activators by Machine Learning Techniques: A Multifaceted Approach.

The Human Pregnane X Receptor (hPXR) is a regulator of drug metabolising enzymes (DME) and efflux transporters (ET). The prediction of hPXR activators and non-activators has pharmaceutical importance to predict the multiple drug resistance (MDR) and drug-drug interactions (DDI). In this study, we developed and validated the computational prediction models to classify hPXR activators and non-activators. We employed four machine learning methods support vector machine (SVM), k-nearest neighbour (k-NN), random forest (RF) and naïve bayesian (NB). These methods were used to develop molecular and fingerprint based descriptors for the prediction of hPXR activators and non-activators. Total 529 molecules consitsting of 317 activators and 212 non-activators were used for model development. The overall prediction accuracy of models was 69% to 99% to classify hPXR activators and nonactivators using RDkit descriptors. In case of 5 and 10-fold cross validation the prediction accuracy for training set is 74% to 82% and 79% to 83% for hPXR activators respectively and 50% to 62% and 49% to 65% non-activators, respectively. The external test prediction is between 59% to 73% for hPXR activators and 55% to 68% for hPXR non-activators. In addition, consensus models were developed in which the best model shows overall 75% to 83% accuracy for fingerprint and RDkit descriptors, respectively. The best developed model will be utilized for the prediction of hPXR activators and non-activators.

Noninvasive monitoring of female reproductive hormone metabolites in the endangered European mink (Mustela lutreola).

This study examined the reproductive physiology of female European mink (Mustela lutreola) to augment the available information on estrus, ovulation, and pregnancy with the long-term goal of supporting ex situ breeding management of this highly endangered species. Fecal reproductive hormone metabolites were measured using EIAs for estrogen and 20-oxo-pregnane metabolites. Seasonal hormone profiles were established. A comparison of hormone fluctuations in pregnant and nonpregnant females showed that both estrogen and 20-oxo-pregnane metabolites were significantly elevated during gestation, which is 42 days in length. Delayed implantation or embryonic diapause does not occur in this species. Litter size was correlated with 20-oxo-pregnane levels but not with estrogen concentrations. During lactation, 20-oxo-pregnane metabolite levels remained higher than in nonpregnant females. The breeding season was characterized by peaks in vaginal cornified cells and fecal estrogen metabolite levels. Up to four peaks in estrogen levels were identified and confirmed that European mink are seasonally polyestrous. The results of 20-oxo-pregnane measurements indicated that hCG can be applied to induce ovulation. With the establishment of this noninvasive method, we present a new tool to support population management of this species.

Steroidogenesis in plants--Biosynthesis and conversions of progesterone and other pregnane derivatives.

In plants androstanes, estranes, pregnanes and corticoids have been described. Sometimes 17ß-estradiol, androsterone, testosterone or progesterone were summarized as sex hormones. These steroids influence plant development: cell divisions, root and shoot growth, embryo growth, flowering, pollen tube growth and callus proliferation. First reports on the effect of applicated substances and of their endogenous occurrence date from the early twenties of the last century. This caused later on doubts on the identity of the compounds. Best investigated is the effect of progesterone. Main steps of the progesterone biosynthetic pathway have been analyzed in Digitalis. Cholesterol-side-chain-cleavage, pregnenolone and progesterone formation as well as the stereospecific reduction of progesterone are described and the corresponding enzymes are presented. Biosynthesis of androstanes, estranes and corticoids is discussed. Possible progesterone receptors and physiological reactions on progesterone application are reviewed.

[Effect of Acupuncture Stimulation of Different Acupoint Groups on Levels of Stress Hormones and Serum Brain-derived Neurotrophic Factor in Depression Rats].

OBJECTIVE: To screen the effective acupoints for anti-depression in depression rats and to explore the mechanisms of acupuncture for relieving depression. METHODS: Fifty rats were randomly divided into normal control, model, "Yin-tang" (EX-HN 3)-"Baihui" (GV 20, 2-acupoints), EX-HN 3-GV 20-"Fengchi" (GB 20)-"Shenshu" (BL 23, 4-acupoints) and medication groups, with 10 rats in each group. The depression model was established by unpredictable chronic mild stress (UCMS) for 28 days. For rats of the 2-acupoints and 4-acupoints groups, EX-HN 3 and GV 20, and EX-HN 3, GV 20, GB 20 and BL 23 were punctured with filiform needles respectively before performing mild stress every time. The acupuncture needles were retained for 30 min during each intervention and the treatment was conducted once daily for 28 days. The rats of the medication group were treated by intragastric administration of Fluoxetine (0.18 mg/kg) once a day for 28 days. The rats' anxiety-like behavior (rearing and crossing times) was detected by open-field test. The contents of adrenocorticotropic hormone (ACTH) in the pituitary, 5-hydroxytryptamine (5-HT) in the hippocampus, the cortisol (CORT) in the adrenal gland, and the brain derived neurotrophic factor (BDNF) in the serum were examined by ELISA. RESULTS: Compared to the normal control group, the numbers of both rearing and crossing motions in the model group were significantly decreased (P<0.01, P<0.05), while in comparison with the model group, the rearing and crossing numbers of rats in the 2-acupoints and 4-acupoints and medication groups were significantly increased (P<0.01). ELISA showed that after modeling, the content of adrenal CORT was significantly increased (P<0.01), and those of hippocampal 5-HT and serum BDNF were obviously down-regulated in the model group (P<0.01). After the treatment, the adrenal CORT levels in the three intervention groups were notably down-regulated, and hippocampal 5-HT and serum BONE evidently up-regulated in these 3 intervention groups (P<0.05, P<0.01). No marked changes were found in the pituitary ACTH contents of the model and 3 intervention groups (P>0.05), and no significant differences were shown among the three intervention groups in the levels of the aforementioned 6 indexes (P>0.05). CONCLUSION: Acupuncture intervention can effectively improve the unprompted activates of the depression rats, which may be related to its effects in up-regulating hippocampal 5-HT and serum BONE levels, and in down-regulating adrenal CORT content.

Important roles of the AKR1C2 and SRD5A1 enzymes in progesterone metabolism in endometrial cancer model cell lines.

Endometrial cancer is the most frequently diagnosed gynecological malignancy. It is associated with prolonged exposure to estrogens that is unopposed by progesterone, whereby enhanced metabolism of progesterone may decrease its protective effects, as it can deprive progesterone receptors of their active ligand. Furthermore, the 5α-pregnane metabolites formed can stimulate proliferation and may thus contribute to carcinogenesis. The aims of our study were to: (1) identify and quantify progesterone metabolites formed in the HEC-1A and Ishikawa model cell lines of endometrial cancer; and (2) pinpoint the enzymes involved in progesterone metabolism, and delineate their roles. Progesterone metabolism studies combined with liquid chromatography-tandem mass spectrometry enabled identification and quantification of the metabolites formed in these cells. Further quantitative PCR analysis and small-interfering-RNA-mediated gene silencing identified individual progesterone metabolizing enzymes and their relevant roles. In Ishikawa and HEC-1A cells, progesterone was metabolized mainly to 20α-hydroxy-pregn-4-ene-3-one, 20α-hydroxy-5α-pregnane-3-one, and 5α-pregnane-3α/ß,20α-diol. The major difference between these cell lines was rate of progesterone metabolism, which was faster in HEC-1A cells. In the Ishikawa and HEC-1A cells, expression of AKR1C2 was 110-fold and 6800-fold greater, respectively, than expression of AKR1C1, which suggests that 20-ketosteroid reduction of 5α-pregnanes and 4-pregnenes is catalyzed mainly by AKR1C2. AKR1C1/AKR1C2 gene silencing showed decreased progesterone metabolism in both cell lines, thus further supporting the significant role of AKR1C2. SRD5A1 was also expressed in these cells, and its silencing confirmed that 5α-reduction is catalyzed by 5α-reductase type 1. Silencing of SRD5A1 also had the most pronounced effects, with decreased rate of progesterone metabolism, and consequently higher concentrations of unmetabolized progesterone. Our data confirm that in model cell lines of endometrial cancer, AKR1C2 and SRD5A1 have crucial roles in progesterone metabolism, and may represent novel targets for treatment.

Identification of cortisol metabolites in the bile of Atlantic cod Gadus morhua L.

Interpretation of plasma cortisol levels in wild-caught fish is confounded by the stress of capture. Measurement of cortisol metabolites in fish bile could provide a method for assessing the stress level of wild fish because the time-lag for metabolism, conjugation and excretion into bile avoids the effects of sampling stress. To determine which biliary metabolite(s) to target, four Atlantic cod, Gadus morhua L., were injected with radioactive cortisol. After 22 h, the bile was collected and found to contain 30% of the injected activity. Cortisol metabolites were extracted from diluted bile samples using solid phase extraction and the radioactive metabolites separated by several different chromatographic procedures. The metabolites were predominantly present as sulfates (95%) with the remainder being glucuronidated. Chromatography split the sulfates into at least seven peaks, and acid solvolysis (which removes sulfate groups from steroids) generated four major radioactive steroids. These were identified, using microchemical reactions and re-crystallization to constant specific activity, as: 11ß,17,21-trihydroxypregn-4-ene-3,20-dione (cortisol), 3α,11ß,17,21-tetrahydroxy-5ß-pregnan-20-one (tetrahydrocortisol; THF), 3α,17,21-trihydroxy-5ß-pregnane-11,20-dione (tetrahydrocortisone; THE) and 3α,17,20ß,21-tetrahydroxy-5ß-pregnan-11-one (ß-cortolone). The last of these was the most abundant, and thus a likely target for a biliary stress assay. Studies were also carried out to determine the best method for extraction and solvolysis of sulfates. Solid phase extraction (i.e. using octadecylsilane) was found to be too unreliable for routine use. Even though the extraction efficiency could be improved by acidifying the bile, this caused premature solvolysis of sulfated steroids. Acid solvolysis of unextracted bile worked best (c. 90% converted to free steroids) on volumes that were 1 µL or lower. Aryl sulfatase digestion of unextracted bile did not work well (only 20% of radioactivity was converted to free steroids).

Novel oxazolinyl derivatives of pregna-5,17(20)-diene as 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitors.

New oxazolinyl derivatives of [17(20)E]-pregna-5,17(20)-diene: 2'-{[(E)-3ß-hydroxyandrost-5-en-17-ylidene]methyl}-4',5'-dihydro-1',3'-oxazole 1 and 2'-{[(E)-3ß-hydroxyandrost-5-en-17-ylidene]methyl}-4',4'-dimethyl-4',5'-dihydro-1',3'-oxazole 2 were evaluated as potential CYP17A1 inhibitors in comparison with 17-(pyridin-3-yl)androsta-5,16-dien-3ß-ol 3 (abiraterone). Differential absorption spectra of human recombinant CYP17A1 in the presence of compound 1 (λmax=422 nm, λmin=386 nm) and compound 2 (λmax=416 nm) indicated significant differences in enzyme/inhibitors complexes. CYP17A1 activity was measured using electrochemical methods. Inhibitory activity of compound 1 was comparable with abiraterone 3 (IC50=0.9±0.1 µM, and IC50=1.3±0.1 µM, for compounds 1 and 3, respectively), while compound 2 was found to be weaker inhibitor (IC50=13±1 µM). Docking of aforementioned compounds to CYP17A1 revealed that steroid fragments of compound 1 and abiraterone 3 occupied close positions; oxazoline cycle of compound 1 was coordinated with heme iron similarly to pyridine cycle of abiraterone 3. Configuration of substituents at 17(20) double bond in preferred docked position corresponded to Z-isomers of compounds 1 and 2. Presence of 4'-substituents in oxazoline ring of compound 2 prevents coordination of oxazoline nitrogen with heme iron and worsens its docking score in comparison with compound 1. These data indicate that oxazolinyl derivative of [17(20)E]-pregna-5,17(20)-diene 1 (rather than 4',4'-dimethyl derivative 2) may be considered as potential CYP17A1 inhibitor and template for development of new compounds affecting growth and proliferation of prostate cancer cells.

Sexual maturation in the Sumatran rhinoceros (Dicerorhinus sumatrensis).

To help save the Sumatran rhino from extinction, the captive breeding program must capitalize on each rhino's reproductive lifespan. Doing so requires knowing when calves are sexually mature. The goal of this study was to monitor physiological changes associated with sexual maturation in two captive born calves (one male and one female) to determine the approximate age of maturity for both sexes of this species. Fecal testosterone metabolites were monitored in the male calf from 6 months to 7 years of age, and fecal pregnane metabolites were measured in the female calf from 6 months to 5.5 years of age. In addition, rectal ultrasonography was employed to monitor changes in ovarian activity from 2 to 5.5 years of age. The male calf's fecal testosterone concentrations reached levels comparable to those detected in samples from adult males when he was 6-6.5 years of age. The first pre-ovulatory sized follicle was observed on the ovaries of the female calf when she was 4.75 years old, but fecal pregnane metabolite concentrations only reached maximum mean concentrations and variability when she was 5-5.5 years of age. Results from this study indicate that male and female Sumatran rhino calves are sexually mature at 6-6.5 and 5-5.5 years of age, respectively.

[Effect of the steroid molecule structure on the direction of its hydroxylation by the fungus Curvularia lunata].

The main and side products of hydroxylation by the C. lunata VKPM F-981 mycelium of fourteen delta(4)-3-ketosteroids of the estrane, androstane, and pregnane series and six of their delta(5)-3beta-hydroxy analogues were identified by H1 PMR spectroscopy and comparison with standard samples. The obtained experimental data are considered in terms of the triangular model of the enzyme-substrate interaction. The dependence of the direction of hydroxylation of steroid molecules and the orientation of hydroxy groups on the structure of the initial substrate was revealed.

Non-targeted metabolomic approach reveals urinary metabolites linked to steroid biosynthesis pathway after ingestion of citrus juice.

Citrus juice intake has been highlighted because of its health-promoting effects. LC-MS based metabolomics approaches are applied to obtain a better knowledge on changes in the concentration of metabolites due to its dietary intake and allow a better understanding of involved metabolic pathways. Eight volunteers daily consumed 400 mL of juice for four consecutive days and urine samples were collected before intake and 24h after each citrus juice intake. Urine samples were analysed by nanoHPLC-q-TOF, followed by principal component analysis (PCA) and Student's t-test (p<0.05). PCA showed a separation between two groups (before and after citrus juice consumption). This approach allowed the identification of four endocrine compounds (tetrahydroaldosterone-3-glucuronide, cortolone-3-glucuronide, testosterone-glucuronide and 17-hydroxyprogesterone), which belonged to the steroid biosynthesis pathway as significant metabolites upregulated by citrus juice intake. Additionally, these results confirmed the importance of using the non-targeted metabolomics technique to identify new endogenous metabolites, up- or down-regulated as a consequence of food intake.

Fecal 20-oxo-pregnane concentrations in free-ranging African elephants (Loxodonta africana) treated with porcine zona pellucida vaccine.

Because of overpopulation of African elephants in South Africa and the consequent threat to biodiversity, the need for a method of population control has become evident. In this regard, the potential use of the porcine zona pellucida (pZP) vaccine as an effective means for population control is explored. While potential effects of pZP treatment on social behavior of African elephants have been investigated, no examination of the influence of pZP vaccination on the endocrine correlates in treated females has been undertaken. In this study, ovarian activity of free-ranging, pZP-treated African elephant females was monitored noninvasively for 1 yr at Thornybush Private Nature Reserve, South Africa, by measuring fecal 5α-pregnan-3ß-ol-20-on concentrations via enzyme immunoassay. A total of 719 fecal samples from 19 individuals were collected over the study period, averaging 38 samples collected per individual (minimum, maximum: 16, 52). Simultaneously, behavioral observations were made to record the occurrence of estrous behavior for comparison. Each elephant under study showed 5α-pregnan-3ß-ol-20-on concentrations rising above baseline at some period during the study indicating luteal activity. Average 5α-pregnan-3ß-ol-20-on concentrations were 1.61 ± 0.46 µg/g (mean ± SD). Within sampled females, 42.9% exhibited estrous cycles within the range reported for captive African elephants, 14.3% had irregular cycles, and 42.9% did not appear to be cycling. Average estrous cycle duration was 14.72 ± 0.85 wk. Estrous behavior coincided with the onset of the luteal phase and a subsequent rise in 5α-pregnan-3ß-ol-20-on concentrations. Average 5α-pregnan-3ß-ol-20-on levels positively correlated with rainfall. No association between average individual 5α-pregnan-3ß-ol-20-on concentrations or cyclicity status with age or parity were detected. Earlier determination of efficacy was established via fecal hormone analysis with no pregnancies determined 22 mo post-treatment and onward. Results indicate the presence of ovarian activity amongst pZP-treated female African elephants in 2 yr after initial immunization. Further study should now be aimed toward investigating the long-term effects of pZP vaccination on the reproductive function of female African elephants.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC-MS and GC-MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ring.

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group. Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1ß-, 2α-, 4ß-, and 6ß-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored. We conclude that GC-MS combined with GC-MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1ß, 15ß, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4ß and 6ß. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5ß-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3ß-hydroxy-5α-anes.

Pattern of faecal 20-oxopregnane and oestrogen concentrations during pregnancy in wild plains zebra mares.

Regulative endocrine mechanisms influence the reproductive behaviour and success of mammals, but they have been studied predominantly in domestic and captive animals. The study aims at describing the pattern of faecal 20-oxopregnane and oestrogen concentrations during pregnancy in wild plains zebra Equus quagga chapmani. Data were collected during wet and dry seasons 2007-2009. Enzyme Immunoassays were used to determine 20-oxopregnane and oestrogen concentrations in faecal samples (n=74) collected from individual mares (n=32) whose dates of foaling were known through long-term monitoring. Hormonal profiles were described with a General Additive Model (GAM: Hormone ∼ Days to Foaling). Faecal 20-oxopregnanes have a complex cycle during pregnancy (GAM, n=70, R(2)=0.616, p<0.001). From -250 days to foaling, faecal 20-oxopregnane concentrations were above the baseline levels found in non-pregnant mares, peaking in the last 50 days. Faecal oestrogen levels showed a clear peak in mid-pregnancy (GAM, n=62, R(2)=0.539, p<0.001). The sex of the foetus and season had no detectable effect on hormone concentrations during pregnancy. High levels (>200 ng/g DW) of faecal 20-oxopregnanes associated with high (>160 ng/g DW) faecal oestrogen levels indicate mid-pregnancy in c.90% of cases (16/17). High faecal 20-oxopregnanes (>200 ng/g DW) and low faecal oestrogen levels (<160 ng/g DW) indicate late pregnancy, again in c.90% of cases. Two faecal samples would allow the stage of pregnancy to be determined with confidence.