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• Influence of ethanol consumption and food intake on serum concentrations of endogenous steroids. • Ethanol consumption increased testosterone/androstenedione ratio in blood. • Meal-induced ACTH and cortisol peaks coincided with pulsatile androstenedione peaks. • Female testosterone levels are more susceptible to perturbations by ethanol or ACTH. Steroid biosynthesis and biotransformation are based on a cascade of enzymatic processes being highly sensitive to various external influences. Amongst those, ethanol was shown to affect testosterone metabolism. For doping analyses, athlete steroid profiles comprise seven urinary steroid metabolites, of which relevant ratios are significantly increased following ethanol consumption. This effect is presumably based on the lack of hepatic NAD+-coenzyme as a consequence of ethanol oxidation. Only recently, testosterone (T) and androstenedione (A4) blood profiles have been introduced as additional approach for doping control. However, a potential influence of ethanol intake on testosterone biosynthesis and thus on blood steroid profiles has not been investigated so far. Therefore, steroid concentrations from 10 males and 10 females receiving an ethanol infusion up to a breath alcohol concentration of 0.5 mg/L which was hold as a plateau for two hours were conducted. Blood samples were drawn every 15 min for steroid quantification. An ethanol-dependent T/A4 increase up to 385 % resulting from A4 suppression was observed in 14 volunteers. In addition, we observed sporadic A4 increases coinciding with cortisol and ACTH pulses pointing to a meal-induced adrenal stimulation. While testosterone levels in males showed diurnal variation solely, testosterone levels in some females were found to be susceptible to ethanol- and ACTH-dependent perturbations, which is thought to be due to its predominant adrenal synthesis in females. In conclusion, the results of the present study emphasize the importance of blood sampling at a sufficient time interval from food and ethanol intake. This is of interest if T and A4 are used for diagnostics in doping control. [ABSTRACT FROM AUTHOR]

Highlights • Progesterone converted by the fungus Aspergillus sojae PTCC 5196 produced testololactone. • The production of testololactone indicated fungal Baeyer-Villiger monooxygenase (BVMO) activity. • Substrate-induced cultures have a decisive impact on the metabolism of progesterone. • Progesterone, a C-21 steroidal compound, induced 17β-acetyl side chain cleavage. • Androstenedione, testosterone, and DHEA, C-19 steroidal substances, induced ring-D oxidation. Abstract Microbial transformations are capable of producing steroid substances difficult to synthesize by chemical methods. Strains belonging to the genus Aspergillus are effective facilitators of microbial biotransformations due to their enzymatic diversity. In this study, the biotransformation of progesterone by the fungus Aspergillus sojae (A. sojae) PTCC 5196 was examined. Analysis of the bioconversion process revealed that progesterone was converted to testololactone through a three-step pathway (17β-acetyl side chain cleavage, 17β-hydroxyl oxidation, and oxygenative lactonization of 17-ketone), indicating the presence of Baeyer-Villiger monooxygenase (BVMO) activity in the fungal strain. GC analysis confirmed the production of testololactone with a yield of 99% in 24 h. Faster testololactone production was induced in the presence of both C-21 (progesterone) and C-19 (androstenedione, testosterone, and dehydroepiandrosterone [DHEA]) steroid substances. Due to the high biotransformation rate observed in the present study, A. sojae may be a novel and promising candidate in the production of testololactone. [ABSTRACT FROM AUTHOR]

The purpose of this study was to identify the C11-oxy C 19 and C11-oxy C 21 steroids in male and female neonate plasma. At birth, the most abundant C11-oxy steroids detected in neonatal plasma were 11β-hydroxyandrostenedione, ∼13 nM, and 11-ketoprogesterone, ∼23 nM. C11-oxy C 19 steroids were higher than C 19 steroids in neonatal plasma, 22.2 nM vs 5.4 nM. The inclusion of C11-oxy C 19 and C 21 steroid reference ranges in routine steroid analyses will assist the characterization of disorders associated with impaired steroidogenic enzyme expression and the identification of potential biomarkers. [ABSTRACT FROM AUTHOR]

Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ 5 -androstene-3,17-dione to Δ 4 -androstene-3,17-dione and of Δ 5 -pregnene-3,20-dione to Δ 4 -pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals. The mRNA encoding the steroid isomerase GST A3-3 was cloned from testis of the horse ( Equus ferus caballus). The concentrations of GSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. EcaGST A3-3 produced in E. coli has been characterized and shown to have highly efficient steroid double-bond isomerase activity, exceeding its activities with conventional GST substrates. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals and approaches the activity of the bacterial ketosteroid isomerase, one of the most efficient enzymes of all categories known today. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones. [ABSTRACT FROM AUTHOR]

The aim of this study was to perform a systematic review of the published literature to determine the available serum/plasma steroid reference intervals generated by mass spectrometry (MS) methods across all age groups in healthy subjects and to suggest recommendations to achieve common MS based reference intervals for serum steroids. MEDLINE, EMBASE and PubMed databases were used to conduct a comprehensive search for English language, MS-based reference interval studies for serum/plasma steroids. Selection of steroids to include was based on those listed in the Royal College of Pathologists of Australasia Quality Assurance Programs, Chemical Pathology, Endocrine Program. This methodology has been registered onto the PROSPERO International prospective register of systematic reviews (ID number: CRD42015029637). After accounting for duplicates, a total of 60 manuscripts were identified through the search strategy. Following critical evaluation, a total of 16 studies were selected. Of the 16 studies, 12 reported reference intervals for testosterone, 11 for 17 hydroxy-progesterone, nine for androstenedione, six for cortisol, three for progesterone, two for dihydrotestosterone and only one for aldosterone and dehydroepiandrosterone sulphate. No studies established MS-based reference intervals for oestradiol. As far as we are aware, this report provides the first comparison of the peer reviewed literature for serum/plasma steroid reference intervals generated by MS-based methods. The reference intervals based on these published studies can be used to inform the process to develop common reference intervals, and agreed reporting units for mass spectrometry based steroid methods. [ABSTRACT FROM AUTHOR]

Ovarian monitoring requires the determination of serum estradiol and progesterone levels. We investigated whole follicular steroidogenesis under rFSH in medically assisted procreation (MAP: 26 IVF, 24 ICSI) compared to 11 controls (IUI). Estrone, estradiol, Δ4-androstenedione, testosterone, progesterone and 17-hydroxyprogesterone were measured by immunoassay and mass spectrometry except for estrogens. At the start of a spontaneous or induced cycle, steroids levels fluctuated within normal ranges: estradiol (314–585 pmol/L), estrone (165–379 pmol/L) testosterone (1.3–1.6 nmol/L), Δ4-androstenedione (4.5–5.6 nmol/L), 17-hydroxyprogesterone (2.1–2.2 nmol/L) and progesterone (1.8–1.9 nmol/L). 17-hydroxyprogesterone, Δ 4-androstenedione and estradiol predominated. Then estradiol and oestrone levels rise, but less markedly for oestrone in IUI. In MAP, rFSH injections induce a sharp increase in estrogens associated with a rise in 17-hydroxyprogesterone and Δ4-androstenedione levels, disrupting oestrogen/androgen ratios. rFSH stimulation induces an ovarian hyperplasia and Δ4pathway which could become abnormal. Determining 17-hydroxyprogesterone and Δ4-androstenedione levels with LC-MS/MS may therefore be useful in managing recurrent MAP failures. [ABSTRACT FROM AUTHOR]

A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18 min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100 μL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis. [ABSTRACT FROM AUTHOR]

Background The simultaneous quantification of a steroid hormones panel provides more clinical information than a single steroid assay. Traditionally, steroids have been quantified with immunoassays which are characterized by high rate of positive results. Aim of this work, was to develop a TurboFlow-LC-MS/MS method for the simultaneous quantification of 17-hydroxyprogesterone, androstenedione, cortisol and testosterone in serum. Methods To 100 μL of serum, 100 μL of internal standard solution in methanol were added; after centrifugation the supernatant was injected in the TurboFlow for further purification. Steroids were determined using a TSQ Vantage operating with an atmospheric pressure chemical ionization source. Method was fully validated and results compared with immunoassay methods. Results Limit of quantification ranged from 0.02 ng/mL to 1 ng/mL. The precision was lower than 11% and accuracy ranged from 93.5 to 121.6%. The correlation was acceptable for all analytes except for low levels of testosterone. However, the Bland-Altman plots display a positive bias for androstenedione and 17-hydroxyprogesterone, and a negative bias for cortisol and testosterone. Conclusions TurboFlow analysis provides a simple and effective clean-up procedure minimizing the interference of the matrix. The presented method is selective, precise, and sensitive being suitable in a clinical laboratory. [ABSTRACT FROM AUTHOR]

In this work, new potent steroidal aromatase inhibitors both in microsomes and in breast cancer cells have been found. The synthesis of the 3,4-(ethylenedioxy)androsta-3,5-dien-17-one ( 12 ), a new steroid containing a heterocycle dioxene fused in the A-ring, led to the discovery of a new reaction for which a mechanism is proposed. New structure–activity relationships were established. Some 5β-steroids, such as compound 4β,5β-epoxyandrostan-17-one ( 9 ), showed aromatase inhibitory activity, because they adopt a similar A-ring conformation as those of androstenedione, the natural substrate of aromatase. Moreover, new chemical features to increase planarity were disclosed, specifically the 3α,4α-cyclopropane ring, as in 3α,4α-methylen-5α-androstan-17-one ( 5 ) (IC 50 = 0.11 μM), and the Δ 9–11 double bond in the C-ring, as in androsta-4,9(11)-diene-3,17-dione ( 13 ) (IC 50 = 0.25 μM). In addition, induced-fit docking (IFD) simulations and site of metabolism (SoM) predictions helped to explain the recognition of new potent steroidal aromatase inhibitors within the enzyme. These insights can be valuable tools for the understanding of the molecular recognition process by the aromatase and for the future design of new steroidal inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Commercially available radioimmunoassays (RIAs) are frequently used to evaluate the effects of endocrine disrupting chemicals (EDCs) on steroidogenesis in rats. Currently there are limited data comparing steroid concentrations in rats as measured by RIAs to those obtained using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). This study evaluates the concordance of serum and urine steroid concentrations as quantified by select RIA kits and LC–MS/MS following exposure to an EDC, atrazine (ATR). Methods: Adult male rats were orally dosed with ATR (200mg/kg/day) or methylcellulose (1%, vehicle control) for 5days. Serum was collected and separated into aliquots for analysis. Serum was assayed by RIA for androstenedione (ANDRO), corticosterone (CORT), estradiol (E2), estrone (E1), progesterone (P4), and testosterone (T). Serum was extracted prior to LC–MS/MS analysis with positive electrospray ionization in multiple-reaction monitoring mode for ANDRO, CORT, P4, and T. E1 and E2 concentrations were quantified similarly by LC–MS/MS, following derivatization with dansyl chloride. To compare CORT values from urine, pregnant adult rats were orally dosed with either ATR (100mg/kg/day) or methylcellulose for 5days (i.e., gestational days 14–18). Urine samples were collected daily and assayed for CORT by RIA and LC–MS/MS as described above. Results: Data analyses demonstrated significant agreement between the two detection methods as assessed by Pearson product-moment correlation coefficient, Bland–Altman analysis, and the interclass correlation coefficient. No statistically significant differences were observed between RIA and LC–MS/MS means for any of the steroids assayed. Discussion: These findings indicate a significant correlation between the measurement of steroids within rat serum and urine using RIA kits and LC–MS/MS. Differences in the absolute measurements existed, but these were not statistically significant. These findings indicate that steroids may be reliably measured in rat biological media using RIAs or LC–MS/MS. [Copyright &y& Elsevier]

Abstract: In the last couple of decades fungal infections have become a significant clinical problem. A major interest into fungal steroid action has been provoked since research has proven that steroid hormones are toxic to fungi and affect the host/fungus relationship. Steroid hormones were found to differ in their antifungal activity in ascomycetous fungi Hortaea werneckii, Saccharomyces cerevisiae and Aspergillus oryzae. Dehydroepiandrosterone was shown to be the strongest inhibitor of growth in all three varieties of fungi followed by androstenedione and testosterone. For their protection, fungi use several mechanisms to lower the toxic effects of steroids. The efficiency of biotransformation in detoxification depended on the microorganism and steroid substrate used. Biotransformation was a relatively slow process as it also depended on the growth phase of the fungus. In addition to biotransformation, steroid extrusion out of the cells contributed to the lowering of the active intracellular steroid concentration. Plasma membrane Pdr5 transporter was found to be the most effective, followed by Snq2 transporter and vacuolar transporters Ybt1 and Ycf1. Proteins Aus1 and Dan1 were not found to be involved in steroid import. The research of possible targets of steroid hormone action in fungi suggests that steroid hormones inhibit ergosterol biosynthesis in S. cerevisiae and H. werneckii. Results of this inhibition caused changes in the sterol content of the cellular membrane. The presence of steroid hormones most probably causes the degradation of the Tat2 permease and impairment of tryptophan import. [Copyright &y& Elsevier]

Abstract: Objective: To evaluate the placental activity of steroid sulfatase (STS), 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD-1) and P450 aromatase (P450arom) in polycystic ovarian syndrome (PCOS) compared to normal pregnant women. Design: Twenty pregnant women with PCOS and 30 control pregnant women who delivered at term were studied. Samples of placental tissue and cord blood were obtained after delivery. A maternal blood sample was obtained during the 34th week of gestation. In placental tissue, the activities of STS, 3β-HSD-1 and P450arom were evaluated. In the blood samples, progesterone, DHEAS, DHEA, androstenedione, testosterone, estrone, estradiol and total estriol were determined. Result: In placental tissue from women with PCOS, higher 3β-HSD-1 and lower P450 aromatase activities were observed compared to control women. Moreover, women with PCOS showed higher androstenedione and testosterone concentrations compared to normal pregnant women (p =0.016 and p =0.025, respectively). In cord blood, female newborns of women with PCOS exhibited lower androstenedione and higher estriol concentrations compared to daughters of control women (p =0.038; p =0.031, respectively). Conclusion: These data suggest that placental tissue from women with PCOS shows changes in the activities of two important enzymes for steroid synthesis, higher 3β-HSD-1 and lower P450, which could increase androgen production during pregnancy. [Copyright &y& Elsevier]

Abstract: It is well known that acute psychosocial stress activates the hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic nervous system (SNS). However, the effect of acute psychosocial stress on the hypothalamic–pituitary–gonadal (HPG) axis and levels of sex steroids are less known. The aim of this study was to investigate the effect of acute psychosocial stress on serum concentrations of sex steroids in healthy men and women. Twenty men and 19 women (age 30–50years) underwent Trier Social Stress Test (TSST), a tool for investigating psychobiological stress responses in a laboratory setting. Blood samples were collected before, directly after the stress test, and after 30min of recovery. Concentrations of androgens were measured with high specificity LC-MS/MS method; concentrations of cortisol, estradiol and sex hormone-binding globulin were determined using immunoassays. In both men and women we observed significantly elevated levels of testosterone, estradiol, androstenedione and sex hormone binding globulin along with significantly increased adrenocorticotropic hormone (ACTH), serum cortisol, heart rate, systolic blood pressure (SBP), and diastolic blood pressure (DBP) as a response to the stressor. Thus, even though the HPG axis and the production of sex steroids may be inhibited during prolonged periods of stress, the sex steroid levels may increase in the initial phase of acute psychosocial stress. [Copyright &y& Elsevier]

Abstract: Background: Salivary steroid measurements have the potential to provide a non-invasive assessment of serum free steroid concentrations. Salivary testing has been proposed as a possible substitute for serum testing partly because of easy sample collection. Methods: We developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for simultaneous measurement of testosterone and androstendione in saliva. We spiked 100μL of saliva with isotope-labeled internal standards and performed extraction with diethyl ether. After evaporation both steroids were analyzed by LC–MS/MS operating in the positive mode electrospray ionization (ESI) after separation on a reversed-phase column. Results: The calibration curves for analysis of testosterone and androstendione exhibited consistent linearity and reproducibility in the range of 20–1000pmol/L. The limit of quantitation (LOQ) was 10pmol/L for both steroids. The mean recovery of the analytes added to saliva ranged from 97 to 102%. Between-run CVs were 3.6–4.0% for testosterone and 4.8–8.0% for androstendione. The upper limit (the 97.5 percentile) of testosterone and androstendione reference ranges for males was 356pmol/L and 344pmol/L and that for females 39pmol/L and 349pmol/L, respectively. Conclusions: This LC–MS/MS method has the required lower limit of quantification for analysis of salivary concentrations of testosterone and androstendione. The assay is rapid, reliable and simple to perform with a routine LC–MS/MS spectrometer with required sensitivity. [Copyright &y& Elsevier]

Abstract: Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to androstenedione and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M r 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-4-ene steroids with androstenedione as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms. [Copyright &y& Elsevier]

Abstract: A new stereoselective synthesis of E-guggulsterone is described starting from androsten-3,17-dione. Protection of the ring A enonic system, followed by regioselective Wittig reaction and C-16 oxidation, affords E-guggulsterone in good yields and high stereoselectivity, making this approach easily accessible and scalable. Moreover, an original normal-phase HPLC method enabling the fast quantitation of the guggulsterone isomeric purity, combined with the suitability for sampling procedures, is detailed. The relying upon the cellulose-based Chiralpak IB column and the chloroform as the “non-standard” component of the eluent mixture, allows to get profitably high chromatographic performances. [Copyright &y& Elsevier]

Abstract: Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2–22h post-administration. However, IRMS could provide positive determinations for up to 55h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2–36h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS. [Copyright &y& Elsevier]

Abstract: Bisphenol A (BPA) is a potential endocrine disruptor. It has been shown that it reduces serum testosterone level in rodents after exposure. However, the mechanism is unclear. The object of the present study is to investigate the effects of BPA on human and rat steroidogenic enzymes including P450 17α-hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3). Human and rat testis microsomes were exposed to various concentrations of BPA (10−8–10−4 M). BPA inhibited human and rat 3β-HSD, CYP17A1 and 17β-HSD3 activities. The half maximal inhibitory concentrations (IC50s) of BPA for human and rat testis 3β-HSD were 7.92±1.03 and 26.49±3.03μM (200μM pregnenolone), respectively. The IC50s for human and rat CYP17A1 (1μM progesterone) were 18.99±3.75 and 64.67±4.04μM, respectively. BPA was a weak HSD17B3 inhibitor with IC50s of about 100μM (200nM androstenedione). BPA also concentration-dependently inhibited testosterone production by rat Leydig cells. In conclusion, BPA is an inhibitor for 3β-HSD, CYP17A1 and 17β-HSD3. Human 3β-HSD and CYP17A1 are more sensitive to BPA than rat 3β-HSD and CYP17A1. [Copyright &y& Elsevier]

Abstract: Background: Newborn screening for congenital adrenal hyperplasia (CAH) is commonly accomplished by measurement of 17-α-hydroxyprogestrone (17-OHP) using enzyme immunoassay (EIA). EIA contributes a significant number of false positives. Therefore, second-tier steroid profile by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is warranted. Methods: Dried blood spots (DBS) were extracted with a mixture of methanol and water containing the deuterium labeled internal standards of d8-17-OHP, d7-androstenedione, and d4-cortisol. The final extracts were analyzed for 17-OHP, androstenedione and cortisol by LC-MS/MS in the multiple reaction monitoring (MRM) mode. Results: Mean recoveries of the target analytes, 17-OHP, androstenedione and cortisol, were between 97 and 115% with an average intra- and inter-assay CVs ranging from 3.9–9.9% to 3.6–10.1%, respectively. The high efficiency of this method enabled us to test 11,598 specimens, identified as indeterminate by EIA in ~6 years; resulting in 809 presumptive positives reducing the false positives rate by 93%. Conclusions: The three steroid profile provided better screening outcomes of CAH than 17-OHP concentration alone. Our sample preparation allowed high throughput using common laboratory chemicals. Using three internal standards significantly improved method precision and accuracy. The reduction in false positives significantly reduces anxiety for newborns and their families. [Copyright &y& Elsevier]

Abstract: Breed differences in steroidogenic activity between primary Leydig cells derived from neonatal purebred Duroc and Norwegian Landrace boars were investigated in vitro. Concentrations of testosterone, estradiol, androstenone, cortisol and progesterone produced into the medium were determined. To explore underlying mechanisms the cellular expression of a suite of genes relevant in steroidogenesis was measured using reverse transcription and quantitative PCR (RT-qPCR). Basal steroid concentrations indicated a larger production capacity for steroids in unstimulated Duroc cells. Stimulation of the cells with LH increased steroid hormone secretion significantly in both breeds in a dose dependent manner. Testosterone and androstenone concentrations increased approximately 50- and 15-fold, respectively, whereas concentrations of estradiol, cortisol and progesterone increased to a lesser extent. At levels of maximal LH stimulation, absolute steroid concentrations were higher in Duroc. However, the relative increase in hormone concentrations was significantly lower in Duroc cells for estradiol, progesterone and cortisol when compared to basal levels. LH exposure was associated with a general up-regulation of mRNA levels for steroidogenic genes, stronger in Duroc than in Norwegian Landrace. This was in agreement with the higher absolute concentrations of steroid hormones measured in culture medium from the LH-stimulated Duroc Leydig cells, but did not concur with the fact that the relative increase in hormone production was lower in Duroc than in Norwegian Landrace Leydig cells for some hormones. It was concluded that breed differences in steroid hormone concentrations and gene expression between Norwegian Landrace and Duroc are complex and cannot be explained by a simple mechanism of action. [Copyright &y& Elsevier]

Abstract: Aromatase is the rate-limiting enzyme in estrogen biosynthesis. As a cytochrome P450, it utilizes electrons from NADPH–cytochrome P450 reductase (CPR) to produce estrogen from androgen. Estrogen is a key factor in the promotion of hormone-dependent breast cancer growth. Aromatase inhibitors (AIs) are drugs that block estrogen synthesis, and are widely used to treat estrogen-dependent breast cancer. Structure–function experiments have been performed to study how CPR and AIs interact with aromatase to further the understanding of how these drugs elicit their effects. Our studies have revealed a strong interaction between aromatase and CPR, and that the residue K108 is situated in a region important to the interaction of aromatase with CPR. The published X-ray structure of aromatase indicates that the F221, W224 and M374 residues are located in the active site. Our site-directed mutagenesis experiments confirm their importance in the binding of the androgen substrate as well as AIs, but these residues interact differently with steroidal inhibitors (exemestane) and non-steroidal inhibitors (letrozole and anastrozole). Furthermore, our results predict that the residue W224 also participates in the mechanism-based inhibition of exemestane, as time-dependent inhibition is eliminated with mutation on this residue. Together with previous research from our laboratory, this study confirms that W224, E302, D309 and S478 are important active site residues involved in the suicide mechanism of exemestane against aromatase. [Copyright &y& Elsevier]

Abstract: Aromatase (CYP19A1) is an integral membrane enzyme that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens. All human estrogens are synthesized from their androgenic precursors by this unique cytochrome P450. The crystal structure of active aromatase purified from human placenta has recently been determined in complex with its natural substrate androstenedione in the high-spin ferric state of heme. Hydrogen bond forming interactions and tight packing hydrophobic side chains closely complement puckering of the steroid backbone, thereby providing the molecular basis for the androgenic specificity of aromatase. In the crystal, aromatase molecules are linked by a head-to-tail intermolecular interaction via a surface loop between helix D and helix E of one aromatase molecule that penetrates the heme-proximal cavity of the neighboring, crystallographically related molecule, thus forming in tandem a polymeric aromatase chain. This intermolecular interaction is similar to the aromatase-cytochrome P450 reductase coupling and is driven by electrostatics between the negative potential surface of the D–E loop region and the positively charged heme-proximal cavity. This loop-to-proximal site link in aromatase is rather unique—there are only a few of examples of somewhat similar intermolecular interactions in the entire P450 structure database. Furthermore, the amino acids involved in the intermolecular contact appear to be specific for aromatase. Higher order organization of aromatase monomers may have implications in lipid integration and catalysis. [Copyright &y& Elsevier]

Abstract: Here we describe further experiments to support our hypothesis that bidirectional 11β-HSD1-dehydrogenase in Leydig cells is a NADP(H) regenerating system. In the absence of androstenedione (AD), substrate for 17β-HSD3, incubation of Leydig cells with corticosterone (B) or several C19- and C21-11β-OH-steroids, in the presence of [3H]-11-dehydro-corticosterone (A), stimulated 11β-HSD1-reductase activity. However, in presence of 30μM AD, testosterone (Teso) synthesis is stimulated from 4 to 197 picomole/25,000 cells/30min and concomitantly inhibited 11β-HSD1-reductase activity, due to competition for the common cofactor NADPH needed for both reactions. Testo production was further significantly increased (p <0.05) to 224–267 picomole/25,000 cells/30min when 10μM 11β-OH-steroids (in addition to 30μM AD) were also included. Similar results were obtained in experiments conducted with lower concentrations of AD (5μM), and B or A (500nM). Incubations of 0.3–6.0μM of corticosterone (plus or minus 30μM AD) were then performed to test the effectiveness of 17β-HSD3 as a possible NADP+ regenerating system. In the absence of AD, increasing amounts (3–44pmol/25,000 cells/30min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30μM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3–5-fold producing 4–210pmol/25,000 cells/30min of 11-dehydro-corticosterone. We conclude that 11β-HSD1 is enzymatically coupled to 17β-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems. [Copyright &y& Elsevier]

Abstract: In this study, we identified a mechanism by which the neuro-steroid, 5-androstene 3β,17α diol (17α-AED) induces autophagy in human malignant glioma cells and transformed fibroblasts. 17α-AED treatment induced endoplasmic reticulum (ER) stress, identified by the partial activation of an unfolded protein response in T98G, U87MG, U251MG, LN-18, LN-229 and LN-Z308 glioma cell lines. In this regard, there were increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein of 78kDa transcripts but no splicing of X-box-binding protein 1 mRNA or processing of activating transcription factor-6 in glioma cells treated with the neuro-steroid. 17α-AED induced eukaryotic translational initiation factor 2α (eIF2α) phosphorylation in glioma cells which correlated with microtubule-associated protein-light chain 3 (LC3) conversion from LC3-I to -II. In transformed murine embryonic fibroblasts (MEFs) that are deficient of eIF2α function or T98G glioma cells transfected with a dominant-negative eIF2α construct, 17α-AED induced LC3 conversion was significantly reduced as compared to control cells. Neuro-steroid treatment caused the activation of the eIF2α kinase, protein kinase-like ER kinase (PERK) but not other eIF2α kinases in glioma cells. Moreover, eIF2α phosphorylation and LC3 conversion, in response to 17α-AED treatment, was blocked in MEFs that lacked PERK activity. T98G cells transfected with a dominant-negative PERK construct exhibited an attenuated response to neuro-steroid treatment in terms of decreases in: eIF2α activation; CHOP expression; the incidence of autophagy; and cytotoxicity. These results demonstrate that ER stress is linked to 17α-AED induced autophagy by PERK/eIF2α signaling in human malignant glioma cells and transformed fibroblasts. [Copyright &y& Elsevier]

Abstract: Mussels (Mytilus galloprovincialis) were exposed to different concentrations of testosterone (T: 20, 200 and 2000ng/L) in a semi-static water regime (1-day dosing intervals) for up to 5 days in an attempt to see whether endogenous steroid levels and steroid metabolism were altered by exogenous exposure to testosterone. Whole tissue levels of total testosterone (free+esterified) sharply increased in a concentration-dependent manner, from 2ng/g in controls to 290ng/g in organisms exposed to the highest concentration. In contrast, levels of free testosterone were only significantly elevated at the high-exposure group (5-fold increase with respect to controls). Increased activity of palmitoyl-CoA:testosterone acyltransferase (ATAT) was detected in organisms exposed to the highest concentration of testosterone, while those exposed to low and medium concentrations showed significant alterations in their polyunsaturated fatty acid profiles. The obtained results suggest that esterification of the excess of T with fatty acids might act as a homeostatic mechanism to maintain endogenous levels of free T stable. Interestingly, a decrease in CYP3A-like activity was detected in T-exposed mussels together with a significant decrease in the metabolism of the androgen precursor androstenedione to dihydrotestosterone (5α-DHT). Overall, the work contributes to the better knowledge of androgen metabolism in mussels. [ABSTRACT FROM AUTHOR]

Abstract: Although β-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of β-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature. [Copyright &y& Elsevier]

Abstract: A series of 2-phenylaliphatic-substituted androsta-1,4-diene-3,17-diones (6) as well as their androstenedione derivatives (5) were synthesized as aromatase inhibitors to gain insights of structure–activity relationships of varying the alkyl moiety (C1 to C4) of the 2-phenylaliphatic substituents as well as introducing a methyl- or trifluoromethyl function to p-position of a phenethyl moiety to the inhibitory activity. The inhibitors examined showed a competitive type inhibition. The 2-phenpropylandrosta-1,4-diene 6c was the most powerful inhibitor (K i: 16.1nM) among them. Compounds 6c along with the phenethyl derivative 6b caused a time-dependent inactivation of aromatase (k inact: 0.0293 and 0.0454min−1 for 6b and 6c, respectively). The inactivation was prevented by the substrate androstenedione, and no significant effect of l-cysteine on the inactivation was observed in each case. Molecular docking of the phenpropyl compound 6c to aromatase was conducted to demonstrate that the phenpropyl group orients to a hydrophobic binding pocket in the active site to result in the formation of thermodynamically stable enzyme–inhibitor complex. [Copyright &y& Elsevier]

Abstract: Redox conversions of exogenic C19-steroids (androstenedione (AD), androstadienedione (ADD), testosterone and 1(2)-dehydrotestosterone) were studied using a mutant strain of Mycobacterium sp. Et1 with high level of 17β-hydroxysteroid dehydrogenase (17-HSD) activity. Factors affecting target 17β-reduction and side reactions by resting and growing cells were estimated. The effects of glucose supplementation, pH, mode of substrate addition were identified. The results confirmed that double reduction of androstadienedione, both of 17-keto group and 1(2)-double bond, is more effective for testosterone formation than a single reduction of 17-keto group of AD. These findings argued for the application of the strain capable of sterol side chain degradation and expressed 17-HSD, 3-ketosteroid-1(2)-dehydrogenase and 1-ene-reductase activities, for testosterone obtaining from sitosterol. Under optimal conditions, the conversion of sitosterol (5g/l) by Mycobacterium sp. VKM Ac-1816D in laboratory fermenter resulted in 50–55% molar yield of testosterone. [Copyright &y& Elsevier]

Objective: To evaluate the effects of the GnRH antagonist cetrorelix on the gonadal axis in patients with polycystic ovary syndrome (PCOS). Design: Observational clinical study. Setting: Academic research center. Patient(s): Ten patients with PCOS and 10 controls with normal ovulation. Intervention(s): Patients received a daily cetrorelix injection (0.25 mg SC at 9:00 am) for 6 days, starting from day 3 of the menstrual cycle. Main Outcome Measure(s): Serum gonadotropin, E2, T, 17-OH-P, and androstenedione plasma levels were evaluated at baseline and at 12 and 24 hours after each daily injection. These hormones were also assayed at days 10, 12, and 14 of the menstrual cycle. Result(s): We observed in patients with PCOS a significantly higher suppression of FSH and LH for the entire length of therapy; LH recovery secretion was significantly higher in the PCOS group. Regarding androgens, we found a greater suppression of T. Androstenedione and 17-OH-P showed a trend toward a higher suppression in PCOS. Conclusion(s): Gonadotropin and androgen suppression by GnRH antagonist is more effective in PCOS than in controls, suggesting a higher sensitivity of GnRH receptors in PCOS to this drug. [Copyright &y& Elsevier]

Abstract: Steroid determination by immunoassays results in significant interferences and inaccurate results. This study describes the development and validation of a new gas chromatographic–mass spectrometric method for the simultaneous quantification of 17α-hydroxyprogesterone (17αOHP), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (Δ4-A), cortisol (F) and pregnenolone (Preg) in serum of neonates. Steroids were extracted and purified from 0.5mL serum using diethyl ether and Extrelut mini NT1 column. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA)/trimethylsilyl iodide (TMSI)/dithioerythritol (DTE) and the resulting trimethylsilyl derivatives were quantified by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The detection limit for all steroids was lower than 0.1ng/mL. The limit of quantification was 0.1ng/mL for all steroids except cortisol which was at 0.25ng/mL. d3-Testosterone and methyltestosterone served as internal standards. Precision for all compounds at the concentrations of 0.5, 1, 5 and 10ng/mL (n =10) in fortified steroid-free serum samples ranged from 0.8% to 16.6%. Accuracy was calculated at the concentrations of 0.5, 1, 5 and 10ng/mL and ranged from −9.2% to 10.6% (n =10). Linear calibration equations were obtained for all five steroids (0.125–31.25ng/mL) and for cortisol (0.125–200ng/mL). Relative recoveries at concentrations 1.0 and 12.5ng/mL ranged from 70.5% to 97.5%. Absolute recoveries at the same concentrations ranged from 73.2% to 96.6%. Reference intervals were estimated for infants aged from 9 to 40 days. The proposed steroid profile is suitable for routine analysis and provides meaningful data for samples within normal range as well as those with elevated levels. [Copyright &y& Elsevier]

Androstenedione is a key intermediate of microbial steroid metabolism. It belongs to the 17-keto steroid family and is used as starting material for the preparation of different steroids. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis. In this review, various methods of biotransformation of sterols to androstenedione are surveyed. It begins with the history and current research status in this field. The existing methods of chemical and biochemical synthesis are examined. Various issues related to these methods and how researchers have addressed them is presented. Among these, the low solubility of sterols in aqueous systems is a critical problem since it limits the product yield. The main content of this review focuses on new methods of biotransformation that are being investigated. Recent biotechnological advances in this field are presented. The review ends with a note on future perspectives. [Copyright &y& Elsevier]

Abstract: We present a fast and reproducible method for steroid analysis (corticosterone, deoxycorticosterone, progesterone, 17α-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, testosterone, dihydrotestosterone and cortisol) in small volumes of serum and in dried blood spot samples by LC-MS/MS. No derivatisation was needed. LC separation was achieved by using an Atlantis® C18 column and water–methanol–formic acid gradient as a mobile phase and a flow rate of 250μL/min over a run time of 6min. Steroids were measured in MRM mode with electrospray interface (positive ion mode). Validation showed excellent precision, sensitivity, recovery and linearity with coefficients of determination r 2 >0.992. [Copyright &y& Elsevier]

Abstract: The use of steroids and their receptors as ligand-gated transcription factors is thought to be an important step in vertebrate evolution. The lamprey is the earliest-evolving vertebrate to date in which sex steroids and their receptors have been demonstrated to have hormonal roles similar to those found in jawed vertebrates. Sex steroids and their receptors have been examined in several lamprey species, and the majority of studies have focused on the sea lamprey, Petromyzon marinus. While classical steroids appear to be present in lampreys, their function, concentrations, and synthesis have not been determined conclusively. The only classical steroid that is thought to act as a hormone in both males and females is estradiol. Recent research has established that lampreys produce and circulate 15α-hydroxylated steroids, and that these steroids respond to upstream stimulation within the hypothalamic–pituitary–gonadal axis. In particular, 15α-hydroxyprogesterone is highly sensitive and responds in great magnitude to stimulation, and is likely a hormone. Lampreys also appear to use androstenedione, a precursor to vertebrate androgens, as their main androgen, and a receptor for androstenedione has recently been described. Non-classical steroids are prevalent in many aquatic vertebrates, and the non-classical steroids found in the sea lamprey may represent an evolutionary artifact, or alternatively may be a way to avoid endocrine disruption when ingesting the body fluids of host fish. The lamprey will continue to be an interesting model for examining the evolution of steroid hormones, steroid receptors, and steroid function. [Copyright &y& Elsevier]

Objective: To establish normal, trimester-specific reference intervals for serum 17beta-estradiol, progesterone (P), 17alpha-hydroxyprogesterone, cortisol, 11-deoxycortisol, androstenedione, DHEA, and DHEAS, measured simultaneously using isotope dilution tandem mass spectrometry.Design: Sequential cohort study.Patient(s): Healthy women undergoing a normal pregnancy (age, 25-38 years; mean, 30 years) attending a prenatal well clinic at gestation weeks 12, 22, and 32 and approximately 1 year postpartum.Main Outcome Measure(s): Trimester-specific reference intervals of endogenous steroid hormones analyzed using an isotope dilution tandem mass spectrometer equipped with an atmospheric pressure photoionization source with deuterium-labeled internal standards.Result(s): Serum estradiol, P, 17alpha-hydroxyprogesterone, and 11-deoxycortisol increased throughout pregnancy; cortisol increased up to the second trimester and then remained steady, while androstenedione increased by 80 percent by gestation week 12, then remained constant. Serum DHEA-S decreased by 50% by the third trimester.Conclusion(s): Trimester-specific reference intervals are reported for eight serum steroids. The ratios of individual serum hormone concentrations during pregnancy relative to their 1-year postpartum concentrations illustrate the expected normal trends of changes in hormone concentrations during pregnancy. [ABSTRACT FROM AUTHOR]

Abstract: 17β-Reduction is one of key and widespread reactions of microbial steroid metabolism. It accompanies various steroid transformations by microorganisms and plays a role in the regulation of reducing equivalent pool and detoxication of exogenic steroids. The ability of microorganisms to reduce C17-ketosteroids has been known for 60 years, but most progress in the biochemistry and biotechnology of the process has been recorded in the past two decades. The regulation of 17β-hydroxysteroid dehydrogenase activity is of importance for the selectivity of various steroid biotransformations. The potential application of microbial 17β-reduction and the reverse reaction – 17β-oxidation – in the pharmaceutical industry for the production of 17β-hydroxy and 17-oxosteroids is shown. Microbial side chain cleavage of sterols in combination with 17β-reduction represents the shortest way to obtain testosterone in a single biotechnological operation thus replacing multistep chemical and/or combined biotech-chemical syntheses. This review highlights progress in the understanding of the biochemical basis of 17β-reduction and the biotechnological applications of the process. [Copyright &y& Elsevier]

Abstract: It is unknown whether androstenedione, a steroidal dietary supplement taken to enhance athletic performance, can affect physiological hormone levels by altering liver enzyme activities that metabolize steroid hormones. Altered hormone levels could be especially devastating during pregnancy. Mature female rats were gavaged with 0, 5, 30 or 60mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60mg/kg/day androstenedione. Livers were removed from dams on gestation day 20 and from non-pregnant rats after five weeks’ treatment. Liver microsomes were incubated with 200μM testosterone, and the reaction products were isolated and analyzed by HPLC. In pregnant rats, formation of 6α-, 15β-, 7α-, 16β-, and 2β-hydroxytestosterone was increased significantly vs. control at the highest dose level only. Formation of 6β-hydroxytestosterone increased significantly at both the 30 and 60mg/kg/day dose levels. In non-pregnant rats, 60mg/kg/day androstenedione significantly increased formation of 15β-, 6β-, 16β-, and 2β-hydroxytestosterone. The data suggest that high oral doses of androstenedione can induce some female rat liver cytochromes P450 that metabolize steroid hormones and that the response to androstenedione does not differ between pregnant and non-pregnant female rats. [Copyright &y& Elsevier]

Abstract: Steroid derivatives V, VI, VII and VIII reacted with Lawesson''s reagent (LR) to produce spiro-oxazaphosphole-4′,17-androstene derivative XI, diazaphospholoandrostane XIV and the thionated derivatives XVI and XVII, respectively. The structures of the new compounds were confirmed by analytical and spectroscopic evidence. A mechanism accounting for the formation of the new compounds was given. The in vitro antimicrobial activity of the new compounds were tested. [Copyright &y& Elsevier]

Abstract: Androstenedione, a naturally occurring steroid hormone, is a dietary supplement used to enhance athletic performance. Little is known, however, about the safety of its use by young adults including women of child bearing age. To test the possible hepatotoxic effects of androstenedione use, this study was undertaken using a rat model. Pregnant rats (six rats/dose) were exposed to androstenedione in corn oil by gastric intubation at 0, 5, 30 or 60mg/kg body weight/day beginning 2weeks before mating and continuing through gestation day 19. On gestation day 20, blood and livers were collected from the pregnant rats for analysis of hepatotoxicity endpoints: serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutathione (GSH) and glutathione S-transferase (GST), total microsomal P450, nuclear DNA damage and lipid peroxidation. Under these experimental conditions, no significant differences were observed in any of these biomarkers over the concentration range examined. [Copyright &y& Elsevier]

Low fertility of cattle during the cool autumn is mostly associated with exposure to thermal stress in the previous summer. This study examined carry-over effects of summer heat stress and induction of enhanced follicular turnover, on the characteristics of preovulatory follicles during autumn. Holstein cows were treated with PGF2α and GnRH to induce six successive 9-day follicular waves during autumn. Intact cows served as controls. Follicular fluid concentrations of androstenedione were higher in treated cows, but those of IGF-I and progesterone did not differ between groups. The concentration of estradiol at the end of the treatment was higher, and the content per follicle increased with time and was numerically higher in treated cows. The volume of follicular fluid increased during the autumn and was greater in treated cows. A delayed effect of heat stress on preovulatory follicles was noted, and induction of follicular waves by GnRH was able to improve follicular characteristics in autumn. [Copyright &y& Elsevier]

: ObjectiveTo determine if insulin or glucose action plays a role in adrenocortical steroidogenesis in the polycystic ovary syndrome (PCOS).: DesignProspective cohort study.: SettingAcademic medical center.: Patient(s)Nine reproductive-aged patients with PCOS and nine age-, race-, and body mass index–matched controls.: Main outcome measure(s)Insulin-modified frequently sampled intravenous glucose tolerance testing and an acute 60-minute ACTH-(1-24) stimulation test. From the glucose tolerance test, glucose and insulin were measured and the insulin sensitivity index, glucose effectiveness, and acute insulin response to glucose were determined. Dehydroepiandrosterone sulfate (DHEAS) basally and 17-hydroxypregnenolone, 17-hydroxyprogesterone, DHEA, androstenedione, and cortisol during ACTH testing at 0 and 60 minute (steroid0 and steroid60) were determined. The net change in steroid during the ACTH test was calculated.: Result(s)The insulin sensitivity index had limited correlation with adrenocortical variables in both groups. In patients with PCOS, glucose effectiveness was positively associated with DHEAS, cortisol0, cortisol60, change in cortisol, DHEA0, DHEA60, change in DHEA, 17-hydroxyprenenolone60, change in 17-hydroxypregnenolone, DHEA0, androstenedione0, 17-hydroxyprenenolone0, 17-hydroxyprogesterone0, 17-hydroxyprenenolone60, and 17-hydroxyprogesterone60.: Conclusion(s)Adrenocortical biosynthesis, basally and in response to ACTH, appears to be closely associated with glucose effectiveness in PCOS. A common factor determining both the effectiveness of glucose to control its own production or uptake and adrenocortical biosynthesis may be aberrant in PCOS. [Copyright &y& Elsevier]

The effect of neurosteroids is mediated through their membrane or nuclear receptors. However, no dehydroepiandrosterone (DHEA)-specific receptors have been evidenced so far in the brain. In this paper, we showed by isothermal titration calorimetry that the DHEA specifically binds to the dendritic brain microtubule-associated protein MAP2C with an association constant of 2.7 × 10[sup 7] M[sup -1] and at a molar ratio of 1:1. By partial tryptic digestions and mass spectrometry analysis, we found that the binding involved the N-terminal region of MAP2C. Interestingly, MAP2C displays homologies with 17β-hydroxysteroid dehydrogenase 1, an enzyme required for estrogen synthesis. Based on these sequence homologies and on the x-ray structure of the DHEAbinding pocket of 17β-hydroxysteroid dehydrogenase 1, we modeled the complex of DHEA with MAP2C. The binding of DHEA to MAP2C involved specific hydrogen bonds that orient the steroid into the pocket. This work suggests that DHEA can directly influence brain plasticity via MAP2C binding. It opens interesting ways for understanding the role of DHEA in the brain. [ABSTRACT FROM AUTHOR]

: ObjectiveTo investigate the effects of reduction of insulin resistance and hyperinsulinemia associated with the polycystic ovary syndrome (PCOS) on FSH-stimulated ovarian aromatase activity.: DesignProspective study.: SettingAcademic health center, Siena, Italy.: Patient(s)Twenty women 18 to 26 years of age in whom PCOS was diagnosed on the basis of oligomenorrhea or amenorrhea and hyperandrogenemia.: Intervention(s)Recombinant FSH was administered. The next day, therapy with metformin (500 mg t.i.d.) was begun. After 35 to 40 days of treatment, the pretreatment protocol was repeated.: Main outcome measure(s)Plasma levels of estradiol (E2), androstenedione (A), and testosterone (T). The ratios of basal levels and areas under the curve (AUCs) of products and substrates were compared before and after metformin administration to detect differences in aromatase activity.: Result(s)Metformin treatment was associated with significant reduction in basal free testosterone plasma levels, insulin plasma levels, and insulin response to oral glucose tolerance testing. Administration of FSH was followed by a significantly lesser E2 response after metformin therapy than before this therapy. The ratios of AUCE2 to AUCA and to AUCT, indicative of aromatase activity in response to FSH, were significantly lower after metformin therapy than before.: Conclusion(s)Metformin therapy in women with PCOS is associated with a reduction in aromatase activity in response to FSH. Insulin affects production of both androgen and estrogen. Insulin therefore plays a central role in regulating the activity of thecal and granulosa cells. [Copyright &y& Elsevier]

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD-3) is a member of the short-chain dehydrogenase/reductase (SDR) family and is essential for the reductive conversion of inactive C19-steroid, androstenedione, to the biologically active androgen, testosterone, which plays a central role in the development of the male phenotype. Mutations that inactivate this enzyme give rise to a rare form of male pseudohermaphroditism, referred to as 17β-HSD-3 deficiency. One such mutation is the replacement of arginine at position 80 with glutamine, compromising enzyme activity by increasing the cofactor binding constant 60-fold. In the absence of a 17β-HSD-3 crystal structure, we have grafted its amino acid sequence for the NADPH binding site on the X-ray crystal structures of glutathione reductase (Protein Data Bank code 1gra) and 17β-HSD type 1 (Protein Data Bank codes 1fdv and 1fdu) where we find the trunk of the arginine 80 side chain forms part of the hydrophobic pocket for the purine ring of adenosine while its guanidinium moiety interacts with the 2′-phosphate to both stabilize cofactor binding and neutralize its intrinsic negative charge through two hydrogen bonds. To qualitatively assess the role arginine 80 plays in both selecting and stabilizing NADPH binding, it was replaced with each amino acid and the mutant enzymes subjected to enzymatic analysis. There are only seven enzymes exhibiting any measurable enzymatic activity with arginine∼lysine>leucine>glutamine>methionine>tyrosine>isoleucine. With an aspartic acid at position 58 in 17β-HSD-3 occupying the equivalent space in the cofactor binding pocket as arginine 224 in glutathione reductase or serine 12 in 17β-HSD-1, there was an expectation that some of the mutants might use NADH as a cofactor. In no case was NADH found to substitute for NADPH. [Copyright &y& Elsevier]

Human hepatocyte incubations were used to study the metabolism of precursors of testosterone and nortestosterone and to evaluate qualitatively the correlation between in vitro and published in vivo urinary metabolic profiles. Both phase I and phase II biotransformations were observed in vitro: oxidoreduction at C-3 and C-17, reduction at C-4,5, hydroxylation at C-6β and C-16, glucuronidation and sulfation. All major metabolites detected in urine following oral administration of androstenedione and norandrostenedione were present in human hepatocyte incubations. The good correlation between in vitro and in vivo metabolic profiles indicates that hepatocyte incubations can be a useful tool to identify and characterize metabolites that could be potential urinary markers for detection of steroid abuse by athletes. [ABSTRACT FROM AUTHOR]

β Androstenes steroid up-regulates immunity to increase resistance against lethal infection and lethal radiation, and mediates a rapid recovery of hematopoietic precursor cells after radiation injury. β Androstenetriol increases the levels of the TH1 cytokines, IL-2, IL-3, IFNγ and counteracts hydrocortisone mediated immune suppression. In contrast, 17α androstenediol inhibits proliferation and mediates apoptosis in tumor cells of murine and human origin. Its epimer 17β androstenediol does not. The antiproliferative functions of 17α androstenediol are not dependent on either the estrogen or androgen receptors.Our findings show that β androstenes and analogs protect the host from lethal infection by DNA or RNA viruses such as, herpesvirus type 2, coxsackievirus B4, influenza, and arthropod borne viruses. These androstenes also protected the host from lethal bacterial infections by Enterococcus faecalis, Pseudomonas aeruginosa, and Klebsiella pneumonia and from parasites infections, i.e. Cryptosporidium parvum, and malaria.In vivo, the level of potency follows the order: dehydroepiandrosterone⋘androstenediol with the latter being up to one hundred thousand times more potent in protecting the host from infections than the first. In vitro, their effects are also dramatically different from one another with only β androstenetriol potentiating the cellular response by increasing lymphocyte activation and counteracting hydrocortisone immune-suppressive activity. Conceptually, the androstenes form a new and different subclass of steroid hormones with unique physiological properties. Following host injury, the balance between the epimers and isomers is a determining factor in the overall regulation of hematopoiesis, THl/TH2 balance, and host resistance to infections and tumor growth. [Copyright &y& Elsevier]

: ObjectivesThe purpose of this study was to determine the effects of norsteroid supplementation (224 mg of 19-nor-4-androstene-3,17-dione and 120 mg of 19-nor-4-androstene-3,17-diol, total daily dose = 344 mg) on body composition and strength in resistance-trained men.: MethodsIn a placebo-controlled, double-blind, randomized fashion, 10 subjects received the norsteroid (11 capsules containing a combination of both norsteroids) or a placebo for 8 wk (five subjects per group). Each subject participated in resistance training an average of 4 d/wk for the duration of the study. Body composition was determined via dual-energy x-ray absorptiometry. Strength was determined using a one-repetition maximum bench press and a one-repetition maximum biceps curl.: ResultsWith regard to all measures in both groups, there were no significant changes between before and after the study.: ConclusionsTherefore, in this small sample of resistance-trained men, 344 mg/d of norsteroid supplementation had no effect on strength or body composition. [ABSTRACT FROM AUTHOR]

Androstenedione is a steroid hormone sold over-the-counter to individuals who expect that it will enhance strength and athletic performance. Endogenous androstenedione is the immediate precursor of testosterone. To evaluate the metabolism of oral androstenedione, we randomly assigned 37 healthy men to receive 0 (group 1), 100 mg (group 2), or 300 mg (group 3) of androstenedione in a single daily dose for 7 days. Eight-hour urines were collected 1 day before the start of androstenedione, and on days 1 and 7. Using gas chromatography-mass spectrometry, we measured excretion rates of glucuronide-conjugated epitestosterone, its putative precursor (E-precursor), and metabolites (EM-1 and EM-2), and we evaluated possible markers of androstenedione administration. Day 1 and 7 rates were not different: the means were averaged. The means (μg/h) for groups 1, 2, and 3, respectively were, for epitestosterone 2.27, 7.74, and 18.0; for E-precursor, 2.9, 2.0, and 1.5; for EM-1/E-precursor 0.31, 1.25, and 2.88; for EM-2/E-precursor 0.14, 0.15, and 1.15; for testosterone/epitestosterone (T/E) 1.1, 3.5, and 3.2. Epitestosterone, EM-1, and EM-2 excretion was greater in groups 2 and 3 versus group 1 (0.0001 < P < 0.03), as were EM-1/E-precursor, EM-2/E-precursor, and T/E. E-precursor excretion was lower in groups 2 (P = 0.08) and 3 (P = 0.047) versus group 1. Androstenedione increases excretion of epitestosterone and its two metabolites, while decreasing that of its precursor. Elevated ratios of EM-1- and EM-2/E-precursor, and the presence of 6α-hydroxyandrostenedione are androstenedione administration markers. [Copyright &y& Elsevier]

Objective: To describe the sources, production rates, circulating concentrations, and regulatory mechanisms of the major androgen precursors and androgens in women.Design: Review of the major published literature.Result(s): Quantitatively, women secrete greater amounts of androgen than of estrogen. The major circulating steroids generally classified as androgens include dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T), and dihydrotestosterone in descending order of serum concentration, though only the latter two bind the androgen receptor. The other three steroids are better considered as pro-androgens. Dehydroepiandrosterone is primarily an adrenal product, regulated by adrenocorticotropic hormone (ACTH) and acting as a precursor for the peripheral synthesis of more potent androgens. Dehydroepiandrosterone is produced by both the ovary and adrenal, as well as being derived from circulating DHEAS. Androstenedione and testosterone are products of the ovary and the adrenal. Testosterone circulates both in its free form, and bound to protein including albumin and sex steroid hormone-binding globulin (SHBG), the levels of which are an important determinant of free testosterone concentration.Conclusion(s): The postmenopausal ovary is an androgen-secreting organ and the levels of testosterone are not directly influenced by the menopausal transition or the occurrence of menopause. Dihydrotestosterone (DHT) is primarily a peripheral product of testosterone metabolism. Severe androgen deficiency occurs in hypopituitarism, but other causes may lead to androgen deficiency, including Addison’s disease, corticosteroid therapy, chronic illness, estrogen replacement (leads to elevated SHBG and, therefore, low free testosterone), premenopausal ovarian failure, or oophorectomy. [Copyright &y& Elsevier]

Microbial bioconversion of sterols into high value steroid precursors, such as 4-androstene-3,17-dione (AD), is an industrial challenge. Genes and enzymes involved in sterol degradation have been proposed, although the complete pathway is not yet known. The genome sequencing of the AD producer strain ‘ Mycobacterium neoaurum ’ NRRL B-3805 (formerly Mycobacterium sp. NRRL B-3805) will serve to elucidate the critical steps for industrial processes and will provide the basis for further genetic engineering. The genome comprises a circular chromosome (5 421 338 bp), is devoid of plasmids and contains 4844 protein-coding genes. [ABSTRACT FROM AUTHOR]