Free and sulfated steroids secretion in postpubertal boars (Sus scrofa domestica).

Sulfated steroids have been traditionally regarded as inactive metabolites. However, they may also serve as precursors for the production of active free steroids in target cells. In this study, we used the boar as a model to study the metabolism, transport, and function of steroid sulfates due to their high production in the porcine testicular-epididymal compartment, of which the role is unknown. To characterize the secretion of free and sulfated steroids, plasma samples were collected from six postpubertal boars over 6  h every 20  min from the jugular vein. Long-term secretion profiles were also established in seven boars stimulated with human chorionic gonadotropin. To directly characterize the testicular output, samples were collected from superficial testicular arterial and venous blood vessels. Testosterone, androstenedione and sulfated pregnenolone, DHEA, estrone (E1), and estradiol-17ß (E2) were determined by liquid chromatography-tandem mass spectrometry. Free E1 and E2 were measured by RIA. Irrespective of a high variability between individuals, the results suggest that i) all steroids assessed are primarily produced in the testis, ii) they exhibit similar profiles pointing to a pulsatile secretion with low frequency (three to five pulses per day), and iii) after synthesis at least a major proportion is immediately released into peripheral circulation. The fact that all steroid sulfates assessed are original testicular products and their high correlations with one another suggest their role as being intermediates of testicular steroidogenesis rather than as being inactivated end products. Moreover, a substantial use of sulfated steroids in porcine testicular steroidogenesis would assign a crucial regulatory role to steroid sulfatase, which is highly expressed in Leydig cells.

Androgen synthesis in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

A hallmark of severe congenital adrenal hyperplasia due to 21-hydroxylase deficiency is pre- and postnatal virilization. The most characteristic biochemical abnormality is the elevation of 17α-hydroxyprogesterone, which is metabolized to the most potent androgen receptor agonist dihydrotestosterone. 17α-Hydroxyprogesterone can be metabolized to dihydrotestosterone via 4-androstenedione through the classical Δ4-pathway or via 17α-hydroxypregnenolone and dehydroepiandrosterone through the classical Δ5-pathway, as well as through an alternative route, called the 'backdoor pathway', that bypasses dehydroepiandrosterone, 4-androstenedione, and testosterone as intermediates. This review article will summarize recent advances in the understanding of the activities of androgen synthesis pathways in patients with 21-hydroxylase deficiency obtained by urinary steroid metabolomics based on gas chromatography-mass spectrometry. Compared with healthy controls, the relative activities of the backdoor and Δ4-pathways increase in patients with congenital adrenal hyperplasia during neonatal age and infancy, whereas the activity of the Δ5-pathway remains unchanged. Thereafter, the activity of the Δ5-pathway dominates, whereas a decreasing 5α-reductase activity leads to a diminished role of the backdoor pathway for androgenic steroid production. Beside the backdoor pathway, the Δ4-pathway seems to be responsible for increased androgen generation in patients with 21-hydroxylase deficiency before the onset of adrenarche, whereas the Δ5-pathway might contribute to the increased androgen formation in those patients only after the onset of adrenarche.

[The alternative androgen synthesis pathway in humans]. / Der alternative Androgensyntheseweg des Menschen.

During the last year, alternative androgen synthesis pathways have been discovered in humans. This review article highlights these new concepts of androgen synthesis.We performed a selective literature research using PubMed.After the discovery of a new androgen synthesis pathway in marsupials, this new path-way of androgen synthesis could be established in humans during the last year from two independent studies. One of them could demonstrate that two pathways of androgen synthesis are needed for male sexual differentiation in humans; the other study established that the new pathway is an important source of androgen synthesis in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Additionally, it has been shown that an alternative androgen synthesis pathway that bypasses testosterone drives castration resistant prostate cancer.New and alternative androgen path-ways occur in humans. Importantly, these path-ways remain cryptic for the clinician, because the androgen synthesis circumvents classical intermediates like dehydroepiandrosterone, androstenedione and testosterone.

Effects of hyperlipidaemia on glucocorticoid metabolism: results of a randomized controlled trial in healthy young women.

OBJECTIVE: It is well established that the hypothalamic-pituitary-adrenal (HPA) axis is altered in obese individuals. Hyperlipidaemia with elevated levels of free fatty acids (FFAs) is also frequently seen in obesity and in the metabolic syndrome. We hypothesized, therefore, that hyperlipidaemia may alter the activity of the HPA axis. PATIENTS AND METHODS: The effects of hyperlipidaemia, including increased circulating FFAs, on ACTH secretion and cortisol metabolism were analysed in 13 healthy young women during the early follicular phase of two subsequent cycles. We administered a 20% lipid/heparin (LHI) or a saline/heparin infusion (SHI) using a crossover design in random order for 330 min. A detailed characterization of glucocorticoid metabolism was performed by measurement of plasma ACTH, cortisol and urinary excretion rates of adrenal glucocorticoids and the glucocorticoid metabolites. RESULTS: We observed that LHI-induced hyperlipidaemia elevated serum cortisol levels compared to SHI. No changes in plasma ACTH levels, daily urinary excretion rates of adrenal glucocorticoids, glucocorticoid precursors/metabolites and the calculated activities of the 5α-reductase, 3ß-hydroxysteroid dehydrogenase (HSD), 11-, 17-, 21-hydroxylase and 11ß-HSD 1 or 2 were found. CONCLUSION: Our randomized controlled trial suggests that the adrenal sensitivity to ACTH may be enhanced by LHI-induced hyperlipidaemia in normal-weight healthy young women. This effect might contribute to the disturbances of the HPA axis described in women with abdominal obesity and impaired lipid metabolism.

Measurement of urinary free cortisol by current immunoassays: need for sex-dependent reference ranges to define hypercortisolism.

Urinary free cortisol (UFC) is used to assess disease activity in hypercortisolemic patients. However, reference ranges are often lacking, especially with respect to potential confounding variables. This study analyzed upper limits of normal (ULN, mean + 2 SD) for 2 newer immunoassays, using gas chromatography-mass spectrometry (GC-MS) as reference method. Each 10 healthy subjects were grouped by age (18-29; 30-49; ≥ 50 years), BMI (< 25; ≥ 25 kg/m2), and sex, resulting in a total of 120 controls (60 males; age: 39.3±1.3 years; BMI: 25.9±0.4 kg/m2). ULN were calculated for a radioimmunoassay (RIA, Immunotech) and an electrochemiluminescence immunoassay (ECLIA, Roche) and applied to 12 hypercortisolemic patients (4 males; age: 53.1±3.1 years; BMI: 29.1±1.8 kg/m2). To determine degradation, samples were stored at 4°C (without light) or 22°C (with and without light) for 0, 24, and 72 h. Cortisol concentrations were significantly correlated: r=0.88 for RIA vs. ECLIA, r=0.75 for RIA vs. GC-MS, and r=0.77 for ECLIA vs. GC-MS (always p<0.0001). For each procedure, multiple stepwise regression analysis identified sex as the only significant predictor, resulting in sex-dependent ULN (males vs. females): 294 vs. 208 nmol/24 h (RIA), and 379 vs. 277 nmol/24 h (ECLIA). These ULN classified samples from patients as hypercortisolemic in 100% (RIA) and 95% (ECLIA). Different storage conditions over 72 h did not alter UFC levels significantly. Results of the 3 procedures were well correlated, and the use of assay- and sex-specific ULN allowed excellent identification of hypercortisolic states. UFC is stable over 72 h irrespective of the storage conditions applied.

Clinical and metabolic findings in a 6-year-old boy with a Leydig cell tumour.

AIM: To analyse the urinary steroid metabolome in a boy who had true precocious puberty after a Leydig cell tumour. METHOD: Case report and detailed description of clinical and metabolic findings in a 7-year-old-boy with a Leydig cell tumour. RESULTS: Before surgery, the urinary steroid metabolome showed an activation of an alternative route to gonadal androgens independent of dehydroepiandrosterone (DHEA). After surgery, the boy entered true precocious puberty. Under leuprolide acetate treatment, clinical and laboratory findings normalized. CONCLUSION: Central precocious puberty after precocious pseudopuberty may be more common than expected and should be considered in children with persistent or recurrent symptoms after initial treatment of precocious pseudopuberty. Patients with a Leydig cell tumour seem to reactivate the so-called 'back door pathway' of androgen production, which is independent of the classical route via DHEA.

Targeted LC-MS/MS analysis of steroid glucuronides in human urine.

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1-3 G), 17ß-estradiol 17-glucuronide (E2-17 G), 17ß-estradiol 3-glucuronide (E2-3 G), estriol 16-glucuronide (E3-16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F-G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F-G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC-MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC-MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC-MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15-20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0-1 month of age: 184.5 nmol/L; 15-20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.

Peptide hormone analysis in diagnosis and treatment of Differences of Sex Development: joint position paper of EU COST Action 'DSDnet' and European Reference Network on Rare Endocrine Conditions.

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.

Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A GC-MS study.

Intact steroid hormone biosynthesis is essential for growth and development of the human fetus and embryo. In the present study, gas chromatography-mass spectrometry was employed to characterize the steroidal milieu in amniotic fluid (n = 65; male: female = 35: 30) of mid-gestation (median: 18.8th week, range: 16.0th - 24.6th week) by a comprehensive targeted steroid hormone metabolomics approach. The levels of 52 steroids including pregnenolone and 17-OH-pregnenolone metabolites, dehydroepiandrosterone (DHEA) and its metabolites, progesterone and 17-OH-progesterone metabolites, sex hormones as well as corticosterone and cortisol metabolites were measured. The dominating steroids were the group of pregnenolone and 17-OH-pregnenolone metabolites (mean ± SD: 138.0 ± 59.3 ng/mL), followed by the group of progesterone and 17-OH-progesterone metabolites (107.3 ± 44.3 ng/mL), and thereafter DHEA and its metabolites (97.1 ± 56.5 ng/mL). With respect to sex steroids, only testosterone showed a significantly higher value in male fetuses (p < 0.0001). Of all estrogen metabolites, estriol showed by far the highest concentrations (33.2 ± 26.1 ng/mL). Interestingly, cortisol metabolites were clearly present (59.6 ± 13.6 ng/mL) though fetal de novo synthesis of cortisol is assumed to start from gestational 28th week onwards. Our comprehensive characterization of the steroidal milieu in amniotic fluid of mid-gestation shows presence of all relevant classes of steroid hormones and provides reference data. We conclude that the steroidal milieu in amniotic fluid mirrors the steroidome of the feto-placental unit.

Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A LC-MS/MS study.

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC-MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ±â€¯SD, 8.6 ±â€¯3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ±â€¯2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ±â€¯3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ±â€¯10.7 ng/mL), androsterone sulfate (AnS: 9.2 ±â€¯7.4 ng/mL), estrone sulfate (E1S: 3.0 ±â€¯3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ±â€¯4.0 ng/mL) and estriol (E3: 1.2 ±â€¯0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.

Prenatal maternal stress is associated with lower cortisol and cortisone levels in the first morning urine of 45-month-old children.

Prenatal stress (PS) has been related to altered hypothalamic-pituitary-adrenal (HPA) axis activity later in life. So far, studies in children assessing HPA axis functioning have focused on salivary cortisol, reflecting daytime activity. The present work is part of a prospective study and aims to extend knowledge about the association between PS and HPA axis regulation in children. To do so, we investigated cortisol, cortisone, and the ratio cortisone/(cortisone + cortisol) in the first morning urine of 45-month-old children in relation to several measures of maternal stress during pregnancy. Urinary cortisol and cortisone were measured by online turbulent flow chromatography coupled with high performance liquid chromatography-tandem mass spectrometry. PS was defined as: perceived stress for aim 1 (Perceived Stress Scale; n = 280); presence of self-reported (n = 371) and expert-rated psychopathology for aim 2 (Mini International Neuropsychiatric Interview; n = 281); continuous measures of anxiety and depression for exploratory aim 3 (State-Trait Anxiety Inventory and Edinburgh Postnatal Depression Scale; n = 280). Aim 1: Perceived maternal PS showed negative associations with cortisol and cortisone levels. Aim 2: The presence of expert-rated maternal psychopathology was associated with reduced morning cortisone. Aim 3: Continuous measures of anxiety and depression showed negative associations with cortisol and cortisone levels. After correcting for multiple testing, perceived maternal PS (aim 1) and prenatal level of anxiety (aim 3) were significant predictors of children's urinary cortisol and cortisone in the morning (and, in the case of cortisone, also prenatal level of depression). The ratio cortisone/(cortisone + cortisol) as a global marker for the balance between the enzymes metabolizing cortisol to cortisone and vice versa (11ß-hydroxysteroid dehydrogenases type 1 and 2; 11ß-HSD1 and 2) was not associated with any measure of maternal PS (aims 1-3). The present study provides insight into possible programming effects of PS on nocturnal HPA axis activity and a proxy of 11ß-HSD in a large sample. The results suggest that the nocturnal rate of cortisol production is lower in children exposed to PS, but do not support the hypothesis of divergent 11ß-HSD activity.

Intravenous lipid and heparin infusion-induced elevation in free fatty acids and triglycerides modifies circulating androgen levels in women: a randomized, controlled trial.

BACKGROUND: The polycystic ovarian syndrome (PCOS) is characterized by hyperandrogenism and associated with obesity and impaired glucose metabolism. Despite the high prevalence of PCOS and the considerable clinical impact, the precise interplay between metabolism and hyperandrogenemia is not entirely clear. OBJECTIVE: The objective of the study was to analyze the effects of iv lipid and heparin infusion on circulating androgen levels in healthy women. DESIGN: This was a randomized, controlled, crossover trial. SETTING: The study was conducted at an endocrinology center. PATIENTS: Patients included 12 healthy young women during the early follicular phase of two subsequent cycles. INTERVENTION: After an overnight fast, a 20% lipid/heparin or a saline/heparin infusion was administered in random order for 330 min. MAIN OUTCOME MEASURES: A detailed characterization of androgen metabolism was performed. RESULTS: Elevations in free fatty acids and triglycerides, induced by lipid/heparin infusion, elevates the levels of androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), testosterone, 5alpha-dihydrotestosterone, estrone, and 17beta-estradiol. Urinary excretion of DHEA, DHEAS, 5-androstene-3beta,17beta-diol, and the sum of urinary excreted DHEA and its 16-hydroxylated downstream metabolites, 16alpha-hydroxy-DHEA and 5-androstene-3beta,16alpha,17beta-triol, were reduced. CONCLUSION: The mechanism of iv lipid and heparin infusion-induced elevation of circulating androgens described here might contribute to the development of hyperandrogenism in women with PCOS and suggests that lowering of hyperlipidemia might be a potential therapeutic target in patients with PCOS to treat hyperandrogenemia.

McCune-Albright syndrome with male premature pubarche of unusual origin.

Premature pubarche in boys is a rare manifestation of McCune-Albright syndrome (MAS). In all cases published so far, it has always been attributed to an excessive testosterone production in the testicles. For the first time we describe a boy with MAS and evidence of premature pubarche of extragonadal origin. Apart from fibrous dysplasia of the forehead and a growth hormone- and prolactin-producing pituitary adenoma, the boy presented with premature pubarche at the age of 6 years and 11 months. The size of his testicles was only 2 ml at that time and remained thus despite a progression of his pubic hair to Tanner stage IV at the age of 10 years. In the basal blood analysis testosterone was not significantly elevated. However, androstenedione and DHEAS were elevated in the serum, and in repetitive 24-hour urine samples DHEAS metabolites were markedly elevated. We therefore concluded that the patient's premature pubarche might have originated in an increased production of DHEAS. This increased production might be due to an activating mutation of a hormone receptor in the zona reticularis of his adrenal glands leading to an increase in sulfotransferase activity and excessive DHEAS production.

The art of measuring steroids: Principles and practice of current hormonal steroid analysis.

Steroids are small and highly important structural or signalling molecules in living organisms and their metabolism is complex. Due to the multiplicity of enzymes involved there are many different steroid related disorders. E.g., an individual enzyme defect is rather rare but can share various clinical symptoms and can thus be hardly diagnosed clinically. Therefore, reliable hormonal determination still presents the most reasonable initial diagnostic approach and helps to avoid uncritical and expensive attempts at molecular diagnostic testing. It also presents a backbone of monitoring these complex patients. In science, reliable hormone measurement is indispensable for the elucidation of new mechanisms of steroid hormone actions. Steroid analytics is highly challenging and should never be considered trivial. Most common methods for steroid determination comprise traditionally immunoassay, or more recently, mass spectrometry based methods. It is absolutely necessary that clinicians and scientists know the methods they are applying by heart. With the introduction of automated direct assays, a loss of quality could be observed over the last two decades in the field of steroid immunoassays. This review wants to meet the need for profound information and orientation in the field of steroid analysis. The pros and cons of the most important methods, such as immunoassays and mass spectrometry based methods will be discussed. The focus of the latter will lie on gas chromatography-mass spectrometry (GC-MS) as well as liquid chromatography-mass spectrometry (LC-MS). Selected analytical applications from our Deutsche Forschungsgemeinschaft Research Group FOR 1369 "Sulfated Steroids in Reproduction" will illustrate the contents. In brief, immunoassays have for long presented the traditional technique for steroid analysis. They are easy to set up. Only one analyte can be measured per immunoassay. Specificity problems can arise and caution has to be exerted especially regarding direct assays lacking purification steps. Mass spectrometry based methods provide structural information on the analyte and thus higher specificity. In combination with chromatographic techniques, they permit the simultaneous determination of a multitude of analytes. Highest specificity can be obtained using GC-MS, a sophisticated but most powerful tool for characterizing steroid metabolomes. LC-MS is a true high throughput technique and highly suited for detecting complex steroids. GC-MS and LC-MS are not competing but complementary techniques. Since reliable steroid determination requires extremely high expertise in the field of analytics as well as steroid biochemistry, it is recommended that collaborations and networking with highly specialized centers of expertise are developed.

Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC-MS/MS.

Sulfonated steroids (s-St) have been usually regarded as inactive metabolites but are progressively considered as precursors for the intra-tissue formation of bioactive steroids. Moreover, independent effects without preceding removal of the sulfate group have been observed. We use the porcine testicular-epididymal compartment as a model to investigate the still largely unknown s-St physiology as the boar exhibits an intriguingly broad s-St spectrum predominantly originating from the testis. The application of LC-MS/MS in steroidomics enables the determination of unconjugated and intact sulfonated steroids with currently highest specificity and good sensitivity, allowing the concurrent measuring of numerous analytes in larger quantities of samples. Profiles (6h, 20min intervals) were generated for sulfonated 5-androstene-3ß,17ß-diol (Adiol-S), androsterone (A-S), dehydroepiandrosterone (DHEA-S), epiandrosterone (EA-S), epitestosterone (ET-S), estrone (E1-S), estradiol-17ß (E2-S), pregnenolone (P5-S), 17αOH-pregnenolone (OHP5-S) and unconjugated testosterone (T) in four unstimulated and four hCG-stimulated boars. Moreover, concentrations were measured in individual samples collected from testicular afferent and efferent blood to differentiate between testicular vs. extratesticular origin. Highest concentrations were found for EA-S, followed by ET-S, Adiol-S and DHEA-S, which mostly exceeded the levels of E1-S and A-S. Lowest concentrations were obtained for E2-S, P5-S and OHP5-S. The analytical profile also included sulfonated T, 5α-dihydrotestosterone and cholesterol. However, their concentrations were below the limit of quantification. Profiles of quantifiable s-St were consistent with a wave-like pattern associated with T pulses. In postpartal females (n=5) concentrations of all analytes assessed were undetectable, suggesting that in pigs the adrenals are not a quantitatively significant source of s-St.

Efficiency of the sulfate pathway in comparison to the Δ4- and Δ5-pathway of steroidogenesis in the porcine testis.

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis.

GENETICS IN ENDOCRINOLOGY: Approaches to molecular genetic diagnosis in the management of differences/disorders of sex development (DSD): position paper of EU COST Action BM 1303 'DSDnet'

The differential diagnosis of differences or disorders of sex development (DSD) belongs to the most complex fields in medicine. It requires a multidisciplinary team conducting a synoptic and complementary approach consisting of thorough clinical, hormonal and genetic workups. This position paper of EU COST (European Cooperation in Science and Technology) Action BM1303 'DSDnet' was written by leading experts in the field and focuses on current best practice in genetic diagnosis in DSD patients. Ascertainment of the karyotpye defines one of the three major diagnostic DSD subclasses and is therefore the mandatory initial step. Subsequently, further analyses comprise molecular studies of monogenic DSD causes or analysis of copy number variations (CNV) or both. Panels of candidate genes provide rapid and reliable results. Whole exome and genome sequencing (WES and WGS) represent valuable methodological developments that are currently in the transition from basic science to clinical routine service in the field of DSD. However, in addition to covering known DSD candidate genes, WES and WGS help to identify novel genetic causes for DSD. Diagnostic interpretation must be performed with utmost caution and needs careful scientific validation in each DSD case.

Steroid hormone analysis in diagnosis and treatment of DSD: position paper of EU COST Action BM 1303 'DSDnet'.

Disorders or differences in sex development (DSD) comprise a heterogeneous group of conditions with an atypical sex development. For optimal diagnosis, highly specialised laboratory analyses are required across European countries. Working group 3 of EU COST (European Cooperation in Science and Technology) Action BM 1303 'DSDnet' 'Harmonisation of Laboratory Assessment' has developed recommendations on laboratory assessment for DSD regarding the use of technologies and analytes to be investigated. This position paper on steroid hormone analysis in diagnosis and treatment of DSD was compiled by a group of specialists in DSD and/or hormonal analysis, either from participating European countries or international partner countries. The topics discussed comprised analytical methods (immunoassay/mass spectrometry-based methods), matrices (urine/serum/saliva) and harmonisation of laboratory tests. The following positions were agreed upon: support of the appropriate use of immunoassay- and mass spectrometry-based methods for diagnosis and monitoring of DSD. Serum/plasma and urine are established matrices for analysis. Laboratories performing analyses for DSD need to operate within a quality framework and actively engage in harmonisation processes so that results and their interpretation are the same irrespective of the laboratory they are performed in. Participation in activities of peer comparison such as sample exchange or when available subscribing to a relevant external quality assurance program should be achieved. The ultimate aim of the guidelines is the implementation of clinical standards for diagnosis and appropriate treatment of DSD to achieve the best outcome for patients, no matter where patients are investigated or managed.

The steroid metabolite 16(ß)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1.

The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(ß)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased Km and decreased kcat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites.