Plasma Steroid Profiles Research Articles

Plasma Steroid Profiling Between Patients With and Without Diabetes Mellitus in Nonfunctioning Adrenal Incidentalomas.

Adrenal incidentalomas, including nonfunctioning adrenal incidentalomas (NFAI), are associated with a high prevalence of diabetes mellitus (DM). While NFAI is diagnosed by exclusion when no hormone excess exists, subtle cortisol secretion may exist and contribute to DM development. However, it alone cannot explain the increased risk, and whether other steroid metabolites are involved remains unclear. To investigate steroid metabolites associated with DM in patients with NFAI using plasma steroid profiles. Using liquid chromatography-tandem mass spectrometry, 22 plasma steroid metabolites were measured in 68 patients with NFAI (31 men and 37 women). Data were adjusted for age before normalization. Discriminant analysis showed that plasma steroid profiles discriminated between patients with and without DM in men (n = 10 and = 21, respectively) but not women: 11β-hydroxytestosterone, an adrenal-derived 11-oxygenated androgen, contributed most to this discrimination and was higher in patients with DM than in those without DM (false discovery rate = .002). 11β-hydroxytestosterone was correlated positively with fasting plasma glucose (r = .507) and hemoglobin A1c (HbA1c) (r = .553) but negatively with homeostatic model assessment of β-cell function (HOMA2-B) (r = -.410). These correlations remained significant after adjusting for confounders, including serum cortisol after the 1-mg dexamethasone suppression test. Bayesian kernel machine regression analysis verified the association of 11β-hydroxytestosterone with HbA1c and HOMA2-B in men. Plasma steroid profiles differed between those with and without DM in men with NFAI. 11β-hydroxytestosterone was associated with hyperglycemia and indicators related to pancreatic β-cell dysfunction, independently of cortisol.

Plasma Steroid Profiling Combined With Machine Learning for the Differential Diagnosis in Mild Autonomous Cortisol Secretion From Nonfunctioning Adenoma in Patients With Adrenal Incidentalomas

ObjectiveTo assess the diagnostic value of combining plasma steroid profiling with machine learning (ML) in differentiating between mild autonomous cortisol secretion (MACS) and nonfunctioning adenoma (NFA) in patients with adrenal incidentalomas. MethodsThe plasma steroid profiles data in the laboratory information system were screened from January 2021 to December 2023. EXtreme Gradient Boosting was applied to establish diagnostic models using plasma 24-steroid panels and/or clinical characteristics of the subjects. The SHapley Additive exPlanation (SHAP) method was used for explaining the model. ResultsSeventy-six patients with MACS and 86 patients with NFA were included in the development and internal validation cohort while the external validation cohort consisted of 27 MACS and 21 NFA cases. Among 5 ML models evaluated, eXtreme Gradient Boosting demonstrated superior performance with an area under the curve of 0.77 using 24 steroid hormones. The SHAP method identified 5 steroids that exhibited optimal performance in distinguishing MACS from NFA, namely dehydroepiandrosterone, 11-deoxycortisol, 11β-hydroxytestosterone, testosterone, and dehydroepiandrosteronesulfate. Upon incorporating clinical features into the model, the area under the curve increased to 0.88, with a sensitivity of 0.77 and specificity of 0.82. Furthermore, the results obtained through SHAP revealed that lower levels of testosterone, dehydroepiandrosterone, low-density lipoprotein cholesterol, body mass index, and adrenocorticotropic hormone along with higher level of 11-deoxycortisol significantly contributed to the identification of MACS in the model. ConclusionsWe have elucidated the utilization of ML-based steroid profiling to discriminate between MACS and NFA in patients with adrenal incidentalomas. This approach holds promise for distinguishing these 2 entities through a single blood collection.

PLASMA STEROID PROFILE IN WOMEN WITH FIBROMUSCULAR DYSPLASIA - THE ARCADIA-GYN STUDY

Objective: Fibromuscular dysplasia(FMD) mostly affects women in reproductive age. It has been suggested that FMD might be related to female hormone levels or contraceptive use. Therefore, the aim of the ARCADIA-POL substudy ARCADIA-GYN was to perform detailed, prospective obstetrics and gynaecological evaluation of women with FMD. Design and method: We enrolled in the ARCADIA-GYN study 42 women with FMD, ages between 18–46 years old and 45 age-matched healthy, normotensive women who have regular menstruation. By means of LC-MS/MS we analysed plasma steroid profile: estrogen, progesterone, testosterone, aldosterone, cortisol, 18-oxo-cortisol, 18-hydrocortisol, cortisone, 11-deoxycortisol, 21-deoxycortisol, corticosterone, 11-deoxycortisone, progesterone, 17-OH progesterone, pregnenolone, androstenedione, DHEA, DHEA-SO4. Blood samples in all subjects were collected in luteal phase while women stayed without any hormonal contraceptive. FMD was diagnosed based on ARCADIA-POL protocol as published previously. No signs of arterial lesions and/or disturbances in blood flow were found in Doppler duplex evaluations of carotid and renal arteries in control subjects. Results: 42 patients with FMD (34.2 ± 8.1years), predominantly with renal FMD, were compared to 45 healthy, normotensive women (34.0 ± 5.4years; p = 0.88). Hypertension was diagnosed in 88.1% (37) women with FMD. In women with FMD we found lower plasma levels of progesterone (5.1 inter quartile range [IQR] 2.0–8.2 vs 7.6 IQR 3.3–12.5ng/ml;p = 0.017),testosterone (0.20 IQR 0.15–0.28 vs 0.25 IQR 0.18–0.36ng/ml; p = 0.036), DHEA (3.6 IQR 2.6–4.3 vs 4.5 IQR 2.9–6.6ng/ml;p = 0.012), DHEAS04 (1167 IQR 819–1535 vs 1579 IQR 1197–1980ng/ml;p = 0.04) and 11-deoxycortisol (0.09 IQR 0.05–0.12 vs 0.14 IQR 0.09–0.18ng/ml;p = 0.002) as compared to control group. There were no significant differences in other analyzed hormones. There were no differences in steroid profile evaluations between patients with unifocal and multifocal FMD. Conclusions: Women with FMD as compared to healthy controls are characterized by hormonal alterations including lower plasma progesterone level. Taking into consideration previous findings of abnormal balance between estrogen and progesterone receptors in renal artery samples of FMD our results suggest that female hormonal disturbances may have a role in FMD development.

Plasma steroid profiles in patients hospitalised with COVID-19 - an ISARIC/WHO CCP-UK cohort study

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Catch-up Growth and Discontinuation of Fludrocortisone Treatment in Aldosterone Synthase Deficiency.

Aldosterone synthase deficiency (ASD) caused by mutations in the CYP11B2 gene is characterized by isolated mineralocorticoid deficiency. Data are scarce regarding clinical and biochemical outcomes of the disease in the follow-up. Assessment of the growth and steroid profiles of patients with ASD at the time of diagnosis and after discontinuation of treatment. Children with clinical diagnosis of ASD were included in a multicenter study. Growth and treatment characteristics were recorded. Plasma adrenal steroids were measured using liquid chromatography-mass spectrometry. Genetic diagnosis was confirmed by CYP11B2 gene sequencing and in silico analyses. Sixteen patients from 12 families were included (8 females; median age at presentation: 3.1 months, range: 0.4 to 8.1). The most common symptom was poor weight gain (56.3%). Median age of onset of fludrocortisone treatment was 3.6 months (range: 0.9 to 8.3). Catch-up growth was achieved at median 2 months (range: 0.5 to 14.5) after treatment. Fludrocortisone could be stopped in 5 patients at a median age of 6.0 years (range: 2.2 to 7.6). Plasma steroid profiles revealed reduced aldosterone synthase activity both at diagnosis and after discontinuation of treatment compared to age-matched controls. We identified 6 novel (p.Y195H, c.1200 + 1G > A, p.F130L, p.E198del, c.1122-18G > A, p.I339_E343del) and 4 previously described CYP11B2 variants. The most common variant (40%) was p.T185I. Fludrocortisone treatment is associated with a rapid catch-up growth and control of electrolyte imbalances in ASD. Decreased mineralocorticoid requirement over time can be explained by the development of physiological adaptation mechanisms rather than improved aldosterone synthase activity. As complete biochemical remission cannot be achieved, a long-term surveillance of these patients is required.

Use of Steroid Profiling Combined With Machine Learning for Identification and Subtype Classification in Primary Aldosteronism

Most patients with primary aldosteronism, a major cause of secondary hypertension, are not identified or appropriately treated because of difficulties in diagnosis and subtype classification. Applications of artificial intelligence combined with mass spectrometry-based steroid profiling could address this problem. To assess whether plasma steroid profiling combined with machine learning might facilitate diagnosis and treatment stratification of primary aldosteronism, particularly for patients with unilateral adenomas due to pathogenic KCNJ5 sequence variants. This diagnostic study was conducted at multiple tertiary care referral centers. Steroid profiles were measured from June 2013 to March 2017 in 462 patients tested for primary aldosteronism and 201 patients with hypertension. Data analyses were performed from September 2018 to August 2019. The aldosterone to renin ratio and saline infusion tests were used to diagnose primary aldosteronism. Subtyping was done by adrenal venous sampling and follow-up of patients who underwent adrenalectomy. Statistical tests and machine-learning algorithms were applied to plasma steroid profiles. Areas under receiver operating characteristic curves, sensitivity, specificity, and other diagnostic performance measures were calculated. Primary aldosteronism was confirmed in 273 patients (165 men [60%]; mean [SD] age, 51 [10] years), including 134 with bilateral disease and 139 with unilateral adenomas (58 with and 81 without somatic KCNJ5 sequence variants). Plasma steroid profiles varied according to disease subtype and were particularly distinctive in patients with adenomas due to KCNJ5 variants, who showed better rates of biochemical cure after adrenalectomy than other patients. Among patients tested for primary aldosteronism, a selection of 8 steroids in combination with the aldosterone to renin ratio showed improved effectiveness for diagnosis over either strategy alone. In contrast, the steroid profile alone showed superior performance over the aldosterone to renin ratio for identifying unilateral disease, particularly adenomas due to KCNJ5 variants. Among 632 patients included in the analysis, machine learning-designed combinatorial marker profiles of 7 steroids alone both predicted primary aldosteronism in 1 step and subtyped patients with unilateral adenomas due to KCNJ5 variants at diagnostic sensitivities of 69% (95% CI, 68%-71%) and 85% (95% CI, 81%-88%), respectively, and at specificities of 94% (95% CI, 93%-94%) and 97% (95% CI, 97%-98%), respectively. The validation series yielded comparable diagnostic performance. Machine learning-designed combinatorial plasma steroid profiles may facilitate both screening for primary aldosteronism and identification of patients with unilateral adenomas due to pathogenic KCNJ5 variants, who are most likely to show benefit from surgical intervention.

Mass spectrometry-based steroid profiling in primary bilateral macronodular adrenocortical hyperplasia.

Biochemical characterization of primary bilateral macronodular adrenocortical hyperplasia (PBMAH) by distinct plasma steroid profiles and its putative correlation to disease has not been previously studied. LC-MS/MS-based steroid profiling of 16 plasma steroids was applied to 36 subjects (22 females, 14 males) with PBMAH, 19 subjects (16 females, 3 males) with other forms of adrenal Cushing's syndrome (ACS), and an age and sex-matched control group. Germline ARMC5 sequencing was performed in all PBMAH cases. Compared to controls, PBMAH showed increased plasma 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycortisol, and aldosterone, but lower progesterone, DHEA, and DHEA-S with distinct differences in subjects with and without pathogenic variants in ARMC5. Steroids that showed isolated differences included cortisol and 18-oxocortisol with higher (P < 0.05) concentrations in ACS than in controls and aldosterone with higher concentrations in PBMAH when compared to controls. Larger differences in PBMAH than with ACS were most clear for corticosterone, but there were also trends in this direction for 18-hydroxycortisol and aldosterone. Logistic regression analysis indicated four steroids - DHEA, 11-deoxycortisol, 18-oxocortisol, and corticosterone - with the most power for distinguishing the groups. Discriminant analyses with step-wise variable selection indicated correct classification of 95.2% of all subjects of the four groups using a panel of nine steroids; correct classification of subjects with and without germline variants in ARMC5 was achieved in 91.7% of subjects with PBMAH. Subjects with PBMAH show distinctive plasma steroid profiles that may offer a supplementary single-test alternative for screening purposes.

Plasma steroid profiles before and after ACTH stimulation test in healthy horses

This study describes steroid profiles in equine plasma before and after ACTH stimulation. In human medicine, other steroids have been shown to have a more pronounced reaction to an ACTH stimulation test than cortisol. This study aimed to determine if the same was true for the horse. A total of 11 clinically healthy horses were selected for this study. Ethylenediaminetetraacetic acid plasma samples were taken before and 60 min after stimulation with 1 μg/kg BW of synthetic ACTH administered intravenously. The samples were analyzed for cortisol, 11-deoxycortisol, 21-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, androstenedione, 17-OH-progesterone, progesterone, and testosterone with a liquid chromatography–tandem mass spectrometry (LC-MS/MS). Cortisol, 11-deoxycortisol, cortisone, corticosterone, and 11-deoxycorticosterone showed a significant increase after ACTH stimulation. In conclusion, the LC-MS/MS represents a viable method to measure glucocorticoids and related precursors or metabolites in equine plasma samples. In addition, we were able to show a more pronounced increase of 11-deoxycorticosterone, 11-deoxycortisol, and corticosterone compared with cortisol. These 3 metabolites could potentially serve as more sensitive biomarkers for stress in horses than cortisol.

Plasma steroid profiles and ovarian response in llamas treated with eCG for superovulation combined with exogenous progesterone during early luteal phase

The objective of this study was to evaluate the effects of plasma progesterone (P4) concentrations during eCG-ovarian follicular superstimulatory treatment performed in early luteal phase and estradiol concentrations during peri-ovulatory period on ovarian response, number and embryo quality. On Day −2, females (n = 75) having a follicle ≥7 mm were treated with GnRH to induce ovulation. On Day 0, females that had ovulations (n = 54) were treated with 1000 IU eCG and were assigned to one of two treatments: (1) intravaginal device (ID) containing 0.5 g P4 (P4 group) and (2) no ID (Control group). On Day 5, females were administered PGF2α and the ID was removed. On Day 7 and 8, females were mated and embryo recovery was performed 7 or 8 days later. Blood samples were collected from Day 0 to 9. Number (± SD) of follicles ≥7 mm on day of mating was greater (P = 0.04) in the control (9.7 ± 4.2) than P4-treated (6.7 ± 4.9) group; number of corpora lutea did not differ (5.5 ± 3.1 and 5.2 ± 3.4 respectively). Ovulation rate was greater (P < 0.01) in the P4-group (77.4%; 130/168) than control group (53.3%; 135/253). Number of embryos with an excellent grade (grade 1) tended to be greater (P = 0.07) in the P4-group (82.4%; 42/51) than control group (65.4%; 36/55). It was concluded that supplementation with exogenous P4 during eCG treatment in early luteal phase inhibits excessive follicular growth, increases ovulation rate and improves embryo quality.

Modeling Corticosteroid Pharmacokinetics and Pharmacodynamics, Part II: Sex Differences in Methylprednisolone Pharmacokinetics and Corticosterone Suppression.

Methylprednisolone (MPL), a corticosteroid of intermediate potency, remains an important immunomodulatory agent for autoimmune diseases. Although sex differences in corticosteroid pharmacokinetics/pharmacodynamics (PK/PD) have been documented in humans, comprehensive preclinical assessments of such differences have not been conducted. Limited in vitro evidence indicates possible sex differences in corticosteroid PK and PD. Therefore, it is hypothesized that comparative PK/PD assessments of MPL disposition and selected PD actions in both sexes will provide insights into factors controlling sex differences in steroid responses. This report focused on the plasma and tissue pharmacokinetics of MPL and its adrenal suppressive effects. Because time-dependent (estrous) regulation of sex hormones in females can influence drug responses, female rats were studied in the proestrus (high estradiol/progesterone) and estrus (low estradiol/progesterone) phases of the reproductive cycle. Cohorts of male and female rats were given a 50 mg/kg bolus dose of MPL intramuscularly. Plasma and liver concentrations of MPL as well as plasma corticosterone concentrations were assayed using high-performance liquid chromatography. An enhanced minimal physiologically-based PK/PD model was developed to characterize MPL kinetics and corticosterone dynamics. The clearance of MPL was ∼3-fold higher in males compared with females, regardless of estrous phase, likely attributable to sex-specific hepatic metabolism in males. Strong inhibitory effects on adrenal suppression were observed in all animals. These temporal steroid profiles in plasma and tissues will be used to drive receptor/gene-mediated PD effects of MPL in both sexes, as described in a companion article (Part III). SIGNIFICANCE STATEMENT: Sex is a relevant factor influencing the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs. Few preclinical PK/PD studies, however, include sex as a variable. Sex differences in the PK and adrenal suppressive effects of the synthetic corticosteroid, methylprednisolone, were assessed in male and female rats as a function of the 4-day rodent reproductive cycle. Drug exposure was 3-fold higher in females, regardless of estrous stage, compared with males. An extended minimal physiologically-based PK/PD model utilizing in vitro and in vivo measurements was developed and applied. These studies provide a framework to account for sex-dependent variability in drug and endogenous agonist (corticosterone) exposures, serving as a prelude to more intricate assessments of sex-related variability in receptor/gene-mediated PD corticosteroid actions.

OR07-4 Preterm Birth and Transgenerational Epigenetic Programming of Hypertension in Mice

Introduction: Prematurity, a worldwide health issue, is often associated with renal tubular immaturity leading to major salt loss, whose mechanisms remain poorly understood. Moreover, these premature infants are prone to develop early hypertension in adulthood. Objective: To study ontogenesis of renal mineralocorticoid and glucocorticoid signaling pathways in preterm mice, and to evaluate their contribution to neonatal adaptation and to the emergence of hypertension in adulthood. Materials and Methods: We have developed a murine model of prematurity induced by intra-peritoneal injection of O111/B4 lipopolysaccharides (LPS) at 18 days of gestation in Swiss CD1 mice. Offspring of injected mice, when LPS did not trigger preterm birth, were used as a control to exclude intrinsic LPS effects. Pups were sacrificed at various developmental stages (D0, D7 and M6). Blood pressure and heart rate were measured in males at M6 and their plasma steroid profiles were measured using LC-MSMS. Renal mRNA and protein expression of major players of corticosteroid signaling pathways were examined using RT-qPCR and western-blot analyses. Second (F2) and third (F3) generations, established by mating prematurely born adult females with wild type males, were also analyzed. We performed Methylation DNA Immunoprecipitation-qPCR to study the methylation status of candidate corticosteroid target genes that were differentially expressed between the 2 populations. Results: As anticipated, preterm newborn mice presented with maladaptation, high neonatal mortality (35%), and a lower birth weight compared to controls (1.29±0.21 vs 1.46±0.15g, P=0.0027). Former preterm males developed hypertension at M6 (123.1±1.43 vs 114.5±0.79mmHg, P<0.0001). We found a robust activation of renal corticosteroid target gene transcription at birth in preterm mice (αENaC (+45%), Sgk1 (+132%), Gilz (+85%)), which was not related to modified expression of the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Interestingly, a significant increased blood pressure was found in the F2 and F3 males, descendants of the preterm group, concomitantly with increased renal Gilz mRNA expression, despite similar MR or GR expression, and plasma aldosterone or corticosterone levels. Gilz promoter methylation was reduced in preterm offspring with a negative correlation between methylation and expression (P=0.008), highly suggestive of an epigenetic Gilz regulation. Conclusion: We provide first evidence for alterations of the renal corticosteroid signaling pathways induced by prematurity, with a trans-generational transmission of blood pressure dysregulation, associated with epigenetic Gilz regulation. This study should allow better understanding of prematurity-related defects, leading hopefully to better management of premature infants from birth to adulthood.

OR02-6 Mass Spectrometry-Based Steroid Profiling Inprimary Bilateral Macronodular Adrenocortical Hyperplasia

Background: Biochemical characterization of primary bilateral macronodular adrenocortical hyperplasia (PBMAH) by distinct plasma steroid profiles and its putative correlation to disease has not been previously studied. Methods: LC-MS/MS-based steroid profiling of 16 plasma steroids was applied to 36 subjects (22 females, 14 males) with PBMAH, 19 subjects (16 females, 3 males) with other forms of adrenal CS (ACS), and an age and sex-matched control group. Germline ARMC5 sequencing was performed in all PBMAH cases. Results: The steroids showing the most dramatic differences between subject groups included 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycortisol, progesterone and DHEA/DHEA-S. Steroids that showed isolated differences included cortisol and 18-oxocortisol with higher (P<0·05) concentrations in ACS than in controls, and aldosterone with higher concentrations in PBMAH when compared to controls. Larger differences in PBMAH than with ACS were most clear for corticosterone, but there were also trends in this direction for 18-hydroxycortisol and aldosterone. Logistic regression analysis indicated four steroids - DHEA, 11-deoxycortisol, 18-oxocortisol, and corticosterone - with the most power for distinguishing the groups. Discriminant analyses with step-wise variable selection indicated correct classification of 95·2% of all subjects of the four groups using a panel of nine steroids; correct classification of subjects with and without germline variants in ARMC5 was achieved in 91·7% of subjects with PBMAH. Conclusion: Subjects with PBMAH show distinctive plasma steroid profiles that may offer a supplementary single-test alternative for screening purposes.

Gonadal steroids and energy availability during ovarian maturation stages of the Amazonian pirarucu Arapaima gigas (Teleostei: Osteoglossidae) in the wild

The Amazonian pirarucu, Arapaima gigas, belongs to a phylogenetically basal group of teleosts (family Osteoglossidae). The commercial importance and the overexploited status of natural stocks of A. gigas brought the interest to rear it in the Amazon region, but rearing techniques are not fully understood to obtain reproduction success. Gonadal steroids (plasma and cephalic liquid), lipids and proteins (muscles, liver and ovary) of wild A. gigas females were analyzed during one year on Mexiana Island (Amazon Basin, Brazil) and ovarian maturation stages were described. The sexual gender determination method based on 11-ketostestosterone (11KT) and estradiol (E2) ratio was applied to check if the value is in agreement of the sex previously determined by direct observation. Adult females were classified into four reproductive stages according to ovary development stage: previtellogenic, maturation, mature, and regression/spawning. Immature females were also analyzed. All mature females were sampled during the rainy season. Lipids stored in the ventrecha (belly) are a possible source of this substrate for the developing ovary. Plasma steroid profile throughout A. gigas female reproductive stages is similar to that found in most teleosts, but plasma 11KT levels peaked at the mature stage, suggesting an important role of this androgen in female ovulation. The 11KT/E2 method was 100% efficient when sexing juveniles and adults at the beginning of their reproductive cycle and can successfully be applied to identify the sexual gender of animals. The steroid profile of the cephalic liquid suggests a possible role of this fluid as a pheromone, due to the high level of 17α-hydroxyprogesterone at the final stages of the reproductive cycle.

Steroid profiles in both blood serum and seminal plasma are not correlated and do not reflect sperm quality: Study on the male reproductive health of fifty young Swiss men

Steroids play an important role in sperm production and quality. These hormones have been extensively studied in blood, but poorly investigated in semen. The purpose of our study was to evaluate the relationship between sperm quality and steroid profiles in blood and semen in a small cohort of young Swiss men. Another objective was to determine whether the presence of xenobiotics or drugs could influence these profiles.Semen analysis was performed according to WHO guidelines, and steroid profiles in blood serum and seminal plasma were determined by two complementary approaches: a targeted investigation involving the quantification of a limited number of relevant steroids for testing putative correlations with sperm parameters and a global “steroidomic” analysis highlighting their complex metabolic relationship.Results showed that steroid profiles are distinct within blood and seminal fluid. No significant correlation was found between individual steroids measured in blood and in semen, demonstrating the relevance of assessing hormone levels in both fluids. Moreover, testosterone and androstenedione levels were significantly correlated in semen but not in blood. None of the evaluated spermiogram parameters was linked to steroid levels measured in any medium. The steroidomic analyses confirmed that the steroids present in both fluids are different and that there is no correlation with spermiogram parameters. Finally, upon toxicological screening, we observed that all the three samples positive for tetrahydrocannabinol, which is known to act as an endocrine disruptor, displayed low seminal testosterone concentrations.In conclusion, we did not find any evidence suggesting using steroid profiles, neither in blood nor in semen, as surrogates for sperm analyses. However, steroid profiles could be useful biomarkers of individual exposure to endocrine disruptors.

Plasma Steroid Metabolome Profiling for Diagnosis and Subtyping Patients with Cushing Syndrome.

Diagnosis of Cushing syndrome requires a multistep process that includes verification of hypercortisolism followed by identification of the cause of adrenocortical hyperfunction. This study assessed whether pituitary, ectopic, and adrenal subtypes of Cushing syndrome were characterized by distinct plasma steroid profiles that might assist diagnosis. In this retrospective cross-sectional study, mass spectrometric measurements of a panel of 15 plasma steroids were applied to 222 patient samples tested for Cushing syndrome. Disease was excluded in 138 and confirmed in 51 patients with pituitary Cushing syndrome, 12 with ectopic adrenocorticotropin secretion, and 21 with adrenal disease. Another 277 age- and sex-matched hypertensive and normotensive volunteers were included for comparison. Compared with patients without disease, the largest increases in plasma steroids among patients with Cushing syndrome were observed for 11-deoxycortisol (289%), 21-deoxycortisol (150%), 11-deoxycorticosterone (133%), corticosterone (124%), and cortisol (122%). Patients with ectopic disease showed the most prominent increases, but there was considerable variation for other steroids according to subtype. Patients with adrenal disease had the lowest concentrations of androgens, whereas those with ectopic and pituitary disease showed the lowest concentrations of aldosterone. Plasma 18-oxocortisol was particularly low in ectopic disease. With the use of 10 selected steroids, subjects with and without different Cushing syndrome subtypes could be discriminated nearly as closely as with the use of salivary and urinary free cortisol, dexamethasone-suppressed cortisol, and plasma adrenocorticotropin (9.5% vs 5.8% misclassification). Patients with different subtypes of Cushing syndrome show distinctive plasma steroid profiles that may offer a supplementary single-test alternative for screening purposes.

Ovaprim, a commercial spawning inducer, stimulates gonadotropin subunit gene transcriptional activity: A study correlated with plasma steroid profile, ovulation and fertilization in the catfish Heteropneustes fossilis

The commercial fish spawning inducer Ovaprim (OVP) containing a salmon gonadotropin-releasing hormone analogue and domperidone (a dopamine receptor-2 antagonist) has been widely used as an effective spawning inducer in artificial breeding of fishes. It induces a preovulatory LH surge resulting in final oocyte maturation (FOM) and ovulation through a mechanism involving a steroidogenic shift to secrete a maturation-inducing steroid (MIS). In the present study, a 0.5μL/g body weight dose of OVP each injected at 0h and 24h intraperitoneally into gravid female catfish, Heteropneustes fossilis resulted in periovulatory changes in gonadotropin (GtH) subunit gene expression and steroid hormone levels. The OVP injections induced ovulation time-dependently from 6h onwards with 100% ovulation recorded from 24h to 48h. The fertilization rate was high from 6h to 18h and declined from 24h onwards. The OVP treatment up regulated the expression of GtH subunit genes differentially. The expression of glycoprotein-α (GPα) and luteinizing hormone (LHβ) peaked at 6h and 12h, and declined at 18h and 24h after the first injection. The second OVP injection at 24h elicited only a transient increase in the GPα expression at 6h and a sustained increase in the LHβ expression from 6h to 18h after the second injection, but both transcripts decreased subsequently. The follicle-stimulating hormone (FSHβ) expression responded to the OVP treatment from 12h onwards and maintained a constant level from 18h to 36h after the first injection; the second dose had little effect. Plasma steroids were differentially altered: the levels of estradiol-17β decreased while that of the MIS 17,20β-dihydroxy-4-pregnen-3-one; 17,20β-DP increased, causing the steroidogenic shift preceding FOM and ovulation. The present results indicate that LHβ expression coincides with the ovulation response and the late induction and maintenance of the FSH expression may be related to post-ovulatory events in the ovary.

Evidence of estrone-sulfate uptake modification in young and middle-aged rat prostate

High plasma exposure to estrogens is often associated with prostate cancer. Reducing this phenomenon may present therapeutic benefits. The involvement of estrone sulphate (E1S), the most abundant circulating estrogen in men, has been partially studied in this age-related pathology. To investigate the consequences of plasma E1S overload on blood and prostate sex steroid levels and inflammatory tissue responses, young and middle-aged male rats were treated with E1S with or without steroid sulfatase (STS) inhibitor STX64 for 21 consecutive days. A plasma and prostate tissue steroid profile was determined. STS activity, mRNA expression of E1S organic anion transporting polypeptides (slco1a2, slco2b1, slco4a1) and pro-inflammatory cytokines (Il1-beta, Il6, TNF-alpha) were evaluated in prostate tissue according to age and treatment group.A significant correlation between plasma and prostate steroid levels related to hormone treatment was observed in all rat age groups. However, while the E1S level in prostate tissue increased in middle-aged treated rats (p<0.0001), no significant variation was observed in young treated rats. The protective effect of STX64 during E1S infusion was observed by the maintenance of low free estrogen concentrations in both plasma and tissue. However, this protection was not associated with mRNA expression stability of pro-inflammatory cytokines in older rat prostate.These results suggest that E1S uptake in rat prostate cells increases during aging. Therefore, if a similar phenomenon existed in men, preventively reducing the STS activity could be of interest to limit uptake of estrogens in prostate when high E1S plasma level is assayed.

Acute Effects of 3,4-Methylenedioxymethamphetamine and Methylphenidate on Circulating Steroid Levels in Healthy Subjects

3,4-Methylenedioxymethamphetamine (MDMA, ‘ecstasy') and methylphenidate are widely used psychoactive substances. MDMA primarily enhances serotonergic neurotransmission, and methylphenidate increases dopamine but has no serotonergic effects. Both drugs also increase norepinephrine, resulting in sympathomimetic properties. Here we studied the effects of MDMA and methylphenidate on 24-hour plasma steroid profiles. 16 healthy subjects (8 men, 8 women) were treated with single doses of MDMA (125 mg), methylphenidate (60 mg), MDMA + methylphenidate, and placebo on 4 separate days using a cross-over study design. Cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycorticosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and testosterone were repeatedly measured up to 24 h using liquid chromatography-tandem mass spectroscopy. MDMA significantly increased the plasma concentrations of cortisol, corticosterone, 11-dehydrocorticosterone, and 11-deoxycorticosterone and also tended to moderately increase aldosterone levels compared with placebo. MDMA also increased the sum of cortisol + cortisone and the cortisol/cortisone ratio, consistent with an increase in glucocorticoid production. MDMA did not alter the levels of cortisone, DHEA, DHEAS, androstenedione, or testosterone. Methylphenidate did not affect any of the steroid concentrations, and it did not change the effects of MDMA on circulating steroids. In summary, the serotonin releaser MDMA has acute effects on circulating steroids. These effects are not observed after stimulation of the dopamine and norepinephrine systems with methylphenidate. The present findings support the view that serotonin rather than dopamine and norepinephrine mediates the acute pharmacologically induced stimulation of the hypothalamic-pituitary-adrenal axis in the absence of other stressors.

The clinical significance of aldosterone synthase deficiency: report of a novel mutation in the CYP11B2 gene

BackgroundAldosterone synthase (CYP11B2) deficiency is a rare autosomal recessive disorder, usually presenting with severe salt-wasting in infancy or stress-induced hyperkalaemia and postural hypotension in adulthood. Neonatal screening for congenital adrenal hyperplasia, another cause of salt wasting, using 17-hydroxyprogesterone measurement would fail to detect aldosterone synthase deficiency, a diagnosis which may be missed until the patient presents with salt-wasting crisis. Due to this potential life-threatening risk, comprehensive hormonal investigation followed by genetic confirmation for suspected patients would facilitate clinical management of the patient and assessment of the genetic implication in their offspring.Case presentationWe describe a 33-year old Chinese man who presented in infancy with life-threatening hyponatraemia and failure to thrive, but remained asymptomatic on fludrocortisone since. Chromosomal analysis confirmed a normal male karyotype of 46, XY. Plasma steroid profile showed high plasma renin activity, low aldosterone level, and elevated 18-hydroxycorticosterone, compatible with type 2 aldosterone synthase deficiency. The patient was heterozygous for a novel CYP11B2 mutation: c.977C > A (p.Thr326Lys) in exon 3. He also carried a heterozygous mutation c.523_525delAAG (p.Lys175del) in exon 6, a known pathogenic mutation causing aldosterone synthase deficiency. Sequencing of CYP11B2 in his parents demonstrated that the mother was heterozygous for c.977C > A, and the father was heterozygous for c.523_525delAAG.ConclusionAlthough a rare cause of hyperreninaemic hypoaldosteronism, aldosterone synthase deficiency should be suspected and the diagnosis sought in patients who present with life-threatening salt-wasting in infancy, as it has a good long-term prognosis when adequate fludrocortisone replacement is instituted. To our knowledge, this is the first Chinese patient in which the molecular basis of aldosterone synthase deficiency has been identified.

The impact of nandrolone decanoate and growth hormone on biosynthesis of steroids in rats

Growth hormone (GH) and anabolic androgenic steroids (AAS) are commonly used in sports communities. Several studies have suggested an association between GH and AAS. We have investigated the impact of GH in rats treated with nandrolone decanoate (ND). Male Wistar rats received ND (15mg/kg) every third day during three weeks and were subsequently treated with recombinant human GH (1.0IU/kg) for ten consecutive days. Plasma samples were collected and peripheral organs (i.e. heart, liver, testis and thymus) were dissected and weighed. Concentration of thirteen endogenous steroids was measured in the rat plasma samples using high specificity LC–MS/MS methods. Seven steroids were detected and quantified, and concentrations of estrone, testosterone, and androstenedione were significantly different among the groups, while concentrations of pregnenolone, DHEA, 17-hydroxyprogesterone and corticosterone were not altered. Administration of rhGH alone altered the plasma steroid distribution, and the results demonstrated significantly increased concentrations of plasma estrone as well as decreased concentrations of testosterone and androstenedione in the ND-treated rats. Administration of rhGH to ND-pretreated rats did not reverse the alteration of the steroid distribution induced by ND. Administration of ND decreased the weight of the thymus, and addition of rhGH did not reverse this reduction. However, rhGH administration induced an enlargement of thymus. Taken together, the plasma steroid profile differed in the four groups, i.e. control, AAS, rhGH and the combination of AAS and rhGH treatment.