Mitochondrial dysfunction results in enhanced adrenal androgen production in H295R cells.

The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2 h) and slower accumulation of androstenedione and testosterone (24 h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.

Multiple androgen pathways contribute to the steroid signature of adrenarche.

CONTEXT: Adrenarche is a normal developmental event in mid-childhood characterized by increasing adrenal androgen secretion. The role of the classic androgen pathway has been well described in adrenarche, but the role of newer active androgens and additional androgen pathways is less clear. OBJECTIVE: To study the contribution of novel androgens and related steroid biosynthesis pathways to the development of adrenarche, and to identify additional steroid biomarkers of adrenarche. DESIGN: A longitudinal study of children aged 6-8 years at baseline, followed up at ages 8-10 and 14-16 years. A total of 34 children (20 girls) with clinical and/or biochemical signs of adrenarche (cases) and 24 children (11 girls) without these signs (controls) at age 8-10 years were included. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. MAIN OUTCOME MEASURES: Thirty-two steroids compartmentalized in progestagens, gluco- and mineralocorticoid pathways, and four androgen related pathways, including the classic, backdoor, 11-oxy, and 11-oxy backdoor pathways. RESULTS: The classic and 11-oxy androgen pathways were more active, and serum concentrations of main androgens in the classic (dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione and androsterone) and 11-oxy (11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, 11-ketoandrostenedione, and 11-ketotestosterone) pathways were higher in cases at ages 6-8 and 8-10 years. Pregnenolone concentrations at adrenarchal age (8-10 years) and cortisol concentrations at adolescence (14-16 years) were higher in cases. 11ß-hydroxyandrosterone and 11-ketoandrosterone tended to be higher in cases with clinical signs compared to cases who had only biochemical evidence of adrenarche, albeit they were detected at low levels. In biomarker analyses, calculated steroid ratios with cortisol, cortisone, or 11-deoxycortisone as dividers were better classifiers for adrenarche than single steroids. Among these ratios, androstenedione/cortisone was the best. CONCLUSIONS: The classic and 11-oxy androgen pathways are active in adrenarche. Children with earlier timing of adrenarche have higher serum cortisol levels at late pubertal age, suggesting that early adrenarche might have long-term effects on adrenal steroidogenesis by increasing the activity of the glucocorticoid pathway. Future studies should employ comprehensive steroid profiling to define novel classifiers and biomarkers for adrenarche and premature adrenarche.

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among them recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-Related Mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, FRM patients often experience worsening or demise following stress associated with infections. We investigated two female FRM patients carrying the novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from three FRM patients' fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant, showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated, but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.

Adrenal Abcg1 Controls Cholesterol Flux and Steroidogenesis.

Cholesterol is the precursor of all steroids, but how cholesterol flux is controlled in steroidogenic tissues is poorly understood. The cholesterol exporter ABCG1 is an essential component of the reverse cholesterol pathway and its global inactivation results in neutral lipid redistribution to tissue macrophages. The function of ABCG1 in steroidogenic tissues, however, has not been explored. To model this, we inactivated Abcg1 in the mouse adrenal cortex, which led to an adrenal-specific increase in transcripts involved in cholesterol uptake and de novo synthesis. Abcg1 inactivation did not affect adrenal cholesterol content, zonation, or serum lipid profile. Instead, we observed a moderate increase in corticosterone production that was not recapitulated by the inactivation of the functionally similar cholesterol exporter Abca1. Altogether, our data imply that Abcg1 controls cholesterol uptake and biosynthesis and regulates glucocorticoid production in the adrenal cortex, introducing the possibility that ABCG1 variants may account for physiological or subclinical variation in stress response.

Activation of endogenous glucocorticoids by HSD11B1 inhibits the antitumor immune response in renal cancer.

Although immune-based therapies have revolutionized the management of cancer, novel approaches are urgently needed to improve their outcome. We investigated the role of endogenous steroids in the resistance to cancer immunotherapy, as these have strong immunomodulatory functions. Using a publicly available database, we found that the intratumoral expression of 11 beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), which regenerates inactive glucocorticoids into active glucocorticoids, was associated with poor clinical outcome and correlated with immunosuppressive gene signatures in patients with renal cell carcinoma (RCC). HSD11B1 was mainly expressed in tumor-infiltrating immune myeloid cells as seen by immunohistochemistry in RCC patient samples. Using peripheral blood mononuclear cells from healthy donors or immune cells isolated from the tumor of RCC patients, we showed that the pharmacological inhibition of HSD11B1 improved the response to the immune checkpoint inhibitor anti-PD-1. In a subcutaneous mouse model of renal cancer, the combination of an HSD11B1 inhibitor with anti-PD-1 treatment increased the proportion of tumor-infiltrating dendritic cells. In an intrarenal mouse tumor model, HSD11B1 inhibition increased the survival of mice treated with anti-PD-1. In addition, inhibition of HSD11B1 sensitized renal tumors in mice to immunotherapy with resiquimod, a Toll-like receptor 7 agonist. Mechanistically, we demonstrated that HSD11B1 inhibition combined with resiquimod increased T cell-mediated cytotoxicity to tumor cells by stimulating the antigen-presenting capacity of dendritic cells. In conclusion, these results support the use of HSD11B1 inhibitors to improve the outcome of immunotherapy in renal cancer and highlight the role of the endogenous glucocorticoid metabolism in the efficacy of immunotherapy.

Endocannabinoid and steroid analysis in infant and adult nails by LC-MS/MS.

A common method to quantify chronic stress is the analysis of stress markers in keratinized matrices such as hair or nail. In this study, we aimed to validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the combined quantification of steroid hormones and endocannabinoids (eCBs) in the keratinized matrix nail. Furthermore, we aimed to investigate the suitability of the nail matrix for the detection of these stress markers in a pilot study. An LC-MS/MS method was used for the simultaneous identification and quantification of four eCBs (2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA)) and five steroid hormones (cortisol, cortisone, androstenedione, progesterone, testosterone) in human nails using a surrogate analyte method for each analyte. The method was validated in terms of selectivity, response factor, linearity, limit of quantification (LOQ), precision, accuracy, matrix effect, recovery, robustness, and autosampler stability. Nail samples were extracted for 1 h with methanol following a clean-up with a fully automated supported liquid extraction (SLE). The influence of nail weight on the quantification was investigated by using 0.5-20 mg of nail sample. As a proof of concept, nail samples (N = 57) were analyzed from a cohort representing newborns (1 month old), children (between 1 and 10 years), and adults (up to 43 years). It could be shown that the established workflow using a 1 hour extraction and clean-up by SLE was very robust and resulted in a short sample preparation time. The LC-MS/MS method was successfully validated. Matrix effects with ion enhancement occurred mainly for 2-AG. Sample weights below 5 mg showed variations in quantification for some analytes. Certain analytes such as PEA and progesterone could be accurately quantified at a sample weight lower than 5 mg. This is the first study where steroids and eCBs could be simultaneously detected and quantified in infant and adult nails. These results show that nails may serve as an alternative keratinized matrix (compared to hair) for the retrospective monitoring of cumulative eCB and steroid hormone levels. The combined assessment of eCBs and steroids from nails could provide a new approach to gain new insights into stress exposure in newborns and adults.

Alterations of Stress-Related Glucocorticoids and Endocannabinoids in Hair of Chronic Cocaine Users.

BACKGROUND: Previous research in animals and humans has demonstrated a potential role of stress regulatory systems, such as the hypothalamic-pituitary-adrenal (HPA) axis and the endocannabinoid (eCB) system, in the development of substance use disorders. We thus investigated alterations of HPA and eCB markers in individuals with chronic cocaine use disorder by using an advanced hair analysis technique. METHODS: We compared hair concentrations of glucocorticoids (cortisone, cortisol) and the eCBs 2-arachidonylglycerol, anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA) between 48 recreational cocaine users (RCU), 25 dependent cocaine users (DCU), and 67 stimulant-naïve controls. Self-reported substance use and hair concentrations of substances were also assessed. RESULTS: Significantly higher concentrations of hair cortisone were found in RCU and DCU compared with controls. Hair concentrations of OEA and PEA were significantly lower in DCU compared with RCU and controls. Additionally, within cocaine users, elevated cocaine hair concentration was a significant predictor for increased glucocorticoid and decreased OEA hair levels. Moreover, higher 3,4-methyl​enedioxymethamphetamine hair concentration was correlated with elevated cortisone and AEA, OEA, and PEA levels in hair within cocaine users, whereas more self-reported cannabis use was associated with lower eCBs levels in hair across the total sample. CONCLUSION: Our findings support the hypothesis that the HPA axis and eCB system might be important regulators for substance use disorders. The mechanistic understanding of changes in glucocorticoid and eCB levels in future research might be a promising pharmacological target to reduce stress-induced craving and relapse specifically in cocaine use disorder.

A novel bedtime pulsatile-release caffeine formula ameliorates sleep inertia symptoms immediately upon awakening.

Sleep inertia is a disabling state of grogginess and impaired vigilance immediately upon awakening. The adenosine receptor antagonist, caffeine, is widely used to reduce sleep inertia symptoms, yet the initial, most severe impairments are hardly alleviated by post-awakening caffeine intake. To ameliorate this disabling state more potently, we developed an innovative, delayed, pulsatile-release caffeine formulation targeting an efficacious dose briefly before planned awakening. We comprehensively tested this formulation in two separate studies. First, we established the in vivo caffeine release profile in 10 young men. Subsequently, we investigated in placebo-controlled, double-blind, cross-over fashion the formulation's ability to improve sleep inertia in 22 sleep-restricted volunteers. Following oral administration of 160 mg caffeine at 22:30, we kept volunteers awake until 03:00, to increase sleep inertia symptoms upon scheduled awakening at 07:00. Immediately upon awakening, we quantified subjective state, psychomotor vigilance, cognitive performance, and followed the evolution of the cortisol awakening response. We also recorded standard polysomnography during nocturnal sleep and a 1-h nap opportunity at 08:00. Compared to placebo, the engineered caffeine formula accelerated the reaction time on the psychomotor vigilance task, increased positive and reduced negative affect scores, improved sleep inertia ratings, prolonged the cortisol awakening response, and delayed nap sleep latency one hour after scheduled awakening. Based on these findings, we conclude that this novel, pulsatile-release caffeine formulation facilitates the sleep-to-wake transition in sleep-restricted healthy adults. We propose that individuals suffering from disabling sleep inertia may benefit from this innovative approach.Trials registration: NCT04975360.

Systemic detectability of dexamethasone and prednisolone after eye drop application in horses.

BACKGROUND: Equine sport agencies list steroids as prohibited substances for competing horses. OBJECTIVES: The objective of this study was to investigate if the controlled substances dexamethasone and prednisolone are detectable in equine serum and urine samples during and after treatment with eye drops and if this can generate a positive doping test. STUDY DESIGN: Prospective cohort study. METHODS: The study cohort included 11 horses. One eye of the horses was treated with either dexamethasone (Maxitrol® 0.1%, n = 5 eyes) or prednisolone (Pred forte® 1%, n = 6 eyes) eye drops 3 times daily for 14 days. Dexamethasone and prednisolone concentrations were determined in serum and urine at day 0 (negative control), 1, 7, 14, 15, 17 and 21 using liquid chromatography-tandem mass spectrometry. Blood samples were collected within 2 hours post application. Urine samples were collected during spontaneous urination. RESULTS: All serum samples (range: 0.7-43 ng/mL, mean 2.1 ng/mL) and urine samples (range 1.2-5 ng/mL, mean 0.8 ng/mL) showed measurable amounts of dexamethasone during the course of treatment. Concentrations in both serum and urine samples were below limit of detection (LOD) 24 hours after the last dexamethasone treatment (day 15). All serum samples (range 1.1-32.5 ng/mL, mean 6.4 ng/mL) and urine samples (range 3.7-19 ng/mL, mean 4.6 ng/mL) were positive for prednisolone during treatment. Urine samples were below LOD on day 15; serum samples on day 21. CONCLUSIONS: Dexamethasone and prednisolone eye drops can induce detectable drug levels in serum and urine samples of horses after a 14-day treatment plan. This can lead to a positive doping result. All samples tested negative (below LOD of the analytical method) for dexamethasone one day and for prednisolone one week after treatment cessation.

Hair cortisol measurement in older adults: Influence of demographic and physiological factors and correlation with perceived stress.

AIMS: This study aimed to investigate correlation between hair cortisol levels and perceived stress scale (PSS) in addition to a range of demographic and physiological factors in a sample of older adults. EXPERIMENTAL: Hair cortisol concentrations were established from 42 older adults aged between 60 and 80 years old. Age, sex, hair colour, smoking status, employment status, daytime sleeping, medication, waist to hip ratio (WHR) and PSS scores were assessed through a questionnaire. Hair cortisol concentration was assessed through liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS). RESULTS: Amongst the older adult group there was no statistically significant correlation between hair cortisol concentration and age, employment status, daytime sleep duration, WHR or PSS. When compared to previous data assessing hair cortisol in toddlers (7 months to 3 years old), adolescents (12-17 years old) and adults (18-60 years old) it is observed that there is a trend for higher hair cortisol in older adults (60-80 years old). Hair cortisol concentrations were significantly higher in males (n = 20) than in females (n = 22) and in participants with dark brown hair (n = 8). No relationship was investigated between hair cortisol concentration and smoking status or medication intake. CONCLUSIONS: The results confirm that hair samples are a useful alternative to the current mediums that are used to analyse biological cortisol. The results also validate the use of LC-MS/MS as an effective analytical method for the quantitation of hair cortisol concentrations.

Endogenous steroid hormones in hair: Investigations on different hair types, pigmentation effects and correlation to nails.

Steroid hormone analysis is widely used in health- and stress-related research to get insights into various diseases and the adaption to stress. Hair analysis has been used as a tool for the long-term monitoring of these steroid hormones. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous identification and quantification of seven steroid hormones (cortisone, cortisol, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, progesterone) in hair. Cortisol, cortisone, androstenedione, testosterone and progesterone were detected and quantified in authentic hair samples of different individuals. Significantly higher concentrations for body hair were found for cortisone and testosterone compared to scalp hair. Furthermore, weak correlations for the majority of steroids between scalp and body hair indicate that body hair is not really suitable as alternative when scalp hair is not available. The influence of hair pigmentation was analyzed by comparing pigmented to non-pigmented hair of grey-haired individuals. The results showed no differences for cortisol, cortisone, androstenedione, testosterone and progesterone concentrations (p > 0.05) implying that hair pigmentation has not a strong effect on steroid hormone concentrations. Correlations between hair and nail steroid levels were also studied. Higher concentrations of cortisol and cortisone in hair were found compared to nails (p < 0.0001). No significant correlation for cortisone, cortisol, androstenedione, testosterone and progesterone concentrations were found between hair and nails. These results demonstrate that matrix-dependent value ranges for hair and nail steroid levels should be established and applied for interpretation.

Systematic investigations of endogenous cortisol and cortisone in nails by LC-MS/MS and correlation to hair.

Hair samples are increasingly used for measuring the long-term stress mediator cortisol. However, hair is not always available and nails (finger or toe), as a keratinized matrix, may be an alternative to hair. In order to measure cortisol and cortisone in the nail matrix, an LC-MS/MS method has been developed and validated using 13C3-labeled surrogate analytes. Both analytes were measured in ESI negative mode as formic acid adducts. Different sample preparation techniques were assessed, and single-step extraction in methanol was established for determination of cortisone and cortisol in the nail matrix. The method was successfully validated with limits of detection (LOD) and limits of quantification (LOQ) of 0.5 and 1.0 pg/mg for cortisol and cortisone, respectively. The calibration curve was linear up to a concentration of 500 pg/mg. Recovery was good for both analytes and showed values over 50%. Matrix effects with ion suppression occurred for both substances but could be corrected by the use of internal standard. Accuracy and precision were in the accepted range of ± 20% for both substances. The method was successfully applied to determine cortisol and cortisone concentrations in authentic nail samples. Cortisol and cortisone concentrations varied significantly among different fingernails, being highest in the little fingernails and lowest in the thumbnails. It could be shown that even in only 1 mg nail sample cortisol and cortisone can be reliably quantified. No correlation between hair and nail cortisol and cortisone concentrations could be found. Furthermore, cortisol and cortisone concentrations were significantly higher in hair. Graphical abstract.