Inhibition of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 drives concurrent 11-oxygenated androgen excess.

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyzes not only the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modeling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone (11KT), by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11KT. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11KT may offset beneficial effects of decreased glucocorticoid activation.

Gonadal androgens are associated with decreased type I interferon production by plasmacytoid dendritic cells and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents.

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Type I interferon (IFN-I) is an integral part of this early innate response that primes several components of the adaptive immune response. Women are widely reported to respond better than men to tri- and quadrivalent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production, and female pDCs produce more IFN-I than male pDCs since the upstream pattern recognition receptor Toll-like receptor 7 (TLR7) is encoded by X chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains several putative androgen response elements, and androgens have been reported to suppress pDC IFN-I in vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this same cohort to determine the impact of IFN-I on anti-spike and anti-receptor-binding domain IgG titres to BNT162b2. Through flow cytometry and least absolute shrinkage and selection operator (LASSO) modelling, we determined that serum-free testosterone was associated with reduced pDC IFN-I, but contrary to the well-described immunosuppressive role for androgens, the most bioactive androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together, these data support a model where systemic IFN-I increases vaccine-mediated immune responses, yet for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently improve vaccine-driven immunity.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism.

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

Tissue Glucocorticoid Metabolism in Adrenal Insufficiency: A Prospective Study of Dual-release Hydrocortisone Therapy.

BACKGROUND: Patients with adrenal insufficiency (AI) require life-long glucocorticoid (GC) replacement therapy. Within tissues, cortisol (F) availability is under the control of the isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). We hypothesize that corticosteroid metabolism is altered in patients with AI because of the nonphysiological pattern of current immediate release hydrocortisone (IR-HC) replacement therapy. The use of a once-daily dual-release hydrocortisone (DR-HC) preparation, (Plenadren®), offers a more physiological cortisol profile and may alter corticosteroid metabolism in vivo. STUDY DESIGN AND METHODS: Prospective crossover study assessing the impact of 12 weeks of DR-HC on systemic GC metabolism (urinary steroid metabolome profiling), cortisol activation in the liver (cortisone acetate challenge test), and subcutaneous adipose tissue (microdialysis, biopsy for gene expression analysis) in 51 patients with AI (primary and secondary) in comparison to IR-HC treatment and age- and BMI-matched controls. RESULTS: Patients with AI receiving IR-HC had a higher median 24-hour urinary excretion of cortisol compared with healthy controls (72.1 µg/24 hours [IQR 43.6-124.2] vs 51.9 µg/24 hours [35.5-72.3], P = .02), with lower global activity of 11ß-HSD2 and higher 5-alpha reductase activity. Following the switch from IR-HC to DR-HC therapy, there was a significant reduction in urinary cortisol and total GC metabolite excretion, which was most significant in the evening. There was an increase in 11ß-HSD2 activity. Hepatic 11ß-HSD1 activity was not significantly altered after switching to DR-HC, but there was a significant reduction in the expression and activity of 11ß-HSD1 in subcutaneous adipose tissue. CONCLUSION: Using comprehensive in vivo techniques, we have demonstrated abnormalities in corticosteroid metabolism in patients with primary and secondary AI receiving IR-HC. This dysregulation of pre-receptor glucocorticoid metabolism results in enhanced glucocorticoid activation in adipose tissue, which was ameliorated by treatment with DR-HC.

The ultra-acute steroid response to traumatic injury: a cohort study.

OBJECTIVE: Trauma-induced steroid changes have been studied post-hospital admission, resulting in a lack of understanding of the speed and extent of the immediate endocrine response to injury. The Golden Hour study was designed to capture the ultra-acute response to traumatic injury. DESIGN: We conducted an observational cohort study including adult male trauma patients <60 years, with blood samples drawn ≤1 h of major trauma by pre-hospital emergency responders. METHODS: We recruited 31 adult male trauma patients (mean age 28 [range 19-59] years) with a mean injury severity score (ISS) of 16 (IQR 10-21). The median time to first sample was 35 (range 14-56) min, with follow-up samples collected 4-12 and 48-72 h post-injury. Serum steroids in patients and age- and sex-matched healthy controls (HCs) (n = 34) were analysed by tandem mass spectrometry. RESULTS: Within 1 h of injury, we observed an increase in glucocorticoid and adrenal androgen biosynthesis. Cortisol and 11-hydroxyandrostendione increased rapidly, whilst cortisone and 11-ketoandrostenedione decreased, reflective of increased cortisol and 11-oxygenated androgen precursor biosynthesis by 11ß-hydroxylase and increased cortisol activation by 11ß-hydroxysteroid dehydrogenase type 1. Active classic gonadal androgens testosterone and 5α-dihydrotestosterone decreased, whilst the active 11-oxygenated androgen 11-ketotestosterone maintained pre-injury levels. CONCLUSIONS: Changes in steroid biosynthesis and metabolism occur within minutes of traumatic injury. Studies that address whether ultra-early changes in steroid metabolism are associated with patient outcomes are now required.

Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride.

BACKGROUND: Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride. METHODS: Following liquid-liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation. RESULTS: Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122-140 % for 3ßOH-Δ5 steroids and 477-1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range -14 % to 11.9 % for 21/23, -21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from -29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured. CONCLUSIONS: UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.

Cardiometabolic Disease Burden and Steroid Excretion in Benign Adrenal Tumors : A Cross-Sectional Multicenter Study.

BACKGROUND: Benign adrenal tumors are commonly discovered on cross-sectional imaging. Mild autonomous cortisol secretion (MACS) is regularly diagnosed, but its effect on cardiometabolic disease in affected persons is ill defined. OBJECTIVE: To determine cardiometabolic disease burden and steroid excretion in persons with benign adrenal tumors with and without MACS. DESIGN: Cross-sectional study. SETTING: 14 endocrine secondary and tertiary care centers (recruitment from 2011 to 2016). PARTICIPANTS: 1305 prospectively recruited persons with benign adrenal tumors. MEASUREMENTS: Cortisol excess was defined by clinical assessment and the 1-mg overnight dexamethasone-suppression test (serum cortisol: <50 nmol/L, nonfunctioning adrenal tumor [NFAT]; 50 to 138 nmol/L, possible MACS [MACS-1]; >138 nmol/L and absence of typical clinical Cushing syndrome [CS] features, definitive MACS [MACS-2]). Net steroid production was assessed by multisteroid profiling of 24-hour urine by tandem mass spectrometry. RESULTS: Of the 1305 participants, 49.7% had NFAT (n = 649; 64.1% women), 34.6% had MACS-1 (n = 451; 67.2% women), 10.7% had MACS-2 (n = 140; 73.6% women), and 5.0% had CS (n = 65; 86.2% women). Prevalence and severity of hypertension were higher in MACS-2 and CS than NFAT (adjusted prevalence ratios [aPRs] for hypertension: MACS-2, 1.15 [95% CI, 1.04 to 1.27], and CS, 1.37 [CI, 1.16 to 1.62]; aPRs for use of ≥3 antihypertensives: MACS-2, 1.31 [CI, 1.02 to 1.68], and CS, 2.22 [CI, 1.62 to 3.05]). Type 2 diabetes was more prevalent in CS than NFAT (aPR, 1.62 [CI, 1.08 to 2.42]) and more likely to require insulin therapy for MACS-2 (aPR, 1.89 [CI, 1.01 to 3.52]) and CS (aPR, 3.06 [CI, 1.60 to 5.85]). Urinary multisteroid profiling revealed an increase in glucocorticoid excretion from NFAT over MACS-1 and MACS-2 to CS, whereas androgen excretion decreased. LIMITATIONS: Cross-sectional design; possible selection bias. CONCLUSION: A cardiometabolic risk condition, MACS predominantly affects women and warrants regular assessment for hypertension and type 2 diabetes. PRIMARY FUNDING SOURCE: Diabetes UK, the European Commission, U.K. Medical Research Council, the U.K. Academy of Medical Sciences, the Wellcome Trust, the U.K. National Institute for Health Research, the U.S. National Institutes of Health, the Claire Khan Trust Fund at University Hospitals Birmingham Charities, and the Mayo Clinic Foundation for Medical Education and Research.

The broad phenotypic spectrum of 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency: a case series.

CONTEXT: 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. OBJECTIVE: To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. DESIGN: Case series. PATIENTS AND RESULTS: We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometry-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous sequence variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortisol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype severity. CONCLUSION: Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenuation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. SIGNIFICANCE STATEMENT: Here we report, supported by careful phenotyping, genotyping and functional analysis, a prismatic case series of patients with congenital adrenal hyperplasia due to 17α-hydroxylase (CYP17A1) deficiency (17OHD). These range in severity from the abolition of function, presenting in early infancy, and unusually mild with isolated sex steroid deficiency but normal ACTH-stimulated cortisol in adult patients. These findings will guide improved diagnostic detection of CYP17A1 deficiency.

A prospective, phase II, single-centre, cross-sectional, randomised study investigating Dehydroepiandrosterone supplementation and its Profile in Trauma: ADaPT.

INTRODUCTION: The improvements in short-term outcome after severe trauma achieved through early resuscitation and acute care can be offset over the following weeks by an acute systemic inflammatory response with immuneparesis leading to infection, multiorgan dysfunction/multiorgan failure (MOF) and death. Serum levels of the androgen precursor dehydroepiandrosterone (DHEA) and its sulfate ester DHEAS, steroids with immune-enhancing activity, are low after traumatic injury at a time when patients are catabolic and immunosuppressed. Addressing this deficit and restoring the DHEA(S) ratio to cortisol may provide a range of physiological benefits, including immune modulatory effects. OBJECTIVE: Our primary objective is to establish a dose suitable for DHEA supplementation in patients after acute trauma to raise circulating DHEA levels to at least 15 nmol/L. Secondary objectives are to assess if DHEA supplementation has any effect on neutrophil function, metabolic and cytokine profiles and which route of administration (oral vs sublingual) is more effective in restoring circulating levels of DHEA, DHEAS and downstream androgens. METHODS AND ANALYSIS: A prospective, phase II, single-centre, cross-sectional, randomised study investigating Dehydroepiandrosterone supplementation and its profile in trauma, with a planned recruitment between April 2019 and July 2021, that will investigate DHEA supplementation and its effect on serum DHEA, DHEAS and downstream androgens in trauma. A maximum of 270 patients will receive sublingual or oral DHEA at 50, 100 or 200 mg daily over 3 days. Females aged ≥50 years with neck of femur fracture and male and female major trauma patients, aged 16-50 years with an injury severity score ≥16, will be recruited. ETHICS AND DISSEMINATION: This protocol was approved by the West Midlands - Coventry and Warwickshire Research Ethics Committee (Reference 18/WM/0102) on 8 June 2018. Results will be disseminated via peer-reviewed publications and presented at national and international conferences. TRIAL REGISTRATION: This trial is registered with the European Medicines Agency (EudraCT: 2016-004250-15) and ISRCTN (12961998). It has also been adopted on the National Institute of Health Research portfolio (CPMS ID:38158). TRIAL PROGRESSION: The study recruited its first patient on 2 April 2019 and held its first data monitoring committee on 8 November 2019. DHEA dosing has increased to 100 mg in both male cohorts and remains on 50 mg in across all female groups.

11ßHSD1 Inhibition with AZD4017 Improves Lipid Profiles and Lean Muscle Mass in Idiopathic Intracranial Hypertension.

BACKGROUND: The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) determines prereceptor metabolism and activation of glucocorticoids within peripheral tissues. Its dysregulation has been implicated in a wide array of metabolic diseases, leading to the development of selective 11ß-HSD1 inhibitors. We examined the impact of the reversible competitive 11ß-HSD1 inhibitor, AZD4017, on the metabolic profile in an overweight female cohort with idiopathic intracranial hypertension (IIH). METHODS: We conducted a UK multicenter phase II randomized, double-blind, placebo-controlled trial of 12-week treatment with AZD4017. Serum markers of glucose homeostasis, lipid metabolism, renal and hepatic function, inflammation and androgen profiles were determined and examined in relation to changes in fat and lean mass by dual-energy X-ray absorptiometry. RESULTS: Patients receiving AZD4017 showed significant improvements in lipid profiles (decreased cholesterol, increased high-density lipoprotein [HDL] and cholesterol/HDL ratio), markers of hepatic function (decreased alkaline phosphatase and gamma-glutamyl transferase), and increased lean muscle mass (1.8%, P < .001). No changes in body mass index, fat mass, and markers of glucose metabolism or inflammation were observed. Patients receiving AZD4017 demonstrated increased levels of circulating androgens, positively correlated with changes in total lean muscle mass. CONCLUSIONS: These beneficial metabolic changes represent a reduction in risk factors associated with raised intracranial pressure and represent further beneficial therapeutic outcomes of 11ß-HSD1 inhibition by AZD4017 in this overweight IIH cohort. In particular, beneficial changes in lean muscle mass associated with AZD4017 may reflect new applications for this nature of inhibitor in the management of conditions such as sarcopenia.

Insights from the genetic characterization of central precocious puberty associated with multiple anomalies.

STUDY QUESTION: Is there an (epi)genetic basis in patients with central precocious puberty (CPP) associated with multiple anomalies that unmasks underlying mechanisms or reveals novel genetic findings related to human pubertal control? SUMMARY ANSWER: In a group of 36 patients with CPP associated with multiple phenotypes, pathogenic or likely pathogenic (epi)genetic defects were identified in 12 (33%) patients, providing insights into the genetics of human pubertal control. WHAT IS KNOWN ALREADY: A few studies have described patients with CPP associated with multiple anomalies, but without making inferences on causalities of CPP. Genetic-molecular studies of syndromic cases may reveal disease genes or mechanisms, as the presentation of such patients likely indicates a genetic disorder. STUDY DESIGN, SIZE, DURATION: This translational study was based on a genetic-molecular analysis, including genome-wide high throughput methodologies, for searching structural or sequence variants implicated in CPP and DNA methylation analysis of candidate regions. PARTICIPANTS/MATERIALS, SETTING, METHODS: A cohort of 197 patients (188 girls) with CPP without structural brain lesions was submitted to a detailed clinical evaluation, allowing the selection of 36 unrelated patients (32 girls) with CPP associated with multiple anomalies. Pathogenic allelic variants of genes known to cause monogenic CPP (KISS1R, KISS1, MKRN3 and DLK1) had been excluded in the entire cohort (197 patients). All selected patients with CPP associated with multiple anomalies (n = 36) underwent methylation analysis of candidate regions and chromosomal microarray analysis. A subset (n = 9) underwent whole-exome sequencing, due to presenting familial CPP and/or severe congenital malformations and neurocognitive abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 36 selected patients with CPP, the more prevalent associated anomalies were metabolic, growth and neurocognitive conditions. In 12 (33%) of them, rare genetic abnormalities were identified: six patients presented genetic defects in loci known to be involved with CPP (14q32.2 and 7q11.23), whereas the other six presented defects in candidate genes or regions. In detail, three patients presented hypomethylation of DLK1/MEG3:IG-DMR (14q32.2 disruption or Temple syndrome), resulting from epimutation (n = 1) or maternal uniparental disomy of chromosome 14 (n = 2). Seven patients presented pathogenic copy number variants: three with de novo 7q11.23 deletions (Williams-Beuren syndrome), three with inherited Xp22.33 deletions, and one with de novo 1p31.3 duplication. Exome sequencing revealed potential pathogenic variants in two patients: a sporadic female case with frameshift variants in TNRC6B and AREL1 and a familial male case with a missense substitution in UGT2B4 and a frameshift deletion in MKKS. LIMITATIONS, REASONS FOR CAUTION: The selection of patients was based on a retrospective clinical characterization, lacking a longitudinal inclusion of consecutive patients. In addition, future studies are needed, showing the long-term (mainly reproductive) outcomes in the included patients, as most of them are not in adult life yet. WIDER IMPLICATIONS OF THE FINDINGS: The results highlighted the relevance of an integrative clinical-genetic approach in the elucidation of mechanisms and factors involved in pubertal control. Chromosome 14q32.2 disruption indicated the loss of imprinting of DLK1 as a probable mechanism of CPP. Two other chromosomal regions (7q11.23 and Xp22.33) represented new candidate loci potentially involved in this disorder of pubertal timing. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grant number 2018/03198-0 (to A.P.M.C.) and grant number 2013/08028-1 (to A.C.V.K) from the São Paulo Research Foundation (FAPESP), and grant number 403525/2016-0 (to A.C.L.) and grant number 302849/2015-7 (to A.C.L.) and grant number 141625/2016-3 (to A.C.V.K) from the National Council for Scientific and Technological Development (CNPq). The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

11ß-Hydroxysteroid dehydrogenase type 1 inhibition in idiopathic intracranial hypertension: a double-blind randomized controlled trial.

Treatment options for idiopathic intracranial hypertension are limited. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 has been implicated in regulating cerebrospinal fluid secretion, and its activity is associated with alterations in intracranial pressure in idiopathic intracranial hypertension. We assessed therapeutic efficacy, safety and tolerability and investigated indicators of in vivo efficacy of the 11ß-hydroxysteroid dehydrogenase type 1 inhibitor AZD4017 compared with placebo in idiopathic intracranial hypertension. A multicenter, UK, 16-week phase II randomized, double-blind, placebo-controlled trial of 12-week treatment with AZD4017 or placebo was conducted. Women aged 18-55 years with active idiopathic intracranial hypertension (>25 cmH2O lumbar puncture opening pressure and active papilledema) were included. Participants received 400 mg of oral AZD4017 twice daily compared with matching placebo over 12 weeks. The outcome measures were initial efficacy, safety and tolerability. The primary clinical outcome was lumbar puncture opening pressure at 12 weeks analysed by intention-to-treat. Secondary clinical outcomes were symptoms, visual function, papilledema, headache and anthropometric measures. In vivo efficacy was evaluated in the central nervous system and systemically. A total of 31 subjects [mean age 31.2 (SD = 6.9) years and body mass index 39.2 (SD = 12.6) kg/m2] were randomized to AZD4017 (n = 17) or placebo (n = 14). At 12 weeks, lumbar puncture pressure was lower in the AZD4017 group (29.7 cmH2O) compared with placebo (31.3 cmH2O), but the difference between groups was not statistically significant (mean difference: -2.8, 95% confidence interval: -7.1 to 1.5; P = 0.2). An exploratory analysis assessing mean change in lumbar puncture pressure within each group found a significant decrease in the AZD4017 group [mean change: -4.3 cmH2O (SD = 5.7); P = 0.009] but not in the placebo group [mean change: -0.3 cmH2O (SD = 5.9); P = 0.8]. AZD4017 was safe, with no withdrawals related to adverse effects. Nine transient drug-related adverse events were reported. One serious adverse event occurred in the placebo group (deterioration requiring shunt surgery). In vivo biomarkers of 11ß-hydroxysteroid dehydrogenase type 1 activity (urinary glucocorticoid metabolites, hepatic prednisolone generation, serum and cerebrospinal fluid cortisol:cortisone ratios) demonstrated significant enzyme inhibition with the reduction in serum cortisol:cortisone ratio correlating significantly with reduction in lumbar puncture pressure (P = 0.005, R = 0.70). This is the first phase II randomized controlled trial in idiopathic intracranial hypertension evaluating a novel therapeutic target. AZD4017 was safe and well tolerated and inhibited 11ß-hydroxysteroid dehydrogenase type 1 activity in vivo. Reduction in serum cortisol:cortisone correlated with decreased intracranial pressure. Possible clinical benefits were noted in this small cohort. A longer, larger study would now be of interest.

The A-ring reduction of 11-ketotestosterone is efficiently catalysed by AKR1D1 and SRD5A2 but not SRD5A1.

Testosterone and its 5α-reduced form, 5α-dihydrotestosterone, were previously thought to represent the only active androgens in humans. However, recent studies have shown that the potent androgen, 11-ketotestosterone, derived from the adrenal androgen precursor, 11ß-hydroxyandrostenedione, may in fact serve as the primary androgen in healthy women. Yet, despite recent renewed interest in these steroids, their downstream metabolism has remained undetermined. We therefore set out to investigate the metabolism of 11-ketotestosterone by characterising the 5α- or 5ß-reduction commitment step. We show that inactivation of 11-ketotestosterone is predominantly driven by AKR1D1, which efficiently catalyses the 5ß-reduction of 11-ketotestosterone, committing it to a metabolic pathway that terminates in 11-ketoetiocholanolone. We demonstrate that 5α-reduction of 11-ketotestosterone is catalysed by SRD5A2, but not SRD5A1, and terminates in 11-ketoandrosterone, but is only responsible for a minority of 11-ketotestosterone inactivation. However, as 11-ketoetiocholanolone is also generated by the metabolism of the glucocorticoid cortisone, 11-ketoandrosterone should be considered a more specific urinary marker of 11-ketotestosterone production.

Urine steroid metabolomics for the differential diagnosis of adrenal incidentalomas in the EURINE-ACT study: a prospective test validation study.

BACKGROUND: Cross-sectional imaging regularly results in incidental discovery of adrenal tumours, requiring exclusion of adrenocortical carcinoma (ACC). However, differentiation is hampered by poor specificity of imaging characteristics. We aimed to validate a urine steroid metabolomics approach, using steroid profiling as the diagnostic basis for ACC. METHODS: We did a prospective multicentre study in adult participants (age ≥18 years) with newly diagnosed adrenal masses. We assessed the accuracy of diagnostic imaging strategies based on maximum tumour diameter (≥4 cm vs <4 cm), imaging characteristics (positive vs negative), and urine steroid metabolomics (low, medium, or high risk of ACC), separately and in combination, using a reference standard of histopathology and follow-up investigations. With respect to imaging characteristics, we also assessed the diagnostic utility of increasing the unenhanced CT tumour attenuation threshold from the recommended 10 Hounsfield units (HU) to 20 HU. FINDINGS: Of 2169 participants recruited between Jan 17, 2011, and July 15, 2016, we included 2017 from 14 specialist centres in 11 countries in the final analysis. 98 (4·9%) had histopathologically or clinically and biochemically confirmed ACC. Tumours with diameters of 4 cm or larger were identified in 488 participants (24·2%), including 96 of the 98 with ACC (positive predictive value [PPV] 19·7%, 95% CI 16·2-23·5). For imaging characteristics, increasing the unenhanced CT tumour attenuation threshold to 20 HU from the recommended 10 HU increased specificity for ACC (80·0% [95% CI 77·9-82·0] vs 64·0% [61·4-66.4]) while maintaining sensitivity (99·0% [94·4-100·0] vs 100·0% [96·3-100·0]; PPV 19·7%, 16·3-23·5). A urine steroid metabolomics result indicating high risk of ACC had a PPV of 34·6% (95% CI 28·6-41·0). When the three tests were combined, in the order of tumour diameter, positive imaging characteristics, and urine steroid metabolomics, 106 (5·3%) participants had the result maximum tumour diameter of 4 cm or larger, positive imaging characteristics (with the 20 HU cutoff), and urine steroid metabolomics indicating high risk of ACC, for which the PPV was 76·4% (95% CI 67·2-84·1). 70 (3·5%) were classified as being at moderate risk of ACC and 1841 (91·3%) at low risk (negative predictive value 99·7%, 99·4-100·0). INTERPRETATION: An unenhanced CT tumour attenuation cutoff of 20 HU should replace that of 10 HU for exclusion of ACC. A triple test strategy of tumour diameter, imaging characteristics, and urine steroid metabolomics improves detection of ACC, which could shorten time to surgery for patients with ACC and help to avoid unnecessary surgery in patients with benign tumours. FUNDING: European Commission, UK Medical Research Council, Wellcome Trust, and UK National Institute for Health Research, US National Institutes of Health, the Claire Khan Trust Fund at University Hospitals Birmingham Charities, and the Mayo Clinic Foundation for Medical Education and Research.

Altered cortisol metabolism in individuals with HNF1A-MODY.

OBJECTIVE AND CONTEXT: Maturity onset diabetes of the young due to variants in HNF1A (HNF1A-MODY) is the most common form of monogenic diabetes. Individuals with HNF1A-MODY usually have a lean phenotype which contrasts with type 2 diabetes (T2DM). Data from hepatocytes derived from Hnf1a knock-out mice demonstrated dysregulation of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which regulates glucocorticoid availability and action in target tissues, together with 11ß-HSD2 and steroid A-ring reductases, 5α- and 5ß-reductase. We proposed that altered glucocorticoid metabolism might underpin some of the phenotypic differences between patients with HNF1A-MODY and those with T2DM. DESIGN: A retrospective matched cohort study. PATIENTS AND MEASUREMENTS: 24-hours urine steroid metabolome profiling was carried out by gas chromatography-mass spectrometry in 35 subjects with HNF1A-MODY, 35 individuals with T2DM and 35 healthy controls matched for age, sex and BMI. The steroid metabolites were expressed as µg/L in all groups and measured in mid-morning urine in diabetic subjects and 24-hour urine collection in healthy controls. Hence, only ratios were compared not the individual steroids. Established ratios of glucocorticoid metabolites were used to estimate 11ß-HSD1/2 and 5α- and 5ß-reductase activities. RESULTS: While 11ß-HSD1 activity was similar in all groups, 11ß-HSD2 activity was significantly lower in subjects with HNF1A-MODY and T2DM than in healthy controls. The ratio of 5ß- to 5α-metabolites of cortisol was higher in subjects with HNF1A-MODY than in T2DM and healthy controls, probably due to increased activity of the 5ß-reductase (AKR1D1) in HNF1A-MODY. CONCLUSIONS: This is the first report of steroid metabolites in HNF1A-MODY. We have identified distinct differences in steroid metabolism pathways in subjects with HNF1A-MODY that have the potential to alter steroid hormone availability. Further studies are required to explore whether these changes link to phenotype.

Natural History of Adrenal Steroidogenesis in Autoimmune Addison's Disease Following Diagnosis and Treatment.

CONTEXT: The natural history of adrenal function in autoimmune Addison disease once diagnosed and treated has not been systematically studied, but several case reports of recovery from established adrenal failure suggest it may not be uniform. OBJECTIVE: To ascertain steroidogenic function in autoimmune Addison disease immediately following diagnosis and during prolonged treatment. DESIGN: We studied peak serum cortisol in response to ACTH1-24 in 20 newly diagnosed autoimmune Addison disease patients at first presentation and then again within a month. We also studied 37 patients with established Addison disease (for between 7 months and 44 years) in a medication-free state, measuring peak serum cortisol responses to ACTH1-24 and the urine LC-MS steroid metabolome. RESULTS: Adrenal steroidogenesis declined rapidly after steroid replacement treatment for newly diagnosed Addison disease was started, with a peak serum cortisol falling from 138 ±â€…19 nmol/L (SEM) at presentation to 63 ±â€…13 nmol/L over 4 weeks (P < 0.003).Six of 37 participants (16%) with established Addison disease had detectable serum cortisol and urine glucocorticoid and mineralocorticoid metabolites during repeat testing, indicating variable degrees of residual adrenal function. CONCLUSION: Autoimmune Addison disease is a heterogeneous condition, showing a rapid decline in adrenal steroidogenesis during the first few weeks following diagnosis, but low-level residual function in a minority of patients, which appears to persist for many years.

Mapping the Steroid Response to Major Trauma From Injury to Recovery: A Prospective Cohort Study.

CONTEXT: Survival rates after severe injury are improving, but complication rates and outcomes are variable. OBJECTIVE: This cohort study addressed the lack of longitudinal data on the steroid response to major trauma and during recovery. DESIGN: We undertook a prospective, observational cohort study from time of injury to 6 months postinjury at a major UK trauma centre and a military rehabilitation unit, studying patients within 24 hours of major trauma (estimated New Injury Severity Score (NISS) > 15). MAIN OUTCOME MEASURES: We measured adrenal and gonadal steroids in serum and 24-hour urine by mass spectrometry, assessed muscle loss by ultrasound and nitrogen excretion, and recorded clinical outcomes (ventilator days, length of hospital stay, opioid use, incidence of organ dysfunction, and sepsis); results were analyzed by generalized mixed-effect linear models. FINDINGS: We screened 996 multiple injured adults, approached 106, and recruited 95 eligible patients; 87 survived. We analyzed all male survivors <50 years not treated with steroids (N = 60; median age 27 [interquartile range 24-31] years; median NISS 34 [29-44]). Urinary nitrogen excretion and muscle loss peaked after 1 and 6 weeks, respectively. Serum testosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate decreased immediately after trauma and took 2, 4, and more than 6 months, respectively, to recover; opioid treatment delayed dehydroepiandrosterone recovery in a dose-dependent fashion. Androgens and precursors correlated with SOFA score and probability of sepsis. CONCLUSION: The catabolic response to severe injury was accompanied by acute and sustained androgen suppression. Whether androgen supplementation improves health outcomes after major trauma requires further investigation.

Urine Steroid Metabolomics as a Novel Tool for Detection of Recurrent Adrenocortical Carcinoma.

CONTEXT: Urine steroid metabolomics, combining mass spectrometry-based steroid profiling and machine learning, has been described as a novel diagnostic tool for detection of adrenocortical carcinoma (ACC). OBJECTIVE, DESIGN, SETTING: This proof-of-concept study evaluated the performance of urine steroid metabolomics as a tool for postoperative recurrence detection after microscopically complete (R0) resection of ACC. PATIENTS AND METHODS: 135 patients from 14 clinical centers provided postoperative urine samples, which were analyzed by gas chromatography-mass spectrometry. We assessed the utility of these urine steroid profiles in detecting ACC recurrence, either when interpreted by expert clinicians or when analyzed by random forest, a machine learning-based classifier. Radiological recurrence detection served as the reference standard. RESULTS: Imaging detected recurrent disease in 42 of 135 patients; 32 had provided pre- and post-recurrence urine samples. 39 patients remained disease-free for ≥3 years. The urine "steroid fingerprint" at recurrence resembled that observed before R0 resection in the majority of cases. Review of longitudinally collected urine steroid profiles by 3 blinded experts detected recurrence by the time of radiological diagnosis in 50% to 72% of cases, improving to 69% to 92%, if a preoperative urine steroid result was available. Recurrence detection by steroid profiling preceded detection by imaging by more than 2 months in 22% to 39% of patients. Specificities varied considerably, ranging from 61% to 97%. The computational classifier detected ACC recurrence with superior accuracy (sensitivity = specificity = 81%). CONCLUSION: Urine steroid metabolomics is a promising tool for postoperative recurrence detection in ACC; availability of a preoperative urine considerably improves the ability to detect ACC recurrence.

Residual Adrenal Function in Autoimmune Addison's Disease-Effect of Dual Therapy With Rituximab and Depot Tetracosactide.

CONTEXT: In autoimmune Addison's disease (AAD), exogenous glucocorticoid (GC) therapy is an imperfect substitute for physiological GC secretion. Patients on long-term steroid replacement have increased morbidity, reduced life expectancy, and poorer quality of life. OBJECTIVE: The objective of this article is to restore adrenocortical steroidogenic function in recent-onset AAD. DESIGN: An open-label, multicenter trial of immunotherapy and trophic stimulation in new-onset AAD was conducted. Serial measurement of serum and urine corticosteroids at baseline and throughout a 72-week follow-up period was performed. SETTING: This study was conducted at the.endocrine departments and clinical research facilities at 5 UK tertiary centers. PATIENTS: Thirteen participants (9 female, 4 male; age 19-64 years) were included with AAD confirmed by high adrenocorticotropin, low circulating cortisol (basal < 100 nmol/L or post-tetracosactide < 300 nmol/L), and positive serum 21-hydroxylase antibodies. INTERVENTION: All participants received dual therapy with B-lymphocyte-depleting immunotherapy (rituximab 1 g given twice) and repeated depot tetracosactide (1 mg on alternate days for 12 weeks). MAIN OUTCOME MEASURE: Restoration of normal GC secretion (stimulated cortisol > 550 nmol/L) at week 48 was the main outcome measure. RESULTS: Ten of 13 (77%) participants had detectable stimulated serum cortisol (26-265 nmol/L) at trial entry. Following intervention, 7 of 13 (54%) had an increase in stimulated cortisol measurement, with a peak response of 325 nmol/L at week 18 in 1 participant. Increased steroid metabolites, assayed by urine gas chromatography-mass spectrometry at week 12 and week 48, was detected in 8 of 13 (62%) individuals, reflecting an increase in endogenous steroidogenesis. Four of 13 had residual adrenal function at 72 weeks. CONCLUSION: Combined treatment with rituximab and depot tetracosactide did not restore normal adrenal function. Nevertheless, adrenocortical plasticity is demonstrated in some patients, and this has the potential to be exploited to improve adrenal function.