Diurnal 11-ketotestosterone and 17-hydroxyprogesterone saliva profiles in paediatric classical congenital adrenal hyperplasia.

OBJECTIVES: The most suitable biochemical markers for therapy adjustment in patients with congenital adrenal hyperplasia are controversial. 11-Oxygenated androgens are a promising new approach. The objective of this study was to investigate the diurnal rhythm of 11-ketotestosterone in children and adolescents in saliva and to correlate it with salivary 17-hydroxyprogesterone. METHODS: Fifty-one samples of steroid day-profiles from 17 patients were additionally analysed for 11-ketotestosterone, retrospectively. All patients were treated in our university outpatient clinic for paediatric endocrinology between 2020 and 2022. Steroid day-profiles of 17 patients could be examined. The cohort showed a balanced sex ratio. The median age was 13 years. The measurements for 17-hydroxyprogesterone were carried out during routine care by immunoassay. The measurements of 11-ketotestosterone were performed from frozen saliva samples using an implemented in-house protocol for liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most important outcome were the absolute values for 11-ketotestosterone, their diurnal rhythmicity and the correlation with 17-hydroxyprogesterone. RESULTS: Both steroids show a circadian diurnal rhythm. 17-hydroxyprogesterone and 11-ketotestosterone correlate significantly. 11-Ketotestosterone showed a positive correlation with BMI at all times of the day. CONCLUSIONS: 11-Ketotestosterone shows circadian rhythmicity in our cohort and correlates with 17-hydroxyprogesterone. These findings serve as an important basis for prospective research into 11-oxygenated androgens as therapeutic markers in paediatrics. However, 11-ketotestosterone appears to be very dependent on BMI.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Discriminatory Value of Steroid Hormones on Polycystic Ovary Syndrome and Clustering of Hyperandrogenism and Metabolic Factors.

OBJECTIVE: We determined (1) if 11-oxygenated androgens better identify polycystic ovary syndrome (PCOS) diagnosis in women with obesity compared to total or free testosterone (T) and free androgen index; (2) how biochemical hyperandrogenism and metabolic factors cluster in a cohort of women with infertility and obesity. METHODS: Women with obesity and PCOS comprised the study group (N = 132). Ovulatory women with obesity and idiopathic, tubal or male factor infertility were the control group (N = 83). Steroid hormones were measured by means of liquid chromatography tandem mass spectrometry. Receiver operating characteristic curves and principal component analysis were used. RESULTS: Women with obesity and PCOS had higher 11-ketotestosterone (11 KT) (1.22 nmol/L [0.84; 1.65] vs 1.05 [0.78; 1.35], P = .04) compared to controls, but not 11ß-hydroxyandrostenedione 4.30 [2.87; 5.92] vs 4.06 [3.22; 5.73], P = .44). 11-ketotestosterone (area under the curve: 0.59) did not better discriminate PCOS in women with obesity compared to: total T (0.84), free T (0.91), and free androgen index (0.85). We identified 4 principal components (PCs) in the PCOS group (72.1% explained variance): (1) insulin resistance status; (2) blood pressure; (3) obesity; (4) androgen status and 4 PCs in the control group (68.7% explained variance) with variables representing metabolism being dispersed in component 2, 3, and 4. CONCLUSIONS: Eleven-oxygenated androgens do not aid in the diagnosis of PCOS in women with obesity. Insulin resistance is the strongest PC in the PCOS group. There is no major dominant characteristic that defines obese non-PCOS women.

Effect of baseline oestradiol serum concentration on the efficacy of anastrozole for preventing breast cancer in postmenopausal women at high risk: a case-control study of the IBIS-II prevention trial.

BACKGROUND: An increased risk of breast cancer is associated with high serum concentrations of oestradiol and testosterone in postmenopausal women, but little is known about how these hormones affect response to endocrine therapy for breast cancer prevention or treatment. We aimed to assess the effects of serum oestradiol and testosterone concentrations on the efficacy of the aromatase inhibitor anastrozole for the prevention of breast cancer in postmenopausal women at high risk. METHODS: In this case-control study we used data from the IBIS-II prevention trial, a randomised, controlled, double-blind trial in postmenopausal women aged 40-70 years at high risk of breast cancer, conducted in 153 breast cancer treatment centres across 18 countries. In the trial, women were randomly assigned (1:1) to receive anastrozole (1 mg/day, orally) or placebo daily for 5 years. In this pre-planned case-control study, the primary analysis was the effect of the baseline oestradiol to sex hormone binding globulin (SHBG) ratio (oestradiol-SHBG ratio) on the development of all breast cancers, including ductal carcinoma in situ (the primary endpoint in the trial). Cases were participants in whom breast cancer was reported after trial entry and until the cutoff on Oct 22, 2019, and who had valid blood samples and no use of hormone replacement therapy within 3 months of trial entry or during the trial. For each case, two controls without breast cancer were selected at random, matched on treatment group, age (within 2 years), and follow-up time (at least that of the matching case). For each treatment group, we applied a multinominal logistic regression likelihood-ratio trend test to assess what change in the proportion of cases was associated with a one-quartile change in hormone ratio. Controls were used only to determine quartile cutoffs. Profile likelihood 95% CIs were used to indicate the precision of estimates. A secondary analysis also investigated the effect of the baseline testosterone-SHBG ratio on breast cancer development. We also assessed relative benefit of anastrozole versus placebo (calculated as 1 - the ratio of breast cancer cases in the anastrozole group to cases in the placebo group). The trial was registered with ISRCTN (number ISRCTN31488319) and completed recruitment on Jan 31, 2012, but long-term follow-up is ongoing. FINDINGS: 3864 women were recruited into the trial between Feb 2, 2003, and Jan 31, 2012, and randomly assigned to receive anastrozole (n=1920) or placebo (n=1944). Median follow-up time was 131 months (IQR 106-156), during which 85 (4·4%) cases of breast cancer in the anastrozole group and 165 (8·5%) in the placebo group were identified. No data on gender, race, or ethnicity were collected. After exclusions, the case-control study included 212 participants from the anastrozole group (72 cases, 140 controls) and 416 from the placebo group (142 cases, 274 controls). A trend of increasing breast cancer risk with increasing oestradiol-SHBG ratio was found in the placebo group (trend per quartile 1·25 [95% CI 1·08 to 1·45], p=0·0033), but not in the anastrozole group (1·06 [0·86 to 1·30], p=0·60). A weaker effect was seen for the testosterone-SHBG ratio in the placebo group (trend 1·21 [1·05 to 1·41], p=0·011), but again not in the anastrozole group (trend 1·18 [0·96 to 1·46], p=0·11). A relative benefit of anastrozole was seen in quartile 2 (0·55 [95% CI 0·13 to 0·78]), quartile 3 (0·54 [0·22 to 0·74], and quartile 4 (0·56 [0·23 to 0·76]) of oestradiol-SHBG ratio, but not in quartile 1 (0·18 [-0·60 to 0·59]). INTERPRETATION: These results suggest that serum hormones should be measured more routinely and integrated into risk management decisions. Measuring serum hormone concentrations is inexpensive and might help clinicians differentiate which women will benefit most from an aromatase inhibitor. FUNDING: Cancer Research UK, National Health and Medical Research Council (Australia), Breast Cancer Research Foundation, and DaCosta Fund.

Salivary androgens in adolescence and their value as a marker of puberty: results from the SCAMP cohort.

Context: Salivary androgens represent non-invasive biomarkers of puberty that may have utility in clinical and population studies. Objective: To understand normal age-related variation in salivary sex steroids and demonstrate their correlation to pubertal development in young adolescents. Design, setting and participants: School-based cohort study of 1495 adolescents at two time points for collecting saliva samples approximately 2 years apart. Outcome measures: The saliva samples were analyzed for five androgens (testosterone, androstenedione (A4), 17-hydroxyprogesterone, 11-ketotestosterone and 11ß-hydroxyandrostenedione) using liquid chromatography-mass spectrometry; in addition, salivary dehydroepiandrosterone (DHEA) and oestradiol (OE2) were analysed by ELISA. The pubertal staging was self-reported using the Pubertal Development Scale (PDS). Results: In 1236 saliva samples from 903 boys aged between 11 and 16 years, salivary androgens except DHEA exhibited an increasing trend with an advancing age (ANOVA, P < 0.001), with salivary testosterone and A4 concentration showing the strongest correlation (r = 0.55, P < 0.001 and r = 0.48, P < 0.001, respectively). In a subgroup analysis of 155 and 63 saliva samples in boys and girls, respectively, morning salivary testosterone concentrations showed the highest correlation with composite PDS scores and voice-breaking category from PDS self-report in boys (r = 0.75, r = 0.67, respectively). In girls, salivary DHEA and OE2 had negligible correlations with age or composite PDS scores. Conclusion: In boys aged 11-16 years, an increase in salivary testosterone and A4 is associated with self-reported pubertal progress and represents valid non-invasive biomarkers of puberty in boys.

Real world evidence supports waking salivary cortisone as a screening test for adrenal insufficiency.

OBJECTIVE: Worldwide, adults and children are at risk of adrenal insufficiency largely due to infectious diseases and adrenal suppression from use of anti-inflammatory glucocorticoids. Home waking salivary cortisone is an accurate screening test for adrenal insufficiency, it has potential to reduce costs, and patients prefer it to the adrenocorticotropin (ACTH) (synacthen) stimulation test. We carried out a service evaluation of home waking salivary cortisone in clinical care to identify implementation barriers. DESIGN, PATIENTS AND MEASUREMENTS: Service evaluation in a centre where 212 patients referred for adrenal insufficiency had a waking salivary cortisone. Problems encountered during testing were recorded and patient feedback, via focus groups, collected. RESULTS: From all patients providing a waking salivary cortisone 55% had a normal test, 23% adrenal suppression, and 22% an equivocal result requiring a clinical centre ACTH stimulation test. The median (interquartile range [IQR]) for the time of the saliva sample was 07:40 (07:00-08:40). The median (IQR) days between collection and (i) delivery to local laboratory was 1 (0.25-2) day; (ii) reporting by local laboratory was 13 (11-18) days. Patients considered the test is "easy to do" and preferred it to the inpatient ACTH stimulation test. The principal challenge to clinical implementation was results reporting to clinicians due to delays at the local laboratory. CONCLUSIONS: This service evaluation provides real-world evidence that home waking salivary cortisone is an effective, practical screening test for adrenal insufficiency. It identified key barriers to testing implementation that need to be addressed when introducing the test to a health service.

Quality of Life in Children and Young People With Congenital Adrenal Hyperplasia-UK Nationwide Multicenter Assessment.

CONTEXT: Quality of life (QoL) has been inconsistently reported in children and young people (CYP) with congenital adrenal hyperplasia (CAH). OBJECTIVE: Assess QoL in CYP with CAH in the UK alongside biometric and androgen profiles. DESIGN: To define the evidence base for health care delivery, we conducted a cross-sectional study in CYP with CAH in the UK. Questionnaire results were compared with normative data and between groups, and modelled for association with sex, height, weight, body mass index, or steroid biomarkers of CAH control. SETTING: Tertiary care in 14 UK centers. PATIENTS: Results from 104 patients, 55% female, mean age 12.7 years (SD 3.0), paired responses from parents. INTERVENTIONS: Strengths and Difficulties questionnaire (SDQ) and pediatric QoL questionnaire. MAIN OUTCOME MEASURE: Total QoL scores as assessed by SDQ and a pediatric QoL questionnaire in comparison to normative data. RESULTS: Total scores were worse in parents than normative data, but similar in patients. Patient QoL was rated better in social functioning but worse in emotional, school, and peer domains by patients, and worse in total scores and domains of peer problems, and psychosocial, emotional, and school functioning by parents. Parents consistently scored QoL of their children lower than their child. Larger height-SD score and lower weight-SD score were associated with better QoL. Girls with lower steroid biomarkers had worse SDQ scores. CONCLUSIONS: In CYP with CAH, reduced height, increased weight, and hormonal biomarkers consistent with overtreatment were associated with worse QoL; addressing these problems should be prioritized in clinical management.Clinical Trials Registration Number: SCH/15/088.

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.

Serum cortisol assay performance following the 1 mg overnight dexamethasone suppression test.

BACKGROUND: The 1 mg overnight dexamethasone suppression test (ONDST) is recommended for the differential diagnosis of Cushing's syndrome and the investigation of adrenal incidentalomas. Despite documented variation in serum cortisol immunoassay performance, little has been published regarding its effect on the ONDST. AIMS: Assess the performance of three immunoassay platforms (Roche Elecsys II, Abbott Alinity & Siemens Centaur) when compared to a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. METHODS: Samples (n = 77) sent to the laboratory as part of an ONDST were retrieved prior to disposal, anonymized, and analysed on all platforms. Samples with factors impacting immunoassay analysis quality were excluded. Results were statistically compared to an LC-MS/MS method that previously demonstrated excellent comparability to a candidate reference method. RESULTS: The Roche gen II showed a mean bias of -2.4 nmol/L and a Passing-Bablok fit of y = -0.9 + 0.97x. This was not affected by sex. The Abbott showed a mean bias -18.8 nmol/L, and a fit of y = -11.3 + 0.88x. This bias was -20.7 nmol/L in females versus -17.2 nmol/L in males. The Siemens had a mean bias of 2.3 nmol/L and a fit of y = 1.4 + 1.07x. This bias was 5.7 nmol/L in males versus -1.0 nmol/L in females. CONCLUSIONS: Clinicians should be aware of the method-dependent variation that exists within serum cortisol analysis during the ONDSTs. Roche and Siemens aligned more closely with LC-MS/MS while the Abbot may cause a reduction in ONDST sensitivity. This data supports assay-specific cut-offs for the ONDST.

The Utility of Salivary Cortisone in the Overnight Dexamethasone Suppression Test in Adrenal Incidentalomas.

CONTEXT: Guidelines recommend the assessment of cortisol secretion in patients with adrenal incidentalomas (AI) using the overnight dexamethasone suppression test (ONDST). This requires attendance at a health care facility and venepuncture. Alternatively, the ONDST can be done by measuring salivary cortisol and cortisone, which can be collected at home. OBJECTIVE: We aimed to assess the utility of these measurements in patients with AI. METHODS: A retrospective analysis of data from 173 patients with AI who underwent an ONDST and salivary cortisol/cortisone diurnal studies. Serum and salivary cortisol and salivary cortisone were collected at 09:00, late night, and at 09:00 the following morning after dexamethasone. Dexamethasone levels were measured in the postdexamethasone samples. Serum and salivary samples were analyzed with liquid chromatography-tandem mass spectrometry. RESULTS: We identified a strong correlation between salivary cortisone and serum cortisol after 1 mg of dexamethasone (r = 0.95). Stepwise multivariate regression showed that postdexamethasone salivary cortisone, baseline serum cortisol, salivary cortisone suppression (predexamethasone/postdexamethasone ratio), and sex were the only significant or near-significant independent variables. Performance of predictive indices using these 4 parameters (sensitivity = 88.5%, specificity = 91.2%; kappa 0.80) and postdexamethasone salivary cortisone alone (sensitivity = 85.3%, specificity = 91.7%; kappa 0.77) were comparable when used to predict an ONDST serum cortisol of ≤50 nmol/L. No correlation was observed with any of the other measured parameters. CONCLUSION: In AI patients, after dexamethasone, salivary cortisone correlates very strongly with serum cortisol in the ONDST and could therefore be used as an alternative sampling method which does not require venepuncture or attendance at hospital.

Classic and 11-oxygenated androgens in serum and saliva across adulthood: a cross-sectional study analyzing the impact of age, body mass index, and diurnal and menstrual cycle variation.

OBJECTIVE: 11-oxygenated androgens significantly contribute to the circulating androgen pool. Understanding the physiological variation of 11-oxygenated androgens and their determinants is essential for clinical interpretation, for example, in androgen excess conditions. We quantified classic and 11-oxygenated androgens in serum and saliva across the adult age and body mass index (BMI) range, also analyzing diurnal and menstrual cycle-dependent variation. DESIGN: Cross-sectional. Morning serum samples were collected from 290 healthy volunteers (125 men, 22-95 years; 165 women, 21-91 years). Morning saliva samples were collected by a sub-group (51 women and 32 men). Diurnal saliva profiles were collected by 13 men. Twelve women collected diurnal saliva profiles and morning saliva samples on 7 consecutive days during both follicular and luteal menstrual cycle phases. METHODS: Serum and salivary steroids were quantified by liquid chromatography-tandem mass spectrometry profiling assays. RESULTS: Serum classic androgens decreased with age-adjusted BMI, for example, %change kg/m2 for 5α-dihydrotestosterone: men -5.54% (95% confidence interval (CI) -8.10 to -2.98) and women -1.62% (95%CI -3.16 to -0.08). By contrast, 11-oxygenated androgens increased with BMI, for example, %change kg/m2 for 11-ketotestosterone: men 3.05% (95%CI 0.08-6.03) and women 1.68% (95%CI -0.44 to 3.79). Conversely, classic androgens decreased with age in both men and women, while 11-oxygenated androgens did not. Salivary androgens showed a diurnal pattern in men and in the follicular phase in women; in the luteal phase, only 11-oxygenated androgens showed diurnal variation. CONCLUSIONS: Classic androgens decrease while active 11-oxygenated androgens increase with increasing BMI, pointing toward the importance of adipose tissue mass for the activation of 11-oxygenated androgens. Classic but not 11-oxygenated androgens decline with age.

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Home Waking Salivary Cortisone to Screen for Adrenal Insufficiency.

BACKGROUND: Worldwide, adults and children are at risk of adrenal insufficiency as a result of adrenal suppression from use of anti-inflammatory glucocorticoids and opiates, as well as infectious diseases. The adrenocorticotropin (ACTH) stimulation test is the reference standard for diagnosis of adrenal insufficiency but requires clinic attendance and venesection. Salivary cortisone reflects free serum cortisol, and samples can be collected at home and posted to a laboratory. We tested whether home waking salivary cortisone level could be used to screen for adrenal insufficiency. METHODS: A prospective, diagnostic accuracy study was performed in patients at high risk of adrenal insufficiency. Patients collected a home salivary sample on waking and then attended the clinical facility for an ACTH stimulation test. Salivary cortisone was measured by liquid chromatography­tandem mass spectrometry. Receiver-operating characteristic curves were computed, and positive and negative predictive values were calculated. RESULTS: Two hundred twenty patients were recruited. As measured by an ACTH stimulation test, the prevalence of adrenal insufficiency was 44%. The area under the receiver-operating characteristic curve for waking salivary cortisone as a predictor of adrenal insufficiency was 0.95 (95% confidence interval [CI], 0.92 to 0.97). Cutoffs to ensure a minimum of 95% sensitivity and specificity gave a negative predictive value of 96% (95% CI, 90 to 99) and a positive predictive value of 95% (95% CI, 87 to 99) to exclude and confirm adrenal insufficiency, respectively. Waking salivary cortisone data provided information similar to that of an ACTH stimulation test in 70% of participants. Eighty-three percent of patients preferred home salivary collection to clinic attendance. CONCLUSIONS: Home waking salivary cortisone sampling has accuracy for the diagnosis of adrenal insufficiency similar to that of a standard ACTH stimulation test. Patients found the at-home test to be more convenient than the hospital-based test. (Funded by the National Institute for Health Research.)

11-Oxygenated androgens are not secreted by the human ovary: in-vivo data from four different cases of hyperandrogenism.

Objective: Differentiation of an adrenal from an ovarian source of hyperandrogenemia can be challenging. Recent studies have highlighted the importance of 11-oxygenated C19 steroids to the androgen pool in humans. The aim of this study was to confirm the origin of 11-oxygenated androgens in females and to explore their potential use in the diagnostics of hyperandrogenic disorders. Methods: We measured testosterone and its precursors (dehydroepiandrosterone-sulfate and androstenedione) and 11-oxygenated androgens (11ß-hydroxyandrostenedione (11-OHA4) and 11-ketotestosterone (11-KT)) in the periphery, adrenal and ovarian veins in four different cases of hyperandrogenism in females (polycystic ovary syndrome (PCOS), primary bilateral macronodular adrenal hyperplasia, Sertoli-Leydig cell tumor and ovarian steroid cell tumor). Results: Two patients demonstrate excessive testosterone secretion in neoplastic ovarian tumors which was not paralleled by a significant secretion of 11-oxygenated androgens as determined by adrenal and ovarian vein sampling. In androgen-secreting bilateral adrenal macronodular hyperplasia, steroid profiles were characterized by elevated 11-KT and 11-OHA4 concentrations in adrenal veins and the periphery. In the patient with PCOS, peripheral 11-KT concentrations were slightly elevated in comparison to the other patients, but the 11-KT and 11-OHA4 concentrations were comparable in ovarian veins and in the periphery. Conclusion: This study confirms that 11-OHA4 and 11-KT are not biosynthesized by the ovary. We propose that the testosterone/11-KT ratio as well as 11-OHA4 could help identify predominant adrenal androgen excess and distinguish neoplastic and non-neoplastic ovarian androgen source. Significance statement: This study confirms that 11ß-hydroxyandrostenedione (11-OHA4) and 11-ketotestosterone (11-KT) are not biosynthesized by the human ovary. We propose that the testosterone/11-KT ratio as well as 11-OHA4 could help to identify predominant adrenal androgen excess and distinguish neoplastic and non-neoplastic ovarian androgen source.

11-Oxygenated C19 steroids are the predominant androgens responsible for hyperandrogenemia in Cushing's disease.

Background: Symptoms of hyperandrogenism are common in patients with Cushing's disease (CD), yet they are not sufficiently explained by androgen concentrations. In this study, we analyzed the contribution of 11-oxygenated C19 steroids (11oxC19) to hyperandrogenemia in female patients with CD. Methods: We assessed saliva day profiles in females with CD pre (n = 23) and post (n = 13) successful transsphenoidal surgery, 26 female controls, 5 females with CD treated with metyrapone and 5 treated with osilodrostat for cortisol, cortisone, androstenedione (A4), 11-hydroxyandrostenedione (11OHA4), testosterone (TS), 11-ketotestosterone (11KT), as well as metabolites of classic and 11-oxygenated androgens in 24-h urine. In addition, morning baseline levels of gonadotropins and estradiol, sex hormone-binding globulin, cortisol and dehydroepiandrosterone sulfate (DHEAS) in serum and adrenocorticotrophic hormone in plasma in patients and controls were investigated. Results: Treatment-naïve females with CD showed a significantly elevated area under the curve of 11OHA4 and 11KT in saliva throughout the day compared to controls (11OHA4 mean rank difference (mrd) 18.13, P = 0.0002; 11KT mrd 17.42; P = 0.0005), whereas A4, TS and DHEAS were comparable to controls. Gonadotropin concentrations were normal in all patients with CD. After transsphenoidal surgery, 11oxC19 and their metabolites dropped significantly in saliva (11OHA4 P < 0.0001; 11KT P = 0.0010) and urine (11-oxo-androsterone P = 0.0011; 11-hydroxy-androsterone P < 0.0001), treatment with osilodrostat and metyrapone efficaciously blocked 11oxC19 synthesis. Conclusion: Hyperandrogenemia in CD is predominantly caused by excess of 11oxC19 steroids.

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.

Salivary Steroid Collection in Children Under Conditions Replicating Home Sampling.

CONTEXT: Measurement of salivary glucocorticoids is an accepted method for testing adrenal function but there are few data on stability during home collection. Current salivary collection techniques require active participation or present a choking hazard and are unsuitable for young children. OBJECTIVE: We sought to compare different salivary collection methods; assess the stability of salivary glucocorticoids under conditions replicating home collection; and assess patient tolerability and caregiver acceptability of a salivary collection device for young children, a swab encased in an infant pacifier (SaliPac). METHODS: Six healthy adults collected salivary samples using a Salivette Cortisol, passive drool, and SalivaBio at night, waking, and 3 Pm for five days. Time to collect 1-mL saliva using the SalivaBio and SaliPac and caregiver acceptability were assessed in 30 children younger than 6 years. Saliva was stored at 4 °C, room temperature (RT), and 50 °C for 24, 48, 72 hours and 1 week to replicate potential postage conditions. Salivary cortisol and cortisone concentrations were measured by mass spectrometry. RESULTS: There was no difference in salivary glucocorticoid concentrations using the 3 collection methods. Salivary cortisol and cortisone were stable for 72 hours at RT and 4 °C, and repeated freeze-thaw cycles did not cause significant degradation. In children younger than 6 years the SalivaBio and SaliPac were well tolerated and collected sufficient saliva for salivary steroid analysis in less than 4 minutes. CONCLUSION: Salivette, passive drool, and SalivaBio collect samples with comparable salivary cortisol and cortisone concentrations, which are stable under conditions replicating home collection. SaliPac is an acceptable device for salivary sampling in young children.

Health status of children and young persons with congenital adrenal hyperplasia in the UK (CAH-UK): a cross-sectional multi-centre study.

Objective: There is limited knowledge on the onset of comorbidities in congenital adrenal hyperplasia (CAH) during childhood. We aimed to establish the health status of children with CAH in the UK. Design and methods: This cross-sectional multicentre study involved 14 tertiary endocrine UK units, recruiting 101 patients aged 8-18 years with classic 21-hydroxylase deficiency and 83 controls. We analysed demographic, clinical and metabolic data, as well as psychological questionnaires (Strengths and Difficulties (SDQ), Paediatric Quality of Life (PedsQL)). Results: Patient height SDS in relation to mid-parental height decreased with age, indicating the discrepancy between height achieved and genetic potential height. Bone age was advanced in 40.5% patients, with a mean difference from the chronological age of 1.8 (±2.3) years. Patients were more frequently overweight (27%) or obese (22%) compared to controls (10.8% and 10.8%, respectively, P < 0.001). No consistent relationship between glucocorticoid dose and anthropometric measurements or hormonal biomarkers was detected. A small number of patients had raised total cholesterol (3.0%), low HDL (3.0%), raised LDL (7.0%) and triglycerides (5.0%). SDQ scores were within the 'high' and 'very high' categories of concern for 16.3% of patients. 'School functioning' was the lowest PedsQL scoring dimension with a median (interquartile range) of 70 (55-80), followed by 'emotional functioning' with a median of 75 (65-85). Conclusions: Our results show an increased prevalence of problems with growth and weight gain in CAH children and suggest reduced quality of life. This highlights the urgent need to optimise management and monitoring strategies to improve long-term health outcomes.