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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.

Whole-genome sequencing of primary breast tumors enabled the identification of cancer driver genes and noncoding cancer driver plexuses from somatic mutations. However, differentiating driver from passenger events among noncoding genetic variants remains a challenge. Herein, we reveal cancer-driver cis-regulatory elements linked to transcription factors previously shown to be involved in development of luminal breast cancers by defining a tumor-enriched catalogue of approximately 100,000 unique cis-regulatory elements from 26 primary luminal estrogen receptor (ER)+ progesterone receptor (PR)+ breast tumors. Integrating this catalog with somatic mutations from 350 publicly available breast tumor whole genomes, we uncovered cancer driver cistromes, defined as the sum of binding sites for a transcription factor, for ten transcription factors in luminal breast cancer such as FOXA1 and ER, nine of which are essential for growth in breast cancer with four exclusive to the luminal subtype. Collectively, we present a strategy to find cancer driver cistromes relying on quantifying the enrichment of noncoding mutations over cis-regulatory elements concatenated into a functional unit.Implications:Mapping the accessible chromatin of luminal breast cancer led to discovery of an accumulation of mutations within cistromes of transcription factors essential to luminal breast cancer. This demonstrates coopting of regulatory networks to drive cancer and provides a framework to derive insight into the noncoding space of cancer.

Supplementary Figure from Mutations in Noncoding Cis-Regulatory Elements Reveal Cancer Driver Cistromes in Luminal Breast Cancer

Supplementary Table from Mutations in Noncoding Cis-Regulatory Elements Reveal Cancer Driver Cistromes in Luminal Breast Cancer

Purpose: There is strong epidemiologic evidence indicating that estrogens may not be the sole steroid drivers of breast cancer. We hypothesize that abundant adrenal androgenic steroid precursors, acting via the androgen receptor (AR), promote an endocrine-resistant breast cancer phenotype. Experimental Design: AR was evaluated in a primary breast cancer tissue microarray (n = 844). Androstenedione (4AD) levels were evaluated in serum samples (n = 42) from hormone receptor–positive, postmenopausal breast cancer. Levels of androgens, progesterone, and estradiol were quantified using LC/MS-MS in serum from age- and grade-matched recurrent and nonrecurrent patients (n = 6) before and after aromatase inhibitor (AI) therapy (>12 months). AR and estrogen receptor (ER) signaling pathway activities were analyzed in two independent AI-treated cohorts. Results: AR protein expression was associated with favorable progression-free survival in the total population (Wilcoxon, P < 0.001). Pretherapy serum samples from breast cancer patients showed decreasing levels of 4AD with age only in the nonrecurrent group (P < 0.05). LC/MS-MS analysis of an AI-sensitive and AI-resistant cohort demonstrated the ability to detect altered levels of steroids in serum of patients before and after AI therapy. Transcriptional analysis showed an increased ratio of AR:ER signaling pathway activities in patients failing AI therapy (t test P < 0.05); furthermore, 4AD mediated gene changes associated with acquired AI resistance. Conclusions: This study highlights the importance of examining the therapeutic consequences of the steroid microenvironment and demonstrable receptor activation using indicative gene expression signatures.

Testosterone (T) affects β-cell function in men and women. T is a prohormone that undergoes intracrine conversion in target tissues to the potent androgen dihydrotestosterone (DHT) via the enzyme 5α-reductase (5α-R) or to the active estrogen 17β-estradiol (E2) via the aromatase enzyme. Using male and female human pancreas sections, we show that the 5α-R type 1 isoform (SRD5A1) and aromatase are expressed in male and female β-cells. We show that cultured male and female human islets exposed to T produce DHT and downstream metabolites. In these islets, exposure to the 5α-R inhibitors finasteride and dutasteride inhibited T conversion into DHT. We did not detect T conversion into E2 from female islets. However, we detected T conversion into E2 in islets from two out of four male donors. In these donors, exposure to the aromatase inhibitor anastrozole inhibited E2 production. Notably, in cultured male and female islets, T enhanced glucose-stimulated insulin secretion (GSIS). In these islets, exposure to 5α-R inhibitors or the aromatase inhibitor both inhibited T enhancement of GSIS. In conclusion, male and female human islets convert T into DHT and E2 via the intracrine activities of SRD5A1 and aromatase. This process is necessary for T enhancement of GSIS.

Background Measurement of testosterone (T), androstenedione (A4) and 17-hydroxyprogesterone (17OHP) usually requires a venous serum sample which may have implications for sample stability or collection. Objective A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for samples collected using Mitra devices. Analytical validation was completed, and sample comparisons were undertaken to assess Mitra versus venous samples. Method Sample was combined with deionized water and internal standard. After mixing, MTBE was added for extraction. The supernatant was transferred to a deep-well plate and dried prior to re-constitution. A HSS T3 column and Waters TQS Micro was used, the detected quantifier transitions were T m/ z 289.2 > 96.95, A4 287.2 > 96.95 and 17OHP 331.25 > 96.95. Results Mean recovery was 102% for T, 98% for A4 and 97% for 17OHP. Lower limit of quantification was 1 nmol/L for T/A4 and 4 nmol/L for 17OHP. T was linear up to 41.6 nmol/L, A4 41.9 nmol/L and 17OHP 72.6 nmol/L. Ion suppression was Discussion A simple, fast and reproducible LC-MS/MS assay has been developed for measurement of blood collected using Mitra devices for T, A4 and 17OHP. Further comparisons with serum and capillary blood collected onto Mitra devices serum may pave the way for future use in a clinical setting.

BackgroundMulti-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 (uPLC-MS/MS) assay for the profiling of 25 steroids using post-column infusion of ammonium fluoride.MethodsFollowing liquid-liquid extraction, steroids were chromatographically separated over 5 minutes using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity uPLC 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.ResultsPost-column infusion (PCI) of ammonium fluoride (NH4F) enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122-140% for 3βOH-Δ5 steroids and 477-1274% for 3-keto-Δ4 steroids). Therefore, we fully analytically validated PCI with NH4F. Lower limits of quantification ranged from 0.28 to 3.42 nmol/L; 23 of 25 analytes were quantifiable with acceptable accuracy (bias range −14% to 11.9%). 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 23.9% for all analytes and was ConclusionsuPLC-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.HighlightsThis multi-steroid profiling assay quantifies 25 steroids in 5.5 minutesPost-column infusion of NH4F enhances the ionisation of 3-keto-Δ4 steroidsThe assay simultaneously quantifies steroids from several biosynthetic pathwaysWe present analytical data validated for serum steroid profiling

Background Symptoms of hyperandrogenism are common in patients with Cushing's disease (CD), but they cannot be sufficiently explained by measured concentrations of circulating androgens. In this study we analyzed the contribution of 11-oxygenated (11oxC19) androgens to hyperandrogenemia in female patients with CD as well as the influence of treatment with steroidogenesis inhibitors osilodrostat and metyrapone on 11oxC19 and classic androgens. Methods In this single-center study, we assessed saliva day profiles of 23 females with treatment naïve CD, 26 female controls, 5 females with CD treated with metyrapone and 5 treated with osilodrostat for cortisol, cortisone, androstenedione (A4), 11-hydroxyandrostenedione (11OHA4), testosterone (T), 11-ketotestosterone (11KT) by by liquid chromatography tandem mass spectrometry as well as morning baseline levels of gonadotropins and estradiol, sex hormone-binding globulin, cortisol anddehydroepiandrosterone sulfate (DHEAS) in serum and adrenocorticotropic hormone in plasma. Results Treatment naïve females with CD showed significantly elevated area under the curve (AUC) of 11OHA4 and 11KT 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, T and DHEAS were comparable to controls. Patients with more symptoms of hyperandrogenism displayed higher concentrations of 11oxC19 androgens and had significantly lower SHBG concentrations. Gonadotropin levels were normal in all patients with CD (LH 7.18 U/l (SD 14.28 U/l); FSH 7.68 U/l (SD 12.0 U/l)) and did not correlate with any other parameters. Treatment with osilodrostat and metyrapone efficaciously blocked 11oxC19 synthesis. In metyrapone but not in osilodrostat treatment a trend towards increased concentrations of T and significantly increased A4-concentrations were observed (A4 mrd 23.07, p = 0.0119). Conclusion Hyperandrogenemia in CD is predominantly caused by excess of 11oxC19 androgens. Due to lower compensatory increase of A4 and T, osilodrostat seems to be more suitable for treatment of females with CD and hyperandrogenism than metyrapone. Presentation: Tuesday, June 14, 2022 9:45 a.m. - 10:00 a.m.

Background Classically, serum testosterone (T) and androstenedione (A4) have been the mainstay for the biochemical assessment of hyperandrogenism. However, recent evidence suggests 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) may also be important. Here, we describe the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitation of total serum T, A4, 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we applied the method to assess pre-analytical stability. Methods An isotopically labelled internal standard was added to samples prior to supported liquid extraction (SLE). Extracts were analysed using LC-MS/MS to detect T/A4/17OHP/11OHA4 and 11KT along with their corresponding internal standards. Samples (n = 7) were collected from healthy volunteers (n = 14) and left incubated at 20 °C for up to 72 h. Tubes were retrieved at select time points, centrifuged, separated and frozen prior to analysis. Results The total run time was 4 min. For all analytes, intra- and inter-assay imprecision did not exceed 7.9% and 5.3%, respectively; matrix effects were negligible and mean recoveries ranged from 95.3 to 111.6%. The limits of quantitation (LOQs) were 0.25 nmol/L for T, A4 and 11OHA4, 0.50 nmol/L for 17OHP, and 0.24 nmol/L for 11KT. No significant change was observed in pre-centrifugation A4 or female T concentrations over 72 h. Significant increases (p Conclusions We developed a robust LC-MS/MS assay for the quantitation of total serum T/A4/17OHP/11OHA4 and 11KT. Applying the method to determine pre-analytical stability suggests samples requiring 11KT need separating from the cells within 2 h.

Whole-genome sequencing of primary breast tumors enabled the identification of cancer driver genes and noncoding cancer driver plexuses from somatic mutations. However, differentiating driver from passenger events among noncoding genetic variants remains a challenge. Herein, we reveal cancer-driver

Context Several studies have highlighted the importance of the 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as potential biomarkers for monitoring patients with 21-hydroxylase deficiency (21OHD). Objective To analyze circadian rhythmicity of 11oxC19 steroids in saliva profiles and evaluate their relevance as potential monitoring parameters in 21OHD. Design, Setting, and Participants Cross-sectional single-center study including 59 patients with classic 21OHD (men = 30; women = 29) and 49 body mass index- and age-matched controls (men = 19; women = 30). Outcome Measures Salivary concentrations of the following steroids were analyzed by liquid chromatography-tandem mass spectrometry: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11β-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). Results Similar to the previously described rhythmicity of 17OHP, 11OHA4 and 11KT concentrations followed a distinct diurnal rhythm in both patients and controls with highest concentrations in the early morning and declining throughout the day (11-OHA4: mean reduction of hormone concentrations between timepoint 1 and 5 (Δ mean) in male patients = 66%; male controls Δ mean = 83%; female patients Δ mean = 47%; female controls Δ mean = 86%; 11KT: male patients Δ mean = 57%; male controls Δ mean = 63%; female patients Δ mean = 50%; female controls Δ mean = 76%). Significant correlations between the area under the curve for 17OHP and 11KT (rpmale = 0.773 Conclusions Adrenal 11oxC19 androgens are secreted following a diurnal pattern. This should be considered when evaluating their utility for monitoring treatment control.

ContextHypothalamus-pituitary-gonadal (HPG)-axis disturbances are a common phenomenon in patients with classic congenital adrenal hyperplasia (CAH). 11-oxygenated androgens have been suggested to play a role in this context.DesignCross-sectional single center study including 89 patients (N=42 men, N=55 women) with classic CAH.Main Outcome MeasuresIndependent predictors for hypogonadism in men and secondary amenorrhea in women with CAH with a special focus on 11-ketotestosterone (11KT) and 11β-hydroxyandrostenedione (11OHA4).ResultsHypogonadotropic hypogonadism was present in 23% of men and 61% of those women currently not on contraceptives suffered from irregular menstrual cycles or amenorrhea. Testicular adrenal rest tumor (TART) was documented in 28% of men. While 11KT (3.5x) and 11OHA4 (5.7x) among other adrenal steroids were significantly elevated in men with hypogonadism, in stepwise logistic regression, the only significant independent predictor for hypogonadism were elevated 17-OHP levels (B = 0.006; p = 0.039). Although 11KT (5.2x) and 11OHA4 (3.7x) levels were also significantly higher in women with amenorrhea in comparison to those with a regular cycle, the only significant predictor for amenorrhea were elevated total testosterone levels (B = 1.806; p = 0.040). 11-oxygenated androgens were not different in those with TART and those without. Of note, there were no significant differences in 11OHA4 or 11KT between those with a regular cycle and those currently on hormonal contraceptives.Conclusions11-oxygenated androgens do not seem to add additional information for explaining menstrual disturbances and hypogonadism in patients with CAH in comparison to established marker of disease control.HighlightsThe 11-oxygenated androgens 11-ketotestosterone and 11β-hydroxyandrostenedione are significantly elevated in men with CAH with hypogonadotropic hypogonadism and women with amenorrhea.Elevated 17-OHP levels in male patients and elevated testosterone levels in female patients are however the dominant predictor of HPG-axis disturbances.11-oxygenated androgens are not predictive for testicular adrenal rest tumors in men with CAH11-ketotestosterone and 11β-hydroxyandrostenedione levels do not seem to be influenced by intake of oral contraceptives.

Context Hypothalamus-pituitary-gonadal (HPG)-axis disturbances are a common phenomenon in patients with classic congenital adrenal hyperplasia (CAH). 11-oxygenated androgens have been suggested to play a role in this context. Design Cross-sectional single center study including 89 patients (N = 42 men, N = 55 women) with classic CAH. Main Outcome Measures Differences in steroid markers in men with hypogonadism and women with secondary amenorrhea with a special focus on 11-ketotestosterone (11KT) and 11β-hydroxyandrostenedione (11OHA4). Results Hypogonadotropic hypogonadism was present in 23 % of men and 61 % of those women currently not on contraceptives suffered from irregular menstrual cycles or amenorrhea. Testicular adrenal rest tumor (TART) was documented in 28 % of men. 11KT (3.5x) and 11OHA4 (5.7x) among other adrenal steroids were significantly elevated in men with hypogonadism and in women with amenorrhea in comparison to those with a regular cycle (11KT: 5.2x; 11OHA4: 3.7x). 11-oxygenated androgens were not higher in men with TART than in those without. There was a negative association of 11KT and 11OHA4 with FSH but not with LH in men. As expected, all steroids were strongly correlated with each other and cases of disproportionally elevated 11-oxygenated androgens that could explain for HPG-disturbances or TART in otherwise controlled patients were rare and also found in eugonadal individuals. Conclusions In CAH, 11-oxygenated androgens are elevated in women with menstrual disturbances and in men with hypogonadotropic hypogonadism. Due to the close correlation of 11-oxygenated androgens with other adrenal steroids it remains to be shown if their measurement is superior to conventional markers of androgen control.

Testosterone (T) affects β cell function in men and women. T is a pro-hormone that undergoes intracrine conversion in target tissues to the potent androgen dihydrotestosterone (DHT) via the enzyme 5α-reductase (5α-R), or to the active estrogen 17β-estradiol (E2) via the aromatase enzyme. Using male and female human pancreas sections, we show that the 5α-R type1 isoform (SRD5A1) and aromatase are expressed in male and female β cells. We show that cultured male and female human islets exposed to T produce DHT and downstream metabolites. In these islets, exposure to the 5α-R inhibitors finasteride and dutasteride inhibited T conversion into DHT. We did not detect T conversion into E2 from female islets. However, we detected T conversion into E2 in islets from one out of four male donors. In this donor, exposure to the aromatase inhibitor anastrozole inhibited E2 production. Notably, in cultured male and female islets, T enhanced glucose-stimulated insulin secretion (GSIS). In these islets, exposure to 5α-R inhibitors or the aromatase inhibitor both inhibited T enhancement of GSIS. In conclusion, male and female human islets convert T into DHT and E2 via the intracrine activities of SRD5A1 and aromatase. This process is necessary for T enhancement of GSIS.

Background Emerging evidence has suggested the 11-oxygenated steroids may be important in the diagnosis and monitoring of hyperandrogenism. Two in particular, 11-ketotestosterone (11KT) and 11β-hydroxyandrostenedione (11OHA4) have been implicated in polycystic ovary syndrome, congenital adrenal hyperplasia, precocious puberty and castration resistant prostate cancer. Despite the interest in these analytes, some of their more fundamental properties have yet to be determined. At present, no data is available that quantifies the biological variation of 11KT and 11OHA4 within individuals over time, this may be important as we look to establish normative reference ranges for these potentially useful analytes. Objective Here we sought to define the intra-individual variability of 11KT and 11OHA4 in serum using LC-MS/MS. Method Blood was collected from 18 healthy volunteers (8 males, 10 females) on the same day each week over a 10 week period using standard venepuncture technique. After collection, the samples were centrifuged within 1 hour, aliquoted and stored at -20°C (-4°F) prior to analysis. All samples from individual volunteers were analysed by LC-MS/MS in triplicate within the same batch so as to limit analytical variability. Results The mean analytical coefficient of variation (CV%) for the triplicate analysis was 3.2% for 11KT and 3.7% for 11OHA4. No significant difference was observed between the variability of 11KT concentrations in the male and female cohorts; total intra-individual variation for 11KT was 18.0%. Concentrations of 11OHA4 were more variable in the male cohort when compared to the female cohort. This was reflected by differences in their respective intra-individual variations of 32.5% vs 24.8%. Summary Intra-individual variation is an important consideration when interpreting patient results. Concentrations of 11KT were tightly regulated in both the male and female cohorts with no clear demarcation between the two groups. Although concentrations of 11OHA4 were prone to greater variation over the 10 week period, considerable overlap was observed between the male and female subjects. Our data suggests that 11KT and 11OHA4 concentrations are not significantly affected by the menstrual cycle.

Background Recent evidence suggests that 11-oxygenated C19 steroids may have an important role in the assessment of hyperandrogenism [1]. Two in particular, 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) have been implicated in polycystic ovarian syndrome [2] and congenital adrenal hyperplasia [3]. At present there are limited methods available to quantitate these potentially useful analytes and no data is available on their stability. Objectives Here, we sought to develop an LC-MS/MS assay to quantify total serum testosterone (T), androstenedione (A4), 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we aimed to apply the method to evaluate the stability of 11OHA4 and 11KT. Method An LC-MS/MS assay that measured total serum T, A4, 17OHP, 11OHA4 and 11KT was developed and validated in accordance with FDA guidelines. The method was applied to assess the stability of each analyte in serum over a 3 day period. Seven BD Vacutainer® SST™ tubes were collected from 14 volunteers (6 males, 8 females) into. The tubes were stored unseparated at 20°C (68°F) and retrieved at the following times points: 0, 2, 8, 12, 24, 48 and 72h. Once retrieved, samples were centrifuged, aliquoted and stored at -20°C (-4°F) prior to analysis. Results The performance characteristics of the method were acceptable when assessed against an industry standard. Although 11OHA4 concentrations did not significantly change over the 3 day period, we observed a progressive and statistically significant increase in 11KT concentrations between day 0 to day 3 (p

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) is a powerful analytical technique that offers exceptional selectivity and sensitivity. Used optimally, LC–MS/MS provides accurate and precise results for a wide range of analytes at concentrations that are difficult to quantitate with other methodologies. Its implementation into routine clinical biochemistry laboratories has revolutionised our ability to analyse small molecules such as glucocorticoids. Whereas immunoassays can suffer from matrix effects and cross-reactivity due to interactions with structural analogues, the selectivity offered by LC–MS/MS has largely overcome these limitations. As many clinical guidelines are now beginning to acknowledge the importance of the methodology used to provide results, the advantages associated with LC–MS/MS are gaining wider recognition. With their integral role in both the diagnosis and management of hypo- and hyperadrenal disorders, coupled with their widespread pharmacological use, the accurate measurement of glucocorticoids is fundamental to effective patient care. Here, we provide an up-to-date review of the LC–MS/MS techniques used to successfully measure endogenous glucocorticoids, particular reference is made to serum, urine and salivary cortisol.

BackgroundMonitoring of hormonal control represents a key part of the management of congenital adrenal hyperplasia (CAH). Monitoring strategies remain suboptimal because they rely on frequent blood tests and are not specific for adrenal-derived hormones. Recent evidence suggests the crucial role of adrenal-specific 11-oxygenated-C19 androgens in the pathogenesis of CAH.ObjectiveTo establish a correlation between plasma and salivary adrenal-specific androgens in CAH as a noninvasive monitoring strategy.DesignThis prospective cross-sectional study recruited patients between 2015 and 2018.SettingMulticenter study including 13 tertiary centers in the United Kingdom.ParticipantsSeventy-eight children with CAH and 62 matched healthy controls.MethodsUsing liquid chromatography–tandem mass spectrometry, plasma and salivary concentrations of five steroids were measured: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). The correlation between plasma and salivary steroids was analyzed to assess their use in clinical practice.ResultsStrong correlations between plasma and salivary steroid concentrations in patients with CAH were detected: 17OHP (rs = 0.871; P < 0.001), A4 (rs = 0.931; P < 0.001), T (rs = 0.867; P < 0.001), 11OH4A (rs = 0.876; P < 0.001), and 11KT (rs = 0.944; P < 0.001). These results were consistent for patient subgroups based on sex and age. Analysis of patient subgroups based on 17OHP concentrations established clear correlations between plasma and salivary concentrations of the adrenal-specific androgen 11KT.ConclusionsThe current study identified tight correlations between plasma and saliva for the adrenal-derived 11-oxygenated C19 androgen 11KT, as well as 17OHP and A4, which are widely used for monitoring treatment in CAH. This combination of steroid hormones will serve as an improved noninvasive salivary test for disease monitoring in patients with CAH.

Background Measurement of serum dexamethasone during the overnight dexamethasone-suppression test has been recommended to reduce false-positive results when investigating Cushing’s syndrome or increasingly commonly found adrenal incidentalomas. Despite this, there remains a paucity of well-validated dexamethasone methods currently available. Here, we describe the development of a rapid and sensitive liquid chromatography tandem mass spectrometry serum dexamethasone assay and verify its utility in a cohort of postmenopausal females. Method Isotopically labelled internal standard was added to samples prior to supported liquid extraction. Extracts were analysed using liquid chromatography tandem mass spectrometry in the positive electrospray ionization mode. Multiple reaction monitoring was used to detect dexamethasone and its corresponding internal standard transitions. Normal healthy postmenopausal women ( n = 95) were recruited and underwent an overnight dexamethasone suppression test, with serum dexamethasone and cortisol measurements at 09:00 after administration of oral dexamethasone 1 mg at 23:00 the night before. Results Mean intra- and inter-assay imprecision were 4.1% and 2.9%, respectively, for dexamethasone concentrations of 1.5, 6.0 and 12.0 nmol/L. Matrix effects were found to be negligible at 106–109% with recovery ranging from 96 to 100%. The limit of quantitation was 0.25 nmol/L, and structural analogue analysis proved the method to be robust against interferences. Applying a serum dexamethasone cut-off of >3.3 nmol/L was associated with a serum cortisol ≤50 nmol/L in 84/95 individuals. Conclusion We have developed a sensitive and robust liquid chromatography tandem mass spectrometry method for the quantitation of serum dexamethasone. The method can be used to identify false-positive results during the overnight dexamethasone suppression test or for pharmacokinetic studies.

IntroductionFormula One returned to the United States on November 16-18, 2012, with the inaugural United States Grand Prix in Austin, Texas. Medical preparedness for motorsports events represents a unique challenge due to the potential for a high number of spectators seeking medical attention, and the possibility for a mass-casualty situation. Adequate preparation requires close collaboration across public safety agencies and hospital networks to minimize impact on Emergency Medical Services (EMS) resources.Hypothesis/ProblemTo report the details of preparation for an inaugural mass-gathering motorsports event, and to describe the details of the medical care rendered during the 3-day event.MethodsA retrospective analysis was completed utilizing postevent summaries, provided by the medical planning committee, by the Federation Internationale de L'Automobile (FIA), and Austin Travis County Emergency Medical Services (ATCEMS). Patient data were collected from standardized patient care records for descriptive analysis. Medical usage rates (MURs) are reported as a rate of patients per 10,000 (PPTT) participants.ResultsA total of 566 patients received medical care over the 3-day period with the on-site care rate of 95%. Overall, MUR was 21.3 PPTT attendees. Most patients had minor problems, and there were no driver injuries or deaths.ConclusionThis mass-gathering motorsport event had a moderate number of patients requiring medical attention. The preparedness plan was implemented successfully with minimal impact on EMS resources and local medical facilities. This medical preparedness plan may serve as a model to other cities preparing for an inaugural motorsports event.SabraJP, CabañasJG, BedollaJ, BorgmannS, HawleyJ, CravenK, BrownC, ZiebellC, OlveyS. Medical support at a large-scale motorsports mass-gathering event: the inaugural Formula One United States Grand Prix in Austin, Texas. Prehosp Disaster Med. 2014;29(4):1-7.