Serum 11-deoxycortisol Research Articles

Steroid hormones, vitamin D and melatonin in rapidly rotating shift female hospital workers

Disruption of circadian rhythm caused by night-shift work has been associated with several disorders, including cancer. Health care personnel often works at night to insure the continuity of care. Aim of this study was to evaluate the influence of night-shift work on serum and saliva levels of steroid hormones, vitamin D, and melatonin in hospital female workers. Ninety-seven female hospital workers were recruited: 46 nurses performing clockwise rapid rotating shift schedule on a 5-day cycle, including one night, and 51 day workers. Thirteen steroid hormones and vitamin D were assessed in morning serum samples; cortisol, cortisone and melatonin were assessed in morning and evening saliva samples. We fitted multiple regression models adjusted for age, BMI, sampling month, ovarian cycle phase, and use of oral contraceptives (OC). Rapidly rotating clockwise shift work was associated with increased levels of serum corticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), and androstenedione, and decreased levels of estradiol and vitamin D. OC modulated the association between serum cortisol, corticosterone and 11-deoxycortisol and work shift. The normal circadian phase of salivary melatonin, cortisol and cortisone was not affected by shift work. In female hospital nurses, the clockwise rapid rotating shift schedule increases the level of some hormones, likely associated with stress. No increase of estradiol, nor modification of salivary hormones was observed.

A-046 Quantification of Corticosteroids and Androgens in Serum, utilizing Waters MassTrak™ Steroid Serum Sets 2 & Sets 3 for Clinical Research

Abstract Background Steroid hormones encompass a large class of small molecules that play a central role in metabolic processes, such as the regulation of sexual characteristics, blood pressure, and inflammation. Enzymes that form part of the steroid biosynthetic pathway are pivotal in these metabolic processes, and their dysfunction can be examined through the correct measurement of steroid hormones in a clinical research setting. The availability of lyophilized calibrators and QCs reduces sample preparation time, aids in method harmonization, and assists with metrological traceability in accordance with ISO15189:2022, when used alongside an analytically selective chromatographic method. Methods A quantitative clinical research method utilizing Waters MassTrak Steroid Serum Sets 2 and 3, Cals & QCs and Waters Oasis™ PRiME HLB µElution plate technology for the extraction of testosterone, androstenedione, 17-hydroxyprogesterone (17- OHP), dehydroepiandrosterone sulfate (DHEAS), Cortisol, 11-deoxycortisol and 21-deoxycortisol from human serum samples. Chromatographic separation was performed on an Waters ACQUITY™ UPLC™ I Class Plus (FL-I) System, using an ACQUITY UPLC HSS T3 Column, accompanied by a Xevo™ TQ-S micro Mass Spectrometer. Results Accuracy (±6%) and precision (±10%) have been confirmed through comparison to External Quality Assurance (EQA) LC-MS/MS schemes, panels and QC material for all seven steroid hormones. All analytes were assessed at the low, medium and high concentrations for each MassTrak Steroid Serum Set, which yielded excellent results across the range (±10%). Deming and Linear regression analysis were performed. No statistically significant bias was observed for each compound, with a mean method bias of ±1.3% for Set 2 and ±1.0% for Set 3. The clinical research method was shown to be linear for all analytes over the calibration ranges specified, furthermore, calibration lines created using Set 2 and Set 3 were analysed over a five-day period and were linear with a co-efficient of determination of (r2) > 0.999 for all analytes. Analytical sensitivity using Signal:Noise (S/N) of the low calibrator (Calibrator 1) of each set, was >10:1 for each analyte across several analytical runs. Conclusions This evaluation has demonstrated that the MassTrak Steroid Serum Calibrator and Quality Control Sets 2 and 3 can provide precise and accurate quantification of steroid hormones in serum. An analytically sensitive and selective clinical research method has been developed for the analysis of testosterone, androstenedione, 17- OHP, DHEAS, cortisol, 11-deoxycortisol and 21-deoxycortisol in serum using Waters I-Class Xevo TQ-S micro system. For Research Use Only. Not for Use in Diagnostic Procedures.

Associations of serum glucocorticoid levels on hypertension and blood pressure-related indicators: a nested case-control study in rural China.

The relationship between glucocorticoids and hypertension has shown inconsistent findings in previous studies. To address this, our study employed a nested case-control design in rural areas to further investigate the association between serum glucocorticoid levels and hypertension, and blood pressure-related indicators. This study employed a nested case-control design, involving 560 pairs of hypertensive cases and matched controls. The concentrations of serum cortisol (F), cortisone (E) and 11-deoxycortisol (S) were determined using liquid chromatography-tandem mass spectrometry. We employed various methods, including generalized linear model (GLM), conditional logistic regression model, restricted cubic spline regression, subgroup analysis, interaction, and joint effects, with adjustments for multiple covariates to analyze the relationships between glucocorticoids, hypertension, and blood pressure-related indicators. After multivariable adjustments, ln-F, ln-F/E, and ln-S were positively associated with SBP, DBP, pulse pressure (PP), and mean arterial pressure (MAP), while ln-E was negatively associated with DBP and MAP ( P < 0.05). Interestingly, ln-S showed no statistically significant association with hypertension prevalence ( P > 0.05), whereas ln-F and ln-F/E were positively associated with it ( P < 0.05). The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were 1.153 (1.011-1.315) for ln-F and 2.072 (1.622-2.645) for ln-F/E, respectively. In contrast, ln-E exhibited a negative association with hypertension prevalence (adjusted OR = 0.837, 95% CI 0.714-0.982). Moreover, a significant association was observed between the combined use of high-dose F/E and high-dose S with hypertension prevalence (adjusted OR = 3.273, 95% CI 2.013-5.321). Blood pressure indicators and hypertension prevalence significantly increased with elevated serum F and F/E concentrations ( P < 0.05). Interaction analysis further revealed that among women, the positive association between F/E and hypertension prevalence was more pronounced than in men ( P < 0.05), and S exhibited a more significant positive association with hypertension prevalence in the overweight population ( P < 0.05). Serum F/E and S levels demonstrated positive associations with hypertension and blood pressure-related indicators, and their combined influence exhibited a synergistic effect on hypertension. Notably, F, F/E, and S were associated with heightened hypertension risk, warranting particular attention in women and overweight populations.

Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample Preparation with Phospholipid Removal and Comparison to Immunoassays.

Steroid analysis in clinical laboratories is dominated by immunoassays (IAs) that have a high sample turnover but are inherently limited in trueness, precision, and sensitivity. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has proved to be a far more capable tool, delivering better sensitivity, specificity, and the possibility of parallel analysis of multiple steroids and metabolites, providing the endocrinologist with more reliable and comprehensive diagnostic information. An LC-MS/MS assay with gradient elution over less than eight minutes and a one-step sample preparation combining protein precipitation with phospholipid removal of off-line solid-phase extraction was developed and validated. It allowed the quantification of 11-deoxycorticosterone (11-DOC), 11-deoxycortisol (11-DF), 17-OH-progesterone (17P), 21-deoxycortisol (21-DF), androstenedione (ANDRO), aldosterone (ALDO), corticosterone (CC), cortisol (CL), cortisone (CN), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol (E2), progesterone (PROG), and testosterone (TES) in human serum. Interday imprecision was generally better than 15%, trueness was proven by recovery experiments with ISO 17034-certified reference materials, proficiency testing (UK NEQAS), and measuring serum reference standards. In-house comparison against IVD-CE-certified immunoassays (IA) for 17P, ANDRO, CL, DHEAS, E2, PROG, and TES was conducted by assessing leftover routine patient samples and purpose-built patient serum pools. None of the compared routine IAs were meeting the standards of the LC-MS/MS. Insufficient overall comparability was found for ANDRO and 17P (mean bias > +65%). Accuracy limitations at lower concentrations were present in IAs for PROG, E2, and TES.

Performance of glucagon stimulation test in diagnosing central adrenal insufficiency in children when utilising the Roche Elecsys® cortisol II assay: a pilot study.

The glucagon stimulation test (GST) is used for the simultaneous assessment of central adrenal insufficiency (CAI) and growth hormone deficiency. The new Roche cortisol II (C II) assay was recently introduced, confounding interpretation of the GST. The performance of the GST in diagnosing central adrenal insufficiency (CAI), utilising the C II assay, was therefore compared with that of the overnight metyrapone test (ONMTPT). A diagnostic accuracy study was performed by retrospectively analysing folders and laboratory records of 25 children and adolescents investigated for hypopituitarism with the GST and the ONMTPT between September 2016 and December 2019. The peak serum cortisol (C)of the GST, the post-metyrapone serum 11-deoxycortisol and adrenocorticotropin levels of the ONMTPT were recorded. Diagnostic performance of the GST at a previously suggested cut-off of 374nmol/L was evaluated. Seventeen boys and 8 girls, aged 1.7-16.3years (median 7.3years) were identified. The sensitivity of the post-GST C-level at 374nmol/L was 0.40 (95% confidence interval [CI] 0.17-0.69), specificity 0.64 (95% CI 0.39-0.84), positive predictive value 0.44 (95% CI 0.19-0.73), negative predictive value 0.60 (95% CI 0.36-0.80), accuracy 0.54 (95% CI 0.35-0.72), positive likelihood ratio (+LR) 0.93 (95% CI 0.49-1.77) and negative LR 1.12 (95% CI 0.40-3.15). The area under the receiver of operating characteristics(ROC) curve was 0.379 (95% CI 0.142-0.615). This study suggests that the GST at any C II cut-off cannot replace the ONMTPT as a diagnostic test for CAI in children. Findings should be confirmed in a larger study.

COVID-19 patients with altered steroid hormone levels are more likely to have higher disease severity.

PurposeThis study aims to evaluate the correlations between the severity of the disease and serum steroid levels by analyzing the serum steroid levels in COVID-19 patients with different levels of disease progression and the control group.MethodsMorning serum Aldosterone, 11-deoxycortisol, Androstenedione, 17-hydroxyprogesterone, Dihydrotestosterone (DHT), Dehydroepiandrosterone (DHEA), Corticosterone, Dehydroepiandrosterone sulfate (DHEAS), Estrone, Estradiol, Progesterone, 11-deoxycorticosterone, Cortisol, Corticosterone, Androsterone, Pregnenolone, 17-hydroxypregnenolone and 21-deoxycortisol levels were measured in 153 consecutive patients were grouped as mild, moderate, and severe based on the WHO COVID-19 disease severity classification and the control group. Steroid hormone levels were analyzed at once with a liquid chromatography-tandem mass spectrometric method (LC-MS/MS).ResultsIn our study, nearly all steroids were statistically significantly higher in the patients’ group than in the control group (p < 0.001). Also, DHEA was an independent indicator of the disease severity with COVID-19ConclusionsOur study reveals that the alteration in steroid hormone levels was correlated with disease severity. Also, steroid hormone levels should be followed up during COVID-19 disease management.

A preliminary study on the relationship between environmental endocrine disruptors and precocious puberty in girls

To explore the associations of environmental endocrine disruptors on precocious puberty in girls. This was a case-control study in which 30 girls with precocious puberty and 46 age- and race-matched prepubertal females were enrolled. The concentrations of 10 environment endocrine disruptors (bisphenol A, bisphenol B, butylparaben, propylparaben, ethvlparaben, methylparaben, mono-butyl phthalate, mono-2-ethylhexyl phthalate, monoethyl phthalate, and monomethyl phthalate) in urine and 10 steroid hormones (dihydrotestosterone, corticosterone, hydrocortisone, 11-deoxycortisol, 17α-hydroxy progesterone, 4-androstene-3,17-dione, estrone, deoxycorticosterone, pregnenolone, and dehydroepiandrosterone) in serum were detected with the liquid chromatography-mass spectrometry (LC-MS). According to the Mann-Whitney U test, urinary levels of bisphenol A, monobutyl phthalate, and monomethyl phthalate were significantly higher in the precocious group than in the prepubertal group, and blood levels of hydrocortisone, 11-deoxycortisol, corticosterone, deoxycorticosterone, and pregnenolone were significantly lower in the precocious group than in the prepubertal group (p<0.05, VIP>1). Our findings confirm the association between phthalate exposure and the incidence of precocious puberty in girls. Control and reduction of children exposure to phthalate esters should be considered as a health priority.

Acton-Prolongatum Stimulated Blood Steroid Profile in Apparently Healthy Asian Indian Women of Reproductive-Age Group.

Blood steroid profile is a recently introduced test in India that is commercially available through a few diagnostic laboratories. In adult women, ACTH-stimulated steroid panel helps to differentiate polycystic ovary syndrome (PCOS) from nonclassical forms of CAH. However, the interpretation of ACTH-stimulated steroid panels is often impeded by the limited availability of reference ranges. Here, we report the adrenal steroid levels after stimulation with Acton Prolongatum in Asian Indian women of reproductive age. This prospective study was conducted at a tertiary health care center in the Southern part of India. Apparently healthy women in the reproductive age group with regular menstrual cycles (21-35days) at least over the last 6months were included. All participants received intramuscular Acton Prolongatum® (Pfizer) in the morning hours during the follicular phase and the steroid profile was analyzed by liquid chromatography-tandem mass spectrometry in a blood sample collected 60-min later. The study included 32 apparently healthy women. The mean age of the study population was 22.19 ± 4.36years. None of the participants experienced any adverse events during the procedure. The median (range) serum cortisol, 17α-hydroxyprogesterone, 11-deoxycortisol, and corticosterone were 22.65 (14.3-37.21) μg/dl, 99.72 (47.21-344.71) ng/dl, 287.2 (74.41-530.61) ng/dl and 728.04 (118.74-1708.2) respectively. In conclusion, this is the first report of the response of adrenal steroids measured by LC-MS/MS at 60min after Acton Prolongatum in Asian Indian women of the reproductive age group. However, further larger studies are warranted to establish more robust ACTH-stimulated reference ranges for steroid profile in Indian women.

Expanding genetic spectrum and discriminatory role of steroid profiling by LC-MS/MS in 11β-hydroxylase deficiency.

To report clinical, hormonal and structural effects of CYP11B1 pathogenic variations in Indian patients with 11β-hydroxylase deficiency (11βOHD) and find hormonal criteria that accurately distinguish 11βOHD from 21α-hydroxylase deficiency (21OHD). Retrospective record review of genetically diagnosed patients with 11βOHD. Clinical features, hormonal parameters at diagnosis (by immunoassay) and recent follow-up of 13 genetically proven 11βOHD patients managed at our centre were retrospectively reviewed. ACTH-stimulated serum adrenal steroids (measured by LC-MS/MS) of 11βOHD were compared with those of simple virilizing and non-classic 21OHD. Structural analysis of the observed pathogenic variations was performed by computational modelling. Nine (four females) and four (all females) patients had classic and non-classic disease, respectively. All 11βOHD patients had elevated ACTH-stimulated serum 11-deoxycortisol (26.5-342.7nmol/L) whereas none had elevated serum 17-hydroxyprogesterone (4.2-21.2nmol/L); both hormonal parameters distinguished 11βOHD from 21OHD with 100% accuracy. ACTH-stimulated serum cortisol, but not 11-deoxycortisol, clearly distinguished classic (<70nmol/L) from non-classic (>160nmol/L) disease. Thirteen (eight novel, two recurrent) pathogenic variants were observed. Only missense mutations were observed among patients with non-classic disease. Computational modelling predicted the possible affection of enzyme structure and function for all the observed missense mutations. This first Indian study describes 13 11βOHD patients, including four with the rarer non-classic variant. A total of eight novel pathogenic variants were identified in our study, highlighting regional genetic heterogeneity. Measurement of ACTH-stimulated adrenal steroids by LC-MS/MS will help avoid the misdiagnosis of 11βOHD as 21OHD and has potential to distinguish classic from non-classic 11βOHD.

732. Posaconazole Serum Drug Levels Associated with Pseudohyperaldosteronism

BackgroundPosaconazole is an extended-spectrum triazole used in the treatment and prophylaxis of many fungal diseases. There have been case reports of posaconazole-induced pseudohyperaldosteronism; however, its occurrence and association with serum posaconazole drug levels need further investigation.MethodsIn this single-center retrospective observational study, we examined the occurrence of posaconazole-induced pseudohyperaldosteronism (PIPH), diagnosed either clinically or via laboratory abnormalities, and evaluated differences in serum posaconazole concentration and clinical characteristics between those with and without this syndrome.ResultsSixty-nine patients receiving posaconazole were included; of whom, 16 (23.2%) met the definition of PIPH. Patients with PIPH were significantly older (61.1 vs. 44.7 years, P = 0.007), more frequently had hypertension prior to starting posaconazole (68.8% vs. 32.1%, P = 0.009), and were more frequently prescribed posaconazole for active treatment instead of prophylaxis compared with patients without PIPH (62.5% vs. 24.5%, P = 0.005). Patients with PIPH had a significantly higher median serum posaconazole level than those without PIPH (3.0 vs. 1.2 µg/mL, P = 0.0001). There was a positive correlation between serum posaconazole level and change in systolic blood pressure (r = 0.37, P = 0.01), a negative correlation between serum posaconazole level and change in serum potassium (r = −0.39, P = 0.006), and a positive correlation between serum posaconazole level and serum 11-deoxycortisol (r = 0.69, P < 0.0001).ConclusionPosaconazole is associated with secondary hypertension and hypokalemia, consistent with pseudohyperaldosteronism, and development is associated with higher serum posaconazole concentrations, older age, and baseline hypertension. Management may include dose reduction, the addition of an aldosterone antagonist, or an alternative triazole agent. Disclosures All authors: No reported disclosures.

Posaconazole Serum Drug Levels Associated With Pseudohyperaldosteronism.

Posaconazole tablets are well tolerated and efficacious in the prophylaxis and treatment of aspergillosis, mucormycosis, and other invasive fungal infections. There have been case reports of posaconazole-induced pseudohyperaldosteronism (PIPH); however, its occurrence and association with serum posaconazole drug levels have not previously been investigated. In this single-center, retrospective, observational study, we examined the occurrence of PIPH in outpatients newly starting posaconazole and evaluated differences in serum posaconazole concentrations and clinical characteristics between those with and without this syndrome. Sixty-nine patients receiving posaconazole were included, of whom 16 (23.2%) met the definition of PIPH. Patients with PIPH were significantly older (61.1 vs 44.7 years, P = .007) and more frequently had hypertension prior to starting posaconazole (68.8% vs 32.1%, P = .009). Patients with PIPH had a significantly higher median serum posaconazole level than those without PIPH (3.0 vs 1.2 µg/mL, P ≤ .0001). There was a positive correlation between serum posaconazole levels and changes in systolic blood pressure (r = .37, P = .01), a negative correlation between serum posaconazole levels and changes in serum potassium (r = -.39, P = .006), and a positive correlation between serum posaconazole levels and serum 11-deoxycortisol (r = .69, P < .0001). Posaconazole is associated with secondary hypertension and hypokalemia, consistent with pseudohyperaldosteronism, and development is associated with higher serum posaconazole concentrations, older age, and baseline hypertension.

Gender-Specific Independent and Combined Effects of the Cortisol-to-Cortisone Ratio and 11-Deoxycortisol on Prediabetes and Type 2 Diabetes Mellitus: From the Henan Rural Cohort Study.

Objective The aim of the study was to investigate the independent and combined effects of the cortisol-to-cortisone ratio (F/E) and 11-deoxycortisol on prediabetes and type 2 diabetes mellitus (T2DM) among different genders. Methods A case-control study was performed including 2676 participants from the Henan Rural Cohort Study. Liquid chromatography-tandem mass spectrometry was used to assess serum cortisol, cortisone, and 11-deoxycortisol. Conditional logistic regression was performed to estimate the associations between hormones and outcomes. Results After adjusting for multiple variables, the negative associations of F/E and 11-dexyocortisol with T2DM were observed in females (T3 vs. T1: OR = 0.56, 95% CI: 0.39-0.80 for F/E; T3 vs. T1: OR = 0.44, 95% CI: 0.27-0.73 for 11-dexyocortisol). However, only 11-dexyocortisol showed a negative association with prediabetes both in males and females. Compared with the combination of low F/E and 11-dexyocortisol, the combination of middle F/E and high 11-dexyocortisol was significantly associated with prediabetes (OR = 0.29, 95% CI: 0.12-0.71) in males. Furthermore, the combination of high F/E and 11-dexyocortisol was associated with the lowest odds of prediabetes (OR = 0.39, 95% CI: 0.21-0.73) and T2DM (OR = 0.25, 95% CI: 0.12-0.52) in females. Conclusions Serum F/E level was negatively associated with T2DM only in females whereas serum 11-deoxycortisol level was negatively associated with prediabetes in males and with prediabetes and T2DM in females. Additionally, their combination has a synergistic effect on T2DM.

MON-369 Treatment of Nonclassic 11-Hydroxylase Deficiency with "Hair, Skin, and Nails," an Over-the-Counter, Insulin Sensitizing Vitamin/Mineral Supplement

Background: Insulin Resistance (IR) is seen in classic (CAH) and non-classic congenital adrenal hyperplasia (NCAH). We previously reported amelioration of biochemical/clinical features in patients with NCAH and Type 2 Diabetes Mellitus (T2DM) or prediabetes using insulin sensitizers (IS) metformin and/or thiazolidinediones. Regrettably, these medications can have undesirable side effects e.g. GI intolerance and peripheral edema, respectively. It would be useful to explore alternative treatments that might function as IS, perhaps with fewer side effects. A few studies suggest that biotin might be an IS. Pre-clinical studies showed improvement of IR with biotin use in biotin deficient rats, and human trials revealed improved fasting glucose and insulin levels after biotin use in diabetics. These outcomes suggest that biotin might be an IS and a possible treatment for T2DM, CAH, NCAH, and PCOS where IR prevails. Clinical Case: Our patient is a 62 year old Afro-Caribbean female with a past history of autoimmune thyroid disease, vitamin D deficiency, prediabetes, and central adiposity (BMI=31.96kg/m²) who complained of recent onset hair loss in a male-pattern. Investigation revealed that she had non-classic 11-hydroxylase deficiency. To treat her alopecia and reduce her risk of developing T2DM, we prescribed lifestyle changes and metformin 500mg daily, which she discontinued after two doses due to GI intolerance. She took a hair, skin & nails supplement once daily for 114 days containing beta carotene 2500 IU, vitamin C 100mg, biotin 2500 mcg, zinc oxide 11mg, and copper oxide 0.9mg. Following this, her serum 11-deoxycortisol levels (11-DOC) fell from 80ng/dl (<37) [2.16xULN] to < 20 ng/dl, with reduction of scalp hair loss. Hemoglobin A1c during this period fell from 5.7% to 5.3%. Conclusion: Improvement of this patient’s 11-DOC and HbA1c levels with use of this product, has led us to explore the role of its components as IS. Biotin, beta-carotene, vitamin C, zinc, and copper, all contained in this preparation, are reported to have IS properties. Biotin regulates insulin receptor transcription and improves beta cell function. Zinc improves alopecia, hirsutism, and plasma malondialdehyde levels; however it did not affect other key parameters in women with PCOS, although a trend toward reduction in CRP level was seen. We conclude that this preparation may be an effective treatment for non-classic 11-hydroxylase deficiency, and suggest that it may ultimately be used in treating other IR conditions e.g. NCAH, CAH, PCOS, non-alcoholic fatty liver disease, T2DM and prediabetes, if our results are corroborated in larger studies.

A 13-Steroid Serum Panel Based on LC-MS/MS: Use in Detection of Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignancy, with an annual incidence of 1 or 2 cases per million. Biochemical diagnosis is challenging because up to two-thirds of the carcinomas are biochemically silent, resulting from de facto enzyme deficiencies in steroid hormone biosynthesis. Urine steroid profiling by GC-MS is an effective diagnostic test for ACC because of its capacity to detect and quantify the increased metabolites of steroid pathway synthetic intermediates. Corresponding serum assays for most steroid pathway intermediates are usually unavailable because of low demand or lack of immunoassay specificity. Serum steroid analysis by LC-MS/MS is increasingly replacing immunoassay, in particular for steroids most subject to cross-reaction. We developed an LC-MS/MS method for the measurement of serum androstenedione, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, and testosterone. Assay value in discriminating ACC from other adrenal lesions (phaeochromocytoma/paraganglioma, cortisol-producing adenoma, and lesions demonstrating no hormonal excess) was then investigated. In ACC cases, between 4 and 7 steroids were increased (median = 6), and in the non-ACC groups, up to 2 steroids were increased. 11-Deoxycortisol was markedly increased in all cases of ACC. All steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC from non-ACC adrenal lesions. Serum steroid paneling by LC-MS/MS is useful for diagnosing ACC by combining the measurement of steroid hormones and their precursors in a single analysis.

Isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of ten steroid metabolites in serum

An isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of the ten steroid metabolites dehydroepiandrosterone sulfate (DHEAS), progesterone, 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), corticosterone, 11-deoxycortisol, cortisol, cortisone, testosterone (T), and estrone 3-sulfate (E1-S) in serum was developed and validated. Limits of quantification, variability (inter- and intra-day), analytical range and linearity were all found to be acceptable for clinical use. Furthermore, sample stability was evaluated including the influence of freeze-thaw cycles and the effects of temperature and storage time.The method was applied to 391 serum samples from healthy, Danish boys 10–18years old. The concentration ranges of the included steroid metabolites for this population are presented.Concentrations of DHEAS, 17-OHP, Adione and T in the 391 serum samples were furthermore compared to results obtained using an existing LC-MS/MS method in our laboratory. Excellent agreement was found between the methods. Furthermore, the improved sensitivity of the new method allowed for quantification of a number of samples found to be below the LOQs of the existing method. Thus, the two instruments and their associated methods were validated as possible back-ups for each other, which we consider an extremely important issue in high-throughput laboratories analyzing clinical samples on a regular basis.The ten analytes included can be analyzed simultaneously but it is also possible only to include some of these analytes for specific diagnostic purposes which make the new method an extremely useful tool in the clinical laboratory.

Clinical perspectives in congenital adrenal hyperplasia due to 11β-hydroxylase deficiency

Congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency is a rare autosomal recessive genetic disorder. It is caused by reduced or absent activity of 11β-hydroxylase (CYP11B1) enzyme and the resultant defects in adrenal steroidogenesis. The most common clinical features of 11 beta-hydroxylase deficiency are ambiguous genitalia, accelerated skeletal maturation and resultant short stature, peripheral precocious puberty and hyporeninemic hypokalemic hypertension. The biochemical diagnosis is based on raised serum 11-deoxycortisol and 11-deoxycorticosterone levels together with increased adrenal androgens. More than 100 mutations in CYP11B1 gene have been reported to date. The level of in-vivo activity of CYP11B1 relates to the degree of severity of 11 beta-hydroxylase deficiency. Clinical management of 11 beta-hydroxylase deficiency can pose a challenge to maintain adequate glucocorticoid dosing to suppress adrenal androgen excess while avoiding glucocorticoid-induced side effects. The long-term outcomes of clinical and surgical management are not well studied. This review article aims to collate the current available data about 11 beta-hydroxylase deficiency and its management.

Performance enhancement in the measurement of 5 endogenous steroids by LC–MS/MS combined with differential ion mobility spectrometry

BackgroundChallenges for steroid analysis by LC–MS/MS include low ionization efficiency, endogenous isobars with similar fragmentation patterns and chromatographic retention. Differential ion mobility spectrometry (DMS) provides an additional degree of separation prior to MS/MS detection, and shows promise in improving specificity of analysis. We developed a sensitive and specific method for measurement of corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and progesterone in human serum and plasma using an ABSciex 5500 mass spectrometer equipped with a differential ion mobility interface. Methods250μL aliquots of serum were spiked with deuterated internal standards and extracted with MTBE. The samples were analyzed using positive mode electrospray LC–DMS–MS/MS. The method was validated and compared with immunoassays and LC–MS/MS methods of reference laboratories. ResultsInter and intra assay imprecision was <10%. Limits of quantification and detection in nmol/L were 0.18, 0.09 for corticosterone and 17-hydroxyprogesterone, 0.30, 0.16 for 11-deoxycortisol, 0.12, 0.06 for progesterone and 0.06, 0.03 for 11-deoxycorticosterone. Comparison for progesterone and 17-hydroxyprogesterone with immunoassay showed slopes of 0.97 and 1.0, intercepts of 0.16 and 0.10 and coefficients of determination (r2) of 0.92 and 0.97, respectively. Progesterone by immunoassay showed positive bias in samples measuring <3.18nmol/L. Reference intervals for progesterone and 11-deoxycorticosterone in post-menopausal women were found to be <2.88 and <0.28nmol/L respectively. ConclusionsWe developed and validated an LC–DMS–MS/MS method for analysis of five endogenous steroids suitable for routine measurements in clinical diagnostic laboratories. Specificity gained with DMS allows reducing the complexity of sample preparation, decreasing LC run times and increasing speed of the analysis.

Effect of Vitamin D in a Patient With Classical Adrenal Hyperplasia due to 11-Hydroxylase Deficiency

Both classical and non-classical CAH, like PCOS, are characterized by insulin resistance (IR) and are also ameliorated by measures which reduce IR. Vitamin D supplementation reduces IR and ameliorates PCOS in Vitamin D deficient/insufficient (VDDI) patients-raising the question: would Vitamin D replacement also ameliorate CAH? Our patient is a 47 year old man with Type 2 diabetes (Type 2 DM) and hypertension hospitalized for cellulitis, abscess, osteomyelitis, and gangrene of the left foot. He was not taking any known insulin sensitizers, and denied any history of acne or hyperpigmentation. The patient has no progeny and never had intercourse due to issues concerning his phallus size. He had hypospadias and micropenis at birth. The former was surgically corrected in childhood. His BMI = 36.45 kg/m 2 with a central fat distribution. On physical examination there was no acanthosis nigricans or hyperpigmentation. He had a stage IVa Hamilton male pattern alopecia that started during his early 20s. He had micropenis (or clitoromegaly), measuring 1.5 - 2cm when flaccid; scrotum (or scrotalized labia majora) was normal in size. Testes is measured 3.4 cm in their long diameter and were firm. On 12-4-2012 his serum 11-deoxycortisol by liquid chromatography tandem mass spectrometry (LC MS/MS) was 2024 ng/dL ( less than or equal to 76) while his serum 25-OH-vitamin D by LC MS/MS was 12 ng/mL( greater than or equal to 30), and his 1,25 (OH)2-Vitamin D3 by LC MS/MS was less than or equal to 8 pg/mL (18 - 72). Supplementation was begun with ergocalciferol 50,000 units orally weekly. Nine days later his serum 11-deoxycortisol fell to 425 ng/dL, while serum 25-OH-Vitamin D rose to 23 ng/mL, and serum 1,25-(OH)2-Vitamin D3 remained ? 8 pg/mL. Seventeen days later his serum 11-deoxycortisol was 194 ng/dL, while his serum 25-OH-Vitamin D was 27 ng/mL, and his 1,25-(OH)2-vitamin D3 remained less than or equal to 8 pg/mL. 28 days after starting supplementation with ergocalciferol the 11-deoxycortisol was < 20ng/dL. The laboratory lost the corresponding sample for Vitamin D metabolites. His lymphocyte karyotype was XY. He refused any biopsies, which might have disclosed mosaicism.Vitamin D might be a means of treating classical 11-hydroxylasedeficiency in patients with a VDDI co-morbidity and suggest that it may work by reducing IR. doi: http://dx.doi.org/10.4021/jmc1352w

A sensitive and rapid mass spectrometric method for the simultaneous measurement of eight steroid hormones and CALIPER pediatric reference intervals

ObjectivesTo develop an accurate assay and establish the normal reference intervals for serum cortisol, corticosterone, 11-deoxycortisol, androstenedione, 21-hydroxyprogesterone, testosterone, 17-hydroxyprogesterone, and progesterone. These steroids are commonly used as biomarkers for the diagnosis and monitoring of endocrine diseases such as congenital adrenal hyperplasia. Appropriate age- and gender-stratified reference intervals are essential in accurate interpretation of steroid hormone levels. Design and methodsThe samples analyzed in this study were collected from healthy, ethnically diverse children in the Greater Toronto Area as part of the CALIPER program. A total of 337 serum samples from children between the ages of 0 and 18years were analyzed. The concentrations were measured by using an LC–MS/MS method. The data were analyzed for outliers and age- and gender-specific partitions were established prior to establishing the 2.5th and 97.5th percentiles for the reference intervals. ResultsReference intervals for all hormones required significant age-dependent stratification while testosterone and progesterone required additional sex-dependent stratification. ConclusionsWe report a sensitive, accurate and relatively fast LC–MS/MS method for the simultaneous measurement of eight steroid hormones. Detailed reference intervals partitioned based on both age and gender were also established for all eight steroid hormones.

Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone

The accurate measurement of cortisol by immunoassay is compromised by the potential for cross-reactivity of reagent antibodies with structurally related steroids present in serum. This susceptibility is potentiated when normal steroid metabolism is altered pharmaceutically by antisteroidogenic drugs utilized in the management of Cushing's syndrome to moderate cortisol production. The clinical implications of falsely elevated cortisol results include over-treatment and unrecognized hypoadrenalism. To investigate the effect of the 11β-hydroxylase inhibitor metyrapone on serum cortisol assay, a comparison of measurement by immunoassay versus liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted. Cortisol was measured in serum from three patient groups: (1) patients receiving metyrapone (n = 112 samples from 10 patients); (2) control group of patients diagnosed with Cushing's syndrome currently receiving no treatment (n = 31); and (3) control group of patients with no adrenal pathology and not receiving medication known to interfere in cortisol immunoassay (n = 67). Bland-Altman plots showed agreement between methods for the control group (bias = 1.1% [-4.3 nmol/L]) and Cushing's control group (bias = 1.3% [-3.7 nmol/L]). This was in stark contrast to the metyrapone therapy group (bias = 23% [59 nmol/L]). The difference between LC-MS/MS versus immunoassay in the metyrapone therapy group positively correlated with metyrapone dose and serum 11-deoxycortisol concentration (Pearson's correlation coefficient r = 0.47, 95% CI 0.32-0.61; P < 0.0001). These data show that liability of immunoassay measurement of serum cortisol to interference in patients receiving metyrapone may lead to erroneous clinical decisions concerning dose titration.