Longitudinal proneuroactive and neuroactive steroid profiles in medication-free women with, without and at-risk for perinatal depression: A liquid chromatography-tandem mass spectrometry analysis.

BACKGROUND: Neuroactive steroids (NAS) are derivatives of cholesterol or steroidal precursors made in the gonads, adrenal gland, placenta and brain. We characterized longitudinal plasma proneuroactive and NAS in healthy perinatal comparison women (HPCW), women at-risk for perinatal depression (AR-PND), and women with PND with/without comorbid anxiety. We hypothesized that AR-PND women who either did or did not go on to develop PND would have elevated NAS concentrations as compared to HPCW and that NAS would be correlated to depressive and anxiety symptoms. METHODS: A prospective cohort study evaluated 75 medication-free perinatal women (HPCW, n = 30; AR-PND, n = 19; PND, n = 26). Standardized depression and anxiety assessments and blood samples were completed across 5 visits. Structured Clinical Interviews for DSM-IV TR Disorders were administered at study entry and exit. Plasma pregnenolone, progesterone, 5α- and 5ß-dihydroprogesterone, pregnanolone, allopregnanolone, deoxycorticosterone and tetrahydrodeoxycorticosterone were quantified by liquid chromatography-tandem mass spectrometry. Longitudinal relationships between risk-group, depression and anxiety symptoms, and NAS concentrations were analyzed using generalized estimating equations to control for repeated measures correlations. RESULTS: Perinatal 5α-dihydroprogesterone, 5ß-dihydroprogesterone, allopregnanolone, deoxycorticosterone, and tetrahydrodeoxycorticosterone concentrations were higher in AR-PND and PND women compared to HPCW (ß = 3.57 ± 1.40 and ß = 2.11 ± 1.12, p = 0.03; ß = 0.18 ± 0.06 and ß = 0.03 ± 0.05, p = 0.02; ß = 1.06 ± 0.42 and ß = 1.19 ± 0.47, p = 0.01; ß = 0.17 ± 0.07 and ß = 0.11 ± 0.06, p = 0.05; ß = 0.03 ± 0.01 and ß = 0.03 ± 0.01, p = 0.05, respectively). Perinatal allopregnanolone, 5α-dihydroprogesterone and tetrahydrodeoxycorticosterone were positively associated with HAM-D17 (all p < 0.02). HAM-A was positively associated with 5α- and 5ß-dihydroprogesterone, pregnanolone, allopregnanolone, deoxycorticosterone and tetrahydrodeoxycorticosterone (all p < 0.05). A history of depression was associated with increased 5α-dihydroprogesterone (2.20 ± 1.09, p = 0.05), deoxycorticosterone (0.13 ± 0.06, p = 0.03) and tetrahydrodeoxycorticosterone (0.03 ± 0.01, p = 0.02). CONCLUSION: To our knowledge, this study represents the largest prospective study of 5-α and 5-ß reductase products of progesterone and deoxycorticosterone in HPCW and women AR-PND. Data suggest that PND is associated with both a reduction of progesterone to 5ß-dihydroprogesterone, 5α-dihydroprogesterone, and allopregnanolone, and the 21-hydroxylation to deoxycorticosterone and tetrahydrodeoxycorticosterone. The shift towards 5α-dihydroprogesterone, deoxycorticosterone and tetrahydrodeoxycorticosterone was associated with a history of depression, a significant risk factor for PND.

Dose- and time-dependent changes in blood and adrenal levels of multiple steroids in rats after administration of ketoconazole with or without ACTH.

To verify simultaneous measurement of blood levels of adrenal steroids as a tool to evaluate drug effects on adrenal steroidogenesis, dose- and time-dependent changes in blood levels of corticosterone and its precursors (pregnenolone, progesterone and deoxycorticosterone), as well as their relationship with the pathological changes in the adrenal gland, were examined in rats dosed with ketoconazole (KET). Also examined were whether effects on adrenal steroidogenesis that were not obvious in the blood steroid levels after sole administration of KET could be revealed by post-administration of ACTH, and the correlation between the blood and adrenal steroid levels. Male rats were dosed with 15, 50, or 150 mg/kg of KET for 1 or 7 days with or without ACTH, and the blood and adrenal concentrations of the steroids were measured. KET increased the blood deoxycorticosterone level even at a dose level and time point at which histopathological changes were not obvious. KET-induced changes in blood levels of other steroids were revealed by ACTH, and the blood and adrenal levels were generally correlated especially after ACTH post-administration. Thus, blood levels of adrenal steroids, including precursors, can be a sensitive and early marker of drug effects on the adrenal steroidogenesis that reflect adrenal levels of steroids. The usefulness of the multiple steroid measurement as a method for mechanism investigation of drug effects on the adrenal gland can be further enhanced by ACTH.

Plasma concentrations of steroid precursors, steroids, neuroactive steroids, and neurosteroids in healthy neonatal foals from birth to 7 days of age.

BACKGROUND: Transient hypothalamic-pituitary-adrenal axis dysfunction occurs in critically ill foals with sepsis and neonatal maladjustment syndrome (NMS). Cortisol is the most commonly measured steroid. However, a complex interaction of various steroid compounds might play a role in pathophysiology of this disorder. OBJECTIVE: To identify steroid compounds present at high concentrations at birth that rapidly and steadily decrease within the first 7 days of life in healthy foals and that might be supportive diagnosis of NMS and other neonatal disorders. ANIMALS: Ten healthy neonatal Quarter Horse foals (5 females and 5 males). METHODS: Prospective study. Blood was collected in heparinized tubes within 30 minutes after birth, and at 12, 24, 48, 72, 96, 120, 144, and 168 hours of age. Plasma was separated and a panel of steroid compounds was analyzed using liquid chromatography-mass spectrometry. A nonlinear regression model was used to determine decay concentrations over time. Confidence intervals (CIs) were calculated and significance was set a P ≤ .05. RESULTS: Five compounds were identified: pregnenolone, progesterone, deoxycorticosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. Pregnenolone and progesterone concentrations rapidly decreased by 24 hours of age and remained low throughout the first 7 days of life. Their half-life (95% CI) was short at 3.7 (3.4, 4.0) and 4.5 (2.8, 6.1) hours, respectively. No statistical differences in the concentrations of these compounds were found between males and females. CONCLUSIONS AND CLINICAL RELEVANCE: Progesterone might be a useful marker for identifying continuous endogenous production of neuroactive steroids in foals with suspected NMS and other neonatal diseases.

Regulation of the corticosteroid signalling system in rainbow trout HPI axis during confinement stress.

This study aims to shed light on corticosteroid regulation of stress in teleost fish with focus on the corticosteroid signalling system. The role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) in fish is still enigmatic, as is the function of the mineralocorticoid receptor, MR. Low plasma DOC levels and ubiquitous tissue distribution of MR question the physiological relevance of the mineralocorticoid-axis. Furthermore, the particular purpose of each of the three corticosteroid receptors in fish, the glucocorticoid receptors, GR1 and GR2, and the MR, is still largely unknown. Therefore we investigate the regulation of cortisol and DOC in plasma and mRNA levels of MR, GR1 and GR2 in the HPI-axis tissues (hypothalamus, pituitary and interrenal gland) during a detailed confinement stress time-course. Here we show a sustained up-regulation of plasma DOC levels during a confinement stress time-course. However, the low DOC levels compared to cortisol measured in the plasma do not favour an activity of DOC through MR receptors. Furthermore, we show differential contribution of the CRs in regulation and control of HPI axis activity following confinement stress. Judged by the variation of mRNA levels negative feedback regulation of cortisol release occurs on the level of the pituitary via MR and on the level of the interrenal gland via GR2. Finally, asa significant effect of confinement stress on CR expressions was observed in the pituitary gland, we completed this experiment by demonstrating that corticosteroid receptors (GR1, GR2 and MR) are co-expressed in the ACTH cells located in the adenohypophysis. Overall, these data suggest the involvement of these receptors in the regulation of the HPI axis activity by cortisol.

Usefulness of Simultaneous Measurement of Plasma Steroids, Including Precursors, for the Evaluation of Drug Effects on Adrenal Steroidogenesis in Rats.

The aim of this study was to evaluate the usefulness of simultaneous measurement of plasma steroids, including precursors, for the evaluation of drug effects on adrenal steroidogenesis in vivo. Plasma concentrations of corticosterone and its precursors were examined in rats dosed with compounds that affect adrenal steroidogenesis via different modes of action as well as the relationships of the changes with blood chemistry and adrenal histopathology. Male rats were dosed with tricresyl phosphate, aminoglutethimide, trilostane (TRL), metyrapone (MET), ketoconazole (KET), or mifepristone for 7 days. In the TRL, MET, and KET groups, precursor levels were markedly increased, while there were no significant changes in the corticosterone level, suggesting that the precursors are more sensitive biomarkers to detect the effect on adrenal steroidogenesis. Also, the precursors with increased levels were those that are normally metabolized by the inhibited enzymes, reflecting the modes of action of the compounds. In addition, different patterns of changes were observed in blood chemistry and histopathology, supporting the mechanism suggested by the steroid changes. These results show that simultaneous measurement of plasma steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

The steroid metabolome in women with premenstrual dysphoric disorder during GnRH agonist-induced ovarian suppression: effects of estradiol and progesterone addback.

Clinical evidence suggests that symptoms in premenstrual dysphoric disorder (PMDD) reflect abnormal responsivity to ovarian steroids. This differential steroid sensitivity could be underpinned by abnormal processing of the steroid signal. We used a pharmacometabolomics approach in women with prospectively confirmed PMDD (n=15) and controls without menstrual cycle-related affective symptoms (n=15). All were medication-free with normal menstrual cycle lengths. Notably, women with PMDD were required to show hormone sensitivity in an ovarian suppression protocol. Ovarian suppression was induced for 6 months with gonadotropin-releasing hormone (GnRH)-agonist (Lupron); after 3 months all were randomized to 4 weeks of estradiol (E2) or progesterone (P4). After a 2-week washout, a crossover was performed. Liquid chromatography/tandem mass spectrometry measured 49 steroid metabolites in serum. Values were excluded if >40% were below the limit of detectability (n=21). Analyses were performed with Wilcoxon rank-sum tests using false-discovery rate (q<0.2) for multiple comparisons. PMDD and controls had similar basal levels of metabolites during Lupron and P4-derived neurosteroids during Lupron or E2/P4 conditions. Both groups had significant increases in several steroid metabolites compared with the Lupron alone condition after treatment with E2 (that is, estrone-SO4 (q=0.039 and q=0.002, respectively) and estradiol-3-SO4 (q=0.166 and q=0.001, respectively)) and after treatment with P4 (that is, allopregnanolone (q=0.001 for both PMDD and controls), pregnanediol (q=0.077 and q=0.030, respectively) and cortexone (q=0.118 and q=0.157, respectively). Only sulfated steroid metabolites showed significant diagnosis-related differences. During Lupron plus E2 treatment, women with PMDD had a significantly attenuated increase in E2-3-sulfate (q=0.035) compared with control women, and during Lupron plus P4 treatment a decrease in DHEA-sulfate (q=0.07) compared with an increase in controls. Significant effects of E2 addback compared with Lupron were observed in women with PMDD who had significant decreases in DHEA-sulfate (q=0.065) and pregnenolone sulfate (q=0.076), whereas controls had nonsignificant increases (however, these differences did not meet statistical significance for a between diagnosis effect). Alterations of sulfotransferase activity could contribute to the differential steroid sensitivity in PMDD. Importantly, no differences in the formation of P4-derived neurosteroids were observed in this otherwise highly selected sample of women studied under controlled hormone exposures.

Conjugated Linoleic Acid Administration Induces Amnesia in Male Sprague Dawley Rats and Exacerbates Recovery from Functional Deficits Induced by a Controlled Cortical Impact Injury.

Long-chain polyunsaturated fatty acids like conjugated linoleic acids (CLA) are required for normal neural development and cognitive function and have been ascribed various beneficial functions. Recently, oral CLA also has been shown to increase testosterone (T) biosynthesis, which is known to diminish traumatic brain injury (TBI)-induced neuropathology and reduce deficits induced by stroke in adult rats. To test the impact of CLA on cognitive recovery following a TBI, 5-6 month old male Sprague Dawley rats received a focal injury (craniectomy + controlled cortical impact (CCI; n = 17)) or Sham injury (craniectomy alone; n = 12) and were injected with 25 mg/kg body weight of Clarinol® G-80 (80% CLA in safflower oil; n = 16) or saline (n = 13) every 48 h for 4 weeks. Sham surgery decreased baseline plasma progesterone (P4) by 64.2% (from 9.5 ± 3.4 ng/mL to 3.4 ± 0.5 ng/mL; p = 0.068), T by 74.6% (from 5.9 ± 1.2 ng/mL to 1.5 ± 0.3 ng/mL; p < 0.05), 11-deoxycorticosterone (11-DOC) by 37.5% (from 289.3 ± 42.0 ng/mL to 180.7 ± 3.3 ng/mL), and corticosterone by 50.8% (from 195.1 ± 22.4 ng/mL to 95.9 ± 2.2 ng/mL), by post-surgery day 1. CCI injury induced similar declines in P4, T, 11-DOC and corticosterone (58.9%, 74.6%, 39.4% and 24.6%, respectively) by post-surgery day 1. These results suggest that both Sham surgery and CCI injury induce hypogonadism and hypoadrenalism in adult male rats. CLA treatment did not reverse hypogonadism in Sham (P4: 2.5 ± 1.0 ng/mL; T: 0.9 ± 0.2 ng/mL) or CCI-injured (P4: 2.2 ± 0.9 ng/mL; T: 1.0 ± 0.2 ng/mL, p > 0.05) animals by post-injury day 29, but rapidly reversed by post-injury day 1 the hypoadrenalism in Sham (11-DOC: 372.6 ± 36.6 ng/mL; corticosterone: 202.6 ± 15.6 ng/mL) and CCI-injured (11-DOC: 384.2 ± 101.3 ng/mL; corticosterone: 234.6 ± 43.8 ng/mL) animals. In Sham surgery animals, CLA did not alter body weight, but did markedly increase latency to find the hidden Morris Water Maze platform (40.3 ± 13.0 s) compared to saline treated Sham animals (8.8 ± 1.7 s). In CCI injured animals, CLA did not alter CCI-induced body weight loss, CCI-induced cystic infarct size, or deficits in rotarod performance. However, like Sham animals, CLA injections exacerbated the latency of CCI-injured rats to find the hidden MWM platform (66.8 ± 10.6 s) compared to CCI-injured rats treated with saline (30.7 ± 5.5 s, p < 0.05). These results indicate that chronic treatment of CLA at a dose of 25 mg/kg body weight in adult male rats over 1-month 1) does not reverse craniectomy- and craniectomy + CCI-induced hypogonadism, but does reverse craniectomy- and craniectomy + CCI-induced hypoadrenalism, 2) is detrimental to medium- and long-term spatial learning and memory in craniectomized uninjured rats, 3) limits cognitive recovery following a moderate-severe CCI injury, and 4) does not alter body weight.

Elevated Steroid Hormone Production in the db/db Mouse Model of Obesity and Type 2 Diabetes.

Obesity and type 2 diabetes have become a major public health problem worldwide. Steroid hormone dysfunction appears to be linked to development of obesity and type 2 diabetes and correction of steroid abnormalities may offer new approaches to therapy. We therefore analyzed plasma steroids in 15-16 week old obese and diabetic db/db mice using liquid chromatography-tandem mass spectrometry. Lean db/+ served as controls. Db/db mice developed obesity, hyperglycemia, hyperleptinemia, and hyperlipidemia. Hepatic triglyceride storage was increased and adiponectin and pancreatic insulin were lowered. Aldosterone, corticosterone, 11-deoxycorticosterone, and progesterone were respectively increased by 3.6-, 2.9-, 3.4, and 1.7-fold in db/db mice compared to controls. Ratios of aldosterone-to-progesterone and corticosterone-to-progesterone were respectively 2.0- and 1.5-fold higher in db/db mice. Genes associated with steroidogenesis were quantified in the adrenal glands and gonadal adipose tissues. In adrenals, Cyp11b2, Cyp11b1, Cyp21a1, Hsd3b1, Cyp11a1, and StAR were all significantly increased in db/db mice compared with db/+ controls. In adipose tissue, no Cyp11b2 or Cyp11b1 transcripts were detected and no differences in Cyp21a1, Hsd3b1, Cyp11a1, or StAR expression were found between db/+ and db/db mice. In conclusion, the present study showed an elevated steroid hormone production and adrenal steroidogenesis in the db/db model of obesity and type 2 diabetes.

Peripartum neuroactive steroid and γ-aminobutyric acid profiles in women at-risk for postpartum depression.

Neuroactive steroids (NAS) are allosteric modulators of the γ-aminobutyric acid (GABA) system. NAS and GABA are implicated in depression. The peripartum period involves physiologic changes in NAS which may be associated with peripartum depression and anxiety. We measured peripartum plasma NAS and GABA in healthy comparison subjects (HCS) and those at-risk for postpartum depression (AR-PPD) due to current mild depressive or anxiety symptoms or a history of depression. We evaluated 56 peripartum medication-free subjects. We measured symptoms with the Hamilton Depression Rating Scale (HAM-D17), Hamilton Anxiety Rating Scale (HAM-A) and Spielberger State-Trait Anxiety Inventory-State (STAI-S). Plasma NAS and GABA were quantified by liquid chromatography-mass spectrometry. We examined the associations between longitudinal changes in NAS, GABA and depressive and anxiety symptoms using generalized estimating equation methods. Peripartum GABA concentration was 1.9±0.7ng/mL (p=0.004) lower and progesterone and pregnanolone were 15.8±7.5 (p=0.04) and 1.5±0.7ng/mL (p=0.03) higher in AR-PPD versus HCS, respectively. HAM-D17 was negatively associated with GABA (ß=-0.14±0.05, p=0.01) and positively associated with pregnanolone (ß=0.16±0.06, p=0.01). STAI-S was positively associated with pregnanolone (ß=0.11±0.04, p=0.004), allopregnanolone (ß=0.13±0.05, p=0.006) and pregnenolone (ß=0.02±0.01, p=0.04). HAM-A was negatively associated with GABA (ß=-0.12±0.04, p=0.004) and positively associated with pregnanolone (ß=0.11±0.05, p=0.05). Altered peripartum NAS and GABA profiles in AR-PPD women suggest that their interaction may play an important role in the pathophysiology of peripartum depression and anxiety.

LC-MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers.

OBJECTIVE: Heterozygosity in 21-hydroxylase deficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry. DESIGN: Eleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared. METHOD: Hormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 58 carriers (35 males, 23 females, age range 6-78 years) and 44 random controls (25 males, 19 females, age range 8-58 years). RESULTS: Heterozygotes could be identified best applying the 17-hydroxyprogesterone+21-deoxycortisol/cortisol×1000 ((17OHP+21S)/F×1000) equation 30  min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-off is 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%. CONCLUSION: Considering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHP+21S)/F×1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC-MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-off and to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHP+21S)/F×1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism.

Assessment of testicular corticosterone biosynthesis in adult male rats.

Corticosterone is synthesized in the adrenal glands and is circulated throughout the body to perform regulatory functions in various tissues. The testis is known to synthesize and secrete testosterone and other androgens. We developed an accurate method to measure steroid content using liquid chromatography-mass spectrometry analysis. In the present study, significant levels of the precursor compounds of testosterone and corticosterone synthesis could be detected in rat testis using this method. After adrenalectomy, corticosterone remained in the blood and testicular tissue at approximately 1% of the amount present in the control testis. When the excised testicular tissue was washed and incubated with NADH, NADPH and progesterone, not only testosterone and its precursors but also 11-deoxycorticosterone and corticosterone were produced; the levels of 11-deoxycorticosterone and corticosterone increased with incubation time. The production rate of 11-deoxycorticosterone from progesterone was estimated to be approximately 1/20 that of 17-hydroxyprogesterone, and the corticosterone level was approximately 1/10 that of testosterone. These ratios coincided with those in the testicular tissue of the adrenalectomized rats, indicating that corticosterone was synthesized in the testis and not in the blood. A primary finding of this study was that corticosterone and testosterone were synthesized in a 1/10-20 ratio in the testis. It is concluded that corticosterone, which has various functions, such as the regulation of glycolysis and mediating spermatogenesis, is produced locally in the testis and that this the local production is convenient and functional to respond to local needs.

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

BACKGROUND: Challenges 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. METHODS: 250µL 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. RESULTS: Inter 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 (r(2)) 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. CONCLUSIONS: We 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.

Limitation of number of strains and persistence of false positive loci in QTL mapping using recombinant inbred strains.

While the identification of causal genes of quantitative trait loci (QTL) remains a difficult problem in the post-genome era, the number of QTL continues to accumulate, mainly identified using the recombinant inbred (RI) strains. Over the last decade, hundreds of publications have reported nearly a thousand QTL identified from RI strains. We hypothesized that the inaccuracy of most of these QTL makes it difficult to identify causal genes. Using data from RI strains derived from C57BL/6J (B6) X DBA/2J (D2), we tested the possibility of detection of reliable QTL with different numbers of strains in the same trait in five different traits. Our results indicated that studies using RI strains of less than 30 in general have a higher probability of failing to detect reliable QTL. Errors in many studies could include false positive loci, switches between QTL with small and major effects, and missing the real major loci. The similar data was obtained from a RI strain population derived from a different pair of parents and a RI strain population of rat. Thus, thousands of reported QTL from studies of RI strains may need to be double-checked for accuracy before proceeding to causal gene identification.

Abiraterone acetate to lower androgens in women with classic 21-hydroxylase deficiency.

CONTEXT: Chronic supraphysiological glucocorticoid therapy controls the androgen excess of 21-hydroxylase deficiency (21OHD) but contributes to the high prevalence of obesity, glucose intolerance, and reduced bone mass in these patients. Abiraterone acetate (AA) is a prodrug for abiraterone, a potent CYP17A1 inhibitor used to suppress androgens in the treatment of prostate cancer. OBJECTIVE: The objective of the study was to test the hypothesis that AA added to physiological hydrocortisone and 9α-fludrocortisone acetate corrects androgen excess in women with 21OHD without causing hypertension or hypokalemia. DESIGN: This was a phase 1 dose-escalation study. SETTING: The study was conducted at university clinical research centers. PARTICIPANTS: We screened 14 women with classic 21OHD taking hydrocortisone 12.5-20 mg/d to enroll six participants with serum androstenedione greater than 345 ng/dL (>12 nmol/L). INTERVENTION: AA was administered for 6 days at 100 or 250 mg every morning with 20 mg/d hydrocortisone and 9α-fludrocortisone acetate. MAIN OUTCOME MEASURE: The primary endpoint was normalization of mean predose androstenedione on days 6 and 7 (< 230 ng/dL [<8 nmol/L)] in greater than 80% of participants. Secondary end points included serum 17-hydroxyprogesterone and testosterone (T), electrolytes, plasma renin activity, and urine androsterone and etiocholanolone glucuronides. RESULTS: With 100 mg/d AA, mean predose androstenedione fell from 764 to 254 ng/dL (26.7-8.9 nmol/L). At 250 mg/d AA, mean androstenedione normalized in five participants (83%) and decreased from 664 to 126 ng/dL (23.2-4.4 nmol/L), meeting the primary end point. Mean androstenedione declined further during day 6 to 66 and 38 ng/dL (2.3 and 1.3 nmol/L) at 100 and 250 mg/d, respectively. Serum T and urinary metabolites declined similarly. Abiraterone exposure was strongly negatively correlated with mean androstenedione. Hypertension and hypokalemia were not observed. CONCLUSION: AA 100-250 mg/d added to replacement hydrocortisone normalized several measures of androgen excess in women with classic 21OHD and elevated serum androstenedione.

Implementation of a liquid chromatography tandem mass spectrometry assay for eight adrenal C-21 steroids and pediatric reference data.

BACKGROUND: Sensitive and accurate determination of steroids is essential for diagnosing congenital and acquired adrenal diseases. Since plasma concentrations change during childhood, age-specific reference ranges are the prerequisite for clinical interpretation. The objectives of this study were to develop a sensitive and reliable method for simultaneous detection and quantification of progesterone, 17-hydroxyprogesterone, deoxycorticosterone (DOC), 11-deoxycortisol, 21-deoxycortisol, corticosterone, cortisol (F) and cortisone (E) by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and to establish age- and sex-specific reference ranges from birth to adulthood. METHODS: All eight steroids were measured simultaneously in 0.1 ml plasma by UPLC-MS/MS. Samples of 905 children were measured and grouped in five age groups. RESULTS: The assay was linear up to 70 ng/ml (700 ng/ml for F; r(2) > 0.992). The limit of detection ranged between 0.01 ng/ml for DOC and 0.07 ng/ml for E. Correlations with radioimmunoassays yielded a coefficient of determination between 0.82 and 0.99. Reference data are reported as a function of age and sex. CONCLUSIONS: The UPLC-MS/MS method presented here for the simultaneous detection of eight C-21 adrenal hormones together with the detailed reference ranges for children provides a valuable methodology for assessing adrenal steroids in clinical routine and research.

Implication of the mineralocorticoid axis in rainbow trout osmoregulation during salinity acclimation.

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol-GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater-seawater (FW-SW) or SW-FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation.

Mnemonic effects of progesterone to mice require formation of 3alpha,5alpha-THP.

Ovarian hormones organize and activate neural circuits for reproduction and may also mediate cognition. Research has focused on estradiol's mnemonic effects, albeit progesterone covaries with estradiol and its mechanisms for cognition require attention. Studies tested the hypothesis that cognitive effects of progesterone occur subsequent to its metabolism to 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP), which does not bind progestin receptors. Cognitive performance and progestogen levels in plasma, hippocampus, and cortex were determined in ovariectomized mice administered vehicle, or progestins that differentially form 3alpha,5alpha-THP and bind progestin receptors (progesterone, 3alpha,5alpha-THP, and/or medroxyprogesterone acetate). Only treatments that increased 3alpha,5alpha-THP levels during memory consolidation (progesterone, 3alpha,5alpha-THP, 3alpha-5alpha-THP plus medroxyprogesterone acetate, but not progesterone plus medroxyprogesterone acetate) improved cognitive performance. Thus, formation of 3alpha,5alpha-THP may be required for progesterone's cognitive-enhancing effects.

Quantitation of 17-OH-progesterone (OHPG) for diagnosis of congenital adrenal hyperplasia (CAH).

Most (90%) cases of congenital adrenal hyperplasia (CAH) are due to mutations in the steroid 21-hydroxylase gene (Cyp21). CAH due to 21-hydroxylase deficiency is diagnosed by confirming elevations of 17-hydroxyprogesterone (OHPG) and androstenedione (ANST) with decreased cortisol. By contrast, in two less common forms of CAH, due to 17-hydroxylase or 11-hydroxylase deficiency, OHPG and ANST levels are not significantly elevated and measurement of progesterone (PGSN) and deoxycorticosterone (DOC), respectively are necessary for diagnosis. Since 21-hydroxylase deficiency is more common and results in remarkable increase in OHPG, this test is most commonly ordered compared to other steroid intermediates. Various methods are used in clinical laboratories for the analysis of OHPG in serum or plasma but mass spectrometric methods are considered gold standard method.

Are we missing a mineralocorticoid in teleost fish? Effects of cortisol, deoxycorticosterone and aldosterone on osmoregulation, gill Na+,K+ -ATPase activity and isoform mRNA levels in Atlantic salmon.

It has long been held that cortisol, acting through a single receptor, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. The recent finding that fish express a gene with high sequence similarity to the mammalian mineralocorticoid receptor (MR) suggests the possibility that a hormone other than cortisol carries out some mineralocorticoid functions in fish. To test for this possibility, we examined the effect of in vivo cortisol, 11-deoxycorticosterone (DOC) and aldosterone on salinity tolerance, gill Na(+),K(+)-ATPase (NKA) activity and mRNA levels of NKA alpha 1a and alpha 1b in Atlantic salmon. Cortisol treatment for 6-14 days resulted in increased, physiological levels of cortisol, increased gill NKA activity and improved salinity tolerance (lower plasma chloride after a 24h seawater challenge), whereas DOC and aldosterone had no effect on either NKA activity or salinity tolerance. NKA alpha 1a and alpha 1b mRNA levels, which increase in response to fresh water and seawater acclimation, respectively, were both upregulated by cortisol, whereas DOC and aldosterone were without effect. Cortisol, DOC and aldosterone had no effect on gill glucocorticoid receptor GR1, GR2 and MR mRNA levels, although there was some indication of possible upregulation of GR1 by cortisol (p=0.07). The putative GR blocker RU486 inhibited cortisol-induced increases in salinity tolerance, NKA activity and NKA alpha 1a and alpha 1b transcription, whereas the putative MR blocker spironolactone had no effect. The results provide support that cortisol, and not DOC or aldosterone, is involved in regulating the mineralocorticoid functions of ion uptake and salt secretion in teleost fish.