Metabolite Cortisone Research Articles

12430 Urine Steroid Profiling In Patients With Unilateral And Bilateral Mild Autonomous Cortisol Secretion

Abstract Disclosure: B. Salama: None. J. Saini: None. V. Fell: None. E.J. Atkinson: None. S.J. Achenbach: None. Background: Mildly autonomous cortisol secretion (MACS) can be unilateral in 80% of cases, or bilateral in 20% of cases, due to either bilateral adrenal adenomas or bilateral macronodular adrenal hyperplasia. Scarce evidence demonstrates the differences in plasma steroid profiling of patients with bilateral versus unilateral MACS, however, these findings have not been confirmed using a more comprehensive 24h urine steroid profiling. Objectives: To characterize 24h urine steroid metabolome in patients with unilateral and bilateral MACS. Methods: We conducted a single-center cross-sectional study between 2019-2022. Eligible participants included consecutive patients with MACS who provided a 24h urine sample. Urine was analyzed for 25 steroids with a high-resolution mass spectrometry assay. MACS was defined as cortisol >1.8 mcg/dL following the overnight administration of 1 mg dexamethasone (DST-cortisol). Partial cohort analysis is presented. Results: Of 72 patients with MACS included in the interim analysis, 36 (50%) had bilateral MACS. The two groups (unilateral vs bilateral) were similar in age (median 58 vs 58 years), sex (women 67% vs 61%), BMI (median 33 vs 32 kg/m2), and menopausal status (36% vs 39%), P>0.05 for all. Prevalence of comorbidities was also similar in both groups: hypertension (89% vs 89%), hyperlipidemia (69% vs 78%), and hyperglycemia (69% vs 61%), P >0.05 for all. While the DST-cortisol (median 3.0 vs 3.2 mcg/dL, P= 0.51) and dehydroepiandrosterone sulfate (DHEAS) (median 37.5 vs 55.0, P = 0.21) were similar between the 2 groups, patients with bilateral MACS had a lower ACTH (median 12.4 vs 8.2 pg/mL, P = 0.012) when compared to patients with unilateral MACS. Median adrenal mass size was similar in patients with both unilateral MACS (2.7 cm, IQR 1.7-3.9) and bilateral MACS (3.0 cm, IQR 2.2-3.6) P = 0.54. Of the 25 steroids measured, only tetrahydrocorticosterone was higher in patients with unilateral MACS (median 245 mcg/24h, IQR 165-416 vs median 184 mcg/24h, IQR 121 -278) P = 0.039. No differences in androgens (median 2229 vs 2036 mcg/24h), cortisol metabolites (median 6566 vs 5191 mcg/24h), cortisone metabolites (median 10300 vs 9429 mcg/24h), or glucocorticoid/androgen ratio (median 7.9 vs 8.9) was demonstrated between the unilateral vs bilateral MACS groups (P>0.05 for all). Conclusion: In this interim analysis of patients with unilateral vs bilateral MACS, we have found that patients had a similar post-DST cortisol and similar tumor bulk. Despite a lower ACTH in patients with bilateral MACS, no differences in androgens were found between the two groups. In addition to expanding steroid profiling analysis to the whole cohort, subgroup analyses will need to distinguish between patients with bilateral adenomas and macronodular adrenal hyperplasia, consider aldosterone co-secretion, and employ a more accurate measurement of adrenal tumor bulk. Presentation: 6/3/2024

Steroid Metabolomic Signature in Term and Preterm Infants.

Adrenal function is essential for survival and well-being of preterm babies. In addition to glucocorticoids, it has been hypothesized that C19-steroids (DHEA-metabolites) from the fetal zone of the adrenal gland may play a role as endogenous neuroprotective steroids. In 39 term-born (≥37 weeks gestational age), 42 preterm (30-36 weeks) and 51 early preterm (<30 weeks) infants 38 steroid metabolites were quantified by GC-MS in 24-h urinary samples. In each gestational age group, three distinctive cluster were identified by pattern analysis (k-means clustering). Individual steroidal fingerprints and clinical phenotype were analyzed at the 3rd day of life. Overall, the excretion rates of C21-steroids (glucocorticoid precursors, cortisol, and cortisone metabolites) were low (<99 μg/kg body weight/d) whereas the excretion rates of C19-steroids were up to 10 times higher. There was a shift to higher excretion rates of C19-steroids in both preterm groups compared to term infants but only minor differences in the distribution of C21-steroids. Comparable metabolic patterns were found between gestational age groups: Cluster 1 showed mild elevation of C21- and C19-steroids with the highest incidence of neonatal morbidities in term and severe intraventricular hemorrhage in early preterm infants. In cluster 2 lowest excretion in general was noted but no clinically unique phenotype. Cluster 3 showed highest elevation of C21-steroids and C19-steroids but no clinically unique phenotype. Significant differences in steroid metabolism between clusters are only partly reflected by gestational age and disease severity. In early preterm infants, higher excretion rates of glucocorticoids and their precursors were associated with severe cerebral hemorrhage. High excretion rates of C19-steroids in preterm infants may indicate a biological significance.

Metabotypes of congenital adrenal hyperplasia in infants determined by gas chromatography-mass spectrometry in spot urine

Biochemical monitoring of treatment in infants with classic congenital adrenal hyperplasia (CAH) is not yet well defined. The aim of this study was to perform a cluster analysis of the urinary steroid metabolome for treatment monitoring of infants with classic salt-wasting CAH. We analyzed spot urine samples obtained from 60 young children ≤ 4 years of age (29 females) with classic CAH due to 21-hydroxylase deficiency treated with hydrocortisone and fludrocortisone by targeted gas chromatography-mass spectrometry (GC-MS). Patients were classified into different groups according to their metabolic patterns (metabotypes) using unsupervised k-means clustering algorithms. Three metabotypes could be discovered. Metabotype #1 (N = 15 (25%)) showed high concentrations of androgen and 17-hydroxyprogesterone (17OHP) precursor steroids, metabotype #2 (N = 28 (47%)) revealed balanced metabolic control, and metabotype #3 (N = 17; 28%) demonstrated severe adrenal suppression with low concentrations of androgen and 17OHP precursor steroids. Daily hydrocortisone doses and urinary concentrations of cortisol and cortisone metabolites did not differ between all three metabotypes. Metabotype #2 had highest daily dose of fludrocortisone (p = 0.006). Receiver operating characteristic curve analysis showed that 11-ketopregnanetriol (area under the curve [AUC] 0.967) and pregnanetriol (AUC 0.936) were most suitable of separating metabotype #1 from #2. For separation between metabotypes #2 vs. #3, the 11-oxygenated androgen metabolite 11-hydroxyandrosterone (AUC 0.983) and the ratio of 11-hydroxyandrosterone to tetrahydrocortisone (AUC 0.970) were most suitable. In conclusion, GC-MS-based urinary steroid metabotyping is a new method to help monitor the treatment of infants with CAH. This method allows classification of under-, over- and adequately treated young children.

Chronic stress causes cortisol, cortisone and DHEA elevations in scales but not serum in rainbow trout

Fish scales have been reported to incorporate cortisol over long periods of time and thus provide a promising means of assessing long-term stress in many species of teleost fish. However, the quantification of other stress related hormones has only been accomplished in our previous study conducted in goldfish (Carassius auratus). DHEA is a precursory androgen with anti-stress effects used alongside cortisol to diagnose chronic stress via the cortisol:DHEA ratio in mammals. Included in DHEA's anti-stress mechanisms are changes in the metabolism of cortisol to its inactive metabolite cortisone suggesting the relationships between cortisol, DHEA and cortisone may be additionally informative in the assessment of long-term stress. Therefore, to further explore these concepts in a native fish species and generate more comprehensive comparisons between scale and serum hormone concentrations than was possible in our previous study we implemented a 14-day stress protocol in adult rainbow trout (Oncorhynchus mykiss) and quantified resulting scale and serum cortisol, cortisone and DHEA concentrations. As predicted, elevations in scale concentrations of all hormones were observed in stressed trout compared to controls but were not reflected in serum samples. Significant differences in the cortisol:DHEA and cortisone:cortisol ratios were also found between control and stressed group scales but not serum. These results suggest not only that scales provide a superior medium for the assessment of long-term stress but also that the addition of scale cortisone and DHEA may provide additional relevant information for such assessments.

PSUN20 The ACSPIRE Trial: 11beta-Hydroxysteroid Dehydrogenase Type 1 (HSD-1) Inhibition for Autonomous Cortisol Secretion and Adrenal Cushing's Syndrome

Abstract Background HSD-1, an intracellular enzyme, converts cortisone to cortisol in tissues where cortisol excess is associated with morbidity including liver, adipose, bone, brain, muscle, skin, and eye. SPI-62 is a potent and specific HSD-1 inhibitor in development for treatment of autonomous cortisol secretion (ACS) and Cushing's syndrome, and as adjunctive therapy to prednisolone in polymyalgia rheumatica. In Phase 1 clinical trials SPI-62 was generally well tolerated and associated with maximal liver and brain HSD-1 inhibition. Single and multiple SPI-62 doses decreased urinary cortisol metabolites indicating a similar decrease of hepatocellular cortisol in this important target tissue. After a corresponding transient decrease, circulating cortisol homeostasis was restored rapidly by ACTH increase which also resulted in a moderate adrenal androgen increase. SPI-62's effects on ACTH and androgens did not result in adverse effects. Urinary free cortisol was not affected. The ACSPIRE trial will assess SPI-62 safety and efficacy in patients with dysregulated cortisol production due to ACS or adrenal Cushing's syndrome (aCs) for the first time. Methods In this randomized, placebo-controlled, multinational, Phase 2 clinical trial, adult patients with ACS or aCs with otherwise benign adrenal adenomas, persistently elevated morning cortisol after verifiably adequate dexamethasone suppression, and at least two morbidities associated with hypercortisolism [A) insulin-resistance/type-2 diabetes mellitus, B) dyslipidemia, C) hypertension, or D) osteopenia] will be randomized to receive SPI-62 or placebo for 12 weeks. Subjects must have declined, delayed, or been deemed ineligible for adrenalectomy and not recently taken approved or experimental medical therapies for cortisol excess. Medical conditions or treatments likely to interfere with study assessments or subject safety are also excluded. Efficacy at 12-weeks is assessed by reduction of cortisol-associated morbidities of hyperglycemia and dyslipidemia while also examining, adiposity, hepatic steatosis, hypertension, inflammatory cytokines, osteopenia, muscle strength, cognition, sleep, and mood. Safety is assessed by adverse events, vital signs, ECGs, clinical laboratory analyses. Pharmacology is assessed by effects on HPA/HPG axis biomarkers and suppression of the urinary ratio of hepatic 5- and 3-steroid reductase metabolites of cortisol and cortisone (tetrahydrocortisol + allotetrahydrocortisol)/tetrahydrocortisone). Assessments include timed up-and-go and hand-grip strength tests, dual-energy x-ray absorptiometry, oral glucose tolerance test, continuous glucose monitoring, and 24-hour ambulatory blood pressure monitoring. Results This trial is ongoing; results are pending. Discussion This Phase 2 explores SPI-62 safety, HSD-1 inhibition, effects on HPA/HPG axes, and clinical effects in patients with ACS and aCs. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

PMON72 The "RESCUE" Trial: 11beta-Hydroxysteroid Dehydrogenase Type 1 (HSD-1) Inhibition for ACTH-Dependent Cushing's Syndrome

Abstract Background HSD-1, an intracellular enzyme, converts cortisone to cortisol in tissues where cortisol excess is associated with morbidity including liver, adipose, bone, brain, muscle, skin, and eye. SPI-62 is a potent and specific HSD-1 inhibitor in development for treatment of Cushing's syndrome and autonomous cortisol secretion, and as adjunctive therapy to prednisolone in polymyalgia rheumatica. In Phase 1 clinical trials SPI-62 was generally well tolerated and associated with maximal liver and brain HSD-1 inhibition. SPI-62 decreased urinary cortisol metabolites indicating a similar decrease of hepatocellular cortisol in this important target tissue. After a corresponding transient decrease, circulating cortisol homeostasis was restored by ACTH increase which also resulted in a moderate adrenal androgen increase. SPI-62's effects on androgens did not result in adverse effects. Urinary free cortisol was unaffected. The RESCUE trial will assess SPI-62 safety and efficacy in patients with a dysregulated HPA axis, ie., ACTH-dependent Cushing's syndrome. Methods In this randomized, placebo-controlled, crossover, multinational, Phase 2 clinical trial, adult patients (N=26) with ACTH-dependent Cushing's syndrome with active and consistently elevated urinary free cortisol (UFC) will be randomized to receive SPI-62 and placebo for 12 weeks each. A diagnosis of an inadequately treated pituitary adenoma (Cushing's disease) or ectopic ACTH or CRH producing tumor based on established criteria is required. Evidence of Cushing's associated morbidities including at least 2 of A) insulin-resistance/type-2 diabetes mellitus, B) dyslipidemia, C) hypertension, or D) osteopenia is required. Subjects must not have had recent Cushing's surgical, radiation other approved or experimental medical therapies for cortisol excess. Medical conditions or treatments likely to interfere with study assessments or subject safety are also excluded. The primary outcome is pharmacological suppression of the urinary ratio of hepatic 5- and 3-steroid reductase metabolites of cortisol and cortisone (tetrahydrocortisol+allotetrahydrocortisol)/tetrahydrocortisone). Safety is assessed by adverse events, vital signs, ECG, and clinical laboratory analyses including effects on HPA/HPG axis biomarkers. Efficacy is assessed by reduction of Cushing's features and morbidities of hyperglycemia, dyslipidemia, adiposity, hepatic steatosis, hypertension, glaucoma, osteopenia, muscle strength, cognition, sleep, and mood. Assessments include tumor-imaging by MRI, ocular tonometry, timed up-and-go and hand-grip strength tests, dual-energy x-ray absorptiometry, oral glucose tolerance, continuous glucose monitoring, and ambulatory blood pressure monitoring. Results This trial is ongoing; results are pending. Discussion This Phase 2 explores SPI-62 safety, HSD-1 inhibition, effects on HPA/HPG axes, and clinical effects in patients with ACTH-dependent Cushing's syndrome. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Inhaled glucocorticoid-induced metabolome changes in asthma.

ObjectiveThe aim of this study was toidentify dose-related systemic effects of inhaled glucocorticoids (GCs) on the global metabolome.Design and methodsMetabolomics/lipidomic analysis from plasma was obtained from 54 subjects receiving weekly escalating doses (µg/day) of fluticasone furoate (FF; 25, 100, 200, 400 and 800), fluticasone propionate (FP; 50, 200, 500, 1000 and 2000), budesonide (BUD; 100, 400, 800, 1600 and 3200) or placebo. Samples (pre- and post-dose) were analysed using ultrahigh-performance liquid chromatography-tandem mass spectroscopy and liquid chromatography-mass spectrometry. Ions were matched to library standards for identification and quantification. Statistical analysis involved repeated measures ANOVA, cross-over model, random forest and principal component analysis using log-transformed data.ResultsQuantifiable metabolites (1971) had few significant changes (% increases/decreases; P < 0.05) vs placebo: FF 1.34 (0.42/0.92), FP 1.95 (0.41/1.54) and BUD 2.05 (0.60/1.45). Therapeutic doses had fewer changes: FF 0.96 (0.36/0.61), FP 1.66 (0.44/1.22) and BUD 1.45 (0.56/0.90). At highest/supratherapeutic doses, changes were qualitatively similar: reduced adrenal steroids, particularly glucuronide metabolites of cortisol and cortisone and pregnenolone metabolite DHEA-S; increased amino acids and glycolytic intermediates; decreased fatty acid β-oxidation and branched-chain amino acids. Notable qualitative differences were lowered dopamine metabolites (BUD) and secondary bile acid profiles (BUD/FF), suggesting CNS and gut microbiome effects.ConclusionsDose-dependent metabolomic changes occurred with inhaled GCs but were seen predominately at highest/supratherapeutic doses, supporting the safety of low and mid therapeutic doses. At comparable therapeutic doses (FF 100, FP 500 and BUD 800 µg/day), FF had the least effect on the most sensitive markers (adrenal steroids) vs BUD and FP.

Increased glucocorticoid metabolism in diabetic kidney disease.

AimsGlomerular damage indicated by proteinuria is a main symptom in diabetic nephropathy. Mineralocorticoid receptor (MR) antagonists (MRAs) are beneficial irrespective of aldosterone availability. Thus, we hypothesized an alternatively activated MR to promote glomerular damage in proteinuric diabetic nephropathy. Specifically, we aimed first to demonstrate the presence of steroid hormones serving as alternative MR targets in type II diabetic patients with proteinuric kidney disease, second whether MR selectivity was modified, third to characterize MR and glucocorticoid receptor (GR) expression and activity in glomerular cell types exposed to eu- and hyperglycemic conditions, fourth to characterize the pro-fibrotic potential of primary human renal mesangial cells (HRMC) upon stimulation with aldosterone and cortisol, and fifth to specify the involvement of the MR and/or GR in pro-fibrotic signaling.Materials and methodsUrinary steroid hormone profiles of patients with diabetic kidney disease were analyzed by gas chromatography–mass spectrometry and compared to an age and gender matched healthy control group taken out of a population study. In both cohorts, the activity of the MR pre-receptor enzyme 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2), which inactivates cortisol to prevent it from binding to the MR, was assessed to define a change in MR selectivity. Expression of HSD11B2, MR and GR was quantified in HRMC and primary human renal glomerular endothelial cells (HRGEC). Activity of MR and GR was explored in HRMC by measuring the MR/GR down-stream signal SGK1 and the pro-fibrotic genes TGFB1, FN1 and COL1A1 in normal and high glucose conditions with the MR/GR agonists aldosterone/cortisol and the MR/GR antagonists spironolactone/RU486.ResultsPatients with diabetic kidney disease excreted more tetrahydroaldosterone than the control group reaching significance in men. The excretion of MR-agonistic steroid hormones was only increased for 18-hydroxytetrahydrocorticosterone in diabetic women. The excretion of most glucocorticoids was higher in the diabetic cohort. Higher apparent systemic HSD11B2 activity suggested less activation of the MR by cortisol in diabetic patients. Both cell types, HRMC and HRGEC, lacked expression of HSD11B2. Hyperglycemic conditions did not change MR and GR expression and activity. Stimulation with both aldosterone and cortisol promoted upregulation of pro-fibrotic genes in HRMC. This effect of MR and/or GR activation was more pronounced in high glucose conditions and partially inhibited by MRAs and GR antagonists.ConclusionsIn patients with diabetic kidney disease alternative MR activation is conceivable as cortisol and cortisone metabolites are increased. Systemic availability of active metabolites is counteracted via an increased HSD11B2 activity. As this cortisol deactivation is absent in HRMC and HRGEC, cortisol binding to the MR is enabled. Both, cortisol and aldosterone stimulation led to an increased expression of pro-fibrotic genes in HRMC. This mechanism was related to the MR as well as the GR and more marked in high glucose conditions linking the benefit of MRAs in diabetic kidney disease to these findings.

Role of glucocorticoid metabolism in childhood obesity-associated hypertension.

ObjectiveChildhood obesity is associated with alterations in hypothalamus–pituitary–adrenal axis activity. We tested the hypothesis that multiple alterations in the metabolism of glucocorticoids are required for the development of hypertension in children who become overweight.MethodsSpot urine for targeted gas chromatography-mass spectrometry steroid metabolome analysis was collected from (1) overweight/hypertensive children (n = 38), (2) overweight/non-hypertensive children (n = 83), and (3) non-overweight/non-hypertensive children (n = 56).ResultsThe mean (± s.d.) age of participants was 10.4 ± 3.4 years, and 53% of them were male. Group 1 and group 2 had higher excretion rates of cortisol and corticosterone metabolites than group 3 (869 (interquartile range: 631–1352) vs 839 (609–1123) vs 608 (439–834) μg/mmol creatinine × m2 body surface area, P < 0.01, for the sum of cortisol metabolites), and group 1 had a higher excretion rate of naive cortisol than group 3. Furthermore, groups differed in cortisol metabolism, in particular in the activities of 11β-hydroxysteroid dehydrogenases, as assessed from the ratio of cortisol:cortisone metabolites (group 2 < group 3), 5α-reductase (group 1 > group 2 or 3), and CYP3A4 activity (group 1 < group 2 or 3).DiscussionThe sequence of events leading to obesity-associated hypertension in children may involve an increase in the production of glucocorticoids, downregulation of 11β-hydroxysteroid dehydrogenase type 1 activity, and upregulation of 5α-reductase activity, along with a decrease in CYP3A4 activity and an increase in bioavailable cortisol.

Differential Changes in Circulating Steroid Hormones in Hibernating Brown Bears: Preliminary Conclusions and Caveats.

Brown bears are obese when they enter the den, and after 6 mo of hibernation and physical inactivity, bears show none of the adverse consequences of a sedentary lifestyle in humans, such as cardiovascular disease, type 2 diabetes, and kidney failure. The metabolic mechanisms that drive hibernation physiology in bears are poorly defined, but systemic endocrine regulators are likely involved. To investigate the potential role of steroid hormones, we quantified the total levels of 12 steroid hormones, the precursor cholesterol, sex hormone-binding globulin (SHBG), and corticosterone-binding globulin (CBG) in paired serum samples from subadult free-ranging Scandinavian brown bears during the active and hibernation states. During hibernation, androstenedione and testosterone were significantly decreased in subadult female bears (), whereas they increased in all males but one () and therefore did not reach a significant difference. Despite this difference, SHBG increased more than 20-fold during hibernation for all bears. Compared with SHBG concentrations in humans, bear levels were very low in the active state, but during hibernation, levels equaled high levels in humans. The increased SHBG levels likely maintain a state of relative quiescence of the reproductive hormones in hibernating bears. Interestingly, the combination of SHBG and testosterone levels results in similar free bioavailable testosterone levels of 70-80 pM in both subadult and adult sexually active male bears, suggesting a role for SHBG in controlling androgen action during hibernation in males. Dehydroepiandrosterone sulfate, dihydrotestosterone, and estradiol levels were below the detection limit in all but one animal. The metabolically active glucocorticoids were significantly higher in both sexes during hibernation, whereas the inactive metabolite cortisone was reduced and CBG was low approaching the detection limit. A potential caveat is that the glucocorticoid levels might be affected by the ketamine applied in the anesthetic mixture for hibernating bears. However, increased hibernating cortisol levels have consistently been reported in both black bears and brown bears. Thus, we suggest that high glucocorticoid activity may support the hibernation state, likely serving to promote lipolysis and gluconeogenesis while limiting tissue glucose uptake to maintain a continuous glucose supply to the brain.

Association between Selected Polymorphisms rs12086634, rs846910, rs4844880, rs3753519 of 11β-Hydroxysteroid Dehydrogenase Type 1 (HSD11B1) and the Presence of Insulin Resistance in the Polish Population of People Living in Upper Silesia.

Background: Many factors influence the development of insulin resistance, among other genetic factors. Cortisol is one of the factors that has a significant impact on the development of insulin resistance. The proteins that have a substantial effect on blood cortisol levels include 11β-hydroxysteroid dehydrogenase type 1. HSD11B1 is a microsomal enzyme that catalyzes the conversion of the stress hormone cortisol to the inactive metabolite cortisone. Gene encoding HSD11B1 is located on 1q32.2. This study was designed to assess the association between four polymorphic sides in HSD11B1 (rs12086634, rs846910, rs4844880, rs3753519) between subjects with and without insulin resistance in the Polish population of people living in Upper Silesia. Methods: The study included a total of 507 consecutive patients, 374 (73.77%) with and 133 (26.23%) without insulin resistance. Results: The results show that there were no statistically significant differences in the distribution of genotypes and alleles of the examined polymorphisms of the 11β-hydroxysteroid dehydrogenase type 1 gene between subjects with and without insulin resistance (determined using the HOMA-IR, insulin resistance index) and that rs846910 and rs1208663 polymorphisms of the 11β-hydroxysteroid dehydrogenase type 1 gene in the examined subjects have a significant effect on the magnitude of the HOMA-IR insulin resistance index. Conclusions: The study results suggested that genetic variation of rs846910 and rs1208663 polymorphism of the HSD11B1 gene is related to the susceptibility to insulin resistance. Our results provide a basis to begin basic research on the role of the HSD11B1 gene in the pathogenesis of insulin resistance.

Evidence of the moderating role of hair cortisol and hair cortisone in the relationship between work stress and depression symptoms among Chinese fishermen

BackgroundStress plays an important role in the pathogenesis of depression through functional changes to the hypothalamic-pituitary-adrenal (HPA) axis, a key output of which is the stress hormone cortisol. But the precise relationship between HPA axis activity and depression remains unclear, in part because mostly cortisol measurement only provides reflect acute or short-term fluctuations. In this study, hair cortisol and its metabolite cortisone were measured in a prospective, pre and post follow-up study of working stress and depressive symptoms in Chinese fishermen. We evaluated HPA activity, work stress, and depression symptoms before and after fishing to determine whether cortisol and cortisone in hair can moderate the work stress and depression symptoms of Chinese fishermen. MethodsHair cortisol, hair cortisone, work stress, and depression were measured in Chinese fishermen (n=229; age 16–65) before and after 1–3 months at sea. A week after the fishermen returned, they provided 2–4 cm hair segments closest to the scalp. We measured hair cortisol and hair cortisone using high-performance liquid chromatography–tandem mass spectrometry. ResultsThe work stress and hair cortisol of fishermen before fishing were significantly higher than those after fishing, while hair cortisone was lower. There was no significant difference in depressive symptoms. Fishermen with low hair cortisol or high hair cortisone were more likely to show depressive symptoms after experiencing stress, and were less likely to show depressive symptoms when faced with lower levels of work stress, consistent with the differential susceptibility model. LimitationsThe assumption of an average hair growth of 1 cm/month may cause time overlap between pre- and post-fishing, and time error between the hormone data and questionnaire collection. The present study evaluated only two observation points. The use of self-report scales may affect interpretability of the results. Finally, the study used a limited sample of marine fishermen working out of a single location in Hainan province, China. ConclusionsHair cortisol and hair cortisone appear to have a moderating role in the relationship between work stress and depressive symptoms among fishermen, which fits with the differential susceptibility model of depression. However, their mechanisms of moderation are not the same, which may reflect the complexity of physiological indicators.

74 Central cortisol regulation in pregnancy

Pregnancy is characterized by changes in the regulation of the maternal hypothalamic-pituitary-adrenal (HPA) axis. Even though it is known that aberrations in this pathway could have implications for mood disorders during pregnancy, the effects of cortisol on the maternal brain in healthy women are poorly understood. Therefore, we aimed to enhance our understanding of central adaptations in cortisol metabolism in healthy pregnant women at term gestation. Plasma and CSF cortisol and cortisone were prospectively collected in 24 women at term gestation prior to scheduled cesarean section and in 24 regularly cycling non-pregnant controls matched for age and BMI. Subjects were healthy, with no history of smoking, alcoholism, psychiatric or neurological disorders. Cortisol and cortisone (inactivated form) were assayed by ELISA. Data were analyzed by two-tailed Student t test. Plasma cortisol levels were 54% higher (248.5±15.9 vs 161.7±10.7 ng/ml, p<0.001) and plasma cortisone levels were 88% higher (110.9±7.7 vs 59.1± 6.1 ng/ml, p <0.001) in pregnant vs non-pregnant women, as expected. In CSF, cortisol (7.7 ± 0.5 vs 6.1 ± 0.3 ng/mL, p = 0.01) and cortisone (8.2 ± 0.9 vs 2.7 ± 0.2 ng/mL, p < 0.001) were also significantly higher in pregnant vs non-pregnant subjects. However, interestingly, in pregnant subjects the relative increase in CSF cortisol was only ½ that observed in plasma, while the relative increase in CSF cortisone was 2.3 times greater than in plasma. Accordingly, the CSF cortisol/cortisone ratio was significantly lower in pregnancy (1.30 ± 0.3 vs 2.4 ± 0.2, p = 0.003), due to the increased concentration of the inactive metabolite cortisone. To our knowledge, this is the first study to measure CSF cortisol and cortisone concentrations in pregnancy. These data demonstrate a pregnancy-specific shift in central cortisol metabolism that favors cortisone. This adaptation may protect the maternal brain from high cortisol levels, and aberrations in this pathway could have implications for mood disorders during pregnancy and post-partum.

The contribution of serum cortisone and glucocorticoid metabolites to detrimental bone health in patients receiving hydrocortisone therapy

BackgroundGlucocorticoid therapy is the most common cause of iatrogenic osteoporosis. Less is known regarding the effect of glucocorticoids when used as replacement therapy on bone remodelling in patients with adrenal insufficiency. Enhanced intracellular conversion of inactive cortisone to active cortisol, by 11 beta-hydroxysteroid dehydrogenase type 1(11β-HSD1) and other enzymes leading to alterations in glucocorticoid metabolism, may contribute to a deleterious effect on bone health in this patient group.MethodsStudy design: An open crossover prospective study randomizing ten hypopituitary men, with severe ACTH deficiency, to three commonly used hydrocortisone dose regimens.Measurements: Following 6 weeks of each regimen, patients underwent 24-h serum cortisol/cortisone sampling, measurement of bone turnover markers, and a 24-h urine collection for measurement of urinary steroid metabolites by gas chromatography-mass spectrometry (GC-MS). Serum cortisone and cortisol were analysed by liquid chromatography-mass spectrometry (LC-MS).ResultsDose-related and circadian variations in serum cortisone were seen to parallel those for cortisol, indicating conversion of ingested hydrocortisone to cortisone. The median area under the curve (AUC) of serum cortisone was significantly higher in patients on dose A (20 mg/10 mg) [670.5 (IQR 621–809.2)] compared to those on dose C (10 mg/5 mg) [562.8 (IQR 520.1–619.6), p = 0.01]. A negative correlation was observed between serum cortisone and bone formation markers, OC [1–49] (r = − 0.42, p = 0.03), and PINP (r = − 0.49, p = 0.01). There was a negative correlation between the AUC of night-time serum cortisone levels with the bone formation marker, OC [1–49] (r = − 0.41, p = 0.03) but there were no significant correlations between day-time serum cortisone or cortisol with bone turnover markers. There was a negative correlation between total urinary cortisol metabolites and the bone formation markers, PINP (r = − 0.39, p = 0.04), and OC [1–49] (r = − 0.35, p = 0.06).ConclusionSerum cortisol and cortisone and total urinary corticosteroid metabolites are negatively associated with bone turnover markers in patients receiving replacement doses of hydrocortisone, with nocturnal glucocorticoid exposure having a potentially greater influence on bone turnover.Trial registrationIrish Medicines Board Clinical Trial Number – CT900/459/1 and EudraCT Number – 2007-005018-37. Registration date: 07-09-2007.

The diagnostic utility of late night salivary cortisol (LNSF) and cortisone (LNSE) in Cushing's syndrome.

Measurement of late night salivary cortisol (LNSF) is useful in the identification of cyclical Cushing's syndrome (CS); the usefulness of its metabolite cortisone (late night salivary cortisone, LNSE) is less well described. The aim of this study was to determine the utility of measuring LNSE in patients with confirmed CS compared with other diagnostic tests and to analyse serial LNSF measurements for evidence of variable hormonogenesis. This was a retrospective observational study including patients with confirmed CS in whom LNSF and LNSE were measured. Twenty-three patients with confirmed CS were included, 21 with Cushing's disease. LNSF had a sensitivity of 92%, LNSE 87% and combined LNSF/LNSE 94% per sample. Four patients had cyclical hormonogenesis, when the definition of one trough and two peaks was applied to LNSF measurements, anda fifth patient fell just outside the criteria. Six patients had evidence of variable hormonogenesis, defined as doubling of LNSF concentration on serial measurements. Sensitivity of 24-h urinary free cortisol (UFC) was 89% per collection. Sixteen patients had simultaneous measurements of LNSF and UFC; in three patients, they provided discordant results. LNSF appears more sensitive than LNSE and UFC in the diagnosis of CS, combining LNSF and LNSE results leads to superior sensitivity. Half of our cohort had evidence of cyclical or variable hormonogenesis. Fluctuations in LNSF did not always correlate with changes in UFC concentration, emphasising the importance of performing more than one screening test, particularly if pretest clinical suspicion is high.

Not the Root of the Problem-Hair Cortisol and Cortisone Do Not Mediate the Effect of Child Maltreatment on Body Mass Index.

BackgroundExperiencing maltreatment during childhood exerts substantial stress on the child and increases the risk for overweight and obesity later in life. The current study tests whether hair cortisol—a measure of chronic stress—and its metabolite cortisone mediate the relation between abuse and neglect on the one hand, and body mass index (BMI) on the other.MethodThe sample consisted of 249 participants aged 8 to 87 years (M = 36.13, SD = 19.33). We collected data on child abuse and neglect using questionnaires, measured cortisol and cortisone concentrations in hair, and BMI. In a structural model, the effects of abuse and neglect on hair cortisol, hair cortisone, and BMI were tested, as well as the covariance between hair cortisol and BMI, and hair cortisone and BMI.ResultsWithin the sample, 23% were overweight but not obese and 14% were obese. Higher levels of experienced abuse were related to higher cortisone concentrations in hair (β = 0.24, p < .001) and higher BMI (β = 0.17, p =.04). Neglect was not related to hair cortisol, hair cortisone, or BMI. Hair cortisol and cortisone did not mediate the association between maltreatment, and BMI. Sensitivity analyses demonstrate the same pattern of results in a subsample of adult participants currently not living with their parents. However, in younger participants who were still living with their parents, the associations between abuse and cortisone (β = 0.14, p =.35) and abuse and BMI (β = 0.02, p =.92) were no longer significant.ConclusionThese findings confirm that experiencing abuse is related to higher BMI but suggest that hair cortisol and cortisone are not the mechanism underlying the association between child maltreatment and BMI. This is the first study to show abuse may be associated to elevated concentrations of hair cortisone—evidence of long-term alterations in chronic stress levels. Future research may benefit from exploring the effects of maltreatment on weight gain in longitudinal designs, including measures of other potential mediators such as eating as a coping mechanism, and more direct indicators of metabolic health.

Salivary cortisol response to psychosocial stress in the late evening depends on CRHR1 genotype

The activation of the hypothalamus-pituitary-adrenal (HPA) axis is induced by stress. Imbalances in this system increase the risk of developing stress related disorders including mental illness. Variants in the single nucleotide polymorphism (SNP) rs110402 of the corticotropin-releasing hormone receptor type I (CRHR1) gene have been shown in interaction with childhood maltreatment to increase the vulnerability to develop depressive symptoms in adulthood. In this study, the direct contribution of polymorphism of the CRHR1 gene (rs110402) to the salivary cortisol response to stress independently from childhood adversity was investigated. Healthy young men between the ages of 18 and 30, free from childhood maltreatment and early trauma, were genotyped (n = 121). To increase the power of the genetic analysis, only homozygous carriers of the common C (n = 31) and of the rare T (n = 21) allele were selected for this study and exposed to a Trier Social Stress Test (TSST) in the late evening (22.30 to 22.40). Salivary samples for the assessment of cortisol and its inactive metabolite cortisone were taken early in the evening (20.00), just before (22.30) and immediately after (22.40) as well as 15 minutes after stress exposure (22.55). Participants with the TT genotype showed higher cortisol levels 15 minutes post stress compared to participants with the CC genotype. No genotype differences were found for cortisone. Interestingly, TT participants reported lower subjective perceived stress levels before the TSST, but not after stress exposure. These results confirm that variants of rs110402 in the CRHR1 gene contribute to an increased stress response. Contrary to previous findings, however, this effect could be observed in subjects reporting no exposure to childhood maltreatment or early trauma.

Simultaneous Determination of Total Cortisol and Cortisone in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry: Method Development, Validation and Preliminary Clinical Application

Background:Cortisol as a major glucocorticosteroid product of the adrenal cortex which has been recognized as a stress biomarker in evaluating stress related disorders for a long time. Plasma concentration of cortisol and its metabolite cortisone are usually changed in physiological and psychological tension, anxiety and depression. In order to study these changes properly, we need a sensitive, accurate and reproducible assay for plasma cortisol and cortisone determination. &lt;/P&gt;&lt;P&gt; Objective: The aim of this study was to develop a sensitive and robust method for the determination of total cortisol and cortisone in human plasma using mass spectrometry.Methods:A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LCMS/ MS) method was developed, validated, and then the levels of cortisol and cortisone were determined. Plasma samples cleanup procedure was composed of two steps: the first was a protein precipitation with 1 % formic acid in acetonitrile, and the second was an on-line solid phase extraction (SPE). Afterwards, cortisol and cortisone were separated using a C18 ACQUITY UPLC BEHTM column with a gradient elution. The mobile phase A was 0.1 % formic acid in water, the mobile phase B was 0.1 % methanol. For the detection we used a XEVO TQ-S mass spectrometer operating in the ESI positive mode.Results:The time of analysis was 6.5 minutes and the quantification range was 5-600 ng/mL for cortisol and cortisone, with &gt; 94% recovery for all analytes (cortisol, cortisone and internal standards). The method was validated according to the EMA guideline for bioanalytical method validation.Conclusion:A simple and sensitive LC-MS/MS method was developed and validated for measurement of cortisol and cortisone in human plasma. Our findings indicate that the proposed analytical method is suitable for routine analysis.

Social defeat stimulates local glucocorticoid regeneration in lymphoid organs.

Stress is an important risk factors for human diseases. It activates the hypothalamic–pituitary–adrenal (HPA) axis and increases plasma glucocorticoids, which are powerful regulators of immune system. The response of the target cells to glucocorticoids depends not only on the plasma concentrations of cortisol and corticosterone but also on their local metabolism. This metabolism is catalyzed by 11β-hydroxysteroid dehydrogenases type 1 and 2, which interconvert glucocorticoid hormones cortisol and corticosterone and their 11-oxo metabolites cortisone and 11-dehydrocorticosterone. The goal of this study was to determine whether stress modulates glucocorticoid metabolism within lymphoid organs – the structures where immune cells undergo development and activation. Using the resident-intruder paradigm, we studied the effect of social stress on glucocorticoid metabolism in primary and secondary lymphoid organs of Fisher 344 (F344) and Lewis (LEW) rats, which exhibit marked differences in their HPA axis response to social stressors and inflammation. We show that repeated social defeat increased the regeneration of corticosterone from 11-dehydrocorticosterone in the thymus, spleen and mesenteric lymphatic nodes (MLN). Compared with the F344 strain, LEW rats showed higher corticosterone regeneration in splenocytes of unstressed rats and in thymic and MLN mobile cells after stress but corticosterone regeneration in the stroma of all lymphoid organs was similar in both strains. Inactivation of corticosterone to 11-dehydrocorticosterone was found only in the stroma of lymphoid organs but not in mobile lymphoid cells and was not upregulated by stress. Together, our findings demonstrate the tissue- and strain-dependent regeneration of glucocorticoids following social stress.

Differential Regulation of 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Patients with Differing Etiologies of Hypopituitarism

Pituitary patients with different etiologies of hypopituitarism exhibit differing phenotypes, despite similar replacement therapy strategies. We hypothesized that differential regulation of the isoenzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which mediates the net autocrine conversion of cortisone to cortisol in adipose tissues and liver, may play a role. We studied 11β-HSD1 activity (using urine cortisol/cortisone metabolites ratio) in 36 hypopituitary patients with treated craniopharyngiomas, treated remitted Cushing disease, and treated nonfunctioning pituitary adenomas + prolactinomas on and off growth hormone (GH) replacement. 11β-HSD1 activity was higher in subjects with craniopharyngioma both on and off GH, as evidenced by increased tetrahydrocortisol to tetrahydrocortisone metabolite ratios compared to other diagnostic groups, but there was no difference in body mass index, insulin levels, serum hormone measurements, or hydrocortisone dose between groups. Craniopharyngiomas are associated with enhanced 11β-HSD1 activity compared to other diagnostic hypopituitary groups, and this may contribute to the adverse phenotypic and metabolic features seen in this condition. BMI = body mass index; Em = cortisone metabolites; Fm = cortisol metabolites; GH = growth hormone; 11β-HSD1 = 11β-hydroxysteroid dehydrogenase type 1; IGF-1 = insulin-like growth factor 1; NFPA = nonfunctioning pituitary adenoma; THE = tetrahydrocortisone; THF = tetrahydrocortisol.