Cortisol Metabolites Research Articles

Effects of social stress on the welfare of captive male Alpine musk deer: Stereotypic behavior, fecal cortisol, and musk secretion

Wild Alpine musk deer (Moschus chrysogaster), which are native to the Qinghai-Tibet Plateau and surrounding areas, have been kept in captivity as an effective way to conserve musk deer ex situ and sustainably utilize musk resources. From 1 November to 31 December of 2019, the behaviors of 40 captive Alpine musk deer in Xinglongshan Musk Deer Farm (XMDF) in Gansu Province of western China were sampled by focal sampling and the all-occurrence recording method. Moreover, harvested musk was analyzed for muscone using gas chromatography (GC), and the adrenal cortisol metabolite levels of fecal samples were detected by radioimmunoassay (RIA). The effects of social housing modes (solitary deer per enclosure [SE] or group enclosures [GE]) on the behavior, hormones, and musk secretion of musk deer were investigated. The results showed that most captive male musk deer (75.0%) in the XMDF exhibited stereotypic behavior, with an average duration of stereotypic behaviors of 8.8% ( ± 0.5%) of the total observation time. The fecal cortisol metabolite level of musk deer with stereotypic behavior (48.1 ± 4.3 ng/g) was significantly higher than that of musk deer without stereotypic behavior (33.5 ± 6.6 ng/g). The proportion of stereotypic behavior was significantly lower in SE musk deer (50.0%) than in GE musk deer (83.3%), and the duration of stereotypic behavior (7.3% ± 0.3%) and fecal cortisol metabolite level (29.9 ± 3.1 ng/g) of the SE deer were significantly lower than those of GE (9.2% ± 0.6%; 49.4 ± 4.5 ng/g). There was no significant difference in musk yield and muscone content between SE musk deer (5.8 ± 1.4 g, 1.5% ± 0.3%) and GE musk deer (7.4 ± 0.8 g, 1.0% ± 0.1%). The results showed that the social housing mode is not significantly related to musk yield, muscone, or other performance indicators. It also showed that the stereotypic behavior and cortisol metabolites are significantly lower in SE musk deer. Therefore, we concluded that the animal welfare of captive musk deer can be improved through changes in social housing modes.

Resident wild koalas show resilience to large-scale translocation of bushfire-rescued koalas.

Wildlife translocation is increasingly utilized as a conservation management action, to mitigate the immediate negative effects of habitat loss and fragmentation (e.g. from land clearing or bushfires). Previous research has shown that stress responses can help or hinder survival in translocated wildlife and determine the efficacy of translocation as a conservation action. Yet these translocated animals are only one side of the equation, with translocation also potentially impacting the animals in the recipient population. We measured physiological markers of stress (faecal cortisol metabolite concentrations and neutrophil-lymphocyte ratios) and assessed health condition in a wild koala population one year after a major translocation of bushfire-rescued koalas on Kangaroo Island. We expected to find a high population density at the site (>0.75 koalas per hectare) and that resident koalas would show signs of chronic stress and ill health as a result of territorial conflict over food trees and reproductive opportunities. In contrast, we found that only one-fifth of the population remaining at the site were translocated koalas. The overall population density was also much lower (0.21 koalas per hectare) than anticipated. With no evidence of mass mortality at the site, we suggest that the majority of translocated koalas dispersed away from the site. Our stress marker measurements did not differ between the wild koalas and a sample of captive (non-display) koalas at the nearby Kangaroo Island Wildlife Park and were generally low compared to other studies. Veterinary examinations found that most koalas were in good body condition with very few diagnostic indicators of systemic ill health. Overall, our results suggest that, if there is adequate landscape-scale habitat connectivity and opportunity for dispersal, translocated koalas are likely to disperse from the site of release, with limited impacts on recipient koala populations at translocation release sites.

Seasonal variation in molecular and physiological stress markers in Asian elephants.

Free-living species exhibit seasonal variation in various life history traits, including vital rates such as birth and death patterns. Different physiological mechanisms are thought to underlie the expression of life history traits that contribute to lifetime fitness. However, although the broad impacts of seasonality on life history traits and trade-offs is well established in many systems, the exact physiological mechanisms responsible for driving differences within and between individuals are poorly understood. Among them, molecular and physiological stress pathways, such as stress hypothalamic-pituitary-adrenal axis and oxidative stress, have potential to mediate relationships between individual survival, reproduction and environmental seasonality. Here, we determine how different physiological markers of stress including faecal cortisol metabolites (FCMs), heterophils/lymphocytes (H/L) ratio, two markers indicating oxidative balance including a marker of oxidative damage (reactive oxygen metabolites, ROM) and a marker of antioxidant defences (superoxide dismutase, SOD) and body weight vary in a large semi-captive population of Asian elephants (Elephas maximus) exposed to extreme seasonality (e.g. elevated temperatures). Individuals showed higher FCM levels and H/L ratios during cold season, indicating increased stress, and the lowest FCM levels during monsoon season and H/L ratios during hot and dry season, but we found no pattern in oxidative stress (ROM and SOD) levels. Hot season also associated with a decline in body weight. The present study shows how different physiological parameters (FCM levels and H/L ratio), molecular (oxidative stress) and body condition vary with seasonal changes, and how these parameters might allow individuals to adapt to such variations. Our results on an endangered long-lived species are crucial in indicating the most productive timing for conservation efforts, predicting how individuals cope with environmental changes, and allow for a more accurate representation of how animal physiology operates in nature.

Comparing the agreement of a commercial cortisol kit with a biologically validated assay in evaluating faecal cortisol metabolite levels in koala joeys

Orphaned koala joeys constitute a substantial number of wildlife rescues. Mortality is highly prevalent in rehabilitating joeys, with little knowledge about the causes of mortality. The hypothalamic–pituitary–adrenal axis plays a vital role in mediating stress by producing glucocorticoids (e.g. cortisol), however, no studies have quantified glucocorticoids in koala joeys. Traditional cortisol enzyme immuno-assay (e.g. R4866) are limited in supply and are process intensive, whereas, modern enzyme immuno-assay (EIA) kits (e.g. Arbor Assay cortisol kit) are available world-wide and provide rapid results. Biological validation is unsuitable to be performed in recuperating joeys due to ethical considerations, hence, we compared the results from biologically validated R4866 assay with the commercially available Arbor Assay cortisol kit. Thirty-four faecal samples were collected, processed and analysed for faecal cortisol metabolites (FCM) using both, R4866 assay and Arbor Assay kit. The joeys presented a suite of clinical conditions which provided the natural variation in stress response for comparing the assay sensitivity and range. The results indicated that there were no significant differences between the FCM values measured by both the assays. Furthermore, the Bland-Altman plot indicated a very strong agreement between the FCM concentrations measured by the two assays. This study is only a step towards recommending the routine use of commercial kit in clinical settings with basic resources, for rapid quantification of stress in koala patients. It is crucial for future studies to perform laboratory validation procedures to confirm the efficacy of the commercial kit before practical use for FCM monitoring in koalas.

Stress hormones are associated with inflammatory cytokines and attenuation of T-cell function in the ascites from patients with high grade serous ovarian cancer

Mounting evidence suggests that chronic stress and subsequent distress can promote ovarian cancer progression. These altered psychological states have been linked to sustained release of stress hormones, activation of the β-adrenergic receptors in ovarian cancer cells, and induction of pro-tumoral signaling pathways. In addition, data suggest that chronic stress promotes an inflammatory landscape highlighted by increased infiltration of tumor-associated macrophages into the ovarian tumor microenvironment (TME). In ovarian cancer, ascites is a unique TME comprised of tumor, and immune cells, which secrete pro-tumoral cytokines and chemokines that modulate tumor-associated immunity. However, our knowledge about how stress hormones impact the ascites TME remains limited. We hypothesized that the ascites harbors measurable levels of stress hormones, and accumulation of these in the ascites generates a pro-tumorigenic, inflammatory, and immunosuppressive TME. We evaluated ascites samples from 49 patients with high grade serous ovarian cancer (HGSOC) and quantified cortisol and stress hormones metabolites, metanephrine (MN), and normetanephrine (NMN) in all samples. We also measured 38 individual cytokines in the ascites, including several pro-inflammatory cytokines, such as IL-6, which were positively correlated to MN or NMN levels of those samples. Conversely, we found cortisol levels were negatively correlated to several pro-inflammatory cytokines. As T-cells are integral to the TME and our analyses identified cytokines in the ascites known to modulate T-cell function, we characterized ascites-derived T-cells and assessed the impact of stress hormones on the T-cell phenotype. Our data show an altered CD4+/CD8+ T-cell ratio and a heterogeneous expression of exhaustion markers in T-cells from the ascites, while ascites-derived CD8+ T-cells exposed to epinephrine had decreased co-expression CD38 and Granzyme B. To extend these findings to animal models, we subjected ovarian cancer-bearing mice to daily restraint stress, which resulted in increased tumor growth in two models. Congruent with our human analyses, we detected corticosterone, MN, and NMN in the ascites from tumor-bearing mice, and these stress hormones correlated with several inflammatory cytokines. Moreover, daily restraint stress leads to increased CD4+PD-1+/CD8+PD-1+ T-cell ratio in the ovarian tumor microenvironment. Overall, these data highlight a role of stress hormones in the ascites TME as a driver of tumor-associated inflammation, T-cell suppression, and disease progression.

Heart rate and faecal cortisol metabolites measurements in horses at the Sechseläuten in Zurich

The Zurich's Sechseläuten is a traditional festival of the Zurich guilds, in which around 500 horses take part. After a parade through the old town of Zurich, the riders gather at a big square to canter around a burning woodpile topped with an exploding effigy (the «Böögg»). The level of stress experienced by the horses partaking in this event is subjected to increasing scrutiny. The aim of this study was to evaluate the stress load of the horses participating in the Sechseläuten procession more objectively by measuring heart rate and faecal cortisol metabolites. Twenty-three horse-rider pairs were voluntarily recruited from the guilds for participation in the study. For comparison purposes, three sections of the procession were simulated in terms of gait and distance travelled in a normal riding environment during a test day and observed during the Sechseläuten itself 16 days later: Counter march Bahnhofstrasse, parade Limmatquai and parade around the burning «Böögg». All horses were equipped with a heart rate measuring system with GPS tracking (Polar Team Pro®, Polar Electro Oy, Finland). Faecal samples for measuring cortisol metabolites were taken rectally immediately before and 24 hours after the events. Thirteen of the 23 horses were sedated during the Sechseläuten (Acepromazine, 0,1-0,2 mg/kg p.o.). The differences between the observation periods and groups (sedated/unsedated) were tested by analysis of variance (P.

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.

RF21 | PSAT100 HPA Axis Modulation by a Potent Inhibitor Indicates 11Beta-Hydroxysteroid Dehydrogenase Type 1 (HSD-1) Is a Significant Contributor to Cortisol Levels

Abstract Background HSD-1, an intracellular enzyme, converts cortisone to cortisol in target tissues in which cortisol excess is associated with morbidity including liver, adipose, bone, brain, muscle, skin, and eye. SPI-62 is a potent HSD-1 inhibitor in Phase 2 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. Methods We analyzed data from a multiple ascending dose trial (1) to characterize the contribution of HSD-1 to intracellular cortisol that can bind to intracellular receptors as well as HPA and HPG axis modulation by SPI-62. Data from all non-elderly subjects who received 10-50 mg SPI-62 (doses that achieved maximal liver HSD-1 inhibition) were combined for analysis. ANCOVA models with a treatment effect and baseline covariate were conducted; reported statistics are least squares means and standard errors. Results Compared to placebo (n=10), single doses of SPI-62 (n=40) were associated with 24-hour urinary tetrahydrocortisol (2.27[0.134] v 4.44[0.269] mmol) and allotetrahydrocortisol (2.98[0.146] v 4.80[0.291] mmol) decreases, and 24-hour urinary tetrahydrocortisone (32.71[1.149] v 9.19[2.300] mmol) increase. Serum cortisol was decreased at 2-hours (152.2[11.64] v 226.6[24.00] nM) but not at 4- or 12-hours post-dose. ACTH was increased at 4- and 12-hours (45.6[1.64] v 32.1[3.29]; 38.5[1.67] v 26.3[3.33] pg/mL) but not at 2-hours post-dose. After 14 daily doses, SPI-62 was associated with 24-hour urinary tetrahydrocortisol (2.05[0.154] v 4.36[0.321] mmol), allotetrahydrocortisol (2.75[0.181] v 4.13[0.377] mmol), and tetrahydrocortisone (42.73[1.968] v 8.51[0.410] mmol) changes. ACTH was increased at pre-dose and 2-, 4-, and 12-hours post-dose (47.3[2.13] v 30.4[4.45]; 45.6[1.71] v 27.0[3.61]; 45.3[1.79] v 35.2[3.72]; 34.4[1.71] v 18.8[3.57] pg/mL). No differences on urinary cortisol or cortisone, serum cortisone, or CRH were observed after single or multiple doses. After multiple doses, SPI-62 was associated with increased DHEA-S (342.2[9.94] v 155.0[20.52] mg/dL) and, in females, testosterone (2.1[0.11] v 1.4[0.25] nM). No differences on aldosterone, estradiol, FSH, LH, progesterone, or SHBG were observed. Discussion Single SPI-62 doses resulted in ∼40-50% decreases of urinary cortisol metabolites which indicate similar decrease of hepatocellular cortisol. Following a corresponding decrease, circulating cortisol homeostasis was restored rapidly by ACTH increase. Urinary cortisol was not affected. As other HSD-1 inhibitors, SPI-62 is associated with moderate androgen increases that, to date, appear not to be associated with adverse effects. As HSD-1 contributes much of the intracellular cortisol that can access intracellular receptors in target tissues for cortisol morbidity, we hypothesize that HSD-1 inhibitors have potential as treatments for conditions of cortisol excess such as Cushing's syndrome and autonomous cortisol secretion. Reference (1) Bellaire et al., Clin. Transl. Sci. 2019 May;12(3): 291-301. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 1:06 p.m. - 1:11 p.m.

Age and spatio-temporal variations in food resources modulate stress-immunity relationships in three populations of wild roe deer

Living in variable and unpredictable environments, organisms face recurrent stressful situations. The endocrine stress response, which includes the secretion of glucocorticoids, helps organisms to cope with these perturbations. Although short-term elevations of glucocorticoid levels are often associated with immediate beneficial consequences for individuals, long-term glucocorticoid elevation can compromise key physiological functions such as immunity. While laboratory works highlighted the immunosuppressive effect of long-term elevated glucocorticoids, it remains largely unknown, especially in wild animals, whether this relationship is modulated by individual and environmental characteristics. In this study, we explored the co-variation between integrated cortisol levels, assessed non-invasively using faecal cortisol metabolites (FCMs), and 12 constitutive indices of innate, inflammatory, and adaptive immune functions, in wild roe deer living in three populations with previously known contrasting environmental conditions. Using longitudinal data on 564 individuals, we further investigated whether age and spatio-temporal variations in the quantity and quality of food resources modulate the relationship between FCMs and immunity. Negative covariation with glucocorticoids was evident only for innate and inflammatory markers of immunity, while adaptive immunity appeared to be positively or not linked to glucocorticoids. In addition, the negative covariations were generally stronger in individuals facing harsh environmental constraints and in old individuals. Therefore, our results highlight the importance of measuring multiple immune markers of immunity in individuals from contrasted environments to unravel the complex relationships between glucocorticoids and immunity in wild animals. Our results also help explain conflicting results found in the literature and could improve our understanding of the link between elevated glucocorticoid levels and disease spread, and its consequences on population dynamics.

The use of fecal cortisol for enrolment of free-roaming dogs in Singapore to a national rehabilitation-rehoming program: A possible indicator of allostasis

Singapore's trap-neuter-release-management program strives to rehome as many free-roaming dogs as possible. However, before rehoming, most free-roaming dogs require rehabilitation and this requires sheltering them for some time. Therefore, an individual's suitability for rehoming hinges on his or her adaptability to shelter conditions. Currently, this is being assessed qualitatively across behavioral contexts. Augmenting the current assessment with a known bioindicator of stress, such as cortisol, would increase its robustness. However, the serum measurement of cortisol can be invasive. Canine fecal samples have presented an attractive alternative for measuring cortisol indirectly in various contexts. Nevertheless, it is unclear whether the resolution of fecal cortisol metabolites (FCM) is sufficient for evaluating Singapore free-roaming dogs’ adaptability to shelter conditions. To elucidate this, we performed linear mixed-effects model on 27 candidate models to compare FCM between pet and recently sheltered dogs, with sex, state of the feces, and time as covariates. Subsequently, we ran generalized mixed-effected model on 12 candidate models to examine the influence of between- and within-subject variations in FCM on the prognosis of sheltered dogs—based on our current assessment. Our analyses showed that FCM levels of recently sheltered dogs were higher than pet individuals. However, unlike several studies, FCM concentrations of recently sheltered dogs continued to rise, indicating that individuals have yet to adapt. Furthermore, the difference in FCM levels between newly sheltered individuals and pet dogs was moderated by sex. Unexpectedly, we found the average FCM levels of dogs with a good prognosis to be higher than those that received a poor prognosis, with sex and state of the fecal samples as important covariates. This may be due to the difference in allostasis between the 2 groups of dogs. Thus, our results showed FCM not only has sufficient resolution but is useful in evaluating dogs’ adaptability to shelter conditions.

Excretion of oxidated cortisol metabolites is markedly lower than previously assumed: An analysis of urinary cortoic acids in healthy children by GC–MS

Discovered about 50 years ago, the four C21 steroidal acids (α-)cortolic acid, β-cortolic acid, (α‑)cortolonic acid and β-cortolonic acid present the oxidative end products of cortisol metabolism. Undergoing renal elimination, these cortoic acids have been assumed to constitute up to 25 % of total urinary cortisol metabolites. However, their analysis has been difficult, only few data has been published in adults, and this class of steroids has become practically forgotten. Since data in children are lacking and nothing is known about their metabolism during human development, we aimed at establishing a more practical analytical method and determined their urinary concentrations in a high number of healthy subjects. In our method, 5-mL-aliquots of 24-hour urine samples were subjected to solid phase extraction (C18 cartridges), followed by strong anion exchange chromatography, and formation of 2-propylester-trimethylsilylether derivatives (2-PR/TMS). The cortoic acids were quantified by targeted gas chromatography–mass spectrometry (GC–MS) using a nonpolar GC column and selected ion monitoring (SIM). Baseline separation of all cortoic acids was achieved. Calibration graphs were linear (R2 > 0.98). Variations in precision and accuracy were less than 15 %, respectively. The detection limit was 100 pg (injected) with a signal-to-noise ratio of 3. 240 specimens from 24-hour urine collections from healthy children (120 boys, 120 girls, aged 3–18 years; DONALD study) were analyzed for cortoic acids and neutral cortisol metabolites to create first reference ranges. The profile of cortoic acids was dominated by α-cortolonic acid with excretion rates up to 70 µg/d. Absolute excretion rates of cortoic acids increased with age, their total excretion rates ranged between 11.0 and 127.3 µg/d (median 45.7 µg/d), but did not show any sexual dimorphism. Since cortoic acids make up only about 1 % of total urinary cortisol metabolites, determination of neutral urinary steroids reliably allows assessment of cortisol production. However, cortoic acids might present potential biomarkers of the body’s redox state.

Heifers don't care: no evidence of negative impact on animal welfare of growing heifers when using virtual fences compared to physical fences for grazing

Virtual fencing (VF) represents a way to simplify traditional pasture management with its high labour and cost requirements for fencing and to make better use of the ‘beneficial’ agronomic and ecological effects of livestock grazing. In this study, the VF technology (® Nofence, AS, Batnfjordsøra Norway) was used with Fleckvieh heifers to investigate possible welfare impacts on the animals compared to conventionally fenced animals when they were trained to respond correctly to the system. The Nofence® collars (attached to the neck of the heifers) send acoustic signals as a warning when the animals approach the VF line, which was set up by GPS coordinates within the Nofence®-App, followed by an electric pulse when they do not stop or return. The heifers had no experience with VF prior to the study. Two treatments (VF versus physical fencing (PF)) were applied to six groups of four heifers each (three groups per treatment) over three 12-day time replicates. One VF line separated the pasture of the VF group into an accessible or non-accessible area. The control group had a PF line. Both groups were equipped with Nofence® collars (deactivated for the PF group). The trial took place on two adjacent paddocks of 1 000 m2 each following a 12-day schedule which was divided into three sections: visual support of the VF line by a physical barrier (first 2 days), only virtual border without visual support, moving the VF line (on day 8). Each time replicate followed the next successively on different paddocks with two new groups of heifers, which were grazed 5 h daily. During the whole experiment, the behaviour of each of the four animals per group was continuously observed; 2 h a.m., 2 h p.m. Exclusion by the VF line was effective in our trial. None of the heifers crossed the virtual boundary, i.e. the time spent in exclusion zone was zero. The heifers received 2.70 ± 2.63 acoustic signals and 0.30 ± 0.36 electric pulses (mean ± SD) per heifer and hour during all time replicates. Main cattle behaviour on pasture was not affected by the fencing system. Live weight gain, herbage consumption and faecal cortisol metabolites also revealed no significant differences. The duration until the heifers restarted grazing after an electric pulse from the Nofence® collar was significantly shorter than after an electric pulse from the physical fence. We can summarise that in our study, cattle well-being on pasture was not negatively affected by VF compared to PF.

Cortisol metabolites vary with environmental conditions, predation risk, and human shields in a wild primate, Cercopithecus albogularis

Environmental challenges are often associated with physiological changes in wildlife that allow animals to maintain homeostasis. Among these, scarcity in resources, and risks from predators, competitors, and humans can all result in psychological and physiological stress. Yet, for habituated species, it is not clear whether this relationship with humans still holds to a lesser degree or is outweighed by the benefits of human presence – such as serving as a buffer from competitors or predators. We investigated how human presence and environmental challenges such as resource availability, weather, predation, and competition may be associated with variation in fecal cortisol metabolite levels (FCMs) in a group of samango monkeys (Cercopithecus albogularis) in the Soutpansberg Mountains, South Africa. FCMs can often broadly track environmental challenges and perturbations. Initially, we employed an exploratory analysis comparing candidate models representing biological hypotheses and found that those incorporating information on human presence had less weight than models for food availability, thermoregulation, and water scarcity. When we examined a subset of the data that included information on intergroup competition and predator alarm calls, we found that FCMs were higher on the day following potential predator encounters but not competitive interactions. As observer numbers increased, responses to predators flattened, indicating that the presence of several humans might deter predators and/or affect samangos' perception of danger – yet we could not distinguish between these possibilities. Together, these results suggest that ecological perturbations track with FCMs in this study population and challenge long-held assumptions that human presence has negligible effects on habituated study animals.

Metformin-Treatment Option for Social Impairment? An Open Clinical Trial to Elucidate the Effects of Metformin Treatment on Steroid Hormones and Social Behavior.

Background: Social behavior is mediated by steroid hormones, whereby various lines of evidence indicate that metformin might improve the symptoms of social withdrawal. This directly yields to the aim of the study to correlate the impact of metformin treatment on the potential alterations in steroid hormone homeostasis, which is ultimately impacting social behavior. Therefore, urinary samples of patients before and after treatment with metformin will be correlated to social behavior to elucidate potential changes in steroid hormone profiles and social behavior. Material and Methods: An observational study in healthy adults with a new indication for metformin. Steroid hormone analysis, including the most prominent androgen, estrogen, progesterone, aldosterone, corticosterone, cortisone and cortisol metabolites analyzed with gas chromatography–mass spectrometry and a questionnaire on social behavior (Autism Spectrum Questionnaire (AQ)) will be administered prior to and after around a 12-week phase of metformin treatment. Discussion: It is likely that due to different pathophysiological mechanisms such as an effect on the respiratory chain in mitochondria or via AMP-activated protein kinase, a general alteration of steroid hormone levels can be detected prior to post treatment. The encompassing measurement of steroid hormones shall give hints concerning the involvement of specific cascades yielding potential pharmacological targets for future research.

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.

A Proposal for Nomenclature Revision of Nonfunctioning Adrenal Incidentalomas as Adrenal Lesions of Undetermined Secretion of Adrenal Steroids (ALUSAS)

In this commentary we propose a change of the nomenclature of non-functioning adrenal incidentalomas (NFAI) to adrenal lesions of undetermined secretion of adrenal steroids (ALUSAS) since there are mounting evidence suggesting that a substantial proportion of so-called NFAIs do actually secrete steroids in excess. In fact, it is known that some NFAIs develop ACS over time. In the commentary we justify the change of term by summarizing the evidence that shows that NFAIs present a higher cardiometabolic risk than the general population, and the metabolomics studies in which an increase in cortisol metabolites is observed in patients with NFAI.

Abstract 2543: Chronic stress promotes tumor-associated inflammation in high-grade serous ovarian cancer

Abstract A cancer diagnosis increases stress hormones levels and leads to altered psychological states. In the context of ovarian cancer (OC), chronic stress promotes tumor growth, chemoresistance and modulates immune cell populations in the tumor microenvironment (TME). Moreover, previous work from our team suggests that chronic stress promotes an increased inflammatory response in OC. Our data show an altered CD4+/CD8+ T-cell ratio and a heterogeneous expression of exhaustion markers in patients with high-grade serous ovarian cancer (HGSOC). Therefore, we hypothesized that chronic stress results in chronic inflammation and an immunosuppressed TME. To address this, we obtained ascites from 66 patients with HGSOC and measured cytokine levels using a comprehensive Cytokine/Chemokine immunoassay. Cortisol, corticosterone, and stress hormone metabolites (metanephrine and normetanephrine) levels from ascites were measured by ELISA. CD8+ T-cells isolated from OC patient ascites were stimulated with epinephrine, and flow cytometry was used to measure co-expression of CD38 activation marker and Granzyme B, an essential mediator of CD8+ T-cell killing capacity. To further establish the impact of chronic stress on tumor progression, we subjected IG10 or ID8 tumor-bearing C57/BL6 female mice to daily restraint stress. Mice were sacrificed 8-12 weeks after inoculation, data collected, and ascites stored. Results showed an increase in inflammatory cytokines (Eotaxin, IL-6, and IL-7) in recurrent tumors of HGSOC patients. IP-10 and IFN-γ were negatively associated with cortisol levels. Moreover, normetanephrine levels positively correlated with inflammatory cytokines: IL-6, MCP-1, MCP-3, VEGF, GRO, and IL-7. Metanephrine was also positively correlated with inflammatory cytokines: SCD40L, FGF-2, and MIP1α. Ascites-derived CD8+ T-cells treated with epinephrine showed decreased co-expression CD38 and Granzyme B. Additionally, data show that daily restraint stress led to increased tumor growth in ID8 and IG10 syngeneic mouse models of OC. Ascites derived from our animal models suggest a positive correlation of inflammatory cytokines VEGF, TNFα and Eotaxin, and corticosterone levels in ID8 tumor-bearing mice. These results suggest a role for stress hormones in inflammation and immunosuppression. In conclusion, inflammatory cytokines are upregulated in recurrent HGSOC ascites samples. Cytokines that regulate T-cell function were negatively associated with cortisol levels, while stress hormone metabolites correlated with higher inflammatory cytokines. Moreover, epinephrine stimulation decreased ascites-derived CD8+ T-cell function. Inflammatory cytokines correlated with corticosterone levels in ID8 tumor-bearing mice. Overall, these data suggest a role for chronic stress in inflammation and immunosuppression, impacting the efficacy of therapies that aim to restore T-cell function. Citation Format: Alexandra N. Aquino-Acevedo, Hope Knochenhauer, Melanie Ortiz-León, Yadiel A. Rivera-López, Margarita Bonilla-Claudio, John S. Yi, Rebecca A. Previs, Guillermo N. Armaiz-Pena. Chronic stress promotes tumor-associated inflammation in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2543.

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.