Plasma Free Cortisol Research Articles

7990 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background: Corticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods: 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution & ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results: AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p<0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion: Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

9225 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background Corticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution & ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p<0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

A Cross-Sectional Survey Study on the Diagnosis and Management of Critical Illness-Induced Corticosteroid Insufficiency in Saudi Arabia.

Background The presence of critical illness-induced corticosteroid insufficiency(CIRCI) is correlated with elevated concentrations of circulating biomarkers associated with inflammation and coagulation in multiple domains. The management of adrenal insufficiency remains a topic of ongoing debate and disagreement among endocrinologists and intensivists. This study aimed to assess the extent of understanding regarding CIRCIamong endocrinologists and intensivists who are actively practicing in Saudi Arabia. Methods This is an online cross-sectional survey study that was conducted between June and August 2023 to assess knowledge of CIRCI among endocrinologists and intensivists working in Saudi Arabia. The questionnaire tool for this study was constructed based on a previous literature review. Binary logistic regression analysis was used to define factors that affect participants' knowledge of CIRCI. Results A total of 76 physicians were involved in this study. Around 32.9% (n= 25) of the participants described CIRCI correctly as an impairment of the hypothalamic-pituitary axis during critical illness. Around 35.5% (n=27) of the participants identified that widespread use of corticosteroids in critically ill patients prompted the need to revisit the concept, diagnosis, and management of CIRCI, and a similar proportion of the participants (35.5%) (n=27) identified that the role of corticosteroids in the management of CIRCI in critically ill patients may be beneficial in selected cases. Around 42.1%(n=32) of the participants identified that CIRCI is specific to critically ill patientswhile AI can occur in any individual. Around 17.1%(n=13) of the participants confirmed that there is no task force agreement on whether corticosteroids should be used in adult patients with sepsis but without shock. The mean knowledge score of the study participants was 3.6 (sd: 2.2) out of 10, which demonstrates a weak level of knowledge of CIRCI (36.0%). Binary logistic regression analysis identified that physicians from the southern and western regions were less likely to be knowledgeable of CIRCI compared to physicians from the central region (p< 0.05). Conclusion The study revealed that the level of familiarity with CIRCI among endocrinologists and intensivists in Saudi Arabia fell short of the desired benchmark. Clinicians may opt to utilize delta cortisol levels following cosyntropin administration and random plasma cortisol levels as diagnostic measures for CIRCI, instead of relying on plasma-free cortisol or salivary cortisol levels in conjunction with plasma total cortisol. Adherence to customized treatment protocols is crucial to attain the most favorable results for patients.

Low Urinary Free Cortisol as a Risk Factor for Patients with Variceal Bleeding.

Background and Objectives: Specificity and reliability issues of the current cortisol assessment methods lead to limitations on the accurate assessment of relative adrenal insufficiency. Although free cortisol provides a more accurate evaluation of adrenal cortisol production, the expense and time-consuming nature of these assays make them impractical for routine use. Research has, thus, focused on alternative methods, such as indirectly measuring free cortisol using Coolens' equation or directly assessing salivary cortisol concentration, which is considered a more favorable approach despite associated challenges like sampling issues and infection risks. The aim of this study was to explore correlations between 24 h urinary free cortisol (UFC), free plasma cortisol, serum total cortisol, and salivary cortisol as potential reliable indices of free cortisol in the setting of variceal bleeding. Additionally, we assessed the predictive value of UFC for 6-week mortality and 5-day treatment failure in patients with liver cirrhosis and variceal bleeding. Materials and Methods: A total of 40 outpatients with liver cirrhosis and variceal bleeding were enrolled. Free cortisol levels in serum, saliva, and urine were assessed using the electrochemiluminescence immunoassay method. For the measurement of plasma-free cortisol, a single quadrupole mass spectrometer was employed. The quantification of free cortisol was fulfilled by analyzing the signal response in the negative ESI-MS mode. Results: UFC was significantly correlated to free plasma cortisol. Negative correlations were demonstrated between UFC, the Child-Pugh (CP) score, and C reactive protein (CRP) levels. In the multivariate analysis, CP stage C was associated with 6-week mortality risk and portal vein thrombosis with 5-day treatment failure using Cox regression and binary logistic regression analyses, respectively. Patients who experienced rebleeding, infection, or death (or any combination of these events) presented with lower levels of UFC. Conclusions: This study suggests that low levels of UFC may impose a risk factor for patients with liver cirrhosis and variceal bleeding. The use of UFC as an index of adrenal cortisol production in variceal bleeding warrants further investigation.

THU436 Small Intestinal Bacterial Overgrowth Causing Osteoporosis In An Adult Male

Abstract Disclosure: S. Aziz: None. S.A. Patrick: None. V. Mupparaju: None. S. Kode: None. Background: Small intestinal bacterial overgrowth (SIBO) is a disease caused by intestinal dysbacteriosis which can lead to nutrients malabsorption and chronic metabolic acidosis which affect bones health and cause osteoporosis. Clinical Case: A 45-year-old man with medical issues of GERD presents to the endocrinology clinic in 02/2022 for evaluation of incidental finding of T5 vertebral fracture noted in the chest x-ray done for evaluation of chest tightness. During the clinic visit the patient reported chronic bloating and gassiness. Denied symptoms suggestive of eating disorders, chronic fatigue, decreased libido, recurrent fractures, elastic skin, hypermobile joints or joint dislocation, and steroid use. Patient is married and has two biological children. He drinks alcohol socially and denied tobacco use. Patient was taking cholecalciferol 1000 IU daily at home. His weight is 64 kg with a BMI of 21 kg/m2. Physical examination did not reveal any abnormality. Bone mineral density showed the worst Z-score of -2.3 in his spine. Workup for osteoporosis included 24-hour urine calcium, CBC, CMP (including serum calcium, phosphorus, electrolytes, ALP, and creatinine), PTH, TSH, free plasma cortisol, ACTH, morning total testosterone, 25-hydroxyvitamin D level, SPEP, UPEP, celiac disease screening tests (tissue transglutaminase IgA and tTG IgG), IGF-1 and HbA1c were all within normal limits. The patient was started on alendronate 70 mg once weekly and was referred to a local gastroenterologist for further evaluation of his GI symptoms; he subsequently was diagnosed with SIBO which is the most likely cause for his osteoporosis. Conclusion: SIBO is a disease caused by intestinal dysbacteriosis, which is caused by replacement of physiologic bacteria by pathogenic bacteria from the large intestine leading to maldigestion and malabsorption of nutrients including fat, carbohydrates and protein leading to vitamins deficiencies and metabolic acidosis. Due to impaired absorption of nutrients it can cause systemic complications including osteoporosis. Fat malabsorption can lead to deficiencies of fat-soluble vitamins A, D, K and E. SIBO can also result in protein-losing enteropathy. The state of chronic metabolic acidosis caused by SIBO increases alkali mobilization from the bone and induces dissolution of the bone thus promotes the development of osteoporosis. Gastrointestinal diseases including SIBO should be considered in cases of osteoporosis of unknown origin. Presentation: Thursday, June 15, 2023

Development and Validation of Liquid Chromatography/Stable Isotope-Dilution Tandem Mass Spectrometry Method for Measurement of Dexamethasone, Total Cortisol and Free Cortisol in Human Plasma

Background Cortisol plays a role in the regulation of metabolic homeostasis. Dexamethasone is a powerful synthetic glucocorticoid that acts as an anti-inflammatory and immunosuppressive agent. Plasma free cortisol, which has a low concentration in the blood, is the cortisol fraction that provides biological activity. It is emphasized that measurement of dexamethasone, total and free cortisol levels can directly affect the results of dexamethasone suppression test, which is routinely used to exclude endogenous hypercortisolemia in patients with adrenal adenoma, and is important in differential diagnosis.&#x0D; Purpose In this study, a precise and rapid method was developed for the measurement of dexamethasone, total and free cortisol levels in plasma samples, besides demonstrating the clinical applicability of the method with samples from adrenal adenoma patients. &#x0D; Methods Method optimization studies were performed using liquid chromatography-tandem mass spectrometry with stable isotope dilution-multiple reaction monitoring. &#x0D; Results The assay demonstrated a good linear dynamic range of 0.5–20 μg/L, 5–200 μg/L and 0.5–100 μg/L for dexamethasone, total and free cortisol, respectively. The values for intra- and inter-day precisions of analytes were ≤6.9% with the accuracies ranging from 91.6% to 113.0%.&#x0D; Conclusion Measurement of these parameters can be used reliably to diagnose diseases causing hypercortisolemia.

PS-C21-3: URINARY FREE CORTISOL EXCRETION AS A MARKER OF DECREASED BONE DENSITY IN PATIENTS WITH ADRENAL CUSHING'S SYNDROME

Objective: Patients with adrenal Cushing's syndrome (CS) have a poor prognosis due to the autonomous hypersecretion of endogenous cortisol from the adrenal gland. Although several clinical examinations have been developed to manifest the autonomous hypersecretion of endogenous cortisol in this syndrome, it is unknown which parameter of cortisol overproduction is best associated with adrenal cortisol content and the bone and vascular complications of this syndrome. Therefore, the present study was undertaken to evaluate the parameter(s) associated with adrenal cortisol content in patients with adrenal CS, as well as to assess the associations between these parameters and the vascular and bone complications of this syndrome. Design and method: Patients with CS who were over 18 years old and had undergone adrenalectomy for this syndrome were enrolled in this study. Patients who were pregnant, had an allergy to iodine, or who were receiving medications that may interfere with the evaluation of 131I-adosterol single-photon emission computed tomography (SPECT) /computed tomography (CT) were excluded. Those taking medications that may interfere with the evaluation for the diagnosis of CS were also excluded. Relationships between office blood pressure and pulse rate, urinary examinations, blood examinations, endocrinological examinations, CT and 131I-adosterol SPECT/CT data, bone density, physiological function tests, and adrenal cortisol concentration/content were examined. In addition, relationships between the parameter associated with adrenal cortisol concentration/content and complications of CS were investigated. Results: Cardiovascular factors, except for diastolic blood pressure, failed to show an association with adrenal cortisol content. 24-h urinary free cortisol excretion (ρ = 0.893, P = 0.007) and plasma cortisol levels after the 1 mg (ρ = 0.857, P = 0.014) or 8 mg (ρ = 0.900, P = 0.037) overnight dexamethasone suppression test, but not plasma cortisol levels in the morning or at late night or indices of 131I-adosterol SPECT/CT, were significantly and positively associated with adrenal cortisol content. 24-h urinary free cortisol excretion, and not plasma cortisol levels after the 1 or 8 mg overnight dexamethasone suppression test was significantly and negatively associated with lumbar spine bone density (lumbar spine T-score, ρ = -0.883, P = 0.009; lumbar spine Z-score, ρ = -0.883, P = 0.009). Conclusions: 24-h urinary free cortisol excretion is best associated with a decrease in bone density via increased adrenal cortisol overproduction and suggested that it could be used as a marker for osteoporosis in these patients.

RF09 | PSAT88 Pro-opiomelanocortin (POMC), ACTH and Cortisol Responses to Pediatric Cardiac Surgery

Abstract Introduction In critically ill adults, high plasma free cortisol in face of suppressed plasma ACTH coincides with high plasma pro-opiomelanocortin (POMC), the ACTH precursor (1). Further augmenting the systemic glucocorticoid availability with hydrocortisone treatment further lowered plasma ACTH, while plasma POMC remained unaffected (2). The dynamics of POMC in relation to ACTH and free cortisol prior to intensive care unit (ICU) admission are unknown, as well as the impact hereon of glucocorticoid treatment. Also, elevated plasma POMC concentrations have not yet been confirmed in pediatric ICU (PICU) patients. In pediatric cardiac surgery-induced critical illness, we hypothesized that plasma POMC is elevated and that plasma ACTH transiently rises per-operatively and becomes suppressed once free cortisol has risen on PICU day 1 and 2. In addition, we hypothesized that in patients receiving glucocorticoids per-operatively, plasma ACTH is further suppressed while plasma POMC is unaffected. Methods From 53 children aged 0-36 months, who underwent cardiac surgery and were admitted to the PICU, blood samples were taken prior to surgery (pre-operatively, available samples N=51), during surgery (per-operatively, N=47), on the following morning (PICU day 1, N=40) and the day hereafter (PICU day 2, N=24). Blood samples were also collected from 24 age- and gender-matched healthy children. To investigate the impact of synthetic glucocorticoid treatment on the endogenous plasma hormone concentrations, patients were categorized into steroid-naive (total N=38) and steroid-treated patients (total N=15). Plasma hormone concentrations were quantified with highly-specific ELISA (POMC), RIA (ACTH, cortisol, CBG) or colorimetric assays (albumin). Cross reactivity with synthetic glucocorticoids within the used cortisol RIA is minimal (&amp;lt;0.27% for methylprednisolone, &amp;lt;0.1% for dexamethasone). Free cortisol was estimated with the adapted Coolens’ formula. Results Plasma POMC was increased pre-operatively (p&amp;lt;0.0001) but no longer thereafter (p&amp;gt;0.05), irrespective of steroid treatment. Plasma ACTH in patients was never higher than in healthy children. While in steroid-naive patients, plasma ACTH became suppressed only by PICU day 1 (p&amp;lt;0.0001), steroid-treated patients had already suppressed plasma ACTH per-operatively (p≤0.0001). In steroid-naive patients, plasma free cortisol was increased from per-operatively onwards (p≤0.002), while in steroid-treated patients, plasma free cortisol (endogenous) concentrations always remained comparable to those of healthy children (p&amp;gt;0.05). Conclusion Pre-operatively high plasma POMC, not followed by increased ACTH, suggests a centrally-activated HPA-axis already prior to surgery and an immature pituitary processing of POMC into ACTH. Systemic glucocorticoid availability is elevated from surgery onwards, likely driven by ACTH-independent mechanisms. Further increasing systemic glucocorticoid availability exogenously with glucocorticoid administration accelerates the suppression of plasma ACTH. The long-term impact of early glucocorticoid treatment in critically ill children remains to be investigated. References (1) Téblick A. et al. Critical Care 2021. (2) Téblick A. et al. Endocrinology 2022. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Saturday, June 11, 2022 1:42 p.m. - 1:47 p.m.

Adrenal Insufficiency in Cirrhosis.

Hypothalamus-pituitary-adrenal axis assessment in patients with cirrhosis is challenging. The phenotype of fatigue, hypotension, electrolyte disarray, and abdominal pain characterizing primary adrenal insufficiency (AI) overlaps significantly with decompensated liver disease. Reliance on total cortisol assays in hypoproteinemic states is problematic, yet abnormal stimulated levels in cirrhosis are associated with poor clinical outcomes. Alternative measures including free plasma or salivary cortisol levels have theoretical merit but are limited by unclear prognostic significance and undefined cirrhosis-specific reference ranges. Further complicating matters is that AI in cirrhosis represents a spectrum of impairment. Although absolute cortisol deficiency can occur, this represents a minority of cases. Instead, there is an emerging concept that cirrhosis, with or without critical illness, may induce a “relative” cortisol deficiency during times of stress. In addition, the limitations posed by decreased synthesis of binding globulins in cirrhosis necessitate re-evaluation of traditional AI diagnostic thresholds.

Urinary free cortisol excretion is associated with lumbar bone density in patients with adrenal Cushing's syndrome.

Patients with adrenal Cushing's syndrome have a poor prognosis due to the autonomous hypersecretion of endogenous cortisol from the adrenal gland. Although several clinical examinations have been developed to manifest the autonomous hypersecretion of endogenous cortisol in this syndrome, it is unknown which parameter of cortisol overproduction is best associated with the adrenal cortisol content and bone and vascular complications of this syndrome. Therefore, the present study was undertaken to evaluate the parameter(s) associated with the adrenal cortisol content in patients with adrenal Cushing's syndrome, as well as to assess the associations between these parameters and the vascular and bone complications of this syndrome. Cardiovascular factors such as blood pressure and pulse rate, glucose metabolism, lipid metabolism, renal function, and indices of arteriosclerosis, except for diastolic blood pressure, failed to show an association with the adrenal cortisol content. Twenty-four-hour urinary free cortisol excretion (ρ = 0.893, P = 0.007) and plasma cortisol levels after the 1-mg (ρ = 0.857, P = 0.014) or 8-mg (ρ = 0.900, P = 0.037) overnight dexamethasone suppression test, but not plasma cortisol levels in the morning or late at night or indices of 131I-adosterol single-photon emission computed tomography-computed tomography, were significantly and positively associated with the adrenal cortisol content. Twenty-four-hour urinary free cortisol excretion, and not plasma cortisol levels after the 1- or 8-mg overnight dexamethasone suppression test, was significantly and negatively associated with lumbar spine bone density (lumbar spine bone mineral density, ρ = -0.786, P = 0.036; lumbar spine T score, ρ = -0.883, P = 0.009; and lumbar spine Z score, ρ = -0.883, P = 0.009). These results indicate that 24-h urinary free cortisol excretion is best associated with a decrease in bone density via increased adrenal cortisol overproduction and suggest that it could be used as a marker for osteoporosis in these patients.

Urinary free cortisol is a reliable index of adrenal cortisol production in patients with liver cirrhosis.

The measurement of total and free cortisol has been studied as a clinical index of adrenal cortisol production in patients with liver cirrhosis. Correlations between free plasma and salivary cortisol have previously been reported in stable cirrhotic patients. Urinary free cortisol constitutes an index of adrenal cortisol production; however, it has never been used in assessing adrenal function in patients with liver cirrhosis. The aim of this observational study was to determine associations between urinary free cortisol, serum total, salivary, measured and calculated plasma free cortisol levels in cirrhotics, determining which of them can be used as an indirect index of free cortisol levels. Moreover, we investigated the potential use of 24 h urinary free cortisol as a prognostic factor for mortality. Seventy-eight outpatients with liver cirrhosis were included. Serum, salivary and urinary free cortisol were measured using the electrochemiluminenscence immunoassay. Plasma free cortisol determination was conducted using a single quadrupole mass spectrometer. The quantification of free cortisol was achieved by determining the signal response on negative ESI-MS mode. Twenty-four hour urinary free cortisol levels correlated with free cortisol determined by mass spectrometer, total cortisol and calculated free cortisol levels. Patients with low levels of urinary free cortisol presented a significantly higher mortality rate compared to those with high levels. The factors associated with death risk were determined by Cox regression. In the multivariate analysis, two models were applied; in the first model, CP score, PVT and urinary free cortisol were found to be significantly related to patients' survival, whereas in the second, MELD score, ascites and urinary free cortisol were independently related to survival. This study suggests that 24 h urinary free cortisol could be considered as a potential index of adrenal cortisol production in patients with liver cirrhosis and it potentially detects patients with a high mortality risk.

Territorial scent-marking effects on vigilance behavior, space use, and stress in female Columbian ground squirrels

Social environments can profoundly affect the behavior and stress physiology of group-living animals. In many territorial species, territory owners advertise territorial boundaries to conspecifics by scent marking. Several studies have investigated the information that scent marks convey about donors' characteristics (e.g., dominance, age, sex, reproductive status), but less is known about whether scents affect the behavior and stress of recipients. We experimentally tested the hypothesis that scent marking may be a potent source of social stress in territorial species. We tested this hypothesis for Columbian ground squirrels (Urocitellus columbianus) during lactation, when territorial females defend individual nest-burrows against conspecifics. We exposed lactating females, on their territory, to the scent of other lactating females. Scents were either from unfamiliar females, kin relatives (a mother, daughter, or sister), or their own scent (control condition). We expected females to react strongly to novel scents from other females on their territory, displaying increased vigilance, and higher cortisol levels, indicative of behavioral and physiological stress. We further expected females to be more sensitive to unfamiliar female scents than to kin scents, given the matrilineal social structure of this species and known fitness benefits of co-breeding in female kin groups. Females were highly sensitive to intruder (both unfamiliar and kin) scents, but not to their own scent. Surprisingly, females reacted more strongly to the scent of close kin than to the scent of unfamiliar females. Vigilance behavior increased sharply in the presence of scents; this increase was more marked for kin than unfamiliar female scents, and was mirrored by a marked 131% increase in free plasma cortisol levels in the presence of kin (but not unfamiliar female) scents. Among kin scents, lactating females were more vigilant to the scent of sisters of equal age, but showed a marked 318% increase in plasma free cortisol levels in response to the scent of older and more dominant mothers. These results suggest that scent marks convey detailed information on the identity of intruders, directly affecting the stress axis of territory holders.

Effects of capture on stress-axis measures in endangered little brown bats (Myotis lucifugus)

Abstract Little brown bats (Myotis lucifugus) are a widely distributed species in North America that have been decimated by the fungal disease white-nose syndrome. As such, little brown bats are the focus of monitoring and research initiatives that often include capturing and handling free-ranging individuals. We examined the stress response of 198 adult female little brown bats after being captured from three bat houses, during the summer. Our objective was to inform best practices to researchers capturing and handling bats in the wild. We compared the stress response among bats held for &amp;lt;3 min (baseline), 15–30 min, or &amp;gt;30 min, and then among bats held alone or in a group with conspecifics. We measured the levels of plasma total and free cortisol, maximum corticosteroid binding capacity (MCBC), and blood glucose. Relative to baseline, total and free cortisol levels were significantly higher in bats held for 15–30 min and higher still in those held for &amp;gt; 30 min. Blood glucose levels were elevated after &amp;gt;30 min of holding. MCBC levels showed no differences among holding times. We detected a weak effect of social holding condition, with solitary-held bats having lower total cortisol levels than group-held bats, but MCBC, free cortisol, and blood glucose levels showed no effect of social holding condition. Our findings demonstrate that capture time should be minimized and suggest that little brown bats should be handled and released within 30 min of capture as means of reducing stress. Further, solitary holding did not appear to increase stress measures, which supports holding bats individually after capture, instead of in groups, to reduce risk of pathogen and parasite transmission.

Assessment of Plasma-Free Cortisol Concentrations by LC-MS/MS in Patients with Autonomous Cortisol Secretion.

Autonomous cortisol secretion (ACS) of an adrenal incidentaloma (AI) is associated with mild cortisol excess that could result in poor metabolic and cardiovascular outcomes. The biological activity of glucocorticoids depends on the unbound, free fraction. We aimed to evaluate plasma free cortisol (FC) concentrations in patients with ACS in this cross-sectional study. One hundred and ten AI patients in 3 groups; non-functioning (NFA, n=33), possible ACS (n=65), ACS (n=12) were enrolled. Following measurements were conducted: Clinical data and total serum cortisol (TC), plasma corticotrophin (ACTH), serum dehydroepiandrosterone sulfate (DHEA-S), cortisol after 1 mg dexamethasone by both immunoassay and LC-MS/MS (DexF), serum corticosteroid binding globulin (CBG), plasma dexamethasone concentration [DEX] and plasma FC by LC-MS/MS. Patients with ACS featured an unfavorable metabolic profile. Plasma [DEX] and serum CBG levels were similar between groups. Plasma FC was significantly higher in ACS when compared to NFA and possible ACS groups p<0.05 and p<0.01, respectively. In multiple regression analysis DexF (beta=0.402, p<0.001) and CBG (beta=-0.257, p=0.03) remained as the independent predictors of plasma FC while age, sex, BMI, smoking habit, and existing cardiovascular disease did not make a significant contribution to the regression model. In conclusion, the magnitude of cortisol excess in ACS could lead to increased plasma FC concentrations. Further studies in AI patients are needed to demonstrate whether any alterations of cortisol affinity for CBG exist and to establish whether plasma FC concentrations predict the unfavorable metabolic profile in ACS.

Оцінка ефективності хірургічного лікування АКТГ-залежного синдрому Кушинга в ранньому і пізньому післяопераційному періоді

Актуальність. АКТГ-залежний синдром Кушинга (СК) — тяжке ендокринне захворювання, що характеризується хронічним надлишком кортизолу внаслідок АКТГ-секретуючої аденоми гіпофіза. Метою роботи є оцінка ефективності проведеного хірурічного лікування АКТГ-залежного СК в ранньому і пізньому післяопераційному періоді. Матеріали та методи. Під спостереженням перебувало 234 пацієнти, середній вік чоловіків становив 26,38 ± 3,40 року, жінок — 27,58 ± 3,40 року. Усім хворим виконувався комплекс досліджень, що містив загальноклінічні, біохімічні, гормональні аналізи, а також магнітно-резонансна томографія головного мозку, рентгенографія органів грудної клітки, денситометрія. Результати. Трансназальна аденомектомія гіпофіза (ТАГ) була виконана первинно у 200 пацієнтів, з них повторно — у 34. При цьому ремісія у пацієнтів, які зазнали ТАГ з приводу АКТГ-залежного СК, настала у 42 %, рецидив розвинувся у 5 %, ремісія не досягнута у 46 %. Визначена вірогідність відмінностей між такими параметрами, як число пацієнтів в період ремісії і число рецидивів після ТАГ, рівень вільного кортизолу плазми і глікемії натще у хворих в період ремісії і рецидиву. Вивчений кореляційний зв’язок між рівнем вільного кортизолу плазми в ранньому постопераційному періоді після ТАГ і частотою рецидивів. Висновки. Найбільш вірогідним, інформативним прогностичним маркером рецидиву пухлини є рівень кортизолу плазми в ранньому післяопераційному періоді: чим нижче рівень кортизолу, тим стійкішою спостерігається ремісія. Безпосередні результати хірургічного лікування СК залежать від розмірів пухлини, ступеня тяжкості загального стану пацієнта, наявності нейроендокринних і кістково-метаболічних ускладнень.

Cushing’s Disease (CD) Due to ACTH-Secreting Pituitary Microadenoma Incidentally Discovered on a Sestamibi Scan for Primary Hyperparathyroidism

Introduction: MIBI scintigraphy is commonly being used for the preoperative localization of parathyroid adenomas. Multiple studies showed MIBI uptake in pituitary adenomas are likely due to higher metabolic activity. When hyperfunctioning pituitary adenomas were reported, both had CD [1,2]. We present the third case of increased pituitary uptake on a MIBI scan later confirmed as CD. CaseA 64-year-old Caucasian female s/p renal transplantation for RPGN who presented for evaluation of hypercalcemia. Evaluation confirmed primary hyperparathyroidism with persistently elevated PTH levels 74-108 pg/ml (11-68), serum calcium levels 10.0-10.4 mg/dl (8.4-10.3), albumin 4.1-4.3 g/dl (3.6-5.1), phosphorus 3.0-3.2 mg/dl (2.5-4.5), creatinine 0.94-1.07 mg/dl. 24-hour urine calcium 60 mg/day (35-250). Vitamin D-25 OH level was 37 ng/dL (30-100). A sestamibi scan showed uptake in the right lower parathyroid, the midsternal chest region and the pituitary gland. MRI of the pituitary revealed a 7mm cystic pituitary microadenoma in the right posterior pituitary. CD was confirmed by the findings of persistently elevated 8 AM serum cortisol levels of 28.4 and 24.2 mcg/dl (4-22), ACTH levels of 59 and 39 pg/ml (10-48), and an elevated plasma free cortisol of 1.43 mcg/dl (0.07-0.93). CT of the abdomen showed L adrenal thickening suggesting adrenal hyperplasia from CD. Plasma cortisol suppressed to 1.2 mg/dl following 1 mg of dexamethasone. 24 urine for free cortisol 26.7 mcg/day (4-50). The patient had no proximal muscle weakness, striae or Cushingoid facial features. She had no hyperglycemia or hypertension. Patient was diagnosed with an ACTH secreting pituitary microadenoma with mild CD and adrenal hyperplasia. Her DXA scan showed osteoporosis. Genetic testing for MEN1 mutation was negative. Patient did not wish surgery for either her hyperparathyroidism or her CD and is being evaluated for medical treatment of hypercortisolism. Conclusion: There are two prior case reports of an incidentally discovered pituitary adenoma on sestamibi scan later diagnosed as CD [1,2]. Corticotrophs may have a strong affinity for sestamibi. Our case is the first, to our knowledge, of pituitary MRI confirmation of the ACTH secreting pituitary incidentaloma initially suspected by pituitary uptake on a sestamibi scan in a patient with hyperparathyroidism. Reference1. Kuhadiya ND et al. Incidentally Discovered ACTH-Secreting Pituitary Adenoma on a Sestamibi Scan in a Patient With Hyperparathyroidism. AACE Clinical Case Reports. 2015;1(3):e152-5. 2. Gierach M et al. The case of Cushing’s disease imaging by SPECT examination without manifestation of pituitary adenoma in MRI examination. Nuclear Medicine Review. 2005;8(2):137-9.

Usefulness of salivary cortisol as a marker of secondary adrenal insufficiency in paediatric patients

Background: The main cause of adrenal insufficiency (AI) in paediatric patients is prolonged treatment with corticosteroids. Determination of plasma cortisol (PC) during ACTH test is the most used adrenal function indicator in clinical practice. However, determination of salivary cortisol (SC), a simple test especially useful in children in order to avoid invasive procedures, can be used as an alternative technique for the diagnosis of adrenal disease. Methods: A two-year prospective study (January 2014-January 2016) in paediatric patients (2-18 years of age) treated with corticosteroids for more than fifteen days, who were investigated for suspected AI. Low-dose ACTH test was used to determine adrenal function and samples for SC and PC were obtained simultaneously in basal situation and during the test (at 30, 60 and 90 minutes). Results: 230 samples (118 PC-112 SC) of 30 studies belonging to 20 patients (4 males), mean age 10.93 years ± 3.69 SD. Pearson’s correlation coefficient showed a positive correlation between PC and SC (r = 0.618, p &lt; 0.001). All the studies with some determination of PC higher than 18 μg/dL (n = 8) had a SC peak higher than 0.61 μg/dL with a specificity of 66.67% and a sensitivity of 93.94% (ROC analysis). Conclusion: Measurement of SC is a less invasive, easier and quicker test than PC to measure plasma free cortisol levels. In our study, a SC peak in low-dose ACTH test higher than 0.61 μg/dL was able to discriminate patients without AI, and proved to be a useful tool in the initial evaluation of children with suspected AI.

The role of pro-opiomelanocortin in the ACTH\u2013cortisol dissociation of sepsis

BackgroundSepsis is typically hallmarked by high plasma (free) cortisol and suppressed cortisol breakdown, while plasma adrenocorticotropic hormone (ACTH) is not increased, referred to as ‘ACTH–cortisol dissociation.’ We hypothesized that sepsis acutely activates the hypothalamus to generate, via corticotropin-releasing hormone (CRH) and vasopressin (AVP), ACTH-induced hypercortisolemia. Thereafter, via increased availability of free cortisol, of which breakdown is reduced, feedback inhibition at the pituitary level interferes with normal processing of pro-opiomelanocortin (POMC) into ACTH, explaining the ACTH–cortisol dissociation. We further hypothesized that, in this constellation, POMC leaches into the circulation and can contribute to adrenocortical steroidogenesis.MethodsIn two human studies of acute (ICU admission to day 7, N = 71) and prolonged (from ICU day 7 until recovery; N = 65) sepsis-induced critical illness, POMC plasma concentrations were quantified in relation to plasma ACTH and cortisol. In a mouse study of acute (1 day), subacute (3 and 5 days) and prolonged (7 days) fluid-resuscitated, antibiotic-treated sepsis (N = 123), we further documented alterations in hypothalamic CRH and AVP, plasma and pituitary POMC and its glucocorticoid-receptor-regulated processing into ACTH, as well as adrenal cortex integrity and steroidogenesis markers.ResultsThe two human studies revealed several-fold elevated plasma concentrations of the ACTH precursor POMC from the acute to the prolonged phase of sepsis and upon recovery (all p < 0.0001), coinciding with the known ACTH–cortisol dissociation. Elevated plasma POMC and ACTH–corticosterone dissociation were confirmed in the mouse model. In mice, sepsis acutely increased hypothalamic mRNA of CRH (p = 0.04) and AVP (p = 0.03) which subsequently normalized. From 3 days onward, pituitary expression of CRH receptor and AVP receptor was increased. From acute throughout prolonged sepsis, pituitary POMC mRNA was always elevated (all p < 0.05). In contrast, markers of POMC processing into ACTH and of ACTH secretion, negatively regulated by glucocorticoid receptor ligand binding, were suppressed at all time points (all p ≤ 0.05). Distorted adrenocortical structure (p < 0.05) and lipid depletion (p < 0.05) were present, while most markers of adrenocortical steroidogenic activity were increased at all time points (all p < 0.05).ConclusionTogether, these findings suggest that increased circulating POMC, through CRH/AVP-driven POMC expression and impaired processing into ACTH, could represent a new piece in the puzzling ACTH–cortisol dissociation.

Physiological and Psychological Stress Responses to Labor and Delivery as Expressed by Salivary Cortisol: A Prospective Study

(Am J Obstet Gynecol. 2019;221:351.e1–351.e7) Pregnant women experience both physiological stress and psychological stress during labor and delivery, but research is limited on measurement of stress levels during labor. Previous studies on physiological stress during labor have found a significant increase of total and free plasma cortisol concentrations; this increase is considered to be crucial for helping labor progression and promoting maternal and fetal well-being. While there are few studies on psychological stress during labor, researchers found epidural anesthesia helps alleviate women’s anxiety. In addition, postpartum stress levels can affect breastfeeding duration and health care service usage. The objective of this prospective, observational study was to accurately record the levels of physiological and psychological stress during labor to determine their effect on obstetric and neonatal outcomes.

SUN-221 Subclinical Alpha-1 Antitrypsin Deficiency Is Associated with Increased Free Cortisol Fraction in Plasma and Altered Glucocorticoid Delivery to Tissues

BackgroundCorticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol and controls the circulating free cortisol pool. Proteolytic cleavage by neutrophil elastase is proposed to reduce CBG binding affinity and increase free cortisol availability to inflamed tissues. The CORtisol NETwork (CORNET) consortium found that genetic variation at a locus spanning SERPINA1 (encoding alpha-1 antitrypsin, A1AT, the endogenous inhibitor of neutrophil elastase) and SERPINA6 (CBG) contributes to morning total plasma cortisol variation. We hypothesised that A1AT deficiency increases CBG cleavage and hence free plasma cortisol, resulting in increased tissue cortisol delivery in adipose and in HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1.Methods16 healthy carriers of one of the two most common A1AT-deficiency single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. Participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 400mg and spironolactone 200mg, or placebo in a double blind randomised crossover design. Plasma free cortisol was measured by isotopic dilution and ultrafiltration, total cortisol by LC-MS/MS, total CBG by ELISA, CBG binding capacity by radioligand displacement assay, and ACTH by immunoassay. Serum A1AT was measured by ELISA. Tissue cortisol (LC-MS/MS) and expression of glucocorticoid dependent transcripts (qPCR) were measured in subcutaneous adipose samples collected by needle biopsy.ResultsSerum A1AT was confirmed lower in those with heterozygous mutations vs wild type controls (411.3 +/- 27.44 vs 565.1 +/- 23.38 mg/dL, p=0.0002). No measurable differences in total CBG or CBG binding capacity were observed. However, plasma free cortisol fraction was higher in those carrying A1AT mutations (16.13 +/- 0.2 vs 13.88 +/- 0.04 %, p<0.0001). Adipose cortisol concentrations were not significantly different but expression of glucocorticoid responsive genes e.g. PER1 was 54% higher (p=0.014) in A1AT-deficient subjects. Plasma cortisol was elevated during CRASH testing in both groups, with the increment versus placebo tending to be lower in A1AT-deficient subjects (82.5 +/- 6.7 vs 126.7 +/- 6.8 nM).ConclusionAlpha-1 antitrypsin mutation heterozygosity, common in the general population, is associated with higher free cortisol fraction, consistent with enhanced cleavage of CBG. This is associated with evidence of enhanced delivery of glucocorticoid to adipose tissues but reduced HPA negative feedback, suggesting tissue-specific control of cortisol delivery by CBG.