Corticosteroid-binding Globulin Research Articles

Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test.

Adrenal insufficiency (AI) is usually diagnosed by low plasma cortisol levels following a short Synacthen test (SST). Most plasma cortisol is bound to corticosteroid-binding globulin, which is increased by estrogen in combined estrogen-progestin oral contraceptives (COCs). Women with AI using COCs are therefore at risk of having an apparently normal plasma cortisol level during SST, which would not adequately reflect AI. This work aimed to test whether salivary cortisol or cortisone during SST is more robust against the COC effect and to calculate the lower reference limits (LRLs) for these to be used as tentative diagnostic cutoffs to exclude AI. Forty-one healthy women on COCs and 46 healthy women without exogenous estrogens underwent an SST with collection of plasma and salivary samples at 0, 30, and 60 minutes after Synacthen injection. The groups were compared using regression analysis with age as covariate and the LRLs were calculated parametrically. SST-stimulated plasma cortisol levels were significantly higher in the COC group vs controls, while mean salivary cortisol and cortisone levels were slightly lower in the COC group. Importantly, COC use did not significantly alter LRLs for salivary cortisol or cortisone. The smallest LRL difference between groups was seen for salivary cortisone. Salivary cortisol and especially salivary cortisone are considerably less affected by COC use than plasma cortisol during SST. Due to similar LRLs, a common cutoff for salivary cortisol and cortisone during SST can be used to exclude AI in premenopausal women irrespective of COC use.

Значение тироксина в процессах взаимодействия кортикостероидов с белками плазмы крови на модели резистентности облучённых животных с аутотрансплантированными надпочечниками

The article is devoted to the study of the thyroxine effect on protein-steroid interaction in irradiated animals with autotransplanted adrenal glands. The relevance of the study is due to the need to study the radiobiological patterns of pituitary-thyroid regulation of protein-steroid interaction in acute radiation sickness in conditions of increased radioresistance of the body. The purpose of the work is to study the effect of thyroxine on the fractional composition of 11-hydroxycorticosteroids (11-OCS) and the binding capacity of corticosteroid binding globulin (CBG) in irradiated animals with autotransplanted adrenal glands. For experimental work 60 male rats weighing 200-220 g were used. Groups of animals with autotransplanted (experiment) and intact (control) adrenal glands were formed. Autotransplantation of the adrenal glands was performed according to the method of D. Ingle and G. Higgins. All animals were irradiated at a dose of 6.0 Gy. Animals in the experimental groups were administered 50 μg of thyroxine LD (Reanal, BP). Animals in the control groups were injected intraperitoneally an equal volume of physiological solution. To assess protein-steroid interactions, the fluorometric method, gel filtration method in the author's modification, a method for measuring the weight of the thymus were used. Conclusions were drawn about the effect of thyroxine on the protein-steroid interaction in radiation sickness using a model of resistance in irradiated animals with autotransplanted adrenal glands: 1) under the influence of thyroxine, the level of free 11-OCS in intact irradiated rats increases to a greater extent than the binding capacity of CBG; 2) administration of thyroxine to animals with autotransplanted adrenal glands causes an increase in the binding capacity of CBGs and a significant decrease in the concentration of free 11-OCS in the blood plasma; 3) the positive effect of thyroxine administration on increasing the weight of the thymus was manifested only in animals with autotransplanted adrenal glands; 4) an increase in the weight of the thymus in irradiated rats with autotransplanted adrenal glands under the influence of thyroxine is associated with a decrease in the content of unbound 11-OCS in the blood of animals.

ЗНАЧЕНИЕ СВЯЗЫВАНИЯ КОРТИКОСТЕРОИДОВ С БЕЛКАМИ ПЛАЗМЫ КРОВИ В МЕХАНИЗМЕ ВЛИЯНИЯ РАДИОПРОТЕКТОРА РС-11 НА ФУНКЦИЮ КОРЫ НАДПОЧЕЧНИКОВ ПРИ ОСТРОЙ ЛУЧЕВОЙ БОЛЕЗНИ

Purpose: To study the role of binding of corticosteroids to blood plasma proteins in the mechanism of influence of the radioprotector RS-11 on the function of the adrenal cortex in irradiated animals. Material and methods: The experiments were carried out on 30 male chinchilla rabbits weighing 2.5–3.0 kg. The first series of experiments is aimed at studying the reaction of the adrenal cortex and the processes of protein-steroid interaction after the administration of PC-11 to intact animals; in the second series – the same indicators in animals irradiated during the prophylactic administration of RS-11. The total content of 11-hydroxycorticosteroids (11-OKS) in blood plasma was determined by the fluorometric method of Guillemin et al in the author’s modification. The amount of free corticosteroids was determined by the difference in their content in whole plasma and in its protein fraction after separation on Sephadex G-25. Binding capacity of corticosteroid binding globulin (CSBG) was determined by gel filtration De Moor at al. in the author’s modification. Statistical analysis of the study results was carried out using the Student–Fisher method. Results: In the group of intact rabbits under conditions of administration of PC-11, the reaction of the adrenal cortex after 2.5–3 hours was significantly higher compared to control animals. The increase in the free fraction of the hormone with the introduction of the radioprotector RS-11 during this period is associated with an increase in the total level of 11-OKS, since the binding ability of CSBG did not change. In the group of irradiated rabbits, 2.5–3 hours after irradiation, the administration of PC-11 also led to a significant increase in the total content of 11-OKS in the blood compared to the initial data. At the height of radiation sickness on the 4th day, RS-11 protected rabbits showed a significantly smaller decrease in the binding capacity of CSBG than control animals, and the total content of 11-OKS was higher than in the control. The level of free hormone on the 4th day was sharply reduced (8 % of the total 11-OKS content versus 41 % in the control). On the 8th day of radiation sickness, free 11-OKS in protected RS-11 and control rabbits was practically absent, however, in protected animals, the binding capacity of CSBG was 2 times higher than in control animals. Conclusions: The influence of the radioprotector RS-11 leads in the first hours to an increase in the total concentration of 11-OX in the blood plasma and to an increase in the amount of free hormone in intact animals. The binding capacity does not change. Prophylactic administration of RS-11 inhibits, at the height of radiation sickness, the decrease in the binding capacity of CSBG in the blood plasma, and as a result, the increase in the level of free physiologically active hormone at a higher overall level of hormones in the blood. In the mechanism for reducing post-radiation hypercortisolism under conditions of protection with the drug RS-11, the main significance is the lesser degree of disruption of the binding ability of CSBG, and not a change in the overall level of hormones in the blood.

Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase–mediated proteolysis of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through the proteolytic cleavage of an exposed reactive centre loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localised Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterisation of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we firstly performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified novel O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modelling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and -T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialo- (disialyl T) and asialo-glycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on strategically-positioned and biologically-relevant CBG RCL glycans, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

Corticosteroid-Binding Globulin (SERPINA6) Consolidates Sexual Dimorphism of Adult Rat Liver.

Produced by the liver, corticosteroid-binding globulin (CBG) regulates the plasma distribution and actions of glucocorticoids. A sex difference in pituitary growth hormone secretion patterns established during puberty in rats results in increased hepatic CBG production and 2-fold higher plasma corticosterone levels in females. Glucocorticoids control hepatic development and metabolic activities, and we have therefore examined how disrupting the SerpinA6 gene encoding CBG influences plasma corticosterone dynamics, as well as liver gene expression in male and female rats before and after puberty. Comparisons of corticosterone plasma clearance and hepatic uptake in adult rats, with or without CBG, indicated that CBG limits corticosterone clearance by reducing its hepatic uptake. Hepatic transcriptomic profiling revealed minor sex differences (207 differentially expressed genes) and minimal effect of CBG deficiency in 30-day-old rats before puberty. While liver transcriptomes in 60-day-old males lacking CBG remained essentially unchanged, 2710 genes were differentially expressed in wild-type female vs male livers at this age. Importantly, ∼10% of these genes lost their sexually dimorphic expression in adult females lacking CBG, including those related to cholesterol biosynthesis, inflammation, and lipid and amino acid catabolism. Another 203 genes were altered by the loss of CBG specifically in adult females, including those related to xenobiotic metabolism, circadian rhythm, and gluconeogenesis. Our findings reveal that CBG consolidates the sexual dimorphism of the rat liver initiated by sex differences in growth hormone secretion patterns and provide insight into how CBG deficiencies are linked to glucocorticoid-dependent diseases.

Discovery and validation of protein biomarkers for monitoring the effectiveness of drug treatment for major depressive disorder

Major depressive disorder (MDD) has a high prevalence worldwide. Although the economic burden of depression increases annually, the proportion of patients with MDD receiving treatment did not increase between 2010 and 2018, suggesting an unmet treatment need. The burden of long-term treatment for depression is borne by patients. In this context, biomarkers associated with drug-treatment responses can be used as reference indicators to reduce unnecessary treatment and costs. Changes in biomolecules in response to drug treatment for depression and drug-treatment response markers have been studied extensively. The Hamilton Depression Rating Scale (HAM-D) is mainly used as an indicator of response and remission; however, it is difficult to determine whether the medication contributes to recovery when evaluating the effect of drug treatment for depression based on this assessment. Therefore, it is necessary to monitor the effect of medication compared to normal health conditions. Here, serum protein levels were compared using liquid chromatography-tandem mass spectrometry among a group of patients with depression who did not receive medication, a group of patients receiving medication, and a control group. Eight selected biomarkers, including Apolipoproteins A-I, Complement factor H, Complement C5, Complement C1q subcomponent subunit B, Alpha-2-HS-glycoprotein, Complement C1q subcomponent subunit C, Vitamin D-binding protein and Corticosteroid-binding globulin were distinguished between disease states, and protein levels in the drug-treated group were similar to those in the control group. These markers can be used to monitor the effectiveness of drug treatment.

Adrenal Suppression in Children During the Treatment for Acute Lymphoblastic Leukemia Beyond Induction.

Adrenal suppression (AS) is an iatrogenic, life-threatening condition that can occur after glucocorticoid exposure. Despite recognition that AS occurs after induction phase treatment in children with acute lymphoblastic leukemia (ALL), the risk of AS in phases beyond induction is unknown. We conducted a pilot study in pediatric patients with ALL to ascertain whether the risk of AS persists in post-induction phases of treatment. Patients diagnosed between 12 months to younger than 18 years with B or T-ALL and starting any new phase of treatment were eligible for the study. Relapsed or infant ALL were excluded. Low dose ACTH stimulation testing (LDST), measurement of albumin and cortisol binding globulin were performed in all patients. Screening for symptoms of AS was done. Twenty-four patients enrolled in the study. One was diagnosed with clear AS. Five others had a borderline cortisol peak, representing possible mild AS. Symptoms were nonspecific and did not help distinguish patients with normal LDST from those with borderline or abnormal results. Patients on treatment for ALL continue to be at risk of AS beyond induction treatment. Although this risk appears small, physicians must be vigilant as patients may be asymptomatic but could develop adrenal crisis during treatment.

Effect of The Rs-10 Radioprotector on Protein-Steroid Interaction in Irradiated Animals with Biphasic Adrenocortical Response to Irradiation

The article is devoted to the influence of RS-10 radioprotector on the protein-steroid interaction in irradiated animals. Introduction. Increasing threats to the radiation safety of the population is now causing the need for comprehensive studies of the anti-beam properties of chemical radioprotectors. The aim of the study is to study the effect of the RS-10 radioprotector on protein-steroid interaction in irradiated guinea pigs with biphasic adrenocortical response to irradiation. Study methods: experiments were conducted on 146 guinea pigs weighing 300-350 g, which were exposed to total ƴ radiation at a dose of 3.5 Gy and 4.5 Gy at a dose rate of 5.76 Gy/min, which caused them radiation disease of II and III severity. Experimental animals were injected intraperitoneally with RS-10 (chitosan bitartrate) 15 minutes before irradiation. Control animals were administered an equal volume of saline. The total plasma content of 11-oxycorticosteroids (11-OKS) was determined by the fluorometric method of Guillemin et al. in the author's modification. The binding capacity of corticosteroid binding globulin (CSG) was determined by gel filtration of De Moor at al. in the author's modification. Conclusions. Prophylactic administration of RS-10 to irradiated animals with a two-phase adreno-cortical reaction to radiation causes a later onset of secondary hypercorticism in the midst of radiation sickness in animals. Prophylactic administration of RS-10 before irradiation reduces the level of free hormone and leads to a decrease in hypercorticism, increasing the reserve capabilities of the binding ability of the CSG at the height of radiation sickness. A leading role in the mechanism of reducing affected hypercorticism under protective conditions has a lesser degree of impairment of the binding capacity of the CSG, rather than a change in the total level of 11-OKS in the blood.

OR30-02 Salivary Cortisol Response To The High Dose ACTH Stimulation Test Provides A Reliable Alternative To Serum Cortisol Measurement In The Evaluation of Suspected Hypoadrenalism, And Is Unaffected By Cortisol-Binding Concentration

Abstract Disclosure: N. El-Farhan: None. S. Rees: None. S. Tennant: None. C. Evans: None. A. Rees: None. Context The serum total cortisol response to the ACTH stimulation test is widely used to assess adrenocortical function but is affected in conditions of altered cortisol-binding globulin (CBG) concentration. Salivary cortisol reflects free cortisol concentrations and may offer a reliable alternative. Objectives: 1. To establish the salivary cortisol response to the high dose ACTH stimulation test in healthy volunteers and in patients with altered CBG concentrations. 2. To evaluate the performance of a lower reference limit (LRL) determined in healthy volunteers in patients with suspected hypoadrenalism. Design: A high dose (250 µg) intravenous ACTH stimulation test was undertaken in 139 (60 male, 79 female) healthy volunteers, 24 women taking an oestradiol-containing oral contraceptive pill (OCP females), 10 patients with low serum protein concentration secondary to the nephrotic syndrome or liver cirrhosis (LP patients) and 29 patients with suspected hypoadrenalism (SH patients). Salivary cortisol was measured by liquid chromatography-tandem mass spectrometry. The mean and estimated LRL of the 30-minute salivary cortisol response to ACTH stimulation (mean - 1.96 SD) were derived from log-transformed concentrations. The LRL was subsequently applied as a diagnostic cut-off in SH patients. Results: CBG concentrations were 58 [42-81] mg/L), 64 mg/L [43-95], 41 [28-60] mg/L and116 [84-159] mg/L in male volunteers, female volunteers, LP patients and OCP females, respectively. The mean 30-minute salivary cortisol concentration was 19.3 [2.5th-97.5th percentile 10.3-36.2] nmol/l in healthy volunteers, with no difference between genders. Corresponding values were not different in OCP females (19.7 [9.5-41.2] nmol/l; p=0.59) and LP patients (19.0 [7.7-46.9] nmol/l; p=0.97), in contrast to 30-minute mean serum cortisol responses which differed between healthy female volunteers (555 [421-731] nmol/l) and OCP females (869 [649-1162] nmol/l; p<0.001). Overall diagnostic agreement between 30-minute salivary and serum responses in patients undergoing evaluation for SH was high (79%). Conclusions: Salivary cortisol response to intravenous ACTH stimulation offers a reliable alternative to serum cortisol measurement in the assessment of patients with suspected hypoadrenalism and may be especially useful in conditions of altered CBG concentration. Presentation: Sunday, June 18, 2023

SAT299 Comparison Of Plasma Cortisol-, Salivary Cortisol- And Salivary Cortisone-response To The Short Synacthen Test In Women Using Oral Contraceptives

Abstract Disclosure: N. Bäcklund: None. S. Lundstedt: None. T. Olsson: None. P.M. Dahlqvist: None. G. Brattsand: None. Introduction: The short Synacthen test (SST) is a cornerstone in the diagnosis of adrenal insufficiency (AI). However, cortisol levels in serum or plasma may be misleadingly elevated by estrogen containing oral contraceptives (OCs), due to increased levels of cortisol binding protein (CBG). This may falsely declare a woman with AI to be cortisol sufficient. Salivary cortisol and cortisone closely correlate with the unbound fraction of plasma cortisol and is thus less likely to be influenced by variations in CBG levels. Our hypothesis was that the salivary cortisol and cortisone response to SST would be similar in women with and without OC use. Materials and methods: A standard intravenous 250 µg SST was performed in 87 women without known diseases or medications affecting the cortisol axis, 41 using OCs containing ethinylestradiol and 46 controls. Plasma cortisol was analyzed by Roche Elecsys Cortisol II assay and salivary cortisol and cortisone by LC-MS/MS in samples collected at baseline (time 0), and 30 and 60 minutes after Synacthen injection. The effect of OCs upon plasma cortisol, salivary cortisol and salivary cortisone was assessed by a two-way ANOVA with sampling time after SST as repeated measure. Results: There was an overall effect of OCs on plasma cortisol, but not on salivary cortisol or cortisone. Plasma cortisol levels were considerably higher in the OC group vs. controls at all time points; +150% at 0 minutes; +90% at 30 minutes; and +86% at 60 minutes. There was a significant interaction between OC use and sample time point for both salivary cortisol and cortisone. Mean salivary cortisol in women using OCs was 37% higher vs. controls at 0 minutes but was instead lower in the OCs group at 30 and 60 minutes after SST (-13% and -11%, respectively). Similarly, mean salivary cortisone was also slightly higher in the OC group than controls at baseline (+13%) and somewhat lower at 30 and 60 minutes (-6% and -7%). Conclusion: Plasma cortisol levels during an SST were significantly elevated in women using OCs containing estrogen whereas OC effects upon salivary cortisol and cortisone levels were modest, especially for salivary cortisone. Analysis of salivary cortisone is a promising alternative to plasma cortisol for diagnosis of AI in women using OCs. Presentation: Saturday, June 17, 2023

FRI268 Late Onset Polyglandular Autoimmune Syndrome Type 2

Abstract Disclosure: M.S. Nicholson: None. A. Barnett: None. A 66-year old white female was admitted for weakness, acute kidney injury, and hypotension. This is her 3rd hospital or ER visit in the last 12 months for similar complaints. She has a 25 year history of type 2 diabetes on insulin and oral therapy and 30 year history of hypothyroidism on levothyroxine 75 mcg daily. She was on oral estrogen replacement therapy for post-menopausal symptoms with a hysterectomy in her early 50s. She has a sister with type 2 diabetes on oral meds and hypothyroidism. She has a brother with type 1 diabetes. Her serum sodium was 135 mmol/L (135-145), potassium was 5.4 mEq/L (3.5 -5.1), and calcium was 9.2 (8.5-10.1). Her Creatinine was 1.2 with a GFR of 48. A random cortisol in the ED was 9.2 (no ref range). Blood pressure was low and she was tachycardic and her weight was 68 kg. She was a well developed female with a a tanned complexion. Areas of vitiligo were noted on her arms. A cosyntropin stimulation test was done midday with a baseline, 30, and 60 minute cortisol levels of 9.2, 8.8, and 9.3 respectively. Baseline ACTH was 877.6 pg/mL (7.2-63.6). Her TSH was 4.09 mIU/mL (0.358-3.74) and free T4 was 1.29 ng/dL (0.76-1.46). DHEA-S was 14 mcg/dL (13-130). She was started on stress dose steroids with rapid improvement in BP, hyponatremia, and symptoms. GAD-65 antibodies were above 250 IU/mL and 21-hydroxylase antibodies were positive. Her TPO antibodies were negative. Non-insulin diabetes medications were stopped and she was discharged home after stabilization on prednisone 5 mg daily and fludrocortisone 0.1 mg daily. This patient has an atypical presentation of polyglandular autoimmune syndrome type 2 (PAS2). She presented with an Addisons crisis at the age of 66. She was already diagnosed with hypothyroidism and insulin requiring diabetes and evidence of vitiligo. A majority of patients with PAS2 are female and present in their 20s to 40s with primary adrenal insufficiency and primary hypothyroidism. Her initial manifestation was hypothyroidism and then diabetes and vitiligo with adrenal insufficiency many years later. Her random cortisol was not suppressed and this lead to a delay in her diagnosis. This was likely due to oral estrogen therapy raising cortisol-binding globulin. Her elevated baseline ACTH and lack of response to ACTH stimulation confirmed her diagnosis of primary adrenal insufficiency. An ACTH stimulation test was repeated as an outpatient with similar response. This case highlights the importance of provocative testing for adrenal disorders and testing for autoimmune causes of endocrine disorders when patients are not responding as expected. Presentation: Friday, June 16, 2023

Salivary Cortisol Response to ACTH Stimulation Is a Reliable Alternative to Serum Cortisol in Evaluating Hypoadrenalism.

The serum total cortisol response to the ACTH stimulation test is widely used to assess adrenocortical function but is affected by changes in cortisol-binding globulin (CBG) concentration. Salivary cortisol reflects free cortisol concentrations and may offer a reliable alternative. (1) To establish the salivary cortisol response to ACTH stimulation in healthy volunteers and patients with altered CBG concentrations; (2) to evaluate the performance of a lower reference limit (LRL) determined in healthy volunteers in patients with suspected hypoadrenalism (SH-patients). A 250 µg ACTH stimulation test was undertaken in 139 healthy volunteers, 24 women taking an estradiol-containing oral contraceptive pill (OCP-females), 10 patients with low serum protein concentration (LP-patients), and 30 SH-patients. Salivary cortisol was measured by liquid chromatography-tandem mass spectrometry. Mean and LRL of the 30-minute salivary cortisol response (mean-1.96 standard deviation) were derived from log-transformed concentrations. The LRL was applied as a diagnostic cut-off in SH-patients, with comparison to the serum response. Mean CBG concentrations (range) were 58 (42-81) mg/L, 64 (43-95) mg/L, 41 (28-60) mg/L, and 116 (84-159) mg/L in males, females, LP-patients, and OCP-females, respectively. The mean 30-minute salivary cortisol concentration was 19.3 (2.5th-97.5th percentile 10.3-36.2) nmol/L in healthy volunteers. Corresponding values were not different in OCP-females [19.7 (9.5-41.2) nmol/L; P = .59] or LP-patients [19.0 (7.7-46.9) nmol/L; P = .97]. Overall diagnostic agreement between salivary and serum responses in SH-patients was 79%. Salivary cortisol response to ACTH stimulation offers a reliable alternative to serum and may be especially useful in conditions of altered CBG concentration.

Stress biomarkers in adult patients with drug-resistant epilepsy on a modified Atkins diet: A prospective study.

Ketogenic diets like the modified Atkins diet (MAD) are increasingly used in patients with refractory epilepsy. For epilepsy patients, stress is a well-known seizure-precipitating factor. New possibilities for measuring biomarkers of stress are now available. The purpose of this study was to investigate the impact of MAD on endocrine stress biomarkers. Forty-nine patients with drug-resistant epilepsy were investigated at baseline and after 12 weeks on MAD. Cortisol and cortisol-binding globulin (CBG) were measured and free cortisol index (FCI) calculated. We also measured metanephrine, normetanephrine, and methoxytyramine, all markers of epinephrine, norepinephrine, and dopamine, respectively. Changes were analyzed according to sex and antiseizure medications. The different markers at baseline and after 12 weeks of MAD treatment were correlated with seizure frequency and weight loss, respectively. The change in total cortisol was modest after 12 weeks on the diet (from 432.9 nmol/L (403.1-462.7)) to 422.6 nmol/L (384.6-461.0), P = 0.6). FCI was reduced (from 0.39 (0.36-0.42) to 0.34 (0.31-0.36), P = 0.001). CBG increased during the study (from 1126.4 nmol/L (1074.5-1178.3) to 1272.5 nmol/L (1206.3-1338.7), P < 0.001). There were no changes in the metanephrines after 12 weeks on the diet. The decrease in FCI was significant only in women, and only observed in patients using nonenzyme-inducing ASMs. We did not find any correlation between cortisol, CBG, or FCI levels and seizure frequency. After being on MAD for 12 weeks, FCI decreased significantly. The reduction in FCI may reflect reduced stress, but it may also be an effect of increased CBG. The reasons behind these alterations are unknown. Possibly, the changes may be a result of a reduction in insulin resistance and thyroid hormone levels. Treatment with MAD does not seem to influence "fight or flight" hormones.

The free cortisol calculated: correlation with the free cortisol concentrations measured with liquid chromatography-tandem mass spectrometry after equilibrium dialysis and establishment of reference intervals

Background Changes in cortisol binding globulin (CBG) impact the total serum cortisol concentration and affect the accurate assessment of adrenal function. Free biologically cortisol can be calculated using different equations or directly measured after complicated procedures. Methods The free cortisol index (FCI) obtained using the Bonte formula as well as the free cortisol concentration calculated (Coolens equation) were first estimated for 45 healthy workers. The CBG level was determined by a competitive radioimmunoassay and the total cortisol concentration, was measured with an electrochemiluminescent assay. The correlations between FCI, the free cortisol concentrations calculated and the free cortisol levels measured with liquid chromatography-tandem mass spectrometry after equilibrium dialysis were studied for those 45 samples. Reference limits were established on 158 healthy hospital workers and patients with serum samples collected between 7:30 am and 10 am. Results The FCI as well as the free cortisol concentrations calculated obtained for the 45 samples correlated significantly with the free cortisol levels measured. Although the cortisol and CBG levels were statistically higher in women using contraceptives compared with women not taking them as well as men, the calculated FCI and free cortisol concentrations did not differ between these groups. The medians (P2.5-P97.5) obtained for the 158 healthy workers were respectively 26.4% (12.3-51.6%) and 10.6 nmol/L (4.3-26.7 nmol/L). Conclusions This study highlighted a significant correlation between the FCI, the free cortisol concentrations calculated and the free cortisol levels measured with LC-MS/MS, it has also allowed the establishment of reference intervals for calculated FCI and free cortisol.

Plasma cortisol-linked gene networks in hepatic and adipose tissues implicate corticosteroid-binding globulin in modulating tissue glucocorticoid action and cardiovascular risk.

Genome-wide association meta-analysis (GWAMA) by the Cortisol Network (CORNET) consortium identified genetic variants spanning the SERPINA6/SERPINA1 locus on chromosome 14 associated with morning plasma cortisol, cardiovascular disease (CVD), and SERPINA6 mRNA expression encoding corticosteroid-binding globulin (CBG) in the liver. These and other findings indicate that higher plasma cortisol levels are causally associated with CVD; however, the mechanisms by which variations in CBG lead to CVD are undetermined. Using genomic and transcriptomic data from The Stockholm Tartu Atherosclerosis Reverse Networks Engineering Task (STARNET) study, we identified plasma cortisol-linked single-nucleotide polymorphisms (SNPs) that are trans-associated with genes from seven different vascular and metabolic tissues, finding the highest representation of trans-genes in the liver, subcutaneous fat, and visceral abdominal fat, [false discovery rate (FDR) = 15%]. We identified a subset of cortisol-associated trans-genes that are putatively regulated by the glucocorticoid receptor (GR), the primary transcription factor activated by cortisol. Using causal inference, we identified GR-regulated trans-genes that are responsible for the regulation of tissue-specific gene networks. Cis-expression Quantitative Trait Loci (eQTLs) were used as genetic instruments for identification of pairwise causal relationships from which gene networks could be reconstructed. Gene networks were identified in the liver, subcutaneous fat, and visceral abdominal fat, including a high confidence gene network specific to subcutaneous adipose (FDR = 10%) under the regulation of the interferon regulatory transcription factor, IRF2. These data identify a plausible pathway through which variation in the liver CBG production perturbs cortisol-regulated gene networks in peripheral tissues and thereby promote CVD.

Moderating effects of impulsivity and morning cortisol on the genotype-phenotype relationship of attention deficit hyperactivity disorder in young adults.

Dysregulation of the morning cortisol response in young adults with attention deficit hyperactivity disorder (ADHD) has been shown to underlie several of the alterations present in their lives. Thus, the interaction of this mechanism with genetic and behavioural characteristics could explain a large proportion of the aetiology of ADHD in this population. For these reasons, the present study explores the associations of 30 single nucleotide polymorphisms (SNPs) previously identified as significant (after correction for multiple comparisons) in the aetiology of ADHD with an assessment of morning cortisol and impulsivity traits in a group of 120 adults aged 18-24years. Participants were recruited through private centres of neuropsychology and psychiatry, as well as through events in local universities. Morning cortisol within 30min of awakening and motor impulsivity traits were shown to moderate the effect of SNP rs10129500 on the severity of the symptoms of ADHD measured by the Adult Self-Report Scale. This variant associated with cortisol-binding globulin would explain the low concentrations of this hormone found in young adults with high symptoms of ADHD, which is accentuated when there are high levels of impulsivity. The proposed model allows for transferring the theoretical relationships between the dimensions that explain the aetiology of ADHD to an applied exploratory model with good performance.

Glucocorticoid dysfunction in children with severe malaria.

Malaria remains a widespread health problem with a huge burden. Severe or complicated malaria is highly lethal and encompasses a variety of pathological processes, including immune activation, inflammation, and dysmetabolism. Previously, we showed that adrenal hormones, in particular glucocorticoids (GCs), play critical roles to maintain disease tolerance during Plasmodium infection in mice. Here, GC responses were studied in Cameroon in children with uncomplicated malaria (UM), severe malaria (SM) and asymptomatic controls (AC). To determine the sensitivity of leukocytes to GC signaling on a transcriptional level, we measured the ex vivo induction of glucocorticoid induced leucine zipper (GILZ) and FK506-binding protein 5 (FKBP5) by GCs in human and murine leukocytes. Targeted tracer metabolomics on peripheral blood mononuclear cells (PBMCs) was performed to detect metabolic changes induced by GCs. Total cortisol levels increased in patients with clinical malaria compared to AC and were higher in the SM versus UM group, while cortisol binding globulin levels were unchanged and adrenocorticotropic hormone (ACTH) levels were heterogeneous. Induction of both GILZ and FKBP5 by GCs was significantly reduced in patients with clinical malaria compared to AC and in malaria-infected mice compared to uninfected controls. Increased activity in the pentose phosphate pathway was found in the patients, but this was not affected by ex vivo stimulation with physiological levels of hydrocortisone. Interestingly, hydrocortisone induced increased levels of cAMP in AC, but not in clinical malaria patients. Altogether, this study shows that patients with SM have increased cortisol levels, but also a decreased sensitivity to GCs, which may clearly contribute to the severity of disease.

The Hypothalamus-pituitary-adrenocortical Response to Critical Illness: A Concept in Need of Revision.

Based on insights obtained during the past decade, the classical concept of an activated hypothalamus-pituitary-adrenocortical axis in response to critical illness is in need of revision. After a brief central hypothalamus-pituitary-adrenocortical axis activation, the vital maintenance of increased systemic cortisol availability and action in response to critical illness is predominantly driven by peripheral adaptations rather than by an ongoing centrally activated several-fold increased production and secretion of cortisol. Besides the known reduction of cortisol-binding proteins that increases free cortisol, these peripheral responses comprise suppressed cortisol metabolism in liver and kidney, prolonging cortisol half-life, and local alterations in expression of 11βHSD1, glucocorticoid receptor-α (GRα), and FK506 binding protein 5 (FKBP51) that appear to titrate increased GRα action in vital organs and tissues while reducing GRα action in neutrophils, possibly preventing immune-suppressive off-target effects of increased systemic cortisol availability. Peripherally increased cortisol exerts negative feed-back inhibition at the pituitary level impairing processing of pro-opiomelanocortin into ACTH, thereby reducing ACTH-driven cortisol secretion, whereas ongoing central activation results in increased circulating pro-opiomelanocortin. These alterations seem adaptive and beneficial for the host in the short term. However, as a consequence, patients with prolonged critical illness who require intensive care for weeks or longer may develop a form of central adrenal insufficiency. The new findings supersede earlier concepts such as "relative," as opposed to "absolute," adrenal insufficiency and generalized systemic glucocorticoid resistance in the critically ill. The findings also question the scientific basis for broad implementation of stress dose hydrocortisone treatment of patients suffering from acute septic shock solely based on assumption of cortisol insufficiency.

Endocrinology for the hepatologist.

Endocrinology for the hepatologist.

(013) Estetrol Combined with Drospirenone: An Investigational Oral Contraceptive With A Selective Impact on Endocrine Parameters

Abstract Introduction Combined oral hormone contraceptive use is associated with decreased circulating androgens (Burrows LJ, et al. J Sex Med. 2012) and increased hormone binding globulins (Mawet M, et al. Eu J Cont Rep Hlth Care. 2015). Estetrol (E4) is a human-specific estrogen produced by the fetal liver, and has selective action in different tissues. The combination of E4 and Drospirenone (DRSP) was evaluated in a Phase-2 study to determine the potential effects on endocrine parameters. Objectives To evaluate the change from baseline in serum concentration of various endocrine and liver protein markers at cycle 3 and 6 in subjects receiving E4/DRSP and two comparison combined oral contraceptive (COC) formulations. Methods In this randomized, open-label, three-arm parallel study, participants received E4 15 mg /DRSP 3mg (n = 38), or ethinyl-estradiol (EE) 30 g/Levonorgestrel (LNG) 150 g (n = 29) or EE 20 g/DRSP 3 mg (n = 31) in a 24/4-day regimen of 6 consecutive, 28-day treatment cycles. Changes from baseline in serum total testosterone (TT), free testosterone (FT), androstenedione, dehydroepiandrosterone sulfate (DHEA-S), cortisol binding globulin (CBG), sex hormone binding globulin (SHBG), and thyroxine binding globulin (TBG) were determined at cycle 3 and 6. Potential differences between baseline and cycle 3 and 6 for all treatments, as well as pairwise comparisons of E4/DRSP and each of the other two formulations are considered statistically significant with an exploratory P-value &amp;lt; 0.05. Results TBG was significantly increased with EE/LNG (47% and 37%) and EE/DRSP (80% and 70%) at cycle 3 and 6. TBG was also increased significantly but to a lesser extent with E4/DRSP (27% and 17%). CBG increased at cycle 6 in all treatments. The increase in CBG was significantly greater with EE/LNG (152%) and EE/DRSP (140%) than that noted for E4/DRSP (40%). A significant increase in SHBG was observed at cycle 3 (240%) and 6 (251%) with EE/DRSP as compared to baseline or E4/DRSP (52% and 55%). At cycle 3, a significant decrease in TT was observed for E4/DRSP (35%), EE/LNG (40.5%), and EE/DRSP (41%). FT was significantly reduced at cycle 3 for E4/DRSP, EE/LNG, and EE/DRSP at 50%, 60%, and 75%, respectively. At cycle 6, a similar pattern of significant reductions in both TT and FT was observed with each treatment. Pairwise comparison, revealed no significant differences in TT and FT among groups. There were significant decreases in androstenedione at cycle 3 and 6, with E4/DRSP (32% and 31%), EE/LNG (44% and 49%), and EE/DRSP (40% and 40%). DHEA-S was significantly reduced from baseline at cycle 3 and 6 in all treatments. There was a significant difference in DHEA-S at cycle 6 with EE/DRSP (27%) compared to E4/DRSP (11%). Conclusions E4/DRSP had lower increases in hormone binding globulins compared to EE/LNG or EE/DRSP. Increases in SHBG were significantly higher for EE/DRSP than E4/DRSP. A decrease in TT and FT with E4/DRSP was not significantly different from EE/LNG or EE/DRSP. E4/DRSP had a significantly lower impact on DHEA-S levels than EE/DRSP. Disclosure Yes - Mithra Women's Health. Industry initiated, executed and funded study – Yes.