Corticosteroid-binding Globulin Research Articles

(059) Estetrol(E4) is a Unique Estrogen With Selective Actions in Tissues Which Are Distinctly Different From the Actions of SERMs

Abstract Introduction Estetrol (E4) acts as a typical estrogen (E) agonist in stimulating the endometrium, and when administered to postmenopausal women in various doses, reverses vaginal atrophy and decreases vasomotor symptoms. However, in contrast to other Es administered orally, E4 demonstrates a minimal hepatic effect and does not stimulate breast proliferation. Data in pre and postmenopausal women show minimal stimulatory effects on triglycerides, sex hormone binding globulin, corticosteroid-binding globulin, and angiotensinogen. Similarly, E4 exerts minimal effects on coagulation markers, and when combined with drospirenone as a combined oral contraceptive does not increase the generation of thrombin as measured by the activated protein C resistance ratio, unlike the use of ethinyl estradiol. Objectives Because of the tissue selectivity of E4, this study sought to better delineate the molecular mode of action of E4. Methods Selective E Receptor Modulators (SERMs) exhibit specific cell and tissue estrogenic, neutral or anti-estrogenic activities. SERMs interact with the ligand binding domain of the E Receptor alpha (ERα) in a way that is distinct from that of E. By preventing the kinking of helix 12 in ERα, SERMs recruit other co-receptor activators and inhibitors that are responsible for SERM action. We compared in replicates the binding pattern and affinities of 154 motifs of ERα co- regulator activators and repressors to complexes of ERα with Estradiol (E2), Estriol (E3) or E4 in a specific Pam Gene assay (Broekema et al. Endocrinology. 2018; 159:2397). Results The pattern of co-receptor binding in the presence of E4 was identical to that elicited by E2 and E3, but with a 50-fold lower potency. This pattern is strikingly distinct from that observed with 4-hydroxytamoxifen or raloxifen. These data together with previously published studies of the crystalline structures of ERα with E2, E3 or E4 complexes confirm that E4 interacts with nuclear ERα in a manner identical to that of the other Es (Abot et al. EMBO Mol Med. 2014; 6:1328). Previous studies have shown that E4 inhibits the activity of the membrane ERα, and blocks the stimulatory action of E2 in this membrane ERα subpopulation. Conclusions In conclusion, E4 is a unique E with specific actions in tissues, demonstrating absence of specific membrane receptor effects, and no interaction with ERα in the way characteristic of SERMs. The unique profile of E4 activity with lower hepatic effects (minimal or no effects on triglycerides and coagulation) may ultimately translate into safer clinical use. Disclosure Yes - Mithra Pharmaceuticals, Women's Health. Industry initiated, executed and funded study – Yes.

O-247 The intrafollicular inflammatory response during ovulation in women is most likely controlled by high concentrations of active cortisol being synthesized shortly before follicular rupture

Abstract Study question How is the anti-inflammatory response in human follicles controlled during the ovulatory process leading to the expulsion of the oocyte? Summary answer Intrafollicular concentrations of cortisol becomes high close to ovulation concomitant with an exceptionally high biological activity securing a timely and efficient termination of inflammatory processes. What is known already Ovulation has been described as a local controlled inflammatory process resulting in the degeneration of the follicle wall to facilitate oocyte extrusion. Ovulation also affects glucocorticoid metabolism of granulosa cells (GC) and although de novo synthesis of cortisol only occurs in the adrenal cortex the mid-cycle surge induces a change from high expression of HSD11b2, inactivating cortisol to cortisone, to high expression of HSD11b1 which reversibly catalyses cortisol production from cortisone. Furthermore, the high concentrations of progesterone and 17-OH-progesterone within follicles cause dislodging of cortisol from cortisol binding protein (CBP) thereby activating the biological activity of cortisol. Study design, size, duration This prospective study included 50 women undergoing fertility treatment according to a standard antagonist protocol at the fertility clinic at the university hospital of Koege, Denmark. Women donated one follicle for research purpose aspirated at one of four time points during the process of final maturation of follicles; T = 0h, T = 12h; T = 17h; T = 32h and then one additional follicle at OPU at T = 36h Participants/materials, setting, methods The concentration of cortisol and cortisone together with sex steroids were measured by LC-MS/MS in follicular fluid (FF) collected at the above five time points. Whole genome microarray data, validated by q-PCT analysis, was used to evaluate gene expression of CYP11b1, CYP21A2, HSD11b1, HSD11b2 and NR3C1 in GC at the five different time points. Main results and the role of chance The expression of HSD11b1 is strongly upregulated during the ovulatory process. From 0-12h expression is increased 690 times reaching more than 20.000 times higher than at T = 0 during the remaining period. In contrast, expression of HSD11b2 is quickly downregulated by 15-20 times through the ovulatory process. The concentration of cortisol is different from the gene expression but increases significantly from a few ng/ml at T = 0h to around 15 ng/ml at T = 12h—17h and peaks at 40-50 ng/ml at T = 32h—36h. In contrast, cortisone is almost constant from 0 to 17h at a concentration of 30-40 ng/ml being significantly reduced to 10-20 ng/ml at T = 32—36h. Concentrations of progesterone and 17-OH-progesterone increased during the ovulatory process to high levels which in essence displaces cortisol from its binding protein CPB, due to similar binding affinities. This will contribute to high levels of biologically active cortisol close to ovulation, which allows the inflammatory reaction to act on the follicle wall and secure extrusion of the oocyte to oviduct before becoming inhibited by cortisol. Furthermore, a significant decrease in 11-deoxycortisol expression was seen but CYP11B1 expression was below detection limit in GCs. This study provides new information on human ovulation. Limitations, reasons for caution Although 50 women were included more observations at each specific time point would have strengthened the conclusions. Wider implications of the findings For the first time, this study collectively evaluates the temporal effect of cortisol and cortisone concentrations in connection with gene expression profiles of enzymes involved in regulation the inflammatory response during human ovulation which now forms a physiological framework for understanding potential dysregulations in the involved processes. Trial registration number NA

Body Temperature Manipulation Increases Salivary Cortisol Independent of Exercise

The vast majority of circulating cortisol in the blood is bound and inactivated by binding proteins like corticosteroid-binding globulin (CBG) and albumin. The cortisol that is free of these binding proteins is secreted via saliva and other body fluids. Ex-vivo studies have shown that CBG binding to cortisol is temperature-dependent within the physiological range of human core body temperatures. Exercise is a standard experimental paradigm for inducing cortisol responses. However, these studies rarely consider that exercise-induced changes in core body temperature might increase free cortisol independent of psychological stress or physical exertion. We tested the effect of heat exposure without exertion or other stressors on saliva cortisol. 26 Participants (18 healthy and 8 with Major Depression MDD) sat motionless in a dry infrared sauna set to 65.5 degrees C (150 F). They had their tympanic membrane temperature and salivary cortisol measured as their body temperature increased. Exposure to exogenous heat significantly increased core body temperature from 36.93 C/98.48 F (95% CI 36.79-37.07 C, 98.23-98.73 F) to 38.42 C /101.16 F (95% CI 38.24-38.6 C, 100.83-101.48 F) after 45 minutes of heat exposure, F(15,42.33)=43.509, p<0.001. Concurrently saliva cortisol also increased significantly from 4.00 nmol/l (95%CI 1.89-6.11) to 9.87 nmol/l (95% CI 5.58-14.17), F(1,33.17)=6.64, p=0.015. Elevations in core body temperature can significantly increase free cortisol levels even in the absence of exercise or overt psychological stressors.

Exploiting Blood Transport Proteins as Carborane Supramolecular Vehicles for Boron Neutron Capture Therapy.

Carboranes are promising agents for applications in boron neutron capture therapy (BNCT), but their hydrophobicity prevents their use in physiological environments. Here, by using reverse docking and molecular dynamics (MD) simulations, we identified blood transport proteins as candidate carriers of carboranes. Hemoglobin showed a higher binding affinity for carboranes than transthyretin and human serum albumin (HSA), which are well-known carborane-binding proteins. Myoglobin, ceruloplasmin, sex hormone-binding protein, lactoferrin, plasma retinol-binding protein, thyroxine-binding globulin, corticosteroid-binding globulin and afamin have a binding affinity comparable to transthyretin/HSA. The carborane@protein complexes are stable in water and characterized by favorable binding energy. The driving force in the carborane binding is represented by the formation of hydrophobic interactions with aliphatic amino acids and BH-π and CH-π interactions with aromatic amino acids. Dihydrogen bonds, classical hydrogen bonds and surfactant-like interactions also assist the binding. These results (i) identify the plasma proteins responsible for binding carborane upon their intravenous administration, and (ii) suggest an innovative formulation for carboranes based on the formation of a carborane@protein complex prior to the administration.

Adrenal Failure: An Evidence-Based Diagnostic Approach.

The diagnosis of adrenal insufficiency (AI) requires a high index of suspicion, detailed clinical assessment including detailed drug history, and appropriate laboratory evaluation. The clinical characteristics of adrenal insufficiency vary according to the cause, and the presentation may be myriad, e.g. insidious onset to a catastrophic adrenal crisis presenting with circulatory shock and coma. Secondary adrenal insufficiency (SAI) often presents with only glucocorticoid deficiency because aldosterone production, which is controlled by the renin angiotensin system, is usually intact, and rarely presents with an adrenal crisis. Measurements of the basal serum cortisol at 8 am (<140 nmol/L or 5 mcg/dL) coupled with adrenocorticotrophin (ACTH) remain the initial tests of choice. The cosyntropin stimulation (short synacthen) test is used for the confirmation of the diagnosis. Newer highly specific cortisol assays have reduced the cut-off points for cortisol in the diagnosis of AI. The salivary cortisol test is increasingly being used in conditions associated with abnormal cortisol binding globulin (CBG) levels such as pregnancy. Children and infants require lower doses of cosyntropin for testing. 21-hydoxylase antibodies are routinely evaluated to rule out autoimmunity, the absence of which would require secondary causes of adrenal insufficiency to be ruled out. Testing the hypothalamic-pituitary-adrenal (HPA) axis, imaging, and ruling out systemic causes are necessary for the diagnosis of AI. Cancer treatment with immune checkpoint inhibitors (ICI) is an emerging cause of both primary AI and SAI and requires close follow up. Several antibodies are being implicated, but more clarity is required. We update the diagnostic evaluation of AI in this evidence-based review.

An impaired pituitary–adrenal signalling axis in stable cirrhosis is linked to worse prognosis

Background & AimsInadequate adrenal function has been described in patients with cirrhosis. We investigated (i) the pituitary–adrenal axis at different clinical stages and (ii) the clinical impact of decreased serum cortisol levels in stable patients with advanced chronic liver disease (ACLD). MethodsWe included 137 outpatients with ACLD undergoing hepatic venous pressure gradient (HVPG) measurement in the prospective VICIS study (NCT03267615). Patients were stratified into six clinical stages: S0: subclinical portal hypertension (PH) (HVPG 6–9 mmHg), S1: clinically significant PH (HVPG ≥10 mmHg) without varices, S2: presence of varices, S3: previous variceal bleeding, S4: previous non-bleeding decompensation, and S5: further decompensation. ResultsFifty-one patients had compensated ACLD (S0: n = 13; S1: n = 12; S2: n = 26), whereas 86 patients had decompensated ACLD (S3: n = 7; S4: n = 46; S5: n = 33). Serum total cortisol (t-Cort) showed a strong correlation with estimated serum free cortisol (f-Cort; Spearman’s ρ: 0.889). With progressive clinical stage, median ACTH levels (from S0: 44.0 pg/ml to S5: 20.0 pg/ml; p = 0.006), t-Cort (from S0: 13.9 μg/dl to S5: 9.2 μg/dl; p = 0.091), and cortisol binding globulin (from S0: 49.3 μg/ml to S5: 38.9 μg/ml; p <0.001) decreased, whereas f-Cort (p = 0.474) remained unchanged. Lower t-Cort levels independently predicted bacterial infections (asHR: 1.11; 95% CI: 1.04–1.19; p = 0.002), further decompensation (asHR: 1.08; 95% CI: 1.02–1.12; p = 0.008), acute-on-chronic liver failure (ACLF; asHR: 1.11; 95% CI: 1.04–1.19; p = 0.002), and liver-related death (asHR: 1.09; 95% CI: 1.01-1.18; p = 0.045). ConclusionsThe pituitary–ACTH–adrenal–cortisol axis is progressively suppressed with increasing severity of cirrhosis. Lower t-Cort is an independent risk factor for bacterial infections, further decompensation of ACLF, and liver-related mortality—even in stable outpatients with cirrhosis. Clinical trial numberVienna Cirrhosis Study (VICIS; NCT: NCT03267615). Impact and ImplicationsIn a cohort of stable outpatients, we observed progressive suppression of the pituitary–adrenal axis with increasing clinical stage of advanced chronic liver disease (ACLD). Increased levels of bile acids and systemic inflammation (assessed by interleukin-6 levels) could be involved in this suppression. Serum total cortisol (t-Cort) was strongly correlated with serum free cortisol (f-Cort) and lower t-Cort levels were independently associated with a higher risk of adverse clinical outcomes, including bacterial infections, further decompensation, acute-on-chronic liver failure, and liver-related death.

Abstract 700: Genetic Deletion Of Neutrophil Elastase Does Not Alter Lesions In Mice With Atherosclerosis Induced By Aav8-mediated Over-expression Of PCSK9

Introduction: Elevated circulating glucocorticoids are causally linked to increased cardiovascular disease. Atherosclerosis is a key cardiovascular pathology, yet the contribution of glucocorticoids underlying atherogenesis is unclear. Corticosteroid binding globulin (CBG) regulates glucocorticoid action through binding, thus controlling its systemic bioavailability. Cleavage of CBG by neutrophil elastase (NE) is proposed to enhance bioavailability of glucocorticoids by reducing its binding affinity. Strikingly, Apoe -/- mice lacking NE (ELA -/- ) display reduced atherosclerosis. Hypothesis: This investigation addresses the hypothesis that NE influences atherogenesis through CBG cleavage, increasing glucocorticoid bioavailability at sites of plaque development. Methods: Adult (8-12wk old) male C57BL/6J, ELA -/- and ELA +/+ mice were injected with AAV8-PCSK9 (IP; 1x10 12 vector genomes) and fed a western diet (21% fat; 0.21% cholesterol) for 18wks (N=10-13). Control mice received diet without viral injection (N=10). Results: In C57BL/6J mice, histological analysis of brachiocephalic arteries demonstrated lesion development with AAV8-PCSK9-treatment, in parallel with increased (P=0.015) circulating NE protein levels (58.0 ± 5.5ng/ml) compared to diet-control mice (NE levels 32.8 ± 6.2ng/ml). AAV8-PCSK9 treatment did not alter circulating glucocorticoids (Corticosterone AM levels P=0.94; AAV8-PCSK9 65 ± 16nM; Control 74 ± 36nM) but hepatic CBG (Serpina6) transcript levels were reduced (1-fold; P=0.01; AAV8-PCSK9 0.57 ± 0.04; Control 1.13 ± 0.20). Interestingly, no differences in lesion size (P=0.8539; 23.2 ± 2.5%) or collagen content (P=0.5156; 17.64 ± 5.6%), or in circulating glucocorticoid levels (corticosterone AM levels; P=0.36; 225 ± 59nM) were observed in ELA -/- mice compared to controls (lesion size 24.2 ± 6.3%; collagen 12 ± 5%; glucocorticoid levels 142 ± 29nM). Conclusions: In contrast to published data using Apoe -/- mice, these results demonstrate that NE does not influence lesion size in this model of atherosclerosis. This suggests that NE inhibition would not be a useful therapeutic target in atherosclerosis. Future studies will directly address the role of CBG in the development of atherosclerosis.

Potential Biomarkers to Distinguish Type 1 Myocardial Infarction in Troponin-Elevated Diseases.

Classifying myocardial infarction by subtype is crucial for appropriate patient management. Although troponin is currently the most commonly used biomarker, it is not a specific marker for myocardial infarction and cannot distinguish subtypes. Furthermore, previous studies have confirmed that proteins known as myocardial infarction markers could function to distinguish the type of myocardial infarction. Therefore, we identify a marker that can distinguish type 1 myocardial infarction from other diseases with elevated troponin. We used mass spectrometry to compare type 1 myocardial infarction with other conditions characterized by troponin elevation and identified new candidate markers for disease classification. We then verified these markers, along with those already known to be associated with cardiovascular disease and plaque rupture. We identified α-1 acid glycoprotein 2, corticosteroid-binding globulin, and serotransferrin as potential distinguishing markers. The presence of these markers and other parameters, such as chest pain, electrocardiogram, and troponin levels from the complementary diagnostic processes, could provide valuable information to specifically diagnose type 1 myocardial infarction.

Abstract 3082: Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer

Abstract SerpinA6 is a main transport protein for cortisol binding, which helps in cortisol tissue delivery particularly at inflammatory sites. Cortisol plays a vital role in adaptation to stress, modulating fuel metabolism, and inflammation. SerpinA6 releases cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. Castration induce inflammation in the prostate increases glucocorticoids (GCs) level and elevates pro-inflammatory cytokine IL-6 levels. European and Asian populations represented association between single nucleotide polymorphisms (SNPs) in CYP17A1 and SERPINA6/A1 and circulating glucocorticoid concentrations. We propose increasing IL-6 levels after castration alters SerpinA6 levels in prostate may results in prostate cancer disease progression. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6 (5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Silencing IL-6 in PC-3 cells increased SerpinA6 expression. Moreover, silencing SerpinA6 in metastatic variant of PC-3 cells increased phospho-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45µg/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36µg/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. In-vivo data represented altered SerpinA6 levels in castrated TRansgenic Adenocarcinoma Mouse Prostate model after dexamethasone (GR agonist) and Mifepristone (GR antagonist) treatment. We suggest decreasing levels of SerpinA6 in prostate cancer patients may have potential role in disease progression. We need to have an effective agent, to prevent maintain SerpinA6 level to inhibit the progression of prostate cancer. Citation Format: Mohammed Al-Toubat, Jatinder Kumar, Shiva Gautam, Kethandapatti Balaji, Sanjeev Shukla. Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3082.

Expanding the use of salivary cortisol as a non-invasive outpatient test in the dynamic evaluation of suspected adrenal insufficiency.

Adrenal insufficiency (AI) is potentially life-threatening, and accurate diagnosis is crucial. The first-line diagnostic test, the adrenocorticotrophic hormone (ACTH) stimulation test, measures serum total cortisol. However, this is affected in states of altered albumin or cortisol-binding globulin levels, limiting reliability. Salivary cortisol reflects free bioactive cortisol levels and is a promising alternative. However, few studies are available, and heterogenous methodologies limit applicability. This study prospectively recruited 42 outpatients undergoing evaluation for AI, excluding participants with altered cortisol-binding states. Serum (immunoassay) and salivary (liquid chromatography tandem mass spectrometry) cortisol levels were sampled at baseline, 30 min, and 60 min following 250 µg synacthen administration. AI was defined as a peak serum cortisol level <500 nmol/L in accordance with guidelines. The study recruited 21 (50%) participants with AI and 21 without AI. There were no significant differences in baseline characteristics, blood pressure, or sodium levels between groups. Following synacthen stimulation, serum and salivary cortisol levels showed good correlation at all timepoints (R2 = 0.74, P < 0.001), at peak levels (R2 = 0.72, P < 0.001), and at 60 min (R2 = 0.72, P < 0.001). A salivary cortisol cut-off of 16.0 nmol/L had a sensitivity of 90.5% and a specificity of 76.2% for the diagnosis of AI. This study demonstrates a good correlation between serum and salivary cortisol levels during the 250 µg synacthen test. A peak salivary cortisol cut-off of 16.0 nmol/L can be used for the diagnosis of AI. It is a less invasive alternative to evaluate patients with suspected AI. Its potential utility in the diagnosis of AI in patients with altered cortisol-binding states should be further studied.

Hormonal Programming / Imprinting Phenomena as Related to the Liver and Pancreas

A short overview is presented on hormonal programming / imprinting phenomena in liver and pancreas. The main focus is made on some data related to the concept of DOHaD. It is proposed to enhance research efforts in this important area in the near future.Keywords: Programming; Imprinting; Glucocorticoids Abbreviations: CBG: Corticosteroid-Binding Globulin; DOHaD: Developmental Origins of Health and Disease; GC: Glucocorticoids; GH: Growth Hormone; IGFBP: Insulin-like Growth Factor-Binding Proteins; SA: Serum Albumin; TRH: Thyrotropin-Releasing Hormone

Insights in the maturational processes influencing hydrocortisone pharmacokinetics in congenital adrenal hyperplasia patients using a middle-out approach.

Introduction: Hydrocortisone is the standard of care in cortisol replacement therapy for congenital adrenal hyperplasia patients. Challenges in mimicking cortisol circadian rhythm and dosing individualization can be overcome by the support of mathematical modelling. Previously, a non-linear mixed-effects (NLME) model was developed based on clinical hydrocortisone pharmacokinetic (PK) pediatric and adult data. Additionally, a physiologically-based pharmacokinetic (PBPK) model was developed for adults and a pediatric model was obtained using maturation functions for relevant processes. In this work, a middle-out approach was applied. The aim was to investigate whether PBPK-derived maturation functions could provide a better description of hydrocortisone PK inter-individual variability when implemented in the NLME framework, with the goal of providing better individual predictions towards precision dosing at the patient level. Methods: Hydrocortisone PK data from 24 adrenal insufficiency pediatric patients and 30 adult healthy volunteers were used for NLME model development, while the PBPK model and maturation functions of clearance and cortisol binding globulin (CBG) were developed based on previous studies published in the literature. Results: Clearance (CL) estimates from both approaches were similar for children older than 1 year (CL/F increasing from around 150 L/h to 500 L/h), while CBG concentrations differed across the whole age range (CBGNLME stable around 0.5 μM vs. steady increase from 0.35 to 0.8 μM for CBG PBPK). PBPK-derived maturation functions were subsequently included in the NLME model. After inclusion of the maturation functions, none, a part of, or all parameters were re-estimated. However, the inclusion of CL and/or CBG maturation functions in the NLME model did not result in improved model performance for the CL maturation function (ΔOFV > -15.36) and the re-estimation of parameters using the CBG maturation function most often led to unstable models or individual CL prediction bias. Discussion: Three explanations for the observed discrepancies could be postulated, i) non-considered maturation of processes such as absorption or first-pass effect, ii) lack of patients between 1 and 12 months, iii) lack of correction of PBPK CL maturation functions derived from urinary concentration ratio data for the renal function relative to adults. These should be investigated in the future to determine how NLME and PBPK methods can work towards deriving insights into pediatric hydrocortisone PK.

Роль связывания кортикостероидов с белками плазмы крови у облучённых животных в реализации эффектов радиопротекторов

The article presents a comparative analysis of radioprotectors RS-10 and RS-11 ability to enhance radioresistance in animals exposed to radiation that is based on results of research on protein-steroid interaction in acute radiation sickness. The importance of the research is caused by the need to conduct a comprehensive analysis of the mechanisms of radioprotective action of synthetic radioprotectors, including the revealing of the role of corticosteroid binding to blood plasma proteins in irradiated animals. The aim of the research was to investigate the impact of RS-10 and RS-11 radioprotectors on the binding of corticosteroids to blood plasma proteins in irradiated animals. 90 male Chinchilla rabbits weighing 2.5-3.0 kg were included in the experimental research. At the initial stage of the research the response of the adrenal cortex and the processes of their binding to blood plasma proteins after administration of RS-10 and RS-11 to intact animals was studied; at the next stage the same rates were used for the follow-up of the animals exposed to radiation after prophylactic administration of the radioprotectors. The rabbits were totally irradiated with gamma rays at a dose of 8 Gy that caused acute radiation sickness of the IV degree. The RS-10 (chitosan bitartrate) at the dose of 10.0 mg/kg and the RS-11 (similar to RS-10, but with a lower average molecular mass) at the dose of 2-7 mg/kg were intravenously injected to rabbits 15 minutes before the irradiation. Control animals were injected with an equal volume of the saline. The total content of 11-oxycorticosteroids (11-OCS) in the blood plasma was quantitated with the use of the fluorometric method in the author's modification. The amount of free corticosteroids was quantified as the difference between their content in the whole plasma and in its protein fraction after the gel-filtration chromatography with Sephadex G-25. The binding capacity of corticosteroid-binding globulin (CBG) was determined by gel filtration in the author's modification. The results of the study demonstrated the rise of the concentration of the blood plasma corticosteroids in the first hours after the RS-10 and RS-11 injection to intact animals, the amount of the free hormone increased as well. The effect of RS-10 on the rise of the free hormone levels was more pronounced as compared to the RS-11 effect. The impact of both radioprotectors on the elevation of the amount of 11-OCS binding to blood plasma proteins was similar. The introduction of RS-10 and RS-11 to the animals did not have effect on the binding capacity of the blood plasma proteins. The mechanism of radioprotective action of RS-10 and RS-11 in irradiated animals is the establishment of the optimal response of the adrenal cortex in the first hours after irradiation. The difference of the early adrenocortical response in animals irradiated and protected due to the injection of RS-10 and RS-11 from the optimal response varying towards strengthening or weakening, is the indicator of the radiation sickness severity. Prophylactic administration of RS-10 and RS-11 to irradiated animals at the height of the disease inhibited the reduction of the binding capacity of the CBG, resulting in the rise of the free hormone level at the higher total level of hormones in the blood is inhibited as well. In the mechanism for reducing post-radiation hypercorticism, with the use of the radioprotectors of RS-10 and RS-11, the main importance is the lower degree of the CBG binding capacity impairment rather than the change in the overall level of blood hormones. The higher prophylactic effect of RS-11 compared to RS-10 is confirmed by the significantly high rate of the binding ability of the CBG at the height of radiation sickness and the lower mortality among the animals involved in the experimental research.

Potential Disruption of Systemic Hormone Transport by Tobacco Alkaloids Using Computational Approaches.

Tobacco/nicotine is one of the most toxic and addictive substances and continues to pose a significant threat to global public health. The harmful effects of smoking/nicotine affect every system in the human body. Nicotine has been associated with effects on endocrine homeostasis in humans such as the imbalance of gonadal steroid hormones, adrenal corticosteroid hormones, and thyroid hormones. The present study was conducted to characterize the structural binding interactions of nicotine and its three important metabolites, cotinine, trans-3'-hydroxycotinine, and 5'-hydroxycotinine, against circulatory hormone carrier proteins, i.e., sex-hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG), and thyroxine-binding globulin (TBG). Nicotine and its metabolites formed nonbonded contacts and/or hydrogen bonds with amino acid residues of the carrier proteins. For SHBG, Phe-67 and Met-139 were the most important amino acid residues for nicotine ligand binding showing the maximum number of interactions and maximum loss in ASA. For CBG, Trp-371 and Asn-264 were the most important amino acid residues, and for TBG, Ser-23, Leu-269, Lys-270, Asn-273, and Arg-381 were the most important amino acid residues. Most of the amino acid residues of carrier proteins interacting with nicotine ligands showed a commonality with the interacting residues for the native ligands of the proteins. Taken together, the results suggested that nicotine and its three metabolites competed with native ligands for binding to their carrier proteins. Thus, nicotine and its three metabolites may potentially interfere with the binding of testosterone, estradiol, cortisol, progesterone, thyroxine, and triiodothyronine to their carrier proteins and result in the disbalance of their transport and homeostasis in the blood circulation.

High salt intake activates the hypothalamic-pituitary-adrenal axis, amplifies the stress response, and alters tissue glucocorticoid exposure in mice.

High salt intake is common and contributes to poor cardiovascular health. Urinary sodium excretion correlates directly with glucocorticoid excretion in humans and experimental animals. We hypothesized that high salt intake activates the hypothalamic-pituitary-adrenal axis activation and leads to sustained glucocorticoid excess. In male C57BL/6 mice, high salt intake for 2-8 weeks caused an increase in diurnal peak levels of plasma corticosterone. After 2 weeks, high salt increased Crh and Pomc mRNA abundance in the hypothalamus and anterior pituitary, consistent with basal hypothalamic-pituitary-adrenal axis activation. Additionally, high salt intake amplified glucocorticoid response to restraint stress, indicative of enhanced axis sensitivity. The binding capacity of Corticosteroid-Binding Globulin was reduced and its encoding mRNA downregulated in the liver. In the hippocampus and anterior pituitary, Fkbp5 mRNA levels were increased, indicating increased glucocorticoid exposure. The mRNA expression of the glucocorticoid-regenerating enzyme, 11β-hydroxysteroid dehydrogenase Type 1, was increased in these brain areas and in the liver. Sustained high salt intake activated a water conservation response by the kidney, increasing plasma levels of the vasopressin surrogate, copeptin. Increased mRNA abundance of Tonebp and Avpr1b in the anterior pituitary suggested that vasopressin signalling contributes to hypothalamic-pituitary-adrenal axis activation by high salt diet. Chronic high salt intake amplifies basal and stress-induced glucocorticoid levels and resets glucocorticoid biology centrally, peripherally and within cells.

ODP584 Corticosteroid-binding Globulin Deficiency Independently Predicts Mortality and is Associated with Norepinephrine Requirements in Septic Shock.

Abstract Context: Hydrocortisone administration in septic shock remains controversial. Corticosteroid-binding globulin (CBG) transports cortisol to inflammatory sites and is depleted in septic shock. Objective To determine if severely deficient plasma CBG &amp;lt;200 nmol/L (Ref range 269-641 nmol/L) independently predicts septic shock mortality. Methods A prospective observational study in patients with septic shock. Patients were categorised into two groups: mean plasma CBG concentrations &amp;lt;200 nmol/L and ≥200 nmol/L (day 1/2), with additional categorisation by nadir CBG. Primary outcome was Intensive Care Unit (ICU) mortality. Secondary outcomes were 28- and 90-day mortality, norepinephrine requirements, renal-replacement therapy, and clinician-instituted hydrocortisone. Results 135 patients were included. Mortality rates in ICU were higher in the CBG &amp;lt;200 nmol/L vs the CBG ≥200 nmol/L group: 32.4% vs 13.9%; Odds ratio (OR), 2.97, (95% confidence intervals (CI) 1.19,7.41); P=0. 02: 28-days; OR 2.25 (95% CI 0.99,5.11): 90-days; OR 2.21 (95% CI 0.99,4.91). Multivariate analysis revealed 4 factors independently associated with ICU mortality: CBG &amp;lt;200 nmol/L (OR 3.23, 95% CI 1. 06,9.88), Acute Physiology and Chronic Health Evaluation (APACHE) II &amp;gt;25 (OR 3.58, 95% CI 1.20,10.68); Sequential [Sepsis-related] Organ Failure Assessment (SOFA) liver score (OR 1.98, 95% CI 1. 04,3.72); and renal-replacement therapy (OR 6.59, 95% CI 2.17,20. 01). Nadir CBG levels were associated with higher SOFA cardiovascular scores, norepinephrine total dose (ug) P&amp;lt;0. 01 and duration (days) P&amp;lt;0. 01. Plasma cortisol concentrations and hydrocortisone administration did not relate to ICU mortality. Conclusion Septic shock patients with CBG &amp;lt;200 nmol/L had higher norepinephrine requirements and 3.2-fold higher ICU mortality. CBG concentration was the only directly reversible independent mortality risk factor. Presentation: No date and time listed

PSAT066 Adrenal Insufficiency Associated with Amyloidosis and Sickle Cell Disease

Abstract Background Amyloidosis causes chronic inflammation by the deposition of amyloid protein fragments in multiple organs, such as kidneys, adrenal, and pituitary glands. It is a challenge to access adrenocortical function in renal amyloidosis patients due to heavy proteinuria induced hypoproteinemia and low corticosteroid-binding globulin (CBG) which may cause false positive adrenocorticotropin (ACTH) test and the reduced cortisol level uncorrelated with free cortisol. On the other hand, adrenal insufficiency also frequently occurs in Sickle Cell Disease (SCD) patients. The hypothalamic-pituitary-adrenal (HPA) axis can be affected by tissue hypoxia during recurrent small vessel occlusion, chronic anemia, and iron-overload related oxidative stress. We report a SCD patient with nephrotic syndrome due to amyloidosis, who has adrenal insufficiency. Case Presentation A 41 years old female with Nephrotic syndrome secondary to Amyloid A (AA) amyloidosis, Adrenal insufficiency, Sickle cell disease (HbSC), Graves’ Hyperthyroidism, CVA, and bilateral femoral head avascular necrosis, presented with sickle cell pain crisis and anasarca with hypotension. She was on Hydrocortisone for 12 years but was not compliant for 2 months. She was on Methimazole for several years before it was discontinued one year ago. She has amenorrhea, severe edema with anasarca from the lower extremities to the abdomen. Labs showed morning cortisol 5.46 ug/dL, low albumin 2.4 g/dL, high TSH 5.43 uIU/mL, normal FT4 1.60 ng/dL, high urine protein 996.2 mg/dL, and eGFR 21.2. CT revealed pericardial effusion, free pelvic fluid, splenomegaly, edema, and thyroid goiter. Previous labs showed persistent low cortisol 0.9 to 3.5 ug/dL, high ferritin 1007 ng/mL, and pituitary workup was normal for Prolactin 4.6 ug/L, LH 5.02 mIU/mL, FSH 9.9 mIU/mL, IGF1 216 ng/mL, and ACTH 13.1 pg/mL. NM thyroid uptake elevated 51.7% and positive TPO 79.7 IU/mL. CT head was unremarkable. The patient was managed with Lasix and Albumin infusion for anasarca, Cyclosporine for amyloidosis, and Midodrine 10 mg TID for hypotension. Hydrocortisone stress dose was given followed by Prednisone 30 mg BID. Her symptoms improved. Discussion In Amyloidosis patients with heavy proteinuria, the low serum CBG levels affect total cortisol level. Therefore, the proper assessment for the adrenal function should be used with albumin-calculated free cortisol or cortisol/CBG ratio in addition to the ACTH stimulation tests. It is essential to differentiate between adrenal insufficiency and amyloidosis-related symptoms to prevent adrenal crisis and refrain from glucocorticoid side effects by unnecessary steroid use. On the other hand, endocrine manifestations of SCD may include hypogonadism, thyroid, adrenal, and pituitary dysfunction due to vaso-occlusive episodes induced multi-organ ischemic damage. This patient has amenorrhea, adrenal insufficiency, and normalized thyroid function test in Graves’ disease. It is crucial to avoid iron-overload induced oxidative stress which would worsen endocrine dysfunctions as well. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

ODP621 Hypertension with Hypokalemia in Pregnancy? Think "Cushing's Syndrome"

Abstract Introduction Pregnancy is uncommon in women with Cushing's syndrome (CS) as hypercortisolism and hyperandrogenism suppress gonadotropins leading to anovulatory cycles. Such pregnancies carry a higher rate of fetal morbidity and mortality. Therefore, it is of paramount importance to diagnose and treat CS in pregnancy. The diagnosis of CS in pregnancy is challenging due to the physiologic changes of the hypothalamic-pituitary-adrenal axis during pregnancy. We describe a case of CS in pregnancy that was a successfully treated with surgery. Case description An asymptomatic 39-year-old pregnant female patient with gestational hypertension was admitted to the hospital after she was found to have uncontrolled hypertension (while on 3 anti-hypertensive medications) and severe hypokalemia with potassium level of 2.8 mEq/L. She was 14 weeks pregnant at the time. She has a family history of primary aldosteronism in her father successfully treated with adrenalectomy at the age of 60. Her work up included blood work (done at 13: 42) which showed sodium level of 146 mEq/L, plasma aldosterone concentration of &amp;lt;1 ng/dL, plasma renin activity of 1.0 ng/mL/hr, total testosterone of 46 ng/dL, DHEA-S of 31 mcg/dL, plasma metanephrines &amp;lt;50 pg/mL, 24-hour urine metanephrines 70 ug, 24-hour urine normetanephrines 164 ug/24 hrs, ACTH 5 pg/mL, and cortisol 28.2 mcg/dl. MRI abdomen without contrast showed a 3.5×3.0×4.2 cm left adrenal nodule consistent with adenoma. Patient was diagnosed with non-ACTH dependent subclinical CS based on her elevated cortisol and low ACTH levels. She underwent left laparoscopic adrenalectomy and was discharged on oral hydrocortisone. Pathology showed adrenal cortical adenoma. Her blood pressure normalized shortly after surgery and her anti-hypertensive medications were stopped. Discussion Pregnancy is associated with multiple changes in the hypothalamic-pituitary-adrenal axis making diagnosis of CS in pregnancy challenging. Those changes include increased Cortisol binding globulin production leading to increased serum cortisol level and lack of suppression with Dexamethasone beside placental production of CRH and ACTH, all leading to increased salivary and urinary free cortisol levels, as well. This case of hypertension and hypokalemia in pregnancy presented a diagnostic dilemma. Furthermore, the clinical presentation was confounded by a family history of primary aldosteronism. However, the low serum ACTH level highly suggested the diagnosis of CS. Unfortunately, there are no pregnancy-specific guidelines for the diagnosis of CS. The dexamethasone suppression test is not recommended as it often yields false-positive results. The use of urinary free cortisol and late-night salivary cortisol is recommended, and the diagnosis is established when results are three to four times the upper limit of normal. ACTH levels &amp;lt;10 pg/ml are suggestive of adrenal CS. MRI without contrast can be used for imaging. To date, there have been over 260 reported cases of CS in pregnancy with more than half of them being. Presentation: No date and time listed

ODP047 Intramuscular ACTH stimulation test in a patient with liver disease

Abstract Introduction High dose (250 mcg) ACTH stimulation test is the gold standard dynamic testing to diagnose primary adrenal insufficiency (PAI). While the current guidelines do not elaborate on the preferable route of administration of corticotropin (intravenous or intramuscular), most published studies have been conducted on healthy people using the intravenous route. We present a patient with non-alcoholic steatohepatitis cirrhosis and hypotension who was thought to have suboptimal response with intramuscular ACTH stimulation test. Case 62-year-old male with cirrhosis, presented with dizziness, and was found to be hypotensive. Lab tests showed normal cell counts, electrolytes, renal function and lactic acid. His albumin was 2.6 g/dL. His blood pressure did not improve with intravenous fluids. Workup initiated for adrenal insufficiency showed 8 am cortisol level of 7. 0 mcg/dL, and ACTH level of 26 pg/mL. He underwent ACTH stimulation test with 250 mcg intramuscular cosyntropin, after which the 30-minute cortisol level was 13.7 mcg/dL and the 60-minute level was 15.1 mcg/dL (level immediately before the test 7.2 mcg/dL). Due to the patient not having signs of adrenal insufficiency and low albumin level due to liver disease, the cortisol response was considered adequate. Discussion Per the current Endocrine Society Clinical Practice Guidelines, the standard method of performing the ACTH stimulation test involves measuring baseline cortisol level, administration of 250 mcg of cosyntropin, and then measuring cortisol level 30 and 60 minutes post administration. Traditionally, an appropriate response is characterized by peak cortisol level above 18 mcg/dL, with lower values indicative of AI. While the responses can vary due to multitude of factors including time of testing etc., the individual response to the intravenous versus intramuscular injections is similar. However, the cut-off points between the two administration methods may differ. In a study on 64 healthy adult men who were administered 250 mcg cosyntropin intramuscularly, Longui et al. demonstrated lower cut-off values of 12.6 mcg/dL and 16.3 mcg/dL at 30 and 60 minutes respectively. In addition, with the introduction of newer monoclonal antibody and LC-MS/MS assays having greater specificity for cortisol, a study by Jarvosky et. al. recommends a new lower cut-off of 14-15 mcg/dL. Furthermore, several studies have shown higher prevalence of low total cortisol levels in cirrhosis and other liver disease leading to overdiagnosis of PAI, due to deficiency of cortisol binding globulin and albumin. In our patient, we hypothesize that the combination of all these factors could have caused a falsely low cortisol peak level without any underlying PAI. Our case highlights the need for additional studies on patients with underlying liver disease and hypoalbuminemia who undergo testing with intramuscular cosyntropin administration, and analysis with the new assays available. Presentation: No date and time listed

Corticosteroid-binding Globulin (SERPINA6) Establishes Postpubertal Sex Differences in Rat Adrenal Development.

Encoded by SerpinA6, plasma corticosteroid-binding globulin (CBG) transports glucocorticoids and regulates their access to cells. We determined how CBG influences plasma corticosterone and adrenal development in rats during the pubertal to adult transition using CRISPR/cas9 to disrupt SerpinA6 gene expression. In the absence of CBG, total plasma corticosterone levels were ∼80% lower in adult rats of both sexes, with a greater absolute reduction in females than in males. Notably, free corticosterone and adrenocorticotropic hormone were comparable between all groups. Between 30 and 90 days of age, wild-type female rats showed increases in adrenal weight and the size of the corticosterone-producing region, the zona fasciculata (zf), in tandem with increases in plasma CBG and corticosterone concentrations, whereas no such changes were observed in males. This sex difference was lost in rats without CBG, such that adrenal growth and zf expansion were similar between sexes. The sex-specific effects of CBG on adrenal morphology were accompanied by remarkable changes in gene expression: ∼40% of the adrenal transcriptome was altered in females lacking CBG, whereas almost no effect was seen in males. Over half of the adrenal genes that normally exhibit sexually dimorphic expression after puberty were similarly expressed in males and females without CBG, including those responsible for cholesterol biosynthesis and mobilization, steroidogenesis, and growth. Rat adrenal SerpinA6 transcript levels were very low or undetectable. Thus, sex differences in adrenal growth, morphology and gene expression profiles that emerge during puberty in rats are dependent on concomitant increases in plasma CBG produced by the liver.