Novel Corticosteroid-Binding Globulin Variant That Lacks Steroid Binding Activity

Abstract Background: Corticosteroid-binding globulin (CBG) is the principal carrier for glucocorticoids in the circulation and a regulator of their bioavailability. Inherited CBG deficiencies are rarely reported, and only three causative mutations in four families have been described. Patients, Methods, and Results: In a 26-yr-old female with hypotension, fatigue, and undetectable serum cortisol at presentation, we have identified a novel homozygous c.776g>t transversion in exon 3 of the CBG (SERPINA6) gene. This results in a p.Gly237Val substitution that is predicted to influence the positioning of two β-sheets that constitute part of the CBG steroid-binding site. Two siblings were also homozygous for the variant, whereas her mother and an unaffected sibling were heterozygous. No other symptomatic family members were identified apart from the proband. Individuals homozygous for the variant had serum CBG levels below the reference range when measured by RIA, but CBG was unmeasurable in cortisol-binding capacity assays. In the same individuals, we observed very low baseline and stimulated serum cortisol levels but normal free serum and salivary cortisol and plasma ACTH. In a study of ultradian cortisol pulsatility, increased pulse frequency was only observed in the proband. Conclusion: We describe a novel CBG variant that lacks steroid binding. All mutant homozygotes have very low serum cortisol, but normal free cortisol levels. The only biochemical feature to distinguish the symptomatic subject was increased cortisol pulsatility, and we suggest that this may influence glucocorticoid signaling and contribute to symptoms previously associated with CBG deficiency.

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The effect of social stress on adrenal axis activity in horses: the importance of monitoring corticosteroid-binding globulin capacity

Journal of Endocrinology ◽

10.1677/joe.0.1570425 ◽

1998 ◽

Vol 157 (3) ◽

pp. 425-432 ◽

Cited By ~ 109

Author(s):

SL Alexander ◽

CH Irvine

Keyword(s):

Social Stress ◽

Plasma Cortisol ◽

Binding Capacity ◽

Plasma Concentrations ◽

Social Instability ◽

Total Cortisol ◽

Adrenal Axis ◽

Free Cortisol ◽

Corticosteroid Binding Globulin ◽

Cortisol Levels

Plasma cortisol is largely bound to corticosteroid-binding globulin (CBG), which regulates its bioavailability by restricting exit from capillaries. Levels of CBG may be altered by several factors including stress and this can influence the amount of cortisol reaching cells. This study investigated the effect of social instability on plasma concentrations of CBG, total and free (not protein bound) cortisol in horses. Horses new to our research herd ('newcomers') were confined in a small yard with four dominant resident horses for 3-4 h daily for 3-4 (n = 5) or 9-14 (n = 3) days. Jugular blood was collected in the mornings from newcomers before the period of stress began ('pre-stress'), and then before each day's stress. Residents were bled before stress on the first and thirteenth day. Residents always behaved aggressively towards newcomers. By the end of the stress period, all newcomers were subordinate to residents. In newcomers (n = 8) after 3-4 days of social stress, CBG binding capacity had fallen (P = 0.0025), while free cortisol concentrations had risen (P = 0.0016) from pre-stress values. In contrast, total cortisol did not change. In residents, CBG had decreased slightly but significantly (P = 0.0162) after 12 days of stress. Residents and newcomers did not differ in pre-stress CBG binding capacity, total or free cortisol concentrations. However, by the second week of stress, CBG binding capacity was lower (P = 0.015) and free cortisol higher (P = 0.030) in newcomers (n = 3) than in residents. Total cortisol did not differ between the groups. In conclusion social stress clearly affected the adrenal axis of subordinate newcomer horses, lowering the binding capacity of CBG and raising free cortisol concentrations. However, no effect of stress could be detected when only total cortisol was measured. Therefore, to assess adrenal axis status accurately in horses, it is essential to monitor the binding capacity of CBG and free cortisol concentrations in addition to total cortisol levels.

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Neutrophil elastase-cleaved corticosteroid-binding globulin is absent in human plasma

Journal of Endocrinology ◽

10.1530/joe-18-0479 ◽

2019 ◽

Vol 240 (1) ◽

pp. 27-39

Author(s):

Lesley A Hill ◽

Dimitra A Vassiliadi ◽

Ioanna Dimopoulou ◽

Anna J Anderson ◽

Luke D Boyle ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutrophil Elastase ◽

Binding Capacity ◽

Venous Blood ◽

Biochemical Properties ◽

Binding Activity ◽

Blood Samples ◽

Activity Measurements ◽

Corticosteroid Binding Globulin ◽

Steroid Binding

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.

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Functional implications of corticosteroid-binding globulin N-glycosylation

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0234 ◽

2018 ◽

Vol 60 (2) ◽

pp. 71-84 ◽

Cited By ~ 4

Author(s):

Marc Simard ◽

Caroline Underhill ◽

Geoffrey L Hammond

Keyword(s):

Neutrophil Elastase ◽

Binding Capacity ◽

Binding Activity ◽

Glycosylation Site ◽

Binding Affinities ◽

Glycosylation Sites ◽

Reactive Center ◽

Corticosteroid Binding Globulin ◽

Pngase F ◽

Steroid Binding

Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six N-glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are N-glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved N-glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one N-glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its N-glycan is removed. When expressed in N-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2–4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an N-glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the Pseudomonas aeruginosa protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, N-glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.

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Fluconazole treatment in severe ectopic Cushing syndrome

Endocrinology Diabetes and Metabolism Case Reports ◽

10.1530/edm-19-0020 ◽

2019 ◽

Vol 2019 ◽

Cited By ~ 2

Author(s):

Teresa M Canteros ◽

Valeria De Miguel ◽

Patricia Fainstein-Day

Keyword(s):

Liver Metastases ◽

Alternative Treatment ◽

Cushing Syndrome ◽

Serum Cortisol ◽

Bilateral Adrenalectomy ◽

Ectopic Acth Syndrome ◽

List Type ◽

Ectopic Acth ◽

Free Cortisol ◽

Cortisol Levels

Summary Severe Cushing syndrome (SCS) is considered an emergency that requires immediate treatment to lower serum cortisol levels. Fluconazole may be considered an alternative treatment in Cushing syndrome when ketoconazole is not tolerated or unavailable. We report a 39-year-old woman with a history of partial pancreaticoduodenectomy due to a periampullary neuroendocrine tumor with locoregional extension. Three years after surgery, she developed liver metastases and was started on 120 mg of lanreotide/month, despite which, liver metastases progressed in the following 6 months. The patient showed extreme fatigue, muscle weakness, delirium, moon face, hirsutism and severe proximal weakness. Laboratory tests showed anemia, hyperglycemia and severe hypokalemia. 24-h urinary free cortisol: 2152 nmol/day (reference range (RR): <276), morning serum cortisol 4883.4 nmol/L (RR: 138–690), ACTH 127.3 pmol/L (RR: 2.2–10). She was diagnosed with ectopic ACTH syndrome (EAS). On admission, she presented with acute upper gastrointestinal tract bleeding and hemodynamic instability. Intravenous fluconazole 400 mg/day was started. After 48 h, her mental state improved and morning cortisol decreased by 25%. The dose was titrated to 600 mg/day which resulted in a 55% decrease in cortisol levels in 1 week, but then had to be decreased to 400 mg/day because transaminase levels increased over 3 times the upper normal level. After 18 days of treatment, hemodynamic stability, lower cortisol levels and better overall clinical status enabled successful bilateral adrenalectomy. This case report shows that intravenous fluconazole effectively decreased cortisol levels in SCS due to EAS. Learning points: Severe Cushing syndrome can be effectively treated with fluconazole to achieve a significant improvement of hypercortisolism prior to bilateral adrenalectomy. Intravenous fluconazole is an alternative treatment when ketoconazole is not tolerated and etomidate is not available. Fluconazole is well tolerated with mild side effects. Hepatotoxicity is usually mild and resolves after drug discontinuation.

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Concentrations, metabolic clearance rates, production rates and plasma binding of cortisol in Antarctic phocid seals

Acta Endocrinologica ◽

10.1530/acta.0.1290356 ◽

1993 ◽

Vol 129 (4) ◽

pp. 356-359 ◽

Cited By ~ 16

Author(s):

Graham C Liggins ◽

John T France ◽

Robert C Schneider ◽

Bruce S Knox ◽

Warren M Zapol

Keyword(s):

Binding Capacity ◽

Plasma Concentrations ◽

Human Values ◽

Metabolic Clearance ◽

Clearance Rates ◽

Production Rates ◽

High Production Rate ◽

Free Cortisol ◽

Phocid Seals ◽

Cortisol Levels

We have reported previously that plasma of the Weddell seal, a member of the phocid family, contains a very high concentration of cortisol. The present study was undertaken to determine whether high cortisol levels were common to seals in the Antarctic environment, or to other phocidae, and to determine the mechanism of the hypercortisolaemia. High levels of cortisol (0.82–2.38 μmol/l) were found in 4 phocidae (Weddell, crabeater, leopard and Southern elephant seals), whereas levels in a member of the otariid family (Antarctic fur seal) were similar to human values. Metabolic clearance rates (MCR) and production rates (PR) of cortisol were determined in the field in Weddell (N = 1), crabeater (N= 3) and leopard (N= 3) seals following bolus injections of [3H] cortisol. The MCR and PR did not differ between the three phocids, but whereas the MCR of 410–590 1/day was twice that of human values, the PR of 460–1180 μmol·m−2·d−1 was up to 40-fold greater. The binding capacity of corticosteroid-binding globulin (CBG) was equal to or greater than the plasma concentrations of cortisol, resulting in relatively low concentrations of free cortisol. We conclude that hypercortisolaemia is maintained in phocid seals mainly by a high production rate—the highest (corrected for surface area) reported in any species. The relatively low cortisol levels in otariid seals studied in the same environment suggest that the high PR in phocidae is unrelated to the harsh climatic conditions, but may be part of their adaptation for diving to extreme depths. The phocid seals and New World primates have similarly high levels of cortisol and a high PR but CBG in the primates has low binding capacity and affinity and cortisol is mainly free.

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N-Glycosylation influences human corticosteroid-binding globulin measurements

Endocrine Connections ◽

10.1530/ec-19-0242 ◽

2019 ◽

Vol 8 (8) ◽

pp. 1136-1148 ◽

Cited By ~ 2

Author(s):

Lesley A Hill ◽

Zeynep Sumer-Bayraktar ◽

John G Lewis ◽

Eva Morava ◽

Morten Thaysen-Andersen ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Liver Diseases ◽

Binding Capacity ◽

Congenital Disorders ◽

Congenital Disorders Of Glycosylation ◽

Blood Samples ◽

Reactive Center Loop ◽

Reactive Center ◽

Corticosteroid Binding Globulin ◽

Steroid Binding

Objective Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. Methods and subjects Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. Results We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Conclusions Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

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Cyclic Cushing's syndrome

Problems of Endocrinology ◽

10.14341/probl201056444-51 ◽

2010 ◽

Vol 56 (4) ◽

pp. 44-51

Author(s):

E I Marova ◽

I A Voronkova

Keyword(s):

Cushing’S Syndrome ◽

Clinical Signs ◽

Serum Cortisol ◽

Cushing's Syndrome ◽

Plasma Cortisol Level ◽

Average Life Expectancy ◽

Ectopic Acth ◽

Number Of Patients ◽

Free Cortisol ◽

Cortisol Levels

The cyclic Cushing's syndrome is a rare disease characterized by multiple episodes of elevated cortisol levels alternating with periods of its normal secretion. The so-called hypercorticism cycles may be either regular or episodic with intercycle intervals as long as a few days to several years. Most researchers agree that the reliable diagnosis of cyclic Cushing's syndrome should be based on laboratory detection of 3 peaks and 2 falls of plasma cortisol level. Cyclic Cushing's syndrome may be either ACTH dependent or independent. A review of 65 verified cases indicates that this condition may be caused by pituitary corticotropinoma (54%), ectopic ACTH-producing tumour (26%), and adrenal tumour (roughly 11%). The cause of the disease remains uncertain in 9% of the patients. Pathophysiological mechanisms of cyclic Cushing's syndrome are poorly known. In certain cases of bilateral macronodular adrenal hyperplasia or adrenal corticosteroma, it may be associated with the presence of ectopic receptors or anomalous expression of normally located receptors. The majority of the patients presenting with cyclic Cushing's syndrome exhibit symptoms of classical hypercorticism that manifest themselves either on a permanent or cyclic basis. In a small number of patients, clinical signs of cyclic Cushing's syndrome are virtually absent. Variations of the clinical picture and conflicting results of hormonal assays taken together make cyclic Cushing's syndrome difficult to diagnose. Therefore, physicians must be aware of this condition and actively search for it in all patients believed to have an enhanced cortisol production despite normal results of laboratory analysis. Frequent changes of urinary or salivary free cortisol levels are reliable and convenient criteria for cyclic Cushing's syndrome in patients suspected to have this condition. Results of cortisol stimulation or suppression tests are likely to lead to a false conclusion due to spontaneous falls and rises in serum cortisol levels at the time of analysis. Given laboratory confirmation of cyclic Cushing's syndrome, subsequent studies should be focused on the elucidation of its cause. The average life expectancy of patients with cyclic Cushing's syndrome remains to be determined.

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Bilateral and unilateral adrenal incidentalomas: biochemical and clinical characteristics

Acta Endocrinologica ◽

10.1530/eje-12-0777 ◽

2013 ◽

Vol 168 (2) ◽

pp. 235-241 ◽

Cited By ~ 23

Author(s):

V Morelli ◽

S Palmieri ◽

A S Salcuni ◽

C Eller-Vainicher ◽

E Cairoli ◽

...

Keyword(s):

Serum Cortisol ◽

Urinary Free Cortisol ◽

Adrenal Incidentalomas ◽

Mineral Density ◽

Femoral Bone Mineral Density ◽

Free Cortisol ◽

Suppression Test ◽

Cortisol Levels ◽

Dexamethasone Suppression

ObjectiveThe possible different prevalence of arterial hypertension (AH), type 2 diabetes mellitus (T2DM), dyslipidaemia (DL) and vertebral fractures (FX) between patients with bilateral and unilateral adrenal incidentalomas (BAI and UAI, respectively) with and without subclinical hypercortisolism (SH) is unknown. In this study we compared the prevalence of AH, T2DM, DL and FX in BAI and UAI patients in relation to SH.DesignProspective study.MethodsIn 175 UAI and 38 BAI patients, we evaluated BMI, spinal and femoral bone mineral density (LS and FN BMD, respectively) and the presence of AH, T2DM, DL and FX. SH was diagnosed in the presence of ≥2 of the following: urinary free cortisol levels >193 nmol/24 h, serum cortisol levels after 1 mg dexamethasone suppression test >83 nmol/l or ACTH levels <2.2 pmol/l.ResultsAge, BMI and cortisol secretion were comparable, while FN BMD was lower in BAI than in UAI patients (−0.45±0.86 vs 0.09±1.07, P=0.004). The prevalence of SH, AH, T2DM, and DL was comparable, while the prevalence of FX was higher in BAI than in UAI (52.6 vs 28%, P=0.007). The presence of FX was associated with BAI (odds ratio (OR) 2.6, 95% confidence interval (95% CI) 1.2–5.6, P=0.016), after adjusting for SH (OR 1.77, 95% CI 0.85–3.7, P=0.12), BMI (OR 1.06, 95% CI 0.98–1.13, P=0.1), age (OR 1.07, 95% CI 1.04–1.11, P=0.0001) and LS BMD (OR 1.31, 95% CI 1.03–1.67, P=0.03).ConclusionBAI patients have an increased FX risk than UAI ones. Further studies should investigate the causes of bone involvement in BAI patients.

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