Plasma Corticosteroid-binding Globulin Research Articles

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 <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 <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 <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 <200 nmol/L (OR 3.23, 95% CI 1. 06,9.88), Acute Physiology and Chronic Health Evaluation (APACHE) II >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<0. 01 and duration (days) P<0. 01. Plasma cortisol concentrations and hydrocortisone administration did not relate to ICU mortality. Conclusion Septic shock patients with CBG <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

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.

Corticosteroid-Binding Globulin Deficiency Independently Predicts Mortality in Septic Shock.

Hydrocortisone administration in septic shock remains controversial. Corticosteroid-binding globulin (CBG) transports cortisol to inflammatory sites and is depleted in septic shock. To determine whether severely deficient serum CBG < 200 nmol/L (reference range 269-641 nmol/L) independently predicts septic shock mortality. A prospective observational study in patients with septic shock. Patients were categorized into 2 groups: mean plasma CBG concentrations <200 nmol/L and ≥200 nmol/L (day 1/2), with additional categorization 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. 135 patients were included. Mortality rates in ICU were higher in the CBG < 200 nmol/L vs the CBG ≥ 200 nmol/L group: 32.4% vs 13.9% [odds ratio (OR) 2.97 (95% CI 1.19, 7.41); P = 0.02] with 28-day mortality OR 2.25 (95% CI 0.99, 5.11) and 90-day mortality OR 2.21 (95% CI 0.99, 4.91). Multivariate analysis revealed 4 factors independently associated with ICU mortality: CBG < 200 nmol/L (adjusted OR 3.23, 95% CI 1.06, 9.88), Acute Physiology and Chronic Health Evaluation II > 25 (adjusted OR 3.58, 95% CI 1.20, 10.68), Sequential Organ Failure Assessment (SOFA) liver score (adjusted OR 1.98, 95% CI 1.04, 3.72), and renal replacement therapy (adjusted OR 6.59, 95% CI 2.17, 20.01). Nadir CBG levels were associated with higher SOFA cardiovascular scores and norepinephrine total dose (μg; P < 0.01) and duration (days; P < 0.01). Plasma cortisol concentrations and hydrocortisone administration did not relate to ICU mortality. Septic shock patients with CBG < 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.

Связывающая способность кортикостероидсвязывающего глобулина плазмы крови как механизм повышения свободной фракции гормона в патогенезе острой лучевой болезни

The research of the hormonal level of the body in the pathogenesis of acute radiation sickness does not lose its relevance due to the expansion of the list of radiation hazard sources. The study results are necessary for clarification of a number of radiobiology concepts on the role of the binding capacity of plasma corticosteroid-binding globulin to blood plasma proteins for increasing the free fraction of the hormone in the dynamics of acute radiation sickness. Determination of the role of free corticoids in the hormonal effect in acute radiation sickness in animals with mono- and biphasic curves of adrenocortical response to radiation was the aim of the work. The experiments were carried out on male rabbits and outbred rats. Animals received a dose of gamma-irradiation, that caused acute radiation sickness of the stage IV. The total content of 11-OX, mcg% was determined by the fluorimetric method Guillemin et al. in the author's modification. To determine the free 11-OX and the binding capacity of CSG, the gel filtration method was used by De Moor et al. in the author's modification. Rabbits were tested 2 hours after irradiation, on days 4 and 8 of radiation sickness. Rats were tested one hour after irradiation, on days 1, 3, 8, and 10 of radiation sickness. The results of the study showed that acute radiation syndrome occurs against the background of an increase in free corticosteroids, which is essential in the pathogenesis of acute radiation sickness. The increase in free corticosteroids in the midst of radiation sickness is primarily due to a decrease in the binding capacity of corticosteroid-binding globulin (CSG or transcortin) and does not depend on the total concentration of the hormone in the blood plasma, which is a general radiobiological pattern. The magnitude of the binding capacity of transcortin is an indicator of protein-steroid interaction.

Effect of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation to lactating sows on growth and indicators of stress in the postweaned pig1,2.

Dietary omega-3 polyunsaturated fatty acids (n-3 PUFA) are precursors for lipid metabolites that reduce inflammation. Two experiments were conducted to test the hypothesis that enriching the sow diet in n-3 PUFA during late gestation and throughout lactation reduces stress and inflammation and promotes growth in weaned pigs. A protected fish oil product (PFO; Gromega) was used to enrich the diet in n-3 PUFA. In the initial experiment, time-bred gilts were fed a gestation and lactation diet supplemented with 0% (control; n = 5), 0.25% (n = 4), 0.5% (n = 4), or 1% (n = 5) PFO from 101 ± 2 d of gestation to day 16 of lactation. Adding 1% PFO to the diet increased the n-3:n-6 PUFA ratio in colostrum and milk compared with controls (P = 0.05). A subsequent experiment was performed to determine whether supplementing the sow diet with 1% PFO improved growth and reduced circulating markers of acute inflammation and stress in the offspring. Plasma was harvested from piglets (16 per treatment group) on day 0 (d of weaning) and days 1 and 3 postweaning. Pigs from the 1% PFO treatment group weighed more (P = 0.03) on day 3 postweaning and had a greater (P ˂ 0.05) n-3:n-6 PUFA ratio in plasma on each day sampled compared with 0% PFO controls. There was an overall treatment effect on plasma total cortisol (P = 0.03) and haptoglobin (P = 0.04), with lesser concentrations in pigs on the 1% PFO diet. Plasma corticosteroid-binding globulin (CBG) concentrations were not different between treatment groups but were less (P ˂ 0.001) on days 1 and 3 when compared with day 0. The resultant free cortisol index [FCI (cortisol/CBG)] was less (P = 0.02) on days 1 and 3 for pigs from the 1% treatment group compared with the controls. An ex vivo lipopolysaccharide (LPS) challenge of whole blood collected on days 0 and 1 was used to determine whether 1% PFO attenuated release of inflammatory cytokines (IL-1β, IL-6, and TNF-α). Blood from pigs within the 1% PFO treatment group tended (P = 0.098) to have a lesser mean concentration of TNF-α in response to LPS compared with blood from controls. These results suggest that providing a PFO supplement as 1% of the diet to sows beginning in late gestation and during lactation can increase the n-3:n-6 PUFA ratio in their offspring, which may improve growth and reduce the acute physiological stress response in the pigs postweaning.

N-Glycosylation influences human corticosteroid-binding globulin measurements.

ObjectiveDiscrepancies 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 subjectsMolecular 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.ResultsWe 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.ConclusionsPlasma 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.

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

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.

High-throughput profiling of the circulating proteome suggests sexually dimorphic corticosteroid signaling following ischemic stroke.

Increasing evidence suggests that there are innate differences between sexes with respect to stroke pathophysiology; however, the molecular mechanisms underlying these differences remain unclear. In this investigation, we employed a shotgun approach to broadly profile sex-associated differences in the plasma proteomes of a small group of male ( n = 6) and female ( n = 4) ischemic stroke patients. Peripheral blood was sampled during the acute phase of care, and liquid chromatography electrospray ionization mass spectrometry was used to quantify plasma proteins. We observed widespread differences in plasma composition, as 77 out of 294 detected proteins were significantly differentially expressed between sexes. Corticosteroid-binding globulin (CBG), a negative acute-phase reactant that inversely regulates levels of bioactive free cortisol, was the most dramatically differentially regulated, exhibiting 16-fold higher abundance in plasma from women relative to men. Furthermore, functional annotation analysis revealed that the remaining differentially expressed proteins were significantly enriched for those involved in response to corticosteroid signaling. Plasma CBG levels were further examined in an additional group of male ( n = 19) and female ( n = 28) ischemic stroke patients, as well as a group of male ( n = 13) and female ( n = 18) neurologically normal controls. CBG levels were significantly reduced in male stroke patients relative to male controls; however, no differences were observed between female stroke patients and female controls, suggesting that women may exhibit an attenuated cortisol response to stroke. Collectively, our findings reinforce the idea that there are sex-associated differences in stroke pathophysiology and suggest that cortisol signaling should be investigated further as a potential molecular mediator.

Corticosteroid-binding globulin is a biomarker of inflammation onset and severity in female rats.

Plasma corticosteroid-binding globulin (CBG) plays a critical role in regulating glucocorticoid bioavailability and is an acute phase 'negative' protein during inflammation. In an adjuvant-induced arthritis model, plasma CBG levels decrease in rats that develop severe inflammation, and we have now determined when and how these reductions in CBG occur. After administering complete Freund's adjuvant or saline intra-dermally at the tail base, blood samples were taken periodically for 16days. In adjuvant-treated rats, decreases in plasma CBG levels matched the severity of inflammation, and decreases were observed 4days before any clinical signs of inflammation. Decreases in CBG levels coincided with an ~5kDa reduction in its apparent size, consistent with proteolytic cleavage, and cleaved CBG lacked steroid-binding activity. At the termination of the experimental period, hepatic Cbg mRNA levels were decreased in rats with severe inflammation. While plasma TNF-α increased in all adjuvant-treated rats, increases in Il-4, IL-6, IL-10, IL-13 and IFN-γ were only observed in rats with cleaved CBG. Rats with cleaved CBG also exhibited increased spleen weights, and strong negative correlations were observed among CBG, IL-6 and spleen weights, respectively. However, there were no differences in hepatic Cbg mRNA levels in relation to the apparent proteolysis of CBG, suggesting that CBG cleavage occurs before changes in hepatic Cbg expression. Our results indicate that the levels and integrity of plasma CBG are biomarkers of the onset and severity of inflammation. Dynamic changes in the levels and function of CBG likely modulate the tissue availability of corticosterone during inflammation.

Plasma steroid-binding proteins: primary gatekeepers of steroid hormone action.

Biologically active steroids are transported in the blood by albumin, sex hormone-binding globulin (SHBG), and corticosteroid-binding globulin (CBG). These plasma proteins also regulate the non-protein-bound or ‘free’ fractions of circulating steroid hormones that are considered to be biologically active; as such, they can be viewed as the ‘primary gatekeepers of steroid action’. Albumin binds steroids with limited specificity and low affinity, but its high concentration in blood buffers major fluctuations in steroid concentrations and their free fractions. By contrast, SHBG and CBG play much more dynamic roles in controlling steroid access to target tissues and cells. They bind steroids with high (~nM) affinity and specificity, with SHBG binding androgens and estrogens and CBG binding glucocorticoids and progesterone. Both are glycoproteins that are structurally unrelated, and they function in different ways that extend beyond their transportation or buffering functions in the blood. Plasma SHBG and CBG production by the liver varies during development and different physiological or pathophysiological conditions, and abnormalities in the plasma levels of SHBG and CBG or their abilities to bind steroids are associated with a variety of pathologies. Understanding how the unique structures of SHBG and CBG determine their specialized functions, how changes in their plasma levels are controlled, and how they function outside the blood circulation provides insight into how they control the freedom of steroids to act in health and disease.

Identification of Avian Corticosteroid-binding Globulin (SerpinA6) Reveals the Molecular Basis of Evolutionary Adaptations in SerpinA6 Structure and Function as a Steroid-binding Protein

Corticosteroid-binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG steroid-binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only ∼40% identity, and their steroid-binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG steroid-binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its steroid-binding affinity, but we present evidence that it may participate in protein folding and steroid-binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct steroid-binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts steroid binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex steroids in these species.

Chronic stress does not further exacerbate the abnormal psychoneuroendocrine phenotype of Cbg-deficient male mice

Chronic stress leads to a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis which can constitute a base for pathophysiological consequences. Using mice totally deficient in Corticosteroid binding globulin (CBG), we have previously demonstrated the important role of CBG in eliciting an adequate response to an acute stressor. Here, we have studied its role in chronic stress situations. We have submitted Cbg ko and wild-type (WT) male mice to two different chronic stress paradigms – the unpredictable chronic mild stress and the social defeat. Then, their impact on neuroendocrine function – through corticosterone and CBG measurement – and behavioral responses – via anxiety and despair-like behavioral tests – was evaluated. Both chronic stress paradigms increased the display of despair-like behavior in WT mice, while that from Cbg ko mice – which was already high – was not aggravated. We have also found that control and defeated (stressed) Cbg ko mice show no difference in the social interaction test, while defeated WT mice reduce their interaction time when compared to unstressed WT mice. Interestingly, the same pattern was observed for corticosterone levels, where both chronic stress paradigms lowered the corticosterone levels of WT mice, while those from Cbg ko mice remained low and unaltered. Plasma CBG binding capacity remained unaltered in WT mice regardless of the stress paradigm. Through the use of the Cbg ko mice, which only differs genetically from WT mice by the absence of CBG, we demonstrated that CBG is crucial in modulating the effects of stress on plasma corticosterone levels and consequently on behavior. In conclusion, individuals with CBG deficiency, whether genetically or environmentally-induced, are vulnerable to acute stress but do not have their abnormal psychoneuroendocrine phenotype further affected by chronic stress.

Corticosteroid-binding globulin (CBG) reactive centre loop antibodies and surface plasmon resonance interrogate the proposed heat dependent “flip-flop” mechanism of human CBG

Corticosteroid-binding globulin (CBG) is the predominant carrier of cortisol in circulation and is a non-inhibitory member of the serpin superfamily of serine protease inhibitors. In the stressed or “S” conformation, CBG possesses an intact exposed reactive centre loop (RCL) that can be irreversibly cleaved by elastase released from activated human neutrophils whereupon it adopts a relaxed or “R” conformation. The latter conformation has decreased affinity for cortisol, allowing the release of the majority of cortisol at sites of inflammation. Recently there has been speculation that mild increments in heat such as found in pyrexia (39–40°C) may also induce a reversible “flip-flop” of the RCL into the body of the protein structure, without cleavage, facilitating a reversible temperature-dependent release of cortisol. Here we raised a new monoclonal antibody to the RCL of human CBG and used this in concert with an existing RCL antibody and show by surface plasma resonance that, at temperatures up to 40°C, the RCL of purified CBG and the RCL of CBG in intact plasma is accessible to these two antibodies. Together, the epitopes of these antibodies span 11 consecutive amino acids (STGVTLNLTSK) of the 18 residues of the RCL. This adequate antibody cover of the RCL sequence leads to the conclusion that the proposed temperature-dependent “flip-flop” of the RCL of CBG is doubtful.

Hypothalamic-pituitary-adrenal axis activity under resting conditions and cardiovascular risk factors in adolescents

BackgroundActivation of the hypothalamic-pituitary-adrenal (HPA) axis has been associated with higher levels of cardiovascular (CVD) risk factors in adults. This study aimed to assess the relation between measures of HPA axis activity under resting conditions and CVD risk factors in a general population of adolescents at 17 years. MethodsA total of 1134 adolescents from the Western Australian Pregnancy Cohort (Raine) Study had phenotypic and socio-demographic data. The associations between HPA axis measures (plasma ACTH, total cortisol, calculated free cortisol, corticosteroid binding globulin (CBG), and salivary cortisol) and a range of cardiovascular risk factors were examined using multivariable linear regression models, with adjustment for gender, adiposity, birth weight, gestational age, and socio-behavioural factors. ResultsPlasma total cortisol was positively associated with systolic blood pressure (SBP) (p=0.011), total cholesterol, HDL-cholesterol, and triglycerides (all p<0.001), and hs-CRP (p=0.047). Salivary cortisol was associated positively with HDL-C (p=0.033) and negatively with LDL-cholesterol (p=0.016); plasma calculated free cortisol was positively associated with triglycerides (p=0.006); plasma CBG was positively associated with total cholesterol and HDL-cholesterol (both p<0.001), LDL-cholesterol (p=0.022), and hs-CRP (p=0.001). After correction for multiple comparisons, significant associations remained for total cortisol with total cholesterol, HDL-C, and triglycerides; for calculated free cortisol with triglycerides; and for CBG with HDL-C, total cholesterol, and hs-CRP. Plasma ACTH was not associated with any cardiovascular risk factor. There was no association between BMI and any measure of HPA axis activity. ConclusionIn an adolescent population, HPA axis measures under resting conditions are associated with a range of CVD risk factors. Clarification of the mechanisms underlying these associations in adolescence would be an important step in understanding the evolution of adult CVD.

Plasma corticosteroid-binding globulin: biomarker of inflammation onset and severity in a rat arthritis model

Plasma corticosteroid-binding globulin: biomarker of inflammation onset and severity in a rat arthritis model

Impact of Corticosteroid-Binding Globulin Deficiency on Pregnancy and Neonatal Sex

Plasma corticosteroid-binding globulin (CBG) transports cortisol but high progesterone levels at the maternal-fetal interface can displace cortisol from its steroid-binding site. A secretion-deficient CBG mutant (A51V) in ∼1 of 36 Chinese causes low circulating CBG levels. Assess the implications of a CBG deficiency on pregnancy outcomes. From 1978 Chinese women screened at 12-16 weeks' gestation, 50 A51V carriers were identified and 46 were followed with 60 controls throughout pregnancy. Blood samples from another 2051 pregnant women were obtained at term to determine the secondary sex ratio (SSR) of newborns in an extended cohort (n = 101) of A51V mothers. Among women recruited at 12-16 weeks' gestation, serum CBG increased progressively during pregnancy but was lower (P < .0001) in heterozygous A51V carriers than controls. Two women homozygous for A51V had very low serum CBG but their pregnancies progressed normally. The A51V mothers did not differ from controls in body mass index, gestational age at delivery, duration of parturition, blood pressure, gravidity, infant birth weight and size, or placental weights, and reported no unusual clinical symptoms. Peripheral CBG and progesterone levels correlated (r = 0.459) during first and second trimesters. Progesterone levels were much higher in intervillous blood and correlated (r = 0.637) with CBG levels. A female-skewed SSR in newborns of A51V mothers (0.77) differed (P < .05) from the SSR (1.17) in a reference cohort. CBG influences progesterone levels in peripheral blood and at the maternal-fetal interface. The female-skewed SSR suggests that male fetal survival is compromised in CBG-deficient mothers.

Naturally occurring mutations of human corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) is encoded by SERPINA6. It is the major plasma binding protein of glucocorticoids and regulates plasma cortisol levels and bioavailability in humans. Several proteases target CBG and disrupt its steroid-binding properties. To date, most genetic deficiencies that alter plasma CBG levels or function have been identified in patients presenting with a variety of clinical conditions. The objective of the study was to test 32 previously uncharacterized nonsynonymous, single-nucleotide polymorphisms in SERPINA6 for their ability to alter CBG production and/or function. Human CBG mutants were produced in Chinese hamster ovary cells for ELISA, cortisol-binding activity measurements, and Western blotting as well as assays of their protease sensitivities. Eight naturally occurring CBG mutants with abnormal production and/or function were identified. Cortisol-binding affinity was markedly reduced for CBG H14Q and CBG H89Y, moderately decreased for CBG I279F, and undetectable for CBG R260L. By contrast, CBG H14R exhibited a decreased cortisol-binding capacity. Comparison of CBG levels in cell extracts and media by Western blotting revealed that CBG I48N and CBG P246Q have secretion defects. Two mutants (CBG I179V and CBG I279F) displayed reduced rates of cortisol-binding activity loss after exposure to three different proteases (neutrophil elastase, chymotrypsin, and LasB produced by Pseudomonas aeruginosa). Our data provide insight into how specific residues affect CBG secretion or function and illustrate the need to consider the various naturally occurring human CBG mutations in clinical evaluations of diseases associated with abnormalities in cortisol levels or activity.

Plasma, salivary and urinary cortisol levels following physiological and stress doses of hydrocortisone in normal volunteers.

BackgroundGlucocorticoid replacement is essential in patients with primary and secondary adrenal insufficiency, but many patients remain on higher than recommended dose regimens. There is no uniformly accepted method to monitor the dose in individual patients. We have compared cortisol concentrations in plasma, saliva and urine achieved following “physiological” and “stress” doses of hydrocortisone as potential methods for monitoring glucocorticoid replacement.MethodsCortisol profiles were measured in plasma, saliva and urine following “physiological” (20 mg oral) or “stress” (50 mg intravenous) doses of hydrocortisone in dexamethasone-suppressed healthy subjects (8 in each group), compared to endogenous cortisol levels (12 subjects). Total plasma cortisol was measured half-hourly, and salivary cortisol and urinary cortisol:creatinine ratio were measured hourly from time 0 (between 0830 and 0900) to 5 h. Endogenous plasma corticosteroid-binding globulin (CBG) levels were measured at time 0 and 5 h, and hourly from time 0 to 5 h following administration of oral or intravenous hydrocortisone. Plasma free cortisol was calculated using Coolens’ equation.ResultsPlasma, salivary and urine cortisol at 2 h after oral hydrocortisone gave a good indication of peak cortisol concentrations, which were uniformly supraphysiological. Intravenous hydrocortisone administration achieved very high 30 minute cortisol concentrations. Total plasma cortisol correlated significantly with both saliva and urine cortisol after oral and intravenous hydrocortisone (P <0.0001, correlation coefficient between 0.61 and 0.94). There was no difference in CBG levels across the sampling period.ConclusionsAn oral dose of hydrocortisone 20 mg is supraphysiological for routine maintenance, while stress doses above 50 mg 6-hourly would rarely be necessary in managing acute illness. Salivary cortisol and urinary cortisol:creatinine ratio may provide useful alternatives to plasma cortisol measurements to monitor replacement doses in hypoadrenal patients.

Development of a formula for estimating plasma free cortisol concentration from a measured total cortisol concentration when elastase-cleaved and intact corticosteroid binding globulin coexist

Cortisol bound to corticosteroid binding globulin (CBG) contributes up to 90% of the total cortisol concentration in circulation. Therefore, changes in the binding kinetics of cortisol to CBG can potentially impact on the concentration of free cortisol, the only form that is responsible for the physiological function of the hormone. When CBG is cleaved into elastase-cleaved CBG (eCBG) by the activity of neutrophil elastase, its affinity for cortisol is reduced. Therefore, when eCBG coexists with intact CBG (iCBG) in plasma, the calculation of free cortisol concentration based on the formulae that considers only one CBG pool with the same affinity for cortisol may be inappropriate. In this study, we developed in vivo and in vitro models of cortisol partitioning which considers two CBG pools, iCBG and eCBG, with different affinities for cortisol, and deduce a new formula for calculating plasma free cortisol concentration. The formula provides better estimates of free cortisol concentration than previously used formulae when measurements of the concentrations of the two CBG forms are available. The model can also be used to estimate the affinity of CBG and albumin for cortisol in different clinical groups. We found no significant difference in the estimated affinity of CBG and albumin for cortisol in normal, sepsis and septic shock groups, although free cortisol was higher in sepsis and septic shock groups. The in vivo model also demonstrated that the concentration of interstitial free cortisol is increased locally at a site of inflammation where iCBG is cleaved to form eCBG by the activity of elastase released by neutrophils. This supports the argument that the cleavage of iCBG at sites of inflammation leads to more lower-affinity eCBG and may be a mechanism that permits the local concentration of free cortisol to increase at these sites, while allowing basal free cortisol concentrations at other sites to remain unaffected.

Corticosteroid-binding globulin contributes to the neuroendocrine phenotype of mice selected for extremes in stress reactivity

Increasing evidence indicates an important role of steroid-binding proteins in endocrine functions, including hypothalamic-pituitary-adrenal (HPA) axis activity and regulation, as they influence bioavailability, local delivery, and cellular signal transduction of steroid hormones. In the plasma, glucocorticoids (GCs) are mainly bound to the corticosteroid-binding globulin (CBG) and to a lesser extend to albumin. Plasma CBG levels are therefore involved in the adaptive stress response, as they determine the concentration of free, biologically active GCs. In this study, we investigated whether male mice with a genetic predisposition for high-reactivity (HR), intermediate-reactivity (IR), or low-reactivity (LR) stress-induced corticosterone (CORT) secretion present different levels of free CORT and CORT-binding proteins, basally and in response to stressors of different intensity. Our results suggest a fine control interaction between plasma CBG expression and stress-induced CORT release. Although plasma CBG levels, and therefore CBG binding capacity, were higher in HR animals, CORT secretion overloaded the CBG buffering function in response to stressors, resulting in clearly higher free CORT levels in HR compared with IR and LR mice (HR>IR>LR), resembling the pattern of total CORT increase in all three lines. Both stressors, restraint or forced swimming, did not evoke fast CBG release from the liver into the bloodstream and therefore CBG binding capacity was not altered in our three mouse lines. Thus, we confirm CBG functions in maintaining a dynamic equilibrium between CBG-bound and unbound CORT, but could not verify its role in delaying the rise of plasma free CORT immediately after stress exposure.