Total Corticosteroid-binding Globulin Research Articles

Total and high-affinity corticosteroid-binding globulin depletion in septic shock is associated with mortality.

Corticosteroid-binding globulin (CBG) and albumin transport circulating cortisol. Cleavage of high-affinity CBG (haCBG) by neutrophil elastase at inflammatory sites causes cortisol release into tissues, facilitating immunomodulatory effects. To determine whether depletion of haCBG is related to mortality in septic shock. A single-center prospective observational cohort study of patients recruited with critical illness or septic shock, using serum samples collected at 0, 8, 24, 48 and 72hours. Serum total and haCBG, and total and free cortisol were assayed directly. Glucocorticoid treatment was an exclusion criterion. Mortality was assessed at 28days from Intensive Care Unit admission. Thirty septic shock (SS) and 42 nonseptic critical illness (CI) patients provided 195 serum samples. SS/CI patients had lower total CBG, haCBG and low-affinity CBG (laCBG) than controls. Total CBG and haCBG were significantly lower in septic shock patients who died than in those that survived (P<0.009, P=0.021, respectively). Total and free cortisol were higher in septic than nonseptic individuals. Free/total cortisol fractions were higher in those with low haCBG as observed in septic shock. However, cortisol levels were not associated with mortality. Albumin levels fell in sepsis but were not related to mortality. Low circulating haCBG concentrations are associated with mortality in septic shock. These results are consistent with an important physiological role for haCBG in cortisol tissue delivery in septic shock.

The role of corticosteroid-binding globulin in the evaluation of adrenal insufficiency.

Cortisol is a hydrophobic molecule that is largely bound to corticosteroid-binding globulin (CBG) in the circulation. In the assessment of adrenal insufficiency, many clinicians measure a total serum cortisol level, which assumes that CBG is present in normal concentrations and with a normal binding affinity for cortisol. CBG concentration and affinity are affected by a number of common factors including oral contraceptive pills (OCPs), fever and infection, as well as rare mutations in the serine protease inhibitor A6 (SERPINA6) gene, and as such, total cortisol levels might not be the ideal way to assess adrenal function in all clinical circumstances. This paper reviews the limitations of immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the measurement of total cortisol, the challenges of measuring free serum cortisol directly as well as the difficulties in calculating an estimated free cortisol from total cortisol, CBG and albumin concentrations. Newer approaches to the evaluation of adrenal insufficiency, including the measurement of cortisol and cortisone in the saliva, are discussed and a possible future role for these tests is proposed.

Differential Effects of Estrogen on Corticosteroid-Binding Globulin Forms Suggests Reduced Cleavage in Pregnancy.

Corticosteroid-binding globulin (CBG) is secreted as high-affinity CBG (haCBG), which may be cleaved by tissue proteases to low-affinity CBG (laCBG), releasing free cortisol. Pregnancy and the estrogen-based combined oral contraceptive pill (COCP) increase CBG concentrations twofold to threefold. The relative effects of these two hyperestrogenic states on the CBG affinity forms are unknown. We performed an observational study in 30 pregnant women, 27 COCP takers and 23 controls. We analyzed circulating total CBG, haCBG, laCBG, and free and total cortisol concentrations. In pregnancy, total CBG and haCBG were increased compared to controls (both P < 0.0001); however, laCBG concentrations were similar. In COCP takers, total CBG and haCBG were increased [802 ± 41 vs compared to controls (both P < 0.0001)], but laCBG was also increased (P = 0.03). Pregnancy and use of COCP were associated with a comparable rise in haCBG, but laCBG was lower in pregnancy (P < 0.0001). These results were consistent with an estrogen-mediated increase in CBG synthesis in both hyperestrogenemic states but with reduced CBG cleavage in pregnancy relative to the COCP, perhaps due to pregnancy-induced CBG glycosylation. Speculatively, increased circulating haCBG concentrations in pregnancy may provide an increased reservoir of CBG-bound cortisol to prepare for the risk of puerperal infection or allow for cortisol binding in the face of competition from increased circulating progesterone concentrations.

Evidence of Reduced CBG Cleavage in Abdominal Obesity: A Potential Factor in Development of the Metabolic Syndrome.

Corticosteroid-binding globulin (CBG) is involved in the regulation of cortisol delivery. Neutrophil elastase-mediated cleavage of high to low affinity CBG (haCBG to laCBG) induces cortisol release at inflammatory sites. Past studies have shown reduced CBG in obesity, an inflammatory state, particularly in central adiposity/metabolic syndrome. We performed an observational, cross-sectional study of the effects of obesity, age and sex on ha/laCBG in 100 healthy volunteers. Total and haCBG levels were 11% higher in women but did not vary with age or menopausal status. Total CBG levels were lower with increased body weight and waist circumference; laCBG levels were lower with increased body weight, waist circumference, body mass index and body fat; higher haCBG levels were seen with increased body fat. The relation between CBG and adiposity appeared to be driven predominantly by the metabolic syndrome group. The results suggest reduced CBG cleavage in central obesity, possibly contributing to the characteristic inflammatory phenotype of the central obesity and metabolic syndrome. The mechanism of gender differences in CBG levels is unclear.

Reduced corticosteroid-binding globulin cleavage in active rheumatoid arthritis.

Corticosteroid-binding globulin (CBG), the cortisol transport protein, is cleaved from high-affinity (haCBG) to low-affinity (laCBG) CBG at sites of inflammation releasing bioavailable, anti-inflammatory cortisol. Rheumatoid arthritis (RA) is a glucocorticoid-responsive disorder, with paradoxically normal cortisol levels despite elevated inflammatory mediators. Our objective was to determine whether CBG cleavage relates to RA disease activity. We hypothesized that impaired CBG cleavage may limit delivery of free cortisol to inflamed joints in RA. Prospective, cross-sectional observational study. Fifty-three patients with RA recruited from a Rheumatology outpatient clinic at a tertiary referral centre in Adelaide, Australia, and 73 healthy controls. Total CBG, haCBG and laCBG, total, free and salivary cortisol, inflammatory markers including interleukin-6 soluble receptor (IL-6sR) and macrophage migration inhibitory factor and clinical measures of disease activity. Among patients with RA, a wide range of disease activity scores was observed (DAS28: range 1·2-6·4). laCBG was lower in patients with RA (mean ± SEM); 153 ± 9, compared with healthy controls; 191 ± 8 nmol/l, P = 0·003. Levels of total and haCBG were higher in patients with more severe RA disease activity. Free and total cortisol, free cortisol:IL-6sR ratio and total cortisol:IL-6sR ratio correlated negatively with disease activity. These results suggest that patients with RA have reduced CBG cleavage compared to healthy controls and that cleavage is reduced further with higher RA disease activity. Hence, impaired CBG-mediated delivery of endogenous cortisol may perpetuate chronic inflammation in RA.

Depletion of high-affinity corticosteroid-binding globulin corresponds to illness severity in sepsis and septic shock; clinical implications.

Corticosteroid-binding globulin (CBG) is cleaved by neutrophil elastase converting the high-affinity (haCBG) conformation of CBG to a low-affinity (laCBG) conformation with a ninefold reduced cortisol-binding affinity. These in vitro data suggest that cortisol release by CBG cleavage results in the targeted delivery of cortisol to areas of inflammation. Our objective was to determine whether CBG cleavage alters circulating levels of haCBG and laCBG in vivo in proportion to sepsis severity. Prospective, observational cohort study in an adult tertiary level Intensive Care Unit in Adelaide, Australia. Thirty-three patients with sepsis or septic shock grouped by illness severity [sepsis, septic shock survivors, septic shock nonsurvivors and other shock]. Plasma levels of haCBG and laCBG were assessed using a recently developed in-house assay in patients. Plasma total and free cortisol levels were also measured. Plasma total CBG and haCBG levels fell significantly, in proportion to disease severity (P < 0·0001 for both). There was a nonsignificant increase in free and total cortisol as illness severity worsened (P = 0·19 and P = 0·39, respectively). Illness severity was better correlated with haCBG levels than either free or total cortisol levels. Increasing illness severity in sepsis and septic shock is associated with markedly reduced circulating haCBG concentrations in vivo. We propose that low levels of haCBG in chronic inflammation may limit the availability of cortisol to inflammatory sites, perpetuating the inflammatory process.

Investigation of Genetic Variants, Birthweight and Hypothalamic-Pituitary-Adrenal Axis Function Suggests a Genetic Variant in the SERPINA6 Gene Is Associated with Corticosteroid Binding Globulin in the Western Australia Pregnancy Cohort (Raine) Study

BackgroundThe hypothalamic-pituitary-adrenal (HPA) axis regulates stress responses and HPA dysfunction has been associated with several chronic diseases. Low birthweight may be associated with HPA dysfunction in later life, yet human studies are inconclusive. The primary study aim was to identify genetic variants associated with HPA axis function. A secondary aim was to evaluate if these variants modify the association between birthweight and HPA axis function in adolescents.MethodsMorning fasted blood samples were collected from children of the Western Australia Pregnancy Cohort (Raine) at age 17 (n = 1077). Basal HPA axis function was assessed by total cortisol, corticosteroid binding globulin (CBG), and adrenocorticotropic hormone (ACTH). The associations between 124 tag single nucleotide polymorphisms (SNPs) within 16 HPA pathway candidate genes and each hormone were evaluated using multivariate linear regression and penalized linear regression analysis using the HyperLasso method.ResultsThe penalized regression analysis revealed one candidate gene SNP, rs11621961 in the CBG encoding gene (SERPINA6), significantly associated with total cortisol and CBG. No other candidate gene SNPs were significant after applying the penalty or adjusting for multiple comparisons; however, several SNPs approached significance. For example, rs907621 (p = 0.002) and rs3846326 (p = 0.003) in the mineralocorticoid receptor gene (NR3C2) were associated with ACTH and SERPINA6 SNPs rs941601 (p = 0.004) and rs11622665 (p = 0.008), were associated with CBG. To further investigate our findings for SERPINA6, rare and common SNPs in the gene were imputed from the 1,000 genomes data and 8 SNPs across the gene were significantly associated with CBG levels after adjustment for multiple comparisons. Birthweight was not associated with any HPA outcome, and none of the gene-birthweight interactions were significant after adjustment for multiple comparisons.ConclusionsOur study suggests that genetic variation in the SERPINA6 gene may be associated with altered CBG levels during adolescence. Replication of these findings is required.

The reactive centre loop of corticosteroid-binding globulin (CBG) is a protease target for cortisol release

Corticosteroid-binding globulin (CBG) binds more than 90% of circulating cortisol and is a non-inhibitory member of the family of serine protease inhibitors (SERPINS) with an exposed elastase sensitive reactive centre loop (RCL). At sites of inflammation neutrophil activation can release elastase which may cleave the RCL and result in cortisol release from CBG. The RCL sequence also has two theoretical chymotrypsin cleavage sites and we used a monoclonal antibody with specificity for the RCL to investigate chymotrypsin cleavage of CBG. Here we show, for the first time, rapid chymotrypsin cleavage of the RCL of CBG, resulting in undetectable levels of intact CBG, whereas total CBG levels were unchanged. Coincident with both chymotrypsin and elastase cleavage there was an increase in the free cortisol fraction of serum to levels similar to when CBG had been inactivated by heat indicating total cortisol release from CBG. These findings demonstrate a new mechanism for cortisol release from its binding globulin.

Intact or “active” corticosteroid-binding globulin (CBG) and total CBG in plasma: Determination by parallel ELISAs using monoclonal antibodies

The predominant carrier of cortisol in circulation is corticosteroid-binding globulin (CBG) which is a non-functional member of the family of serine protease inhibitors. Corticosteroid-binding globulin possesses an exposed elastase sensitive loop and upon cleavage it adopts a “relaxed” conformation promoting the delivery of cortisol to sites of inflammation. Recently we have developed monoclonal antibodies which recognise only the intact exposed elastase loop, including an N-glycosylation site, which, in concert with another monoclonal antibody to CBG, offered the potential for the determination of intact and total CBG which may both be present in circulation. Here we validate these parallel ELISAs and show that like total CBG there is little diurnal variation of intact plasma CBG. Furthermore in a normal reference population the majority of CBG is in the intact or active form but a significant level of apparently cleaved CBG is evident. In some subjects there is gross discordance between total CBG and intact CBG implying a predominance of apparently cleaved CBG in circulation and this significantly affects calculated free cortisol levels. Gross differences in total and intact CBG levels may not be due to differences in N-glycosylation affecting antibody binding as CBG levels are unaffected by PNGase F treatment.

Corticosteroid-binding globulin reactive centre loop antibodies recognise only the intact natured protein: Elastase cleaved and uncleaved CBG may coexist in circulation

Corticosteroid-binding globulin (CBG) is the principal carrier of cortisol in circulation and is a non-inhibitory member of the serpin family of serine proteinase inhibitors. It possesses an exposed elastase specific site which, when cleaved, allows a conformational change promoting the delivery of cortisol to sites of inflammation. Previously there was no ability to independently distinguish between the uncleaved, stressed, conformer of CBG and total CBG in circulation. Here we raised and characterized monoclonal antibodies generated against a synthetic peptide spanning the elastase cleavage site within the exposed reactive centre loop (RCL) and measured changes in CBG by ELISA following treatment with human neutrophil elastase. The antibodies recognized the synthetic peptide as well as intact CBG and the epitope (STGVTLNL) spanned the elastase cleavage site. Treatment of plasma with elastase resulted in a complete loss of CBG levels determined using these RCL antibodies whereas CBG levels measured with an unrelated CBG monoclonal antibody were unaffected. We also compared plasma levels of CBG measured by RCL antibodies and an unrelated CBG antibody and showed discordance in some samples. This study shows for the first time the ability to measure the intact, stressed conformer of CBG. We report discordance with total CBG in some samples implying the presence of cleaved CBG in circulation. This is an important finding as it has implications for free cortisol which hitherto have been determined from total cortisol and total CBG levels. This antibody could be used for determining the time course of intact CBG in various relevant patient cohorts and for structure/function studies on the biology of human CBG.

A Longitudinal Study of Plasma and Urinary Cortisol in Pregnancy and Postpartum

There is a paucity of longitudinal data on plasma and urinary cortisol levels during pregnancy using modern assays. Furthermore, conflicting data exist as to the effect of the low-dose oral contraceptive pill (OCP) on cortisol. DESIGN, SUBJECTS, AND MEASUREMENTS: We conducted a prospective longitudinal study on morning plasma cortisol (total and free), corticosteroid-binding globulin (CBG), and 24-h urinary free cortisol (UFC) levels in 20 pregnant women during the first, second, and third trimesters and 2-3 months postpartum compared with 12 subjects on low-dose OCP and 15 nonpregnant subjects not taking the OCP (control group). A progressive rise in total plasma cortisol, CBG, and 24-h UFC was demonstrated during pregnancy, peaking during the third trimester (mean 3-fold rise compared with controls). Plasma free cortisol increased 1.6-fold by the third trimester. In the OCP group, total plasma cortisol and CBG were 2.9- and 2.6-fold elevated, respectively, whereas 24-h UFC and plasma free cortisol were not significantly different from controls. Compared with liquid chromatography-mass spectrometry, a commercial immunoassay underestimated mean total plasma cortisol concentrations by 30% during second and third trimesters and in OCP users and overestimated UFC levels by 30-35% during pregnancy. Our study demonstrated elevations in total plasma cortisol and CBG concentrations during pregnancy and with low-dose OCP use. Pregnancy was also associated with significant increases in plasma free cortisol and UFC, suggesting that the rise in total plasma cortisol is contributed to by up-regulation of the maternal hypothalamic-pituitary-adrenal axis in addition to elevated CBG.

Reproductive experience alters corticosterone and CBG levels in the rat dam

Reproductive experience has significant effects on the brain, behavior and hormone profiles of the mother. Recent work has demonstrated that primiparous rats exhibit decreased dendritic arborizations in the hippocampus, and enhanced hippocampus-dependent spatial memory performance at the time of weaning compared to nulliparous and, to a lesser degree, multiparous rats. Interestingly, enhanced spatial learning and reduced dendritic arbors are seen in nulliparous female rats exposed to chronic stress or repeated corticosterone administration. Based on these observations, we hypothesized that corticosterone may be altered in primiparous rats compared to multiparous and nulliparous rats. The present study investigated whether the levels of circulating corticosterone and its binding protein, corticosteroid binding globulin (CBG), are altered with reproductive experience and pup-exposure during late pregnancy and the postpartum. Total serum corticosterone and CBG were assayed from five groups; multiparous, primiparous, nulliparous, primip-no-pups, and sensitized rats during gestation (days 14 and 19) and the postpartum period (days 1, 5, 14, 21, and 35). Results show that primiparous rats had significantly elevated total corticosterone on postpartum day 1. In addition, primiparous and multiparous rats had significantly lower CBG throughout the postpartum period than all other groups, with primiparous rats exhibiting lower levels than multiparous rats during mid-lactation. These data suggest that free corticosterone is elevated in both primiparous and multiparous dams and is elevated to a greater degree in primiparous compared to multiparous dams during lactation. Corticosterone and CBG levels were positively correlated with specific maternal behaviors during the first week postpartum in parturient rats, but not in sensitized rats, suggesting a role for corticosterone in the modulation of maternal behavior in parturient rats alone.

Reduced maternal corticosteroid‐binding globulin and cortisol levels in pre‐eclampsia and gamete recipient pregnancies

To measure and contrast maternal cortisol and corticosteroid-binding globulin (CBG) levels in pregnancies with normal outcomes, pre-eclampsia, intrauterine growth restriction (IUGR) and in gamete recipients. Prospective study of 93 women at high risk of pre-eclampsia, including gamete recipients (n = 22) and 33 controls. Plasma total and free cortisol and CBG were measured every 2 weeks from 16 weeks' gestation until delivery. Forty-two per cent of the high-risk group had complications, including pre-eclampsia (n = 11), gestational hypertension (n = 16) and small-for-gestational-age (SGA) neonates (n = 12). There were no complications in the controls. In all groups, plasma CBG concentrations increased progressively across gestation (P < 0.05), in parallel to total cortisol, but fell significantly from 36 weeks' gestation onwards, with a corresponding rise in free cortisol concentrations. In pre-eclampsia and gestational hypertension, plasma CBG, and total and free cortisol concentrations were lower from 36 weeks onwards (P < 0.05). In IUGR, plasma CBG concentrations were suppressed from 28 weeks' gestation until delivery (P < 0.05), but with no significant difference in plasma total and free cortisol. Gamete recipients had significantly lower plasma CBG from 20 weeks' gestation onwards, and plasma total and free cortisol were reduced at 24 and 32 weeks onwards, respectively. Maternal plasma CBG, total and free cortisol concentrations are reduced in pre-eclampsia/gestational hypertension, and markedly reduced in gamete recipients. Low CBG may be due to reduced synthesis or enhanced inflammation-driven degradation. Low maternal cortisol may be due to a lack of placental corticotropin-releasing hormone or reduced maternal ACTH, driving cortisol production. Low maternal cortisol may influence the foetal hypothalamic-pituitary-adrenal axis and disease patterns later in life following complicated pregnancy.

COMPARISON OF QUADRATIC AND CUBIC EQUILIBRIUM SOLUTIONS OF FREE CORTISOL FOR PREDICTION OF SALIVARY CORTISOL IN HEALTHY ELDERLY HUMAN SUBJECTS.

Purpose Free cortisol is released into the serum compartment where it is in equilibrium with serum binding proteins, corticosteroid-binding globulin (CBG), and albumin. The distribution of serum cortisol among free, CBG-bound, and albumin-bound compartments is influenced by association and disassociation rate constants for CBG and albumin, the number of unoccupied protein binding sites, and concentrations of CBG and albumin. Free cortisol in serum or plasma is difficult to assay; thus, salivary cortisol has been proposed as an alternative measure of free cortisol. In this study, we sought to assess and compare numerical methods, using both quadratic (Coolens et al, 1987) and cubic (Keenan et al, 2004) equations to calculate serum free cortisol and compare these findings with salivary cortisol in healthy elderly subjects. Methods Serum and saliva samples were collected from 45 healthy, elderly subjects at 0700 h as part of the New Mexico Aging study (UNM HRRC 01-023). The salivary cortisol and total cortisol were measured using radioimmunoassay and ultrasensitive chemiluminescence, respectively. Serum CBG was measured by radioimmunoassay and serum albumin was measured using a bromcresol green method (UNM RAC 51). The cubic equation implied by Keenan et al and the quadratic equation described by Coolens et al were used to calculate free cortisol using our measured values for total cortisol, total CBG, and total albumin. Calculated free cortisol, total cortisol, and CBG concentration were then compared to salivary cortisol using Pearson9s and Spearman9s correlations. Results There was a significant linear correlation between salivary cortisol and free cortisol calculated using the cubic equation (r = .377, p = .033) and Coolen9s quadratic equation (r = .416, p = .018). In addition, we noted a linear correlation between CBG concentration and total cortisol (r = .441, p = .0049). Conclusion Our data indicate that the free cortisol calculated using either quadratic or cubic equations is correlated significantly with salivary cortisol but that other variables appear to influence measured salivary cortisol concentration. We did not observe the previously reported correlation between total and salivary cortisol, which suggests that aging may influence the relative distribution of cortisol in serum or salivary compartments.

Septic Shock and Sepsis: A Comparison of Total and Free Plasma Cortisol Levels

Severe systemic infection leads to hypercortisolism. Reduced cortisol binding proteins may accentuate the free cortisol elevations seen in systemic infection. Recently, low total cortisol increments after tetracosactrin have been associated with increased mortality and hemodynamic responsiveness to exogenous hydrocortisone in septic shock (SS), a phenomenon termed by some investigators as relative adrenal insufficiency (RAI). Free plasma cortisol may correspond more closely to illness severity than total cortisol, comparing SS and sepsis (S). This was a prospective study. This study took place in a tertiary teaching hospital. Patients had SS (n = 45) or S (n = 19) or were healthy controls (HCs; n = 10). The aim of the study was to compare total with free cortisol, measured directly and estimated by Coolens' method, corticosteroid-binding globulin (CBG), and albumin in patients with SS (with and without RAI) and S during acute illness, recovery, and convalescence. Comparing SS, S, and HC subjects, free cortisol levels reflected illness severity more closely than total cortisol (basal free cortisol, SS, 186 vs. S, 29 vs. HC, 13 nmol/liter, P < 0.001 compared with basal total cortisol, SS, 880 vs. S, 417 vs. HC, 352 nmol/liter, P < 0.001). Stimulated free cortisol increments varied greatly with illness category (SS, 192 vs. S, 115 vs. HC, 59 nmol/liter, P = 0.004), whereas total cortisol increments did not (SS, 474 vs. S, 576 vs. HC, 524 nmol/liter, P = 0.013). The lack of increase in total cortisol with illness severity is due to lower CBG and albumin. One third of patients with SS (15 of 45) but no S patients met a recently described criterion for RAI (total cortisol increment after tetracosactrin < or = 248 nmol/liter). RAI patients had higher basal total cortisol (1157 vs. 756 nmol/liter; P = 0.028) and basal free cortisol (287 vs. 140 nmol/liter; P = 0.017) than non-RAI patients. Mean cortisol increments in RAI were lower (total, 99 vs. 648 nmol/liter, P < 0.001; free, 59 vs. 252 nmol/liter, P < 0.001). These differences were not due to altered CBG or albumin levels. Free cortisol levels normalized more promptly than total cortisol in convalescence. Calculated free cortisol by Coolens' method compared closely with measured free cortisol. Free cortisol is likely to be a better guide to cortisolemia in systemic infection because it corresponds more closely to illness severity. The attenuated cortisol increment after tetracosactrin in RAI is not due to low cortisol-binding proteins. Free cortisol levels can be determined reliably using total cortisol and CBG levels.

Pregnancy-associated corticosteroid-binding globulin: high resolution separation of glycan isoforms.

Corticosteroid-binding globulin (CBG) is a glycoprotein that functions as a specific carrier of cortisol in the circulation. CBG contains six sites for N-glycosylation with, on average, five sites occupied by a mixture of biantennary and triantennary oligosaccharides with variable additional terminal sialic acid residues leading to glycoforms with significant heterogeneity in mass and isoelectric points. During pregnancy, a form of CBG possessing only triantennary oligosaccharides comprising approximately 10 % of total CBG appears specifically. We describe the first application of two-dimensional gel electrophoresis to the separation of human CBG glycoforms. This technique resolved a greater degree of charge heterogeneity than previous studies, and allowed simultaneous visualization of changes to the size and isoelectric points of CBG during pregnancy. Profiles of CBG glycoforms during pregnancy showed a general increase in size followed by a shift to lower pI in a large proportion of the glycoprotein. This may result from the enhancement of triantennary glycosylation, with the extent of incorporation of sialic acid increasing with the number of available sites for its addition. The pregnancy-specific CBG previously defined probably represents a subset of the acidic and high molecular weight glycoforms we have resolved by two-dimensional electrophoresis and now describe as pregnancy-associated CBG.

Plasma total and glycosylated corticosteroid-binding globulin levels are associated with insulin secretion.

In humans, steroid hormones circulate in the blood mainly bound to specific steroid transport proteins, namely corticosteroid-binding globulin (CBG) for cortisol and sex hormone-binding globulin (SHBG) for testosterone and estradiol. The binding activities of these proteins are believed to modulate the biodisposal of steroids to target cells. It has been shown in vitro that insulin is a potent inhibitor of both CBG and SHBG secretion by a human hepatoblastoma cell (HepG2) line. To further investigate this potential effect of insulin in vivo, we prospectively studied three groups of lean subjects, obese subjects, and obese subjects with glucose intolerance, all of whom were otherwise healthy. The three groups were comparable in sex and age, and in the two obese groups, body mass index, waist to hip ratio, and blood pressure were similar. Plasma total CBG concentrations (38.2 +/- 5.4 vs. 31.7 +/- 4.05 mg/L; P = 0.016) and glycosylated CBG levels (37.3 +/- 5.2 vs. 31 +/- 3.9 mg/L; P = 0.018) were significantly increased in obese subjects with glucose intolerance. Plasma CBG correlated positively with fasting glucose levels (r = 0.49; P = 0.002), hemoglobin A1c levels (r = 0.35; P = 0.03), and area under the curve of glucose after an oral glucose tolerance test (r = 0.45; P = 0.005) and correlated negatively with the insulin response to i.v. glucose (AIRg; -0.38, P = 0.02) as well as to oral glucose (r = -0.40; P = 0.01) challenge tests. CBG levels did not covariate with insulin sensitivity. Multiple linear regression analysis showed that only AIRg contributed to the variability of the CBG concentration (P = 0.03), explaining 41% of its variance. Morning cortisol levels did not differ between the groups and did not correlate to any of the glucose or insulin metabolism parameters. Because carbohydrate chains influence the biological activity and half-life of glycoproteins, we analyzed the migration profile of CBG by Western blot and the interaction of CBG with lectin, Con A. The results indicated that the CBG mol wt and interaction with Con A did not differ between lean and obese patients. These data favor the hypothesis that the inhibitory effect of insulin on CBG liver secretion might be relevant in vivo and therefore contribute to decrease CBG levels in obese patients with enhanced insulin secretion. In both men and women, SHBG levels correlated negatively with fasting glucose (r = -0.55; P < 0.0001) and hemoglobin A1c (r = -0.38; P = 0.02) and positively with insulin sensitivity (S(I); r = 0.65; P = 0.003 and r = 0.63; P = 0.007 in men and women, respectively), but not with insulin secretion. The disposition index (S(I) x AIRg) was significantly decreased in the obese, glucose-intolerant subjects, suggesting that AIRg was inadequate for their degree of insulin resistance. The disposition index correlated positively with plasma SHBG levels (r = 0.52; P = 0.001) and negatively with plasma CBG levels (r = -0.54; P = 0.001). Our data suggest that CBG is a marker of insulin secretion in a similar way as SHBG is a marker of insulin sensitivity. As high plasma CBG levels have been associated with increased incidence of type 2 diabetes, this important issue merits further investigations.

Corticosteroid-binding globulin and free cortisol in the early postoperative period after cardiac surgery

Objectives: To characterize concentrations of corticosteroid-binding globulin (CBG), total and free serum cortisol, and free urinary cortisol in patients during the postoperative period of cardiac surgery. Design and Methods: In 24 patients serum was sampled on the first and second postoperative day after cardiac surgery (21 procedures with thoracotomy, 3 thoracoscopic procedures); urine was collected for two 10-h periods (8 p.m. until 6 a.m.) on the respective postoperative days. Total serum cortisol and free urinary cortisol were measured with an automated chemiluminescence assay (analysis of urine after extraction with dichloromethane), and CBG using a coated-tube RIA. Free serum cortisol was calculated from the concentrations of total serum cortisol and CBG as decribed previously. Thirty healthy volunteers were studied as controls. Results: CBG was reduced to about one-half of the normal concentration on both postoperative days. Whereas total cortisol was about two-fold increased on the first postoperative day compared to controls extremely high concentrations of free serum cortisol were calculated from CBG and total cortisol [median 136 nmol/L (interquartile range 100–185); controls 21.8 nmol/L (interquartile range 16.9–29.8)]. On the second postoperative day, median total serum cortisol was within the interquartile range of the controls, free serum cortisol in contrast was still two-fold increased. Free serum cortisol and free urinary cortisol were significantly correlated (r = 0.60). Conclusions: Extremely high concentrations of free serum cortisol are typically found in the postoperative period of cardiac surgery; under these conditions the mere consideration of total cortisol does not appropriately display the activation of the adrenal cortex.

Hamster Uterine Tissues Accumulate Corticosteroid-Binding Globulin during Decidualization1

Although corticosteroid-binding globulin (CBG) is known to be a serum steroid-binding protein, its function outside of the vascular space is not well understood. To prove an extravascular role for CBG, it must first be established that CBG occurs in steroid target tissues. We sought information on the occurrence of CBG in the cytosol, nuclear, and membrane fractions of 6 tissues during decidualization in the hamster. Our objectives were to determine if CBG is distributed in a tissue-specific manner, and to investigate the relationship between serum CBG and tissue CBG. Hamsters were given progesterone pellets s.c. on cycle Day 1 and decidualization was induced on Day 4. A 3H-cortisol-binding assay, which distinguished between CBG and glucocorticoid receptor, was used to determine CGB levels in the serum and in the cytosol, nuclear, and membrane fractions of deciduoma, myometrium, liver, kidney, muscle, and small intestine. Cytosol CBG accounted for greater than 97% of the total CBG detected in all tissues except liver, where nuclei contained 11% of the measurable CBG. For all cell fractions, CBG levels showed consistent tissue-specific differences. Cytosol CBG was highest in deciduoma and myometrium, 2-fold less in liver and kidney, and 5-fold less in muscle and small intestine. Nuclear CBG concentration was greatest in liver and approximately 10-fold less in other tissues, except for small intestine, where nuclear CBG was undetectable. Membrane CBG was highest in liver, 5-fold less in deciduoma, 10-fold less in myometrium, and about 20-fold less in other tissues. Serum CBG increased 7-fold from Day 4 to Day 9 in decidualized hamsters, but not in nondecidualized sham-operated hamsters. In all tissues, serum CBG was correlated with cytosol CBG. The high levels of CBG in uterine tissues were not the result of serum contamination because whole-body perfusion with buffered saline failed to remove the majority of cytosol CBG under conditions where over 70% of 51Cr-labeled red blood cells were removed. The identity of uterine cytosol CBG with serum CBG was established by ion-exchange chromatography (O-(diethylaminoethyl)-cellulose) and by immunoprecipitation with an antibody generated against serum CBG. These data demonstrate that uterine tissues accumulate substantial amounts of CBG during decidualization, thus raising the possibility of a functional role of CBG in uterine tissues during early pregnancy.

Glucocorticoid and estrogen regulation of corticosteroid-binding globulin production by rat liver.

We have recently demonstrated that rat liver can synthesize and secrete corticosteroid-binding globulin (CBG). The present study extends these observations and examines the hormonal regulation of hepatic CBG production. Male rats were pretreated by adrenalectomy and/or glucocorticoid or estrogen administration and the rate of CBG production was measured in vitro. The production rates were assessed by the generation of specific corticosterone binding sites in both a liver-slice preparation and in an isolated perfused liver. The two techniques showed qualitatively similar results. Adrenalectomy enhanced and glucorticoid administration inhibited the rate of CBG production and secretion. Pretreatment of male rats with estradiol stimulated the rate of CBG production. The production rates were 20- to 40-fold higher in the perfused liver demonstrating its superiority over the liver-slice system. The livers from intact rats secreted CBG binding sites at a rate of approximately 18 pmol/g liver per hours, generating an estimated 20% of the total CBG content of a rat each day. The possible clinical implications of the therapeutic use of glucocorticoids that bind to CBG, yet inhibit CBG production, are discussed.