Glycosylation may influence sex hormone-binding globulin measurements.

A discordance between sex hormone-binding globulin (SHBG) measurements by 2-site ELISAs was investigated using pairings of various "in house" SHBG antibodies together with a concordant control. The 2-site monoclonal ELISAs used the same base coat (11F11) and discordance was observed with one top coat monoclonal antibody (7H9) and also when a polyclonal SHBG antibody was paired with the basecoat antibody (11F11). Sialidase treatment of the discordant sample and purified SHBG revealed increased levels using 7H9 whereas there was no change in SHBG in the concordant sample. Conversely, following sialidase treatment, the discordant sample showed no change in SHBG measured using the other monoclonal antibody pairings whereas the SHBG levels in the concordant sample declined following sialidase using the same monoclonal antibody pairings. This implicated glycosylation as a factor in antibody recognition and synthetic peptides spanning the two N-linked and one O-linked glycosylation regions showed that SHBG recognition by monoclonal antibody 7H9 could be disrupted by a peptide spanning the O-linked glycosylation site. Hence rather than immunoassay discordance being attributed to heterophile antibodies or other circulating antibodies here it can be likely attributed to glycosylation affecting antibody recognition and hence the measurement of SHBG.

Effect of genetic factors on the response to vitamin D3 supplementation in the VIDARIS randomized controlled trial.

OBJECTIVES: Supplementation provides the best means of improving vitamin D status; however, individual responses vary partly owing to genetics. The aim of this study was to determine whether 28 single nucleotide polymorphisms (SNPs) in six key vitamin D pathway genes (GC, DHCR7, CYP2 R1, CYP24 A1, CYP27 B1, VDR) were associated with differences in response to supplementation. METHODS: Participants (N = 313; n = 160 vitamin D, n = 153 placebo) were part of VIDARIS (Vitamin D and Acute Respiratory Infections Study), a double-blind, randomized controlled trial involving oral monthly supplementation of either vitamin D3 (200 000 IU each for the first 2 mo, thereafter 100 000 IU monthly) or placebo for 18 mo. Circulating 25-hydroxyvitamin D (25[OH]D) concentrations at baseline and 2, 6, 12, and 18 mo, and vitamin D binding protein (Gc-globulin) and calculated free 25(OH)D concentrations at baseline and 2 mo were obtained. Multiple regression was used to model associations between genetic variants and 25(OH)D, Gc-globulin, and free 25(OH)D concentrations. RESULTS: SNPs within GC, CYP2 R1, and CYP27 B1 were associated with 25(OH)D concentrations following supplementation. However, only two GC gene SNPs (rs2282679, rs1155563) were significant after adjustment for multiple testing. This effect disappeared after more than 2 mo of supplementation. None of the SNPs were significantly associated with Gc-globulin concentrations; however, there was a significant interaction with one SNP in DHCR7 (rs12785878), which was associated with reduced free 25(OH)D concentrations in the supplemented arm. CONCLUSION: Only variants of GC were associated with 25(OH)D concentrations after supplementation. This effect was modest and disappeared after >2 mo of supplementation, suggesting it may be time/dose-dependent and saturable.

Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis.

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 µM, cross-linked calprotectin (5 µM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 µM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.

A monoclonal antibody sandwich ELISA for vitamin D-binding protein (VDBP) is unaffected by Gc-globulin phenotype peptides and actin and demonstrates reduced levels in sepsis and non-sepsis intensive care patients.

The measurement of vitamin D-binding protein (VDBP) by immunoassay has been confounded by variable antibody recognition of the Gc1s, Gc1F and Gc2 phenotypes. This has led to spurious conclusions regarding vitamin D status in different ethnic groups. In order to overcome these problems there is a requirement for VDBP antibodies that are unaffected by phenotype status. Here we report the generation and testing of three monoclonal antibodies to VDBP which recognise linear epitopes and are unaffected by vast molar excesses of synthetic peptides spanning these phenotypic domains. These IgG1 kappa antibodies were purified and biotinylated to allow suitable pairings to develop a sandwich ELISA for circulating VDBP. The VDBP ELISA is unaffected by actin and confirms that VDBP levels are significantly reduced in sepsis patients and non-sepsis intensive care patients compared to normal healthy subjects. Levels of VDBP along with total 25OH vitamin D3 can be used to calculate free 25OH vitamin D3 levels and these compare well with consensus values determined independently. The VDBP ELISA meets acceptable performance criteria and as such can be used in conjunction with total 25OH vitamin D3 to determine the free 25OH vitamin D3 status in various cohorts.

Serum fibroblast growth factor 23 concentrations in dogs with chronic kidney disease.

The aim of this study was to determine if serum fibroblast growth factor (FGF23) concentrations were increased in dogs with chronic kidney disease (CKD). Serum samples submitted to a commercial laboratory were collected over a 15-month period, 14 samples were from dogs with a history of polyuria/polydipsia, azotaemia and low urine specific gravity, 20 samples were from non-azotaemic dogs. Serum FGF23, parathyroid hormone, total calcium and phosphorus, urea and creatinine were measured. Mann-Whitney test was used to determine differences between non-azotaemic and CKD groups; a one-way ANOVA with Tukey pairwise comparisons was used to determine any differences between International Renal Interest Society stages; and regression models were used to determine predictors of International Renal Interest Society stage, serum phosphorus and FGF23 concentrations. The median serum FGF23 concentration of dogs with CKD was 5194.6pg/mL, which was significantly greater (P<0.001) than the median serum FGF23 concentration of non-azotaemic dogs (259.2pg/mL). Log serum FGF23 and age were significantly associated with IRIS stage (P=0.027 and P=0.032 respectively), while log serum phosphorus concentration (P<0.001) was significantly associated with log serum FGF23 concentration. In summary, serum FGF23 concentration is increased in dogs with CKD, and is associated with serum phosphorus concentration. This phosphatonin pathway may be a useful target for the development of future treatments to control plasma phosphorus concentrations in chronic kidney disease.

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation.

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.

The half-lives of intact and elastase cleaved human corticosteroid-binding globulin (CBG) are identical in the rabbit.

Corticosteroid-binding globulin (CBG) is a non-inhibitory member of the serpin superfamily of serine protease inhibitors and carries the majority of cortisol in circulation. It can be cleaved by neutrophil elastase at its exposed reactive centre loop which decreases its affinity for cortisol allowing the release of most of the cortisol at sites of inflammation. Intact and elastase cleaved CBG can be distinguished from each other and can coexist in circulation but with unknown half-lives. Here we treated a portion of purified human CBG with elastase, terminated the digestion and then combined this portion with intact human CBG and measured their respective half-lives in rabbits by ELISA. This investigation shows for the first time that the half-lives of intact and elastase cleaved CBG are identical (∼10h). This is an important finding as it implies that in conditions such as sepsis and septic shock where levels of intact CBG are low and the proportion of cleaved CBG is high that this is likely sustained which may affect the CBG mediated targeted delivery of cortisol to sites of inflammation. Furthermore the residual binding of cortisol to cleaved CBG may alter the overall buffering capacity of CBG for cortisol resetting the baseline concentration of free cortisol.

Differential extraction of endogenous and exogenous 25-OH-vitamin D from serum makes the accurate quantification in liquid chromatography-tandem mass spectrometry assays challenging.

BACKGROUND: Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the method of choice when it comes to the accurate quantification of 25-OH-vitamin D in blood samples. It is generally assumed that the addition of exogenous internal standard allows for the determination of the endogenous analyte concentration. In this study we investigated the extraction properties of endogenous and exogenous 25-OH-vitamin D. METHODS: Eight samples were used for the evaluation of the extraction procedure and 59 patients' samples for a method comparison. The methanol-to-sample ratio (v/v) and the sample-to-hexane ratio (v/v) were varied and the LC-MS/MS signals of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and internal standard of the extracts recorded. The optimized 'in-house' LC-MS/MS assay was compared to two automated chemiluminescence immunoassays from DiaSorin and Abbott. RESULTS: Mathematical analysis of the data revealed a differential extraction of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and non-equilibrated internal standard. Exogenous 25-OH-vitamin D can be measured accurately if a definite methanol-to-sample ratio is used. Endogenous 25-OH-vitamin D is affected by critical quantification issues due to a differential slope in the extraction profile. The actual 25-OH-vitamin D concentration can be one-third above the measured extractable concentration. Results confirm that the 'in-house' LC-MS/MS assay provides reproducible 25-OH-vitamin D results. CONCLUSIONS: Discordant concentrations of 25-OH-vitamin D from LC-MS/MS assays can be caused by selection of suboptimal extraction conditions. Furthermore, a different sample pretreatment or solvent extraction system may result in a different dissociation and extraction yield of endogenous 25-OH-vitamin D and therefore contribute to variations of LC-MS/MS results.

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.

Plasma retinol-binding protein is not a marker of insulin resistance in overweight subjects: a three year longitudinal study.

OBJECTIVES: This longitudinal study investigated whether or not plasma retinol-binding protein (RBP), recently referred to as RBP4, was a marker of insulin resistance in overweight subjects. METHODS: We measured anthropometric markers as well as RBP, fasting glucose and insulin in 206 overweight subjects and repeated these measurements 36 months later. Subjects were grouped according to fasting plasma glucose concentration at baseline and 36 months. RESULTS: Subjects (n=51) with a normal basal fasting glucose (<5.6 mmol/L) who developed impaired fasting glucose (IFG) 3 years later (>or=5.6 mmol/L) showed a highly significant increase in both fasting insulin and insulin resistance, but importantly no change in plasma RBP. This group had a significant increase in body mass index (BMI). Subjects (n=101) with a normal fasting glucose at both baseline (<5.6 mmol/L) and 36 months showed no significant change in fasting insulin, insulin resistance, RBP or BMI. The remaining subjects had impaired basal fasting glucose and were not analysed on a group-wise basis. Overall, RBP correlated significantly, but inversely, with anthropometric measures, but not with fasting glucose, insulin or insulin resistance. CONCLUSIONS: This is the first report of a long-term longitudinal study on RBP and the major finding is that subjects who developed insulin resistance showed no change in plasma RBP. On the basis of our results we consider that RBP cannot be construed as a marker of insulin resistance in overweight humans.

Plasma retinol-binding protein is unlikely to be a useful marker of insulin resistance.

To assess whether plasma retinol-binding protein (RBP) is a marker of insulin resistance we measured RBP, insulin and glucose in 285 fasting subjects attending a Lipid Disorders Clinic as outpatients. They were grouped as either subjects without diabetes mellitus and with varying degrees of insulin resistance or subjects with diabetes mellitus according to the WHO criteria. We show that there was no association between plasma RBP and insulin-resistance, insulin, glucose, % body fat, waist circumference or BMI whether analysed together or in groups. We confirm, using the largest study cohort to date, that plasma RBP is unlikely to be a useful marker of insulin resistance.

An ELISA for plasma retinol-binding protein using monoclonal and polyclonal antibodies: plasma variation in normal and insulin resistant subjects.

OBJECTIVES: Plasma retinol-binding protein (RBP) has been linked to insulin resistance and cardiovascular risk, yet little is know of its natural variation in plasma. We examined this in normal subjects and compared plasma levels and variability in lean subjects and subjects with the metabolic syndrome. METHODS: We established an "in house" ELISA for plasma RBP and measured levels in 20 normal subjects over daylight hours and 2 subject groups, either lean or classified with the metabolic syndrome. RESULTS: Plasma RBP in normal subjects did not vary over the day with no differences between males and females. There was also no difference in plasma RBP levels and between the age- and sex-matched lean subjects compared to the metabolic syndrome group. CONCLUSION: The lack of variation in plasma RBP in normal subjects and the lack of difference between plasma RBP in normal and metabolic syndrome subjects suggest the link between plasma RBP and insulin resistance is tenuous. Investigating a large cohort over the diabetic non-diabetic spectrum may clarify this issue.

Plasma free cortisol fraction reflects levels of functioning corticosteroid-binding globulin.

BACKGROUND: In normal plasma free cortisol accounts for less than 6% of the total with 80-90% bound to corticosteroid-binding globulin (CBG) and the remainder associated albumin. However little is known about the distribution of free cortisol in plasma where CBG is inactivated or in congenital CBG deficiency. METHODS AND RESULTS: Here we describe ligand binding experiments revealing that while free cortisol in unstressed individuals is less than 6% of total cortisol this rises markedly to 25% when CBG is totally inactivated by heat. Similar elevations of the free cortisol fraction were noted in a patient with a rare genetically determined complete lack of CBG (mean 32% on frequent circadian sampling). Following heat inactivation of CBG or in the congenital absence of CBG, there is a shift in cortisol binding from CBG to albumin. That this shift occurs is further supported by experiments adding [3H]-cortisol to physiological human serum albumin solutions, where 25% of cortisol remained in the free fraction. CONCLUSION: Taken together the data provide strong evidence that when CBG is inactivated or congenitally absent then more than 25% of the total cortisol appears in the free fraction with the remainder associated with albumin. The proportion of free cortisol measured in plasma thus reflects a simple measure of functional corticosteroid-binding globulin.