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.

Effect of genetic factors on the response to vitamin D 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 D (200 000 IU each for the first 2 mo, thereafter 100 000 IU monthly) or placebo for 18 mo.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

An enzyme-linked immunosorbent assay (ELISA) for methylphenidate (Ritalin ) in urine.

A direct enyzme-linked immunosorbent assay (ELISA) for urinary immunoreactive methylphenidate (Ritalin), in which a standard 96-well microtiter plate is used, is described. For this ELISA, a methylphenidate-thyroglobulin conjugate is immobilized to the microtiter plate and competes with methylphenidate in the standard or urine sample for antibody-binding sites. After washing, the sheep methylphenidate antibody bound to immobilized methylphenidate is detected with peroxidase-labelled goat antisheep IgG.

An enzyme-linked immunosorbent assay for corticosteroid-binding globulin using monoclonal and polyclonal antibodies: decline in CBG following synthetic ACTH.

Background: Adrenal function is commonly assessed by measuring plasma cortisol following synthetic ACTH (synacthen) challenge. Generally little regard is given to plasma levels of corticosteroid-binding globulin (CBG). We have developed and validated an enzyme-linked immunosorbent assay (ELISA) for CBG and together with plasma cortisol calculated the "free cortisol index" as an additional parameter for assessing adrenal function.

Caution on the use of saliva measurements to monitor absorption of progesterone from transdermal creams in postmenopausal women.

Objectives: To determine the levels of progesterone in plasma, red cells and saliva as well as pregnanediol-3-glucuronide excretion in postmenopausal women using transdermal progesterone creams.

Methods: A double-blind placebo controlled study was carried out using 24 postmenopausal women. Creams (placebo, 20 or 40 mg progesterone/g) were applied twice daily for 3 weeks followed by 1 week without before a further 3-week treatment.

The effect of isoflavone extract ingestion, as Trinovin, on plasma steroids in normal men.

Plasma testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone sulfate, androsterone and epiandrosterone sulfates, cortisol and sex hormone binding globulin were measured in six adult men before and during daily isoflavone extract ingestion (40 mg) in the form of Trinovin tablets. Although modest plasma genistein levels were achieved following three weeks of Trinovin ingestion (106-356 nmol/l) there were no significant changes in most of the analytes tested. However plasma levels of dihydrotestosterone showed an increase that reached significance when combined basal levels were compared to levels following Trinovin treatment.