Steroid and Immunobiochemistry Laboratory

Christchurch, New Zealand

Steroid and Immunobiochemistry Laboratory

Christchurch, New Zealand
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Lindsay A.,University of Canterbury | Healy J.,University of Canterbury | Mills W.,University of Canterbury | Lewis J.,Steroid and Immunobiochemistry Laboratory | And 5 more authors.
Scandinavian Journal of Medicine and Science in Sports | Year: 2016

Muscle damage caused through impacts in rugby union is known to increase oxidative stress and inflammation. Pterins have been used clinically as markers of oxidative stress, inflammation, and neurotransmitter synthesis. This study investigates the release of myoglobin from muscle tissue due to force-related impacts and how it is related to the subsequent oxidation of 7,8-dihydroneopterin to specific pterins. Effects of iron and myoglobin on 7,8-dihydroneopterin oxidation were examined in vitro via strong cation-exchange high-performanceliquid chromatography (SCX-HPLC) analysis of neopterin, xanthopterin, and 7,8-dihydroxanthopterin. Urine samples were collected from 25 professional rugby players pre and post four games and analyzed for myoglobin by enzyme-linked immunosorbent assay, and 7,8-dihydroneopterin oxidation products by HPLC. Iron and myoglobin oxidized 7,8-dihydroneopterin to neopterin, xanthopterin, and 7,8-dihydroxanthopterin at concentrations at or above 10μM and 50μg/mL, respectively. All four games showed significant increases in myoglobin, neopterin, total neopterin, biopterin, and total biopterin, which correlated between each variable (P<0.05). Myoglobin and iron facilitate 7,8-dihydroneopterin oxidation to neopterin and xanthopterin. In vivo delocalization of myoglobin due to muscle damage may contribute to oxidative stress and inflammation after rugby. Increased concentrations of biopterin and total biopterin may indicate production of nitric oxide and monoamine neurotransmitters in response to the physical stress. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nguyen P.T.T.,Agresearch Ltd. | Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Sneyd J.,University of Auckland | Lee R.S.F.,Agresearch Ltd. | And 3 more authors.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2014

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. © 2014 Elsevier Ltd.

Burt M.G.,Repatriation General Hospital | Burt M.G.,Flinders University | Mangelsdorf B.L.,Repatriation General Hospital | Rogers A.,Flinders University | And 6 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2013

Context: Measurement of plasma cortisol by immunoassay after ACTH 1-24 stimulation is used to assess the hypothalamic-pituitary-adrenal (HPA) axis. Liquid chromatography-tandem mass spectrometry (LCMS) has greater analytical specificity than immunoassay and equilibrium dialysis allows measurement of free plasma cortisol. Objective: We investigated the use of measuring total and free plasma cortisol by LCMS and total cortisol by immunoassay during an ACTH1-24 stimulation test to define HPA status in pituitary patients. Design and Setting: This was a case control study conducted in a clinical research facility. Participants: Westudied 60 controls and 21 patients with pituitary disease in whom HPA sufficiency (n = 8) or deficiency (n = 13) had been previously defined. Intervention: Participants underwent 1 μg ACTH1-24 intravenous and 250 μg ACTH 1-24 intramuscular ACTH1-24 stimulation tests. Main Outcome Measures: Concordance of ACTH1-24-stimulated total and free plasma cortisol with previous HPA assessment. Results: Total cortisol was 12% lower when measured by immunoassay than by LCMS. Female sex and older age were positively correlated with ACTH1-24-stimulated total and free cortisol, respectively. Measurements of total cortisol by immunoassay and LCMS and free cortisol 30 minutes after 1 μg and 30 and 60 minutes after 250 μg ACTH1-24 were concordant with previous HPA axis assessment in most pituitary patients. However, free cortisol had greater separation from the diagnostic cutoff than total cortisol. Conclusions: Categorization of HPA status by immunoassay and LCMS after ACTH1-24 stimulation was concordant with previous assessment in most pituitary patients. Free cortisol may have greater clinical use in patients near the diagnostic threshold. Copyright © 2013 by The Endocrine Society.

Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Elder P.A.,Steroid and Immunobiochemistry Laboratory
Clinica Chimica Acta | Year: 2013

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. © 2012 Elsevier B.V.

Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Elder P.A.,Steroid and Immunobiochemistry Laboratory
Molecular and Cellular Endocrinology | Year: 2014

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. © 2014 Elsevier Ireland Ltd.

Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Saunders K.,University of Otago | Dyer A.,University of Otago | Elder P.A.,Steroid and Immunobiochemistry Laboratory
Journal of Steroid Biochemistry and Molecular Biology | Year: 2015

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 (∼10 h). 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. © 2015 Elsevier Ltd.

Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Elder P.A.,Steroid and Immunobiochemistry Laboratory
Journal of Steroid Biochemistry and Molecular Biology | Year: 2011

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. © 2010 Elsevier Ltd. All rights reserved.

Lindsay A.,University of Canterbury | Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Scarrott C.,University of Canterbury | Gill N.,University of Auckland | And 3 more authors.
International Journal of Sports Medicine | Year: 2015

Rugby union is a sport involving high force and frequency impacts making the likelihood of injury a significant risk. The aim of this study was to measure and report the individual and group acute and cumulative physiological stress response during 3 professional rugby games through non-invasive sampling. 24 professional rugby players volunteered for the study. Urine and saliva samples were collected pre and post 3 matches. Myoglobin, salivary immunoglobulin A, cortisol, neopterin and total neopterin (neopterin+7,8-dihydroneopterin) were analysed by high performance liquid chromatography or enzyme linked immunosorbent assay. Significant increases in cortisol, myoglobin, neopterin and total neopterin when urine volume was corrected with specific gravity were observed (p<0.05). Significant decreases in salivary immunoglobulin A concentration were observed for games 1 and 2 while secretion rate decreased after games 2 and 3. Significant decreases were seen with the percent of 7,8-dihydroneopterin being converted to neopterin following games 2 and 3. The intensity of 3 professional rugby games was sufficient to elicit significant changes in the physiological markers selected for our study. Furthermore, results suggest the selected markers not only provide a means for analysing the stress encountered during a single game of rugby but also highlight the unique pattern of response for each individual player © Georg Thieme Verlag KG Stuttgart New York.

Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Fredericks R.,University of Canterbury | Fee C.J.,University of Canterbury | Elder P.A.,Steroid and Immunobiochemistry Laboratory
Journal of Steroid Biochemistry and Molecular Biology | Year: 2016

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. © 2016 Elsevier Ltd. All rights reserved.

Simard M.,University of British Columbia | Hill L.A.,University of British Columbia | Lewis J.G.,Steroid and Immunobiochemistry Laboratory | Hammond G.L.,University of British Columbia
Journal of Clinical Endocrinology and Metabolism | Year: 2015

Context: 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. Objective: 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. Design: Human CBG mutants were produced in Chinese hamster ovary cells for ELISA, cortisolbinding activity measurements, and Western blotting as well as assays of their protease sensitivities. Results: 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 thatCBGI48N andCBGP246Qhave secretion defects.Twomutants (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). Conclusion: 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. © 2015 by the Endocrine Society.

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