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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.

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.

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.

Nenke M.A.,Endocrine and Metabolic Unit | Nenke M.A.,University of Adelaide | Rankin W.,Chemical Pathology Direct. | Chapman M.J.,Intensive Care Unit | And 7 more authors.
Clinical Endocrinology | Year: 2015

Objective 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. Design Prospective, observational cohort study in an adult tertiary level Intensive Care Unit in Adelaide, Australia. Patients Thirty-three patients with sepsis or septic shock grouped by illness severity [sepsis, septic shock survivors, septic shock nonsurvivors and other shock]. Measurements 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. Results 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. Conclusions 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. © 2015 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.

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