Protein carbonyl measurement by enzyme-linked immunosorbent assay.
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Aldehydes↗
Biomedical subjects
C C Winterbourn
Publications and source records attributed to C C Winterbourn.
Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide.
The reactivities of glutathione, cysteine, cysteamine, penicillamine, N-acetylcysteine, dithiothreitol and captopril with superoxide generated from xanthine oxidase and hypoxanthine, and with reagent hydrogen peroxide, have been investigated. Rates of thiol loss on adding hydrogen peroxide, and superoxide-dependent thiol loss and oxygen uptake were measured. The relative reactivities of the different thiols with both oxidants were inversely related to the pK of the thiol group, such that at pH 7.4, penicillamine was the most reactive. N-acetylcysteine weakly reactive and no reaction was seen with captopril. For hydrogen peroxide, the calculated rate constants for the reaction with the thiolate anion all fell within the range 18-26 M(-1) s(-1). With superoxide, our results are consistent with each thiol reacting via a short chain that consumes oxygen and regenerates superoxide. Only with some of the thiols, was the consumed oxygen recovered as hydrogen peroxide. Reported values for the rate constant for the reaction of thiols with superoxide vary over four orders of magnitude, with the highest being > 10(5) M(-1) s(-1). Due to the complexity of the chain reaction, no study so far has been able to obtain accurate values and we consider the best estimates to be in the 30 to 1000 M(-1) s(-1) range.
Acetylcysteine↗
Early ascorbic acid depletion is related to the severity of acute pancreatitis.
BACKGROUND: Ascorbic acid (AA) is an important endogenous antioxidant in plasma and has been shown to be decreased at the time of hospital admission in patients with acute pancreatitis. The aim of this study was to determine whether plasma AA concentration continues to decrease after admission and whether the extent of decrease is related to the severity of pancreatitis. METHODS: Consecutive patients with mild (n = 62) and severe (n = 23) acute pancreatitis had plasma AA concentration measured on the day of recruitment and on days 2 and 5 by high-performance liquid chromatography. RESULTS: The plasma AA concentration in patients with acute pancreatitis was significantly less than that in normal volunteers on days 0, 2 and 5 (P < 0.0001) and this was more marked in those with severe disease. There was a decrease in plasma AA concentration from day 0 to day 2 in patients with mild (P < 0.0001) and severe (P = 0.0005) pancreatitis, and from day 2 to day 5 in patients with severe pancreatitis (P = 0.023). CONCLUSION: Endogenous plasma AA continues to decrease over the first 5 days in hospital and the extent is related to the severity of acute pancreatitis. Presented to a meeting of the Australasian Surgical Research Society, Auckland, New Zealand, August 1995 and published in abstract form as Aust N Z J Surg 1996; 66: 243
Acute Disease↗
Small bowel ischaemia-reperfusion increases plasma concentrations of oxidised proteins in rats.
OBJECTIVES: To find out whether plasma concentrations of protein carbonyl (a specific marker of oxidative damage of proteins) are increased during intestinal ischaemia-reperfusion and whether they are correlated with von Willebrand's factor (vWF, a marker of endothelial injury) or myeloperoxidase (a marker of neutrophil activation). DESIGN: Randomised experimental study. SETTING: University department of surgery, New Zealand. ANIMALS: Thirty anaesthetised adult Wistar rats. INTERVENTIONS: The sham operated group (n = 10) had laparotomy and isolation of the superior mesenteric artery without clamping. The ischaemia-reperfusion group (IR, n = 10) had the superior mesenteric artery clamped for 1 hour and reperfusion for 15 minutes. The control group (n = 10) had direct puncture of the heart to sample blood. MAIN OUTCOME MEASURES: Plasma concentrations of protein carbonyl, vWF, and myeloperoxidase. RESULTS: Plasma protein carbonyl concentrations were significantly higher in the IR group than in the sham group (p < 0.02, Mann-Whitney test, median (range) 0.187 (0.141-0.242) compared with 0.144 (0.121-0.185) nmol/mg) and in the control group (p < 0.01, Mann-Whitney test, median (range) 0.187 (0.141-0.242) compared with 0.136 (0.108-0.175) nmol/mg). There was a significant correlation between protein carbonyl and vWF concentrations (r = 0.54, F = 10.9, p < 0.003, linear regression) but not with those of myeloperoxidase. CONCLUSION: Intestinal ischaemia-reperfusion caused an increase in the plasma protein carbonyl concentration, which is possibly produced by endothelial cells.
Animals↗
Loss of GSH and thiol enzymes in endothelial cells exposed to sublethal concentrations of hypochlorous acid.
We investigated the effect of sublethal concentrations of hypochlorous acid (HOCl) on intracellular thiol groups. Exposure of human umbilical vein endothelial cells to HOCl caused a decrease in cell viability, with concentrations of </=25 microM HOCl being sublethal. At these concentrations, we saw a loss of glutathione and total protein thiol groups. Of the thiol enzymes we investigated, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was particularly susceptible to inactivation, creatine kinase was moderately susceptible, and lactate dehydrogenase was unaffected by HOCl at the concentrations used. Similar results were obtained with HOCl generated over 30 min by myeloperoxidase. GAPDH activity could be regenerated on reincubation of cells in Hanks' balanced salt solution or reduction with dithiothreitol. In contrast, glutathione loss was not reversible, and further decreased with time. Cellular ATP levels decreased with sublethal HOCl concentrations and this appeared to be unrelated to the inactivation of GAPDH. Our results demonstrate that intracellular thiol groups differ in their reactivity with HOCl and suggest that HOCl may be able to regulate specific cellular functions.
Adenosine Triphosphate↗
Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing.
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Bacteria↗
Activation of NF-kappaB in human neutrophils during phagocytosis of bacteria independently of oxidant generation.
We have exposed human neutrophils to opsonized Staphylococcus aureus and used an electrophoretic mobility shift assay to show activation of the transcription factor NF-kappaB above basal levels. Activation was evident within 10 min and was increased with higher bacteria:neutrophil ratios. The neutrophil NADPH oxidase inhibitor diphenylene iodonium, catalase, and other oxidant scavengers did not inhibit NF-kappaB activation, and no activation was seen with added hydrogen peroxide. Oxidants produced during phagocytosis, therefore, are not involved in the activation mechanism.
Humans↗
Differential reactivities of hypochlorous and hypobromous acids with purified Escherichia coli phospholipid: formation of haloamines and halohydrins.
Hypochlorous (HOCl) and hypobromous (HOBr) acids are strong oxidants derived from myeloperoxidase and eosinophil peroxidase, the major antimicrobial enzymes of neutrophils and eosinophils, respectively. These oxidants are highly reactive with a wide range of biomolecules. At physiological pH, both HOCl and HOBr react readily with amines to form haloamines and with the unsaturated bonds of fatty acids to form halohydrins. We have investigated which of these reactions occur with phosphatidylethanolamine (PE), the predominant phospholipid of Escherichia coli. The formation of haloamines was determined by TLC and colorimetrically and the formation of halohydrins was determined by TLC and GC-MS. With HOCl, chloramines were much the preferred product and chlorohydrins were formed in substantial amounts only when HOCl was in excess of the amount required to convert the amine to the dichloramine. With HOBr at all concentrations, bromamines and bromohydrins were formed concurrently, indicating a greater relative reactivity with unsaturated fatty acids than with HOCl. The bromamine derivatives of PE, and other primary amines, were found to be more reactive than the equivalent chloramines, and were able to brominate the unsaturated bonds of fatty acids. Bromohydrins (formed directly or through the action of bromamines) may, therefore, be suitable biomarkers for the production of HOBr in vivo.
Alcohols↗
Plasma vitamin A levels in the very low birthweight infant--relationship to respiratory outcome.
OBJECTIVE: To examine the association between plasma vitamin A levels and outcome measures in very low birthweight (VLBW) infants, including meta-analysis of all observational studies. DESIGN: A prospective observational longitudinal study of plasma vitamin A levels measured in the cord blood; maternal blood in the first 48 h after delivery; and the infants' blood at 48 h, 7 days and 28 days of age and correlated with antenatal and postnatal events. A meta-analysis of all published observational studies on the association of vitamin A with respiratory outcome in the VLBW infant was undertaken. PATIENTS: Fifty-seven infants (88% of all eligible) VLBW infants (< 1500 g) admitted from January through October 1993 to one of two regional neonatal intensive care units in the South Island of New Zealand. RESULTS: Exposure to antenatal steroids led to a significant increase in infant cord plasma vitamin A levels (P = 0.003), but no influence on infant plasma vitamin A levels at any other time. Exposure to postnatal steroids produced a significant rise in infant plasma vitamin A levels between 7 and 28 days (P = 0.008). After controlling for gestational age, antenatal and postnatal steroid exposure, low vitamin A levels at 48 h increased the risk of developing chronic lung disease (odds ratio for 50 microg/l decrease: 2.04, 95% CI 1.19-5.77) and bronchopulmonary dysplasia (odds ratio 1.96, 95% CI 1.14-6.87). On combining our results in meta-analysis with those of other published prospective observational studies, infants with chronic lung disease had lower plasma vitamin A levels at all times. CONCLUSIONS: Our results support an association between low plasma vitamin A levels and adverse outcome in the VLBW infant.
Chronic Disease↗
Thiocyanate and chloride as competing substrates for myeloperoxidase.
The neutrophil enzyme myeloperoxidase uses H2O2 to oxidize chloride, bromide, iodide and thiocyanate to their respective hypohalous acids. Chloride is considered to be the physiological substrate. However, a detailed kinetic study of its substrate preference has not been undertaken. Our aim was to establish whether myeloperoxidase oxidizes thiocyanate in the presence of chloride at physiological concentrations of these substrates. We determined this by measuring the rate of H2O2 loss in reactions catalysed by the enzyme at various concentrations of each substrate. The relative specificity constants for chloride, bromide and thiocyanate were 1:60:730 respectively, indicating that thiocyanate is by far the most favoured substrate for myeloperoxidase. In the presence of 100 mM chloride, myeloperoxidase catalysed the production of hypothiocyanite at concentrations of thiocyanate as low as 25 microM. With 100 microM thiocyanate, about 50% of the H2O2 present was converted into hypothiocyanite, and the rate of hypohalous acid production equalled the sum of the individual rates obtained when each of these anions was present alone. The rate of H2O2 loss catalysed by myeloperoxidase in the presence of 100 mM chloride doubled when 100 microM thiocyanate was added, and was maximal with 1mM thiocyanate. This indicates that at plasma concentrations of thiocyanate and chloride, myeloperoxidase is far from saturated. We conclude that thiocyanate is a major physiological substrate of myeloperoxidase, regardless of where the enzyme acts. As a consequence, more consideration should be given to the oxidation products of thiocyanate and to the role they play in host defence and inflammation.
Binding, Competitive↗
The destabilisation of total parenteral nutrition by heparin. How real is the problem?
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Calcium↗
Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid.
Neutrophils, when stimulated, generate reactive oxygen species including myeloperoxidase-derived HOCl. There is an associated decrease in reduced glutathione (GSH) concentration. We have shown that neutrophil GSH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell lysis, was not reversible, and was accompanied by the loss of an equivalent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with 35S-labelled cells indicated that much of the GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the reaction of HOCl and pure GSH. The properties of this compound are consistent with an intramolecular sulphonamide. Neutrophils stimulated with PMA lost 30-40% of their GSH and a similar proportion of protein thiols. Little glutathione disulphide was formed and the products were the same as seen with HOCl-treated cells. From the results and studies with inhibitors and scavengers, we conclude that HOCl was responsible for the GSH loss. Propargylglycine and buthionine sulphoximine, inhibitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of GSH synthesis. The lack of thiol selectivity by HOCl and irreversibility of oxidation means that GSH will provide limited antioxidant protection for thiol enzymes in stimulated neutrophils.
Alkynes↗
Characterization of the oxidation products of the reaction between reduced glutathione and hypochlorous acid.
Reduced glutathione (GSH) is one of the most preferred biological substrates of myeloperoxidase-derived hypochlorous acid and is a likely target for neutrophil oxidants. We have used HPLC to show that the oxidation of GSH by hypochlorous acid gives two major, stable products in addition to glutathione disulphide (GSSG). The most prevalent product lacks free amine and thiol groups, and was shown by electrospray MS to have a molecular mass of 337 Da. This corresponds to GSH with a gain of two oxygen atoms and a loss of two hydrogen atoms, and is consistent with the product being an internal sulphonamide. The other novel product has a molecular mass of 644 Da, and has amine groups but no free thiols. These properties are consistent with it being glutathione thiolsulphonate. Whereas GSSG in the cell is recycled enzymically, formation of these higher oxidation products is likely to be irreversible. Hypochlorous acid, therefore, could compromise the cell by depleting GSH. The putative sulphonamide may be unique for oxidation by hypochlorous acid and thus provide a useful marker of neutrophil oxidant activity.
Amines↗
Myeloperoxidase-dependent generation of a tyrosine peroxide by neutrophils.
It has recently been shown that tyrosyl radicals react with superoxide to form a peroxide adduct of tyrosine. Since myeloperoxidase oxidizes tyrosine to its radical, and neutrophils and monocytes contain myeloperoxidase as well as produce superoxide, we have investigated whether tyrosine peroxide could be a significant product of tyrosine oxidation by these cells. Oxidation of tyrosine by purified myeloperoxidase and a superoxide-generating system, and by stimulated human neutrophils, was found to generate peroxide adducts as detected in the xylenol orange (FOX) assay and by HPLC. Superoxide, hydrogen peroxide, and myeloperoxidase were required for formation of the peroxide. Dityrosine was also formed in each system, and in the presence of superoxide dismutase, suppression of tyrosine peroxide formation gave elevated formation of dityrosine. Quantitative estimates indicate that at physiological tyrosine concentration the peroxide is likely to be formed in preference to dityrosine and to be a significant product of neutrophils. This metastable peroxide therefore has the potential to contribute to neutrophil- or monocyte-mediated tissue injury.
Chromatography, High Pressure Liquid↗
Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide.
Hypochlorous acid is the most powerful oxidant generated by neutrophils and is likely to contribute to the damage mediated by these inflammatory cells. The haem enzyme myeloperoxidase catalyses its production from hydrogen peroxide and chloride. 4-Aminobenzoic acid hydrazide (ABAH) is a potent inhibitor of hypochlorous acid production. In this investigation we show that, in the presence of hydrogen peroxide, ABAH irreversibly inactivates myeloperoxidase. ABAH was oxidized by myeloperoxidase, and kinetic analysis of the inactivation conformed to that for a mechanism-based inhibitor. Inactivation was exacerbated by concentrations of hydrogen peroxide greater than 50 microM and by the absence of oxygen. Hydrogen peroxide alone caused minimal inactivation. Reduced glutathione inhibited the oxidation of ABAH as well as the irreversible inhibition of myeloperoxidase. In the presence of oxygen, ABAH and hydrogen peroxide initially converted myeloperoxidase into compound III, which subsequently lost haem absorbance. In the absence of oxygen, the enzyme was converted into ferrous myeloperoxidase and its haem groups were rapidly destroyed. We propose that myeloperoxidase oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous myeloperoxidase reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation.
Aniline Compounds↗
Endotracheal suctioning of the neonate: comparison of two methods as a source of mucus material for research.
Endotracheal suctioning in the neonatal intensive care setting is a routine procedure performed to maintain patency of the airway in ventilated infants. Harvested material can also be a source of mucus for research into neonatal respiratory disorders. We aimed to investigate whether the composition of material obtained by our clinically preferred technique of dry shallow suctioning differed significantly from that obtained with saline lavage and deep suctioning. Eleven pairs of dry and saline lavage aspiration samples were compared for neutrophil enzyme myeloperoxidase, total and active alpha(1)-antitrypsin, alpha(1)-antitrypsin complexed with elastase, and secretory leukoprotease inhibitor. Even though individual values of each analyte, expressed per gram of albumin, varied over at least a fivefold range, there was no difference between mean values of dry and lavage samples for any of the constituents. We conclude that the yield of material for research obtained by dry shallow suctioning is adequate and the quality at least as satisfactory as that provided by saline lavage.
Bronchoalveolar Lavage Fluid↗
Nuclear magnetic resonance characterization of 6 alpha-chloro-5 beta-cholestane-3 beta,5-diol formed from the reaction of hypochlorous acid with cholesterol.
Hypochlorous acid generated by neutrophil myeloperoxidase has been shown to convert cholesterol into three different chlorohydrin isomers which previously had not been fully characterized. We have reacted hypochlorous acid with cholesterol/1,2-dipalmitoyl phosphatidylcholine liposomes to give these three major products and established that they are 6 beta-chloro-5 alpha-cholestane-3 beta,5-diol (chlorohydrin 1), 5 alpha-chloro-6 beta-cholestane-3,6-diol (chlorohydrin 2) and 6 alpha-chloro-5 beta-cholestane-3 beta,5-diol (chlorohydrin 3). These products were separated by thin-layer chromatography and fully characterized by 1H, 13C, attached proton test, doublequantum correlation spectroscopy, total correlation spectroscopy, heteronuclear multiple bond correlation and heteronuclear multiple quantum coherence nuclear magnetic resonance spectroscopy.
Cholestanols↗
Protein carbonyl measurement by a sensitive ELISA method.
We describe a new immunoassay for measuring protein carbonyls as an index of oxidative injury. Protein samples were reacted with dinitrophenylhydrazine then adsorbed to wells of an ELISA plate before probing with a commercial antibody raised against protein-conjugated dinitrophenylhydrazine. The biotin-conjugated primary antibody was then reacted with streptavidin-biotinylated horseradish peroxidase for quantification. The method was calibrated using oxidized albumin and results correlated well with the colorimetric carbonyl assay. The method required only 60 microg protein and was used to analyze the amount of protein carbonyls in plasma and lung aspirate samples. It was sensitive in the 0-2.5 nmol/mg protein range within which clinical samples fell and was linear up to 10 nmol/mg protein. The ELISA method for protein carbonyls is more sensitive and discriminatory than the colorimetric assay and should have wide application for analysing experimental and clinical samples, especially where concentrations are low and where only small amounts of sample are available.
2,4-Dinitrophenol↗