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.
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↗
Chlorination of cholesterol in cell membranes by hypochlorous acid.
Neutrophils and monocytes produce the highly cytotoxic hypochlorous acid (HOCl) via the myeloperoxidase (MPO)-catalyzed reaction of H2O2 with Cl-. We have investigated the reaction of reagent and MPO-derived HOCl with cholesterol in a purified liposome system, as well as progressively more complex biological systems. The products were identified by thin-layer chromatography (TLC) and characterized by mass spectrometry (MS). TLC of the HOCl-treated samples gave four major cholesterol products with color development typical of oxysterols. Two of the products coeluted with authentic alpha- and beta-chlorohydrin standards. As was the case with the standards, they were readily converted into their respective epoxides during analysis by MS. Gas chromatography-mass spectrometry analysis of the other major product (band 3) gave peaks consistent with epoxides as well as a lateeluting peak with a distinct mass spectrum. Electrospray MS of this product confirmed its identity as a chlorohydrin based on the presence of the predicted molecular ion and 3:1 Cl isotope ratios. Lipids extracted from red cells and isolated red cell membranes were exposed to HOCl and gave identical products to the purified cholesterol liposome system as determined by TLC and MS. Higher concentrations of HOCl were required due to competition by other unsaturated lipids and protein molecules. When intact red cells, neutrophils, and MCF7 mammary carcinoma cells were exposed to HOCl, cholesterol chlorohydrins were formed, as detected by TLC. The formation of cholesterol chlorohydrins could be potentially disruptive to cell membranes and result in cell lysis and death. They could also be potential biomarkers for oxidative damage associated with neutrophil/monocyte activation.
Cell Membrane↗
Peroxidase-mediated bromination of unsaturated fatty acids to form bromohydrins.
Eosinophil peroxidase and myeloperoxidase (MPO) catalyze the oxidation of bromide by hydrogen peroxide to produce hypobromous acid (HOBr). Hypochlorous acid, which is also generated by MPO, reacts with unsaturated fatty acids to form chlorohydrins. In this study the equivalent reaction of HOBr, produced from MPO, bromide, and hydrogen peroxide, with oleic (18:1), linoleic (18:2), and arachidonic (20:4) acids has been investigated. Thin-layer chromatography detected one major product of higher polarity than the unmodified fatty acids and additional more polar products with the polyunsaturated fatty acids. Similar results were observed with N-bromosuccinimide-derived HOBr. Gas chromatography-mass spectrometry (GC-MS) and electrospray MS identified the major products of 18:1 as the isomeric 9,10-bromohydrins based on retention time and mass spectrometric isotope and fragmentation patterns. The products of 18:2 and 20:4 were too unstable for analysis by GC-MS. Electrospray MS identified the mono- and bisbromohydrins formed from 18:2 and 20:4 based on mass/charge ratios of the molecular ions and the presence of bromine isotope patterns. Other oxidation products not containing bromine, such as dihydroxy derivatives, were detected as well. Fatty acid bromohydrins could contribute to the antimicrobial activity and inflammatory tissue damage by eosinophils and neutrophils, and could potentially be useful specific markers for HOBr production in vivo.
Alcohols↗
The correlation of elevated levels of an index of lipid peroxidation (MDA-TBA) with adverse outcome in the very low birthweight infant.
The objective of this study was to examine the relationship between malondialdehyde-thiobarbituric acid (MDA-TBA) levels, as a measure of lipid peroxidation, in very low birthweight (VLBW) infants and outcome measures. A prospective observational longitudinal study was carried out in two level III neonatal units in the South Island of New Zealand measuring MDA-TBA levels in 61 VLBW infants in 1993. MDA-TBA levels were measured in (i) maternal plasma within 48 h of parturition, (ii) cord plasma, and (iii) infants' plasma at 2, 7, 14 and 28 days of age and correlated with antenatal and postnatal factors. Elevated levels of plasma MDA-TBA at 7 days were associated with adverse respiratory and ophthalmological outcome in the VLBW infants. Elevated MDA-TBA levels were measured at sample times close to the time of death in the infants who died. These results substantiate previously reported preliminary observations and support the hypothesis that oxidative injury, particularly within the first 7 days of life, is associated with the development of the long-term complications of the pre-term infant. MDA-TBA levels appear to be a useful measure to continue to explore the role of free radical mediated disease in the VLBW infant.
Fat Emulsions, Intravenous↗
Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.
We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.
Azides↗