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C C Winterbourn

Publications and source records attributed to C C Winterbourn.

At least 73 records · Page 4Linked to original sources
PubMed1995

The relationship of selenium status to respiratory outcome in the very low birth weight infant.

OBJECTIVE: To examine the relationship between plasma and erythrocyte selenium and glutathione peroxidase (GPx) levels in premature infants and outcome measures. DESIGN: Prospective observational longitudinal study. SETTING: Two regional neonatal intensive care units in the South Island of New Zealand, an area with low soil selenium. PATIENTS: Seventy-nine infants with birth weights less than 1500 g or gestation less than 32 weeks admitted within 48 hours of birth from November 1992 through November 1993. MAIN OUTCOME MEASURES: Oxygen requirement at 28 days (chronic lung disease), or 36 weeks postmenstrual age and for all or most of the time from birth (bronchopulmonary dysplasia), total days in oxygen, retinopathy of prematurity, periventricular hemorrhage, or ventricular dilatation. RESULTS: Initial infant plasma selenium and GPx levels were about two thirds of maternal levels and fell a further 30% in 28 days. In contrast to adults, there was a poor correlation in infant plasma between selenium and GPx at birth and 28 days. Plasma selenium at 28 days was significantly lower in infants with chronic lung disease and bronchopulmonary dysplasia. After controlling for gestational age and age when fully fed orally, 28-day plasma selenium was significantly associated with the log of total days of oxygen requirement, each drop of 0.1 mumol/L in 28-day selenium being associated with a 58% increase in days of oxygen dependency. No significant associations of other parameters of selenium status and respiratory outcome were found, and there were no significant associations of any parameters of selenium status with other outcome measures. CONCLUSIONS: This study demonstrates for the first time in human infants that low plasma selenium levels are significantly associated with an increased respiratory morbidity. Whether selenium deficiency is etiologically important in determining the respiratory outcome or the result of sickness in the infant should be investigated in a randomized, controlled trial.

Bronchopulmonary Dysplasia↗
PubMed1994

The reaction of superoxide with reduced glutathione.

Superoxide, generated by a xanthine oxidase/hypoxanthine system, reacts with reduced glutathione (GSH) to cause an increase in oxygen consumption and oxidized glutathione (GSSG) formation, both of which are fully inhibited by superoxide dismutase. In this study we have shown that little, if any, of the additional oxygen consumed is converted to hydrogen peroxide. We have confirmed that approximately 90% of the GSH is oxidized to GSSG, the remainder being converted to the sulfonic acid. Approximately 1.2 mol of GSSG was formed for each additional mole of oxygen consumed in the presence of GSH. The efficiency of the reaction increased with increasing GSH concentration (1-8 mM), pH, and pO2 and with decreasing superoxide generation rate. The results are consistent with a superoxide-dependent chain that does not produce hydrogen peroxide and that is terminated primarily by superoxide dismutation. We propose that this occurs via an initial reaction of superoxide with GSH to produce a sulfinyl radical rather than hydrogen transfer to give the thiyl radical. Our data suggest a rate constant for the superoxide/GSH reaction in the 10(2)-10(3) M-1s-1 range. GSH at the millimolar concentrations found intracellular should react with superoxide, but because superoxide is regenerated, it will not be an effective scavenger. Physiologically, superoxide dismutase is required to prevent chain oxidation of GSH.

Catalase↗
PubMed1994

Superoxide-dependent hydroxylation by myeloperoxidase.

When stimulated, neutrophils undergo a respiratory burst converting oxygen to superoxide. Although superoxide is critical for microbial killing by phagocytic cells, the precise role it plays has yet to be established. It has been proposed to optimize their production of hypochlorous acid and to be required for the generation of hydroxyl radicals. Superoxide is also involved in the hydroxylation of salicylate by neutrophils. However, the mechanism of this reaction is unknown. We found that neutrophils stimulated with opsonized zymosan hydroxylated salicylate to produce mainly 2,5-dihydroxybenzoate. Its formation was dependent on superoxide and a heme protein but was independent of hydrogen peroxide and hydroxyl radicals. Production of 2,5-dihydroxybenzoate was enhanced by methionine, which scavenges hypochlorous acid. Neutrophils from an individual with myeloperoxidase deficiency hydroxylated salicylate at only 13% of the level of control cells. Purified human myeloperoxidase and xanthine oxidase plus hypoxanthine hydroxylated salicylate to produce 2,5-dihydroxybenzoate. As with neutrophils, the reaction required superoxide but not hydrogen peroxide and was unaffected by hydroxyl radical scavengers. Thus, myeloperoxidase catalyzes superoxide-dependent hydroxylation. This newly recognized reaction may be relevant to the in vivo functions of superoxide and myeloperoxidase.

Humans↗
PubMed1994

A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils.

We have developed a method that enables the rates of phagocytosis and killing of bacteria by neutrophils to be measured in a single assay. Neutrophils were incubated with bacteria, and at specific intervals were separated from uningested bacteria by low speed centrifugation. Rates of phagocytosis and killing were calculated from the decrease in number of extracellular bacteria and change in the number of intracellular bacteria. Both phagocytosis and killing were shown to follow first-order kinetics, and rate constants were calculated without having to separate the assay into two phases. In contrast to two-step methods, our method measures killing from the moment the neutrophils start ingesting the bacteria, and also eliminates the need to halt neutrophil activity temporarily and restart the assay after the extracellular bacteria have been removed. We obtained reproducible results for the phagocytosis and killing of Staphylococcus aureus (t1/2 = 9 min and 6 min respectively) and Escherichia coli (t1/2 = 10 min and 2 min respectively). We also were able to detect a 56% impairment in the rate of killing of S. aureus by neutrophils from an individual with a low level of myeloperoxidase.

Cell Separation↗
PubMed1994

Assays using horseradish peroxidase and phenolic substrates require superoxide dismutase for accurate determination of hydrogen peroxide production by neutrophils.

We used horseradish peroxidase and either scopoletin, homovanillic acid, or phenol red to measure hydrogen peroxide generated by human neutrophils. With these assays, superoxide dismutase significantly increased the amount of hydrogen peroxide detected. In contrast, it had no effect when the accumulation of hydrogen peroxide was measured with a hydrogen peroxide electrode. We propose that superoxide interferes with horseradish peroxidase-dependent assays so that hydrogen peroxide is underestimated. Thus, when using these assays, superoxide dismutase must be added to neutrophils to ensure that all the hydrogen peroxide they produce is detected.

Horseradish Peroxidase↗
PubMed1994

Membrane changes associated with lysis of red blood cells by hypochlorous acid.

This study was carried out to investigate HOCl-induced lysis of human erythrocytes. Using reagent HOCl with isolated red cells, we showed that the rate of lysis was dependent on the dose of HOCl per red cell rather than on the concentration of oxidant. The process was inhibited by scavengers such as methionine and taurine, but only if they were present at the time of addition of HOCl. Lysis was preceded by a decrease in cell density, a change in the deformability of the membrane as evidenced by ektacytometry, and an increase in K(+)-leak. Electron microscopy showed extensive disruption of the membrane. Increasing doses of HOCl caused progressive loss of membrane thiols, but complete thiol oxidation by N-ethylmaleimide did not result in an equivalent rate of lysis. Restoration of oxidised thiols by incubation with glucose did not significantly alter the pattern of lysis. Taken together, these results suggest that thiol oxidation was not responsible for HOCl-mediated lysis. There was evidence of increasing crosslinking of membrane proteins on electrophoresis, only some of which was due to the formation of disulfides. TLC of the membrane lipids indicated that there may be formation of chlorohydrins by reaction of HOCl with the fatty acid double bonds. This reaction results in the formation of a more polar species which, if formed, would be extremely disrupting to the lipid bilayer. The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.

Diamide↗
PubMed1994

Bacterial killing by neutrophils in hypertonic environments.

The effects of hypertonic sodium chloride and urea solutions on the phagocytosis and killing of Escherichia coli by neutrophils were examined. Neutrophil function was progressively inhibited by increasing osmolarity, with sodium chloride more inhibitory than urea. However, neutrophils killed at 25% of normal rates in 500 mosmol/L NaCl, and in 300 mosmol/L NaCl with 800 mosmol/L urea. These concentrations would rarely be exceeded in the human urinary tract. Inhibition of neutrophil superoxide production and degranulation was similar to that observed with phagocytosis and killing. Neutrophils also phagocytosed and killed E. coli in isotonic and hypertonic urine, but no improvement in function attributable to the uptake of urinary osmoprotectants was evident. It is concluded that physiologic levels of sodium chloride and urea will slow, but not prevent, neutrophil activity at a hypertonic site of infection.

Cell Degranulation↗
PubMed1994

Proteinase-antiproteinase balance in tracheal aspirates from neonates.

We wanted to identify the inhibitors of neutrophil elastase, quantify their activities in the upper airways of neonates, and relate these to the presence of active elastase and the likelihood of elastolytic injury occurring due to inhibitory capacity being overwhelmed. Activities of neutrophil elastase and its inhibitors were measured in tracheal aspirates from 17 infants, 10 of whom subsequently developed bronchopulmonary dysplasia. All aspirates contained immunologically detectable alpha 1-proteinase inhibitor (alpha 1-PI), but their inhibitory capacity against neutrophil elastase ranged from being undetectable to being in excess of the amount of alpha 1-PI detected immunologically. When the alpha 1-PI was removed from each of the aspirates, using a specific antibody, from 0-50% of the original activity remained, indicating the presence of another elastase inhibitor. Its properties were consistent with it being the low molecular mass, secretory leucoproteinase inhibitor (SLPI), also known as bronchial antileucoproteinase. The alpha 1-PI was from 0-100% active. Most of the inactive inhibitor was shown by western blotting to be complexed with elastase, with a small amount of cleaved material. There was no evidence of major oxidative inactivation. Free elastase was detected in only three of the aspirates; these had little or no detectable elastase inhibitory capacity, and most of their alpha 1-PI was complexed. Elastase load, comprising the sum of free and complexed elastase, correlated closely with myeloperoxidase activity, a recognized marker of inflammatory activity. Active SLPI levels showed a positive correlation with gestational age (r = 0.66). We conclude that most neutrophil elastase in the upper airways of ventilated infants is complexed.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗
PubMed1993

The hydrolysis product of ICRF-187 promotes iron-catalysed hydroxyl radical production via the Fenton reaction.

d-1,2-Bis(3,5-dioxopiperazine-1-yl)propane (ICRF-187) (ADR-529) is a drug that ameliorates the cardiotoxicity of Adriamycin. The drug enters cells where hydrolysis leads to its diacid diamide product, dl-N,N'-dicarboxamidomethyl-N,N'-dicarboxymethyl-1,2-diamino propane (ICRF-198) (ADR-925), which is structurally similar to ethylenediaminetetraacetic acid (EDTA). The protective mechanism of ICRF-187 is unknown, but a plausible explanation is that ICRF-198 chelates iron intracellularly to prevent iron-dependent free radical reactions such as hydroxyl radical (.OH) production. We have compared Fe(ICRF-198) with Fe(EDTA) in its ability to promote .OH formation in several Fenton reaction systems. The Fenton reaction was studied with H2O2 and Fe2+ chelates or catalytic amounts of the iron chelates in the presence of Adriamycin radicals, paraquat radicals, superoxide anion radicals (O2-), and ascorbate as reducing species. .OH was detected with deoxyribose and dimethyl sulfoxide. The two methods gave comparable results. Fe(ICRF-198) was 80-100% as effective as Fe(EDTA) at promoting .OH production in the presence of the organic radicals and ascorbate, 30-70% in the presence of O2-, and 150% with non-cycling Fe2+. Fe(EDTA) is a more efficient catalyst of .OH production than physiological chelates such as ADP, ATP and citrate. Therefore, by comparing previous work which examined physiological chelates and Fe(EDTA) with the present work, Fe(ICRF-198) appears to be a better .OH catalyst than the physiological chelates. These results suggest that ICRF-198 generated in vivo from ICRF-187 would not protect against intracellular .OH production. They also imply that .OH production may not be as important in Adriamycin cardiotoxicity as other radical reactions, such as lipid peroxidation and thiol oxidation, that are inhibited by ICRF-198.

Ascorbic Acid↗
PubMed1993

Superoxide is an antagonist of antiinflammatory drugs that inhibit hypochlorous acid production by myeloperoxidase.

Myeloperoxidase, the most abundant enzyme in neutrophils, catalyses the conversion of hydrogen peroxide and chloride to hypochlorous acid. This potent oxidant has the potential to cause considerable tissue damage in many inflammatory diseases. We have investigated the ability of dapsone, diclofenac, primaquine, sulfapyridine and benzocaine to inhibit hypochlorous acid production by stimulated human neutrophils. The drugs were also tested against purified myeloperoxidase using xanthine oxidase to generate hydrogen peroxide and superoxide. The inhibitory effects of the drugs on hypochlorous acid production, either by cells stimulated with phorbol myristate acetate or by myeloperoxidase and xanthine oxidase, were significantly less than those determined with myeloperoxidase and reagent hydrogen peroxide. Comparable potency was observed only when superoxide dismutase was present to remove superoxide. We also observed that with the xanthine oxidase system, inhibition of hypochlorous acid production by dapsone decreased markedly as the concentration of myeloperoxidase increased. Dapsone was a poor inhibitor of hypochlorous acid production by neutrophils stimulated with opsonized zymosan, regardless of the presence of superoxide dismutase. With this phagocytic stimulus, catalase inhibited hypochlorous acid formation by only 60%, which indicates that a substantial amount of the hypochlorous acid detected originated from within phagosomes. Thus, it is apparent that dapsone is unable to affect intraphagosomal conversion of hydrogen peroxide to hypochlorous acid. All the drugs inhibit myeloperoxidase reversibly by trapping it as its inactive redox intermediate, compound II. We propose that superoxide limits the potency of the drugs by reducing compound II back to the active enzyme. Furthermore, under conditions where the activity of myeloperoxidase exceeds that of the hydrogen peroxide-generating system, which is most likely to occur in phagosomes, partial inhibition of myeloperoxidase need not affect hypochlorous acid production. We conclude that drugs that inhibit myeloperoxidase by converting it to compound II are unlikely to be effective against hypochlorous acid-mediating tissue damage.

Anti-Inflammatory Agents↗
PubMed1993

Myeloperoxidase-dependent loss of malondialdehyde: a limitation for detecting neutrophil-mediated lipid peroxidation.

Lipid peroxidation is commonly measured using the thiobarbituric acid (TBA) assay. We have examined how this assay is affected by hypochlorite, which is generated by the neutrophil enzyme myeloperoxidase. The TBA reactivity of phospholipid liposomes that had been partially peroxidized with iron/ascorbate was destroyed by low concentrations of sodium hypochlorite. Since most of the TBA reactivity in peroxidized liposomes is due to malondialdehyde, its reactivity was investigated. Addition of sodium hypochlorite destroyed the uv absorbance of malondialdehyde with a 2:1 stoichiometry and eliminated its TBA reactivity. The TBA reactivity of malondialdehyde and peroxidized liposomes was also lost after treatment with myeloperoxidase. The reaction with myeloperoxidase required chloride and was inhibited by catalase and methionine, indicating the involvement of hypochlorite. Neutrophils stimulated with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine destroyed the TBA reactivity of malondialdehyde and peroxidized liposomes by a hypochlorite-dependent mechanism. The ability of hypochlorite to break down malondialdehyde explains why lipid peroxidation by stimulated neutrophils, as measured with TBA, is apparently inhibited by myeloperoxidase. Myeloperoxidase may not, however, inhibit the peroxidation process. The TBA assay and other assays of malondialdehyde may be of limited value, therefore, for assessing lipid peroxidation in systems where neutrophils or myeloperoxidase are involved.

Chlorides↗
PubMed1993

Superoxide as an intracellular radical sink.

A pathway is proposed for superoxide to act as a sink for intracellularly generated radicals. A variety of radicals, either directly or via reduced glutathione (GSH) as an intermediate, can transfer their unpaired electron to oxygen to give superoxide. It is proposed that in a cellular environment, superoxide can undergo chain reactions involving GSH with or without another redox cycling agent, converting GSH to oxidized glutathione (GSSG) and oxygen to hydrogen peroxide far in excess of the initial radical. This places an oxidative stress on the cell, depleting reducing equivalents and energy reserves. Superoxide dismutase is necessary to prevent this oxidative stress, as well as any direct damage by superoxide. Through this metabolic pathway, GSH and superoxide dismutase can be linked in antioxidant function, and superoxide dismutase, by reacting with superoxide, can provide general protection against radical reactions in the cell. The pathway also provides a mechanism for superoxide and superoxide dismutase to influence the redox state of the cell and regulate functions that are under redox control.

Antioxidants↗
PubMed1993

Hypochlorous acid-mediated modification of cholesterol and phospholipid: analysis of reaction products by gas chromatography-mass spectrometry.

Oxidative modification of membrane lipids by hypochlorous acid could be an important element in the mechanism of membrane disruption by activated neutrophils. We have previously shown that hypochlorous acid reacts with unsaturated fatty acids of membrane phospholipids to give fatty acid chlorohydrins (Winterbourn et al. 1992. Arch. Biochem. Biophys. 296: 547-555). In the present study, we examined the reaction of cholesterol in bilayers with an inert phospholipid carrier. Product separation and identification was performed using gas chromatography-mass spectrometry after trimethylsilyl-derivatization. Unlike the reaction of hypochlorous acid with unsaturated fatty acids, no chlorohydrin derivatives were found with cholesterol. Instead, the main oxidation products were identified as the epimeric cholesterol 5,6-epoxides and 4-hydroxycholesterol, while several other hydroxy- and keto-derivatives were also found in smaller amounts. Analysis of the products obtained after reaction of vesicles composed of a mixture of several unsaturated phospholipid species plus cholesterol revealed that the individual fatty acids and cholesterol all exhibit comparable susceptibilities toward hypochlorous acid. Using myeloperoxidase to generate hypochlorous acid, basically the same products and product distribution were obtained. These studies show that unsaturated phospholipids and cholesterol can be profoundly modified by reaction with hypochlorous acid. This warrants further investigation to define the role of lipid modifications in neutrophil-mediated membrane disruption.

Cholesterol↗
PubMed1992

Increase in selenium status of Christchurch adults associated with deregulation of the wheat market.

AIMS: to determine whether there have been changes in the plasma selenium status of Christchurch adults between 1981 and 1992, and likely causes of any such changes. METHOD: selenium analyses were performed on plasma samples collected during the period. Changes in agricultural practices and importation policies were also examined. RESULTS: mean plasma selenium levels ranged between 46 and 54 micrograms/L (0.59-0.69 mumol/L) until 1987, after which there was a dramatic and sustained increase to between 66 and 70 micrograms/L (0.84-0.88 mumol/L) for 1988-91, with a 1992 mean of 80 micrograms/L (1.01 mumol/L). This increase closely follows the deregulation of the New Zealand wheat market and greater South Island consumption of wheat imported from Australia and the United States. Whereas flour was made from South Island wheat prior to 1988 and contained about 15 micrograms/kg selenium, flour manufactured by Christchurch mills in 1991 contained between 80 and 140 micrograms/kg. CONCLUSIONS: these results imply that as a result of current government policy, the population of Christchurch no longer has particularly low selenium levels. It follows that if there is an association between low selenium and any form of ill health, a declining incidence or severity in this population might be expected.

Adult↗
PubMed1992

Oxidative metabolism of amsacrine by the neutrophil enzyme myeloperoxidase.

Oxidative metabolism of the anti-cancer drug amsacrine 4'-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.

Amsacrine↗
PubMed1992

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.

Arachidonic Acid↗