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C C Winterbourn

Publications and source records attributed to C C Winterbourn.

At least 55 records · Page 3Linked to original sources
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1995

Generation of superoxide and tyrosine peroxide as a result of tyrosyl radical scavenging by glutathione.

Enzymatically generated tyrosyl radicals are effectively scavenged by reduced glutathione (GSH), thereby generating glutathione thiyl radicals and superoxide radicals, subsequently. Here, we have used horseradish peroxidase to generate tyrosyl radicals and investigated the fate of the superoxide radicals. At low GSH concentrations (with a maximum effect at 250 microM) a major reaction was between superoxide and the phenoxyl radical leading to a tyrosine peroxide. Formation of the peroxide was confirmed using a peroxide-specific colorimetric assay and detection of a new HPLC-peak. Its formation was inhibited by superoxide dismutase (SOD). The peroxide decomposed slowly in a reaction that was accelerated by GSH to give a new chromatographic peak. Increasing the GSH concentration decreased the amount of tyrosine peroxide formed and caused increases in rates of oxygen uptake and GSH oxidation. These increases were not seen in the presence of SOD and are consistent with GSH scavenging superoxide and leading to oxygen-dependent chain oxidation of GSH. Both pathways are undesirable for the cell and are effectively suppressed only if GSH as a radical scavenger acts in concert with SOD.

Disulfides↗
PubMed1995

Decreased thermal stability of red blood cell glu100-->gly superoxide dismutase from a family with amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis is a degenerative motor neuron disease associated in some cases with the presence of a mutant form of Cu/Zn superoxide dismutase. We have studied the stability of the gly100-->glu mutant in extracts of red cells obtained from members of a family with a history of the disease. Extracts containing the mutant had an average 68% of normal superoxide dismutase activity. On heating at 65 degrees C, these extracts lost activity at twice the rate of extracts containing only the normal enzyme. Decreased heat stability was also evident on native polyacrylamide gel electrophoresis with activity staining. This showed selective loss of first the mutant homodimer and then the heterodimer of the enzyme. Decreased stability intracellularly could be a factor in motor neuron degeneration.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Chlorination of tyrosyl residues in peptides by myeloperoxidase and human neutrophils.

Hypochlorous acid is the major strong oxidant generated by human neutrophils, and it has the potential to cause much of the tissue damage that these inflammatory cells promote. It is produced from hydrogen peroxide and chloride by the heme enzyme myeloperoxidase. To unequivocally establish that hypochlorous acid contributes to inflammation, a stable and unique marker for its reaction with biomolecules needs to be identified. In this investigation we have found that reagent hypochlorous acid reacts with tyrosyl residues in small peptides and converts them to chlorotyrosine. Purified myeloperoxidase in combination with hydrogen peroxide and chloride, as well as stimulated human neutrophils, chlorinated tyrosine in the peptide Gly-Gly-Tyr-Arg. Rather than reacting directly with the aromatic ring of tyrosine, hypochlorous acid initially reacted with an amine group of the peptide to form a chloramine. The chloramine then underwent an intramolecular reaction with the tyrosyl residue to convert it to chlorotyrosine. This indicates that tyrosyl residues in proteins that are close to amine groups will be susceptible to chlorination. Peroxidases are the only enzymes capable of chlorinating an aromatic ring. Furthermore, myeloperoxidase is the only human enzyme that produces hypochlorous acid under physiological conditions. Therefore, chlorotyrosine will be a specific marker for the production of hypochlorous acid in vivo and for the involvement of myeloperoxidase in inflammatory tissue damage.

Amino Acid Sequence↗
PubMed1995

Inhibition of myeloperoxidase by benzoic acid hydrazides.

Myeloperoxidase is the most abundant protein in neutrophils and catalyses the conversion of H2O2 and chloride into HOCl. To help clarify the role of this enzyme in bacterial killing and inflammation, a specific and potent inhibitor needs to be identified. We have studied a series of benzoic acid hydrazides and found that in general they inhibit the peroxidation activity of myeloperoxidase with an IC50 value of less than 10 microM. The IC50 values of derivatives with substituents containing oxygen or nitrogen were related to their Hammett substituent constants. This indicates that myeloperoxidase oxidizes the hydrazide group of these compounds, and the degree to which they inhibit the enzyme is dependent on the ease of their oxidation. Unsubstituted benzoic acid hydrazide and its 4-chloro derivative were poor inhibitors of peroxidation. Thus it is likely that hydrogen-bonding of the enzyme to substituents containing oxygen or nitrogen increases the binding affinity of the hydrazides and enhances their oxidation by myeloperoxidase. 4-Aminobenzoic acid hydrazide (ABAH) was the most potent inhibitor of peroxidation. It irreversibly inhibited HOCl production by the purified enzyme, having an IC50 value of 0.3 microM. With neutrophils stimulated with opsonized zymosan or phorbol myristate acetate, ABAH inhibited HOCl production by up to 90% and the IC50 values were 16 microM and 2.2 microM respectively. In the presence of superoxide dismutase, these values decreased to 6.4 microM and 0.6 microM respectively. ABAH had no effect on superoxide radical (O2-.) production and degranulation by neutrophils, nor did it inhibit catalase or glutathione peroxidase. Thus ABAH is an effective and selective inhibitor that should be useful for determining the contribution of myeloperoxidase to oxidant-mediated reactions of neutrophils.

Aniline Compounds↗
PubMed1995

Superoxide dismutase (glu100-->gly) in a family with inherited motor neuron disease: detection of mutant superoxide dismutase activity and the presence of heterodimers.

Superoxide dismutase glu100-->gly, a mutation known to be associated with familial motor neuron disease (familial amyotrophic lateral sclerosis) has been detected in one symptomatic and five of seven asymptomatic members of a family with a history of this disease. On average, the individuals with the mutation had 75% of normal red blood cell superoxide dismutase activity. Native polyacrylamide gels stained for superoxide dismutase activity showed two abnormal bands in the family members identified as carrying the mutation. This indicates that active mutant enzyme is present in red cells and forms stable homodimers and heterodimers with the normal chain. A silent mutation in exon 4, not associated with motor neuron disease, was also detected in one family member.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid.

Exposure of human red blood cells to low doses of hypochlorous acid (HOCl) resulted in the loss of intracellular GSH. Oxidation occurred less than 2 min after the addition of HOCl, and required approx. 2.5 mol of HOCl per mol of GSH lost. Loss of GSH preceded oxidation of membrane thiols, the formation of chloramines and haemoglobin oxidation. The susceptibility of intracellular GSH to oxidation by HOCl was two-thirds that of GSH in cell lysates. These results indicate that HOCl can penetrate the red cell membrane, which provides little barrier protection for cytoplasmic components, and that GSH oxidation by HOCl may be a highly selective process. Virtually all of the GSH lost was converted into GSSG. If glucose was added to the medium, most of the GSH oxidized by low doses of HOCl was rapidly regenerated. At higher doses, recovery was less efficient. However, when HOCl was added as a slow infusion rather than in a single bolus, there was increased recovery at higher doses. This indicates that in metabolically active cells regeneration is rapid and GSH may protect cell components from damage by HOCl. HOCl-induced lysis was only slightly delayed by adding glucose to the medium, indicating that lytic injury is not ameliorated by GSH.

Cell Membrane Permeability↗
PubMed1995

Toxicity of iron and hydrogen peroxide: the Fenton reaction.

Iron and hydrogen peroxide are capable of oxidizing a wide range of substrates and causing biological damage. The reaction, referred to as the Fenton reaction, is complex and capable of generating both hydroxyl radicals and higher oxidation states of the iron. The mechanism and how it is affected by different chelators, and the interpretation of results obtained in biological systems, are discussed.

Animals↗
PubMed1995

Modification of neutrophil oxidant production with diphenyleneiodonium and its effect on bacterial killing.

Diphenyleneiodonium (DPI), an inhibitor of the NADPH oxidase, has been used to distinguish between oxidative and nonoxidative killing of Staphylococcus aureus and Escherichia coli by neutrophils. The rate of killing of S. aureus was inhibited by 77% in the presence of 10 microM DPI, compared to 81% measured under anaerobic conditions. DPI represents a convenient and accessible alternative to an anaerobic environment or using neutrophils from patients with chronic granulomatous disease, for eliminating oxidative killing. The killing of E. coli was also inhibited by DPI. The effect was more apparent at 30 min than at 10 min, suggesting that E. coli can be killed rapidly by nonoxidative mechanisms that become less efficient at later times. DPI was used at concentrations less than 10 microM to determine how this affected production of the three major neutrophil oxidants, superoxide, hydrogen peroxide, and hypochlorous acid, and to determine the effect of partial inhibition of oxidant production on the killing of S. aureus. Unexpectedly, lower concentrations of DPI (0.1-2 microM) inhibited hydrogen peroxide and hypochlorous acid production 10-30% more than they inhibited superoxide production. Correlation of hydrogen peroxide or hypochlorous acid production with the killing of S. aureus showed that up to 30% inhibition had no effect on the rate of killing, implying that agents that impair neutrophil oxidant production less than this will not compromise bacterial killing. Higher inhibition of oxidant production led to a linear decline in the rate of killing.

Bacteria↗
PubMed1995

Oxidation of parenteral lipid emulsion by ambient and phototherapy lights: potential toxicity of routine parenteral feeding.

Vitamin E can be a prooxidant in isolated lipoprotein suspensions. Because lipid emulsions used in parenteral nutrition are lipoprotein-like suspensions rich in polyunsaturated fatty acids and vitamin E, we hypothesized that vitamin E may act as a prooxidant in lipid emulsions, as it is in lipoprotein suspensions. We therefore exposed an intravenously administered lipid emulsion (Intralipid) to a single spotlight commonly used in the treatment of neonatal jaundice, and measured the formation of triglyceride hydroperoxides by using high-performance liquid chromatography with postcolumn chemiluminescence detection. Concentrations of these hydroperoxides in different batches of fresh intralipid were usually approximately 10 mumol/L but increased up to 60 times after exposure to phototherapy light for a period of 24 hours, even though significant amounts of vitamin E were present at the end of the exposure. Triglyceride hydroperoxides were formed during phototherapy light exposure whether the intralipid was in plastic tubing used routinely for infusion or in glass containers. Ambient light also caused significant peroxidation of the formula lipids, although to a much lesser extent than observed with phototherapy light. For infants in the neonatal intensive care unit who were receiving intralipid but not phototherapy, solutions being infused at the end of 24 hours contained a mean of 40 mumol/L hydroperoxides. For infants receiving phototherapy, the mean was 97 mumol/L. Phototherapy light-induced formation of triglyceride hydroperoxides was prevented by covering the intralipid with aluminum foil or supplementation with sodium ascorbate before light exposure. We conclude that intralipid is highly susceptible to oxidation and that elevated levels of oxidized lipids can be formed during its clinical use, especially when intralipid infusion is combined with phototherapy. Because lipid hydroperoxides are cytotoxic and can cause adverse effects, inadvertent infusion of rancid intralipid may add to the numerous problems encountered by premature neonates.

Aluminum↗
PubMed1995

Vitamin A and E status in very low birth weight infants: development of an improved parenteral delivery system.

Plasma vitamin A and E levels were inadequate in very low birth weight infants receiving a continuous infusion of a parenteral multivitamin preparation, 1.5 ml/kg per day, in dextrose-amino acid solution. A new delivery system using 2 ml/kg per day, infused for 6 hours from the first day of life, avoided loss during infusion and significantly improved plasma vitamin A and E levels during the first 28 days of life in very low birth weight infants.

Avitaminosis↗
PubMed1995

Free radical toxicology and antioxidant defence.

1. A large number of compounds that have toxic effects can be metabolized to free radicals and secondary reactive oxygen species. These may be directly damaging or may affect cell function by altering regulatory mechanisms through changing redox status. 2. Protection is provided by an integrated system of antioxidant defences. This includes reduced glutathione, one of the functions of which is to scavenge free radicals. It acts by channelling radicals to superoxide so that the one enzyme, superoxide dismutase, has a major control over radical reactions in the cell.

Antioxidants↗
PubMed1995

Selenium status of New Zealand infants fed either a selenium supplemented or a standard formula.

OBJECTIVE: New Zealand soils are deficient in the essential micronutrient, selenium. New Zealand infants have low selenium levels at birth and experience a further decline if fed cows milk based formula. This study examined the selenium status of infants fed with a new commercially available selenium supplemented formula. METHODOLOGY: Forty-four newborn infants, whose mothers wished to formula feed, were randomized in an open controlled trial to be fed a commercially available selenium supplemented cows milk formula (containing 17 micrograms Se/L) or an unsupplemented formula (containing 4.6 micrograms Se/L). Cord, 1 and 3 month blood samples were obtained for selenium status (plasma and red cell selenium and glutathione peroxidase) and thyroid function. RESULTS: Mean plasma selenium and glutathione peroxidase values were significantly higher in supplemented than unsupplemented infants at 1 month (unpaired t-tests; P < 0.0001 and P = 0.001 respectively) and 3 months (P < 0.0001 and P = 0.0005). Analysis within treatment groups between time points (paired t-tests) showed that selenium supplementation prevented the fall in plasma selenium from birth to 1 month seen in unsupplemented infants and was associated with a rise in levels between 1 and 3 months (P = 0.002). CONCLUSIONS: Supplementing cows milk formula with selenium to replicate the levels found in breast milk is nutritionally sound. Feeding from a few days of age with a formula containing 17 micrograms Se/L in infants with low selenium status at birth is sufficient to cause a rise to 80% of adult levels at 3 months of age.

Analysis of Variance↗