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C C Winterbourn

Publications and source records attributed to C C Winterbourn.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Oxidation of hydroquinone by myeloperoxidase. Mechanism of stimulation by benzoquinone.

Myeloperoxidase (MPO) is a prime candidate for mediating the inflammatory tissue damage of neutrophils because it converts Cl- to the potent oxidant hypochlorous acid. It also oxidizes xenobiotics to reactive free radicals. We have found that the kinetics of oxidation of hydroquinone by myeloperoxidase are inadequately explained by the classical peroxidase mechanism. Peroxidation of hydroquinone displayed a distinct lag phase, which was practically abolished by excluding O2 and was eliminated by adding benzoquinone at the start of the reaction. Superoxide dismutase increased the rate of peroxidation by 40% but did not eliminate the lag phase. Spectral investigations revealed that during the initial phase of the reaction, MPO was converted to oxy-MPO, or compound III, by a mechanism that was not reliant on superoxide. Benzosemiquinone, however, was able to convert ferric-MPO to compound III. Both compound III and ferro-MPO reacted with benzoquinone to regenerate ferric-MPO. We propose that the lag phase occurs because benzosemiquinone reduces ferric-MPO to ferro-MPO, which rapidly binds O2 to form compound III. Since compound III is outside the peroxidation cycle, conversion of hydroquinone to benzoquinone is retarded. However, as benzoquinone accumulates, it oxidizes ferro-MPO and compound III to ferric-MPO, thereby increasing the rate of peroxidation. There is a minimal lag phase under an atmosphere of N2 because ferro-MPO would be rapidly oxidized by benzoquinone, without formation of compound III. We conclude that when substrates produce radicals capable of reducing ferric-MPO, they will be peroxidized efficiently only if oxy-MPO is readily recycled. Furthermore, these radicals will prevent MP3+ from reacting with H2O2, and thereby prevent the enzyme from oxidizing Cl- to hypochlorous acid. Thus, this mechanism could be exploited to prevent hypochlorous acid-mediated inflammatory tissue damage.

Animals↗
PubMed1992

Selenium status of Christchurch infants and the effect of diet.

OBJECT: New Zealanders, because of a soil deficiency, have a low intake of selenium. To determine the impact of this on the infant population in Christchurch. METHODS: we have measured red cell and plasma selenium and the selenoenzyme, glutathione peroxidase, in 70 infants less than 12 months old and related these to age and diet. RESULTS: the infant population as a whole had mean plasma levels of selenium and glutathione peroxidase of 33 micrograms/L and 97 U/L compared with adult values of 74 micrograms/L and 150 U/L. Infant red cell levels of 0.30 mu g selenium and 9.0 U glutathione peroxidase per g haemoglobin were similar to those in adults. The selenium status of most breast fed infants after birth remained similar to that of cord blood. Mean plasma selenium and glutathione peroxidase levels in formula fed infants were about half those of breast fed infants, and their red cell selenium was also significantly lower. These did not increase until solids were introduced into the diet. The status of the infants reflected their diet, with the concentration of selenium in formulae being 3.9-5.2 micrograms/mL compared with a mean of 13.4 micrograms/mL in breast milk. CONCLUSIONS: since infants in more replete selenium areas show a gradual rise in blood selenium parameters after birth, this study suggests that formula fed and some breast fed infants in Christchurch receive an inadequate selenium intake. Consideration should be given to supplementing infant formulae and perhaps also the diet of pregnant and/or breast feeding mothers.

Adult↗
PubMed1992

Different effects of hypochlorous acid on human neutrophil metalloproteinases: activation of collagenase and inactivation of collagenase and gelatinase.

Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOCl activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inactivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOCl and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 +/- 1.2%) at HOCl/enzyme molar ratios less than 30 and was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-stimulated cells and almost all of the proenzyme was destroyed by HOCl after 60 min stimulation. Our results illustrate that only collagenase can be activated by HOCl in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOCl could occur in vivo where various HOCl scavengers are present.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Selenium and glutathione peroxidase levels in premature infants in a low selenium community (Christchurch, New Zealand).

By world standards, the selenium status of the adult population of Christchurch, New Zealand is low. To determine the status of infants undergoing neonatal intensive care, plasma and red cell selenium and glutathione peroxidase levels were measured in infants admitted to the regional neonatal unit. Plasma levels in all newborn infants were one third to one half those in adults. Premature infants had levels significantly lower than those in cord blood from term infants, but their levels were not different from those of term infants admitted to the unit. There were no differences between adult and infant red cell levels. The premature infants remaining in the neonatal unit showed dramatic decreases in plasma selenium and glutathione peroxidase with age, with many infants having selenium levels of less than 0.13 mumol/L (10 micrograms/L). Low levels were seen in infants fed orally as well as those on parenteral nutrition. Thus, the low selenium status of New Zealanders is associated with particularly low selenium levels in premature infants. Because these infants have a high risk for oxidative diseases such as bronchopulmonary dysplasia (chronic lung disease) and retinopathy of prematurity, the possibility that these conditions are more serious in the New Zealand population needs to be assessed and consideration given to dietary supplementation.

Adult↗
PubMed1991

Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs.

Hypochlorous acid (HOCl) is the most powerful oxidant produced by human neutrophils, and should therefore be expected to contribute to the damage caused by these inflammatory cells. It is produced from H2O2 and Cl- by the heme enzyme myeloperoxidase (MPO). We used a H2O2-electrode to assess the ability of a variety of anti-inflammatory drugs to inhibit conversion of H2O2 to HOCl. Dapsone, mefenamic acid, sulfapyridine, quinacrine, primaquine and aminopyrine were potent inhibitors, giving 50% inhibition of the initial rate of H2O2 loss at concentrations of about 1 microM or less. Phenylbutazone, piroxicam, salicylate, olsalazine and sulfasalazine were also effective inhibitors. Spectral investigations showed that the inhibitors acted by promoting the formation of compound II, which is an inactive redox intermediate of MPO. Ascorbate reversed inhibition by reducing compound II back to the active enzyme. The characteristic properties that allowed the drugs to inhibit MPO reversibly were ascertained by determining the inhibitory capacity of related phenols and anilines. Inhibition increased as substituents on the aromatic ring became more electron withdrawing, until an optimum reduction potential was reached. Beyond this optimum, their inhibitory capacity declined. The best inhibitor was 4-bromoaniline which had an I50 of 45 nM. An optimum reduction potential enables inhibitors to reduce MPO to compound II, but prevents them from reducing compound II back to the active enzyme. Exploitation of this optimum reduction potential will help in targeting drugs against HOCl-dependent tissue damage.

Aminobenzoates↗
PubMed1991

Oxidative damage to fibronectin. I. The effects of the neutrophil myeloperoxidase system and HOCl.

Exposure of purified human plasma fibronectin to the myeloperoxidase-H2O2-Cl- system of neutrophils or to reagent HOCl resulted in extensive changes to its primary and tertiary structures. When 1.14 microM fibronectin was exposed to 50-400 microM HOCl or 50-400 microM H2O2 plus myeloperoxidase and Cl-, there was progressive loss of tryptophan fluorescence and cysteines, and an increase in bityrosine fluorescence and carbonyl content. Analysis by SDS-PAGE indicated extensive crosslinking of the fibronectin, the crosslinks being stable under reducing conditions. The coincident increase of bityrosine fluorescence suggests that crosslinking may be largely due to intermolecular bityrosines rather than disulfides. All changes observed with the myeloperoxidase system were inhibited by azide or methionine, and were dependent upon the presence of chloride, indicating that they are mediated by HOCl. The reaction between HOCl and fibronectin resulted in the formation of long-lived chloramines. Exposure to increasing amounts of oxidant resulted in an increase in the susceptibility of fibronectin to proteolytic attack by purified neutrophil elastase. Analysis by SDS-PAGE showed a different fragmentation pattern for oxidant-treated fibronectin compared with the native protein. This suggests that regions of the molecule which were previously resistant to proteolysis were denatured to create susceptible sites for elastase. This demonstration that fibronectin is extensively modified by the myeloperoxidase system has implications for the mechanism of tissue injury by neutrophils in inflammation, since a loss of functional fibronectin would result in cell detachment and a distortion of normal tissue organization.

Azides↗
PubMed1991

Oxidative damage to fibronectin. II. The effect of H2O2 and the hydroxyl radical.

The effect of H2O2 and the hydroxyl radical (.OH) on fibronectin was investigated. .OH was generated in three ways: (i) by radiolysis with 60Co under N2O, or by the Fenton system using either (ii) equimolar Fe(2+)-EDTA and H2O2 or (iii) H2O2 and catalytic amounts of Fe(2+)-EDTA recycled with ascorbate. Each system had a different effect. H2O2 alone caused no changes, even at an 800-fold molar excess. Radiolytic .OH caused a rapid loss of tryptophan fluorescence, an increase in bityrosine fluorescence, and extensive crosslinking. The Fenton system using Fe-EDTA, H2O2, and ascorbate caused a loss in tryptophan fluorescence, a smaller increase in bityrosine than was seen with radiolytic .OH, and a threefold increase in carbonyl groups. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis fragmentation of fibronectin was seen. In contrast, when .OH was generated with equimolar Fe-EDTA and H2O2, the only change was a small increase in bityrosine fluorescence at the highest dose of oxidant. None of the systems used affected cysteine. All the changes except the loss of tryptophan by radiolytic .OH were completely inhibited with mannitol. The differences seen with radiolytic .OH and the Fe-EDTA, H2O2, ascorbate system were not solely due to O2 in the latter system since similar results were obtained under N2. The differences between radiolytic .OH and the Fenton systems could be partly due to the components of the latter systems reacting with .OH and thus competing with fibronectin. Our results demonstrate that the extent and type of fibronectin damage by .OH is dependent on the mode of radical generation.

Ascorbic Acid↗
PubMed1991

Factors that influence the deoxyribose oxidation assay for Fenton reaction products.

The mechanism of oxidation of deoxyribose to thiobarbituric acid-reactive products by Fenton systems consisting of H2O2 and either Fe2+ or Fe2+ (EDTA) has been studied. With Fe2+ (EDTA), dependences of product yield on reactant concentrations are consistent with a reaction involving OH.. With Fe2+ in 5-50 mM phosphate buffer, yields of oxidation products were much higher and increased with increasing deoxyribose concentration up to 30 mM. The product yield varied with H2O2 and Fe2+ concentrations in a way to suggest competition between deoxyribose and both reactants. Deoxyribose oxidation by Fe2+ and H2O2 was enhanced 1.5-fold by adding superoxide dismutase, even though superoxide generated by xanthine oxidase increased deoxyribose oxidation. These results are not as expected for a reaction involving free OH. or site localized OH. product on the deoxyribose. They can be accommodated by a mechanism of deoxyribose oxidation involving an iron(IV) species formed from H2O2 and Fe2+, but the overall conclusion is that the system is too complex for definitive identification of the Fenton oxidant.

Deoxyribose↗
PubMed1991

The influence of superoxide on the production of hypochlorous acid by human neutrophils.

Human neutrophils stimulated with opsonized zymosan promoted hypochlorous acid (HOCl)-dependent loss of monochlorodimedon. Formation of HOCl was completely inhibited by catalase, and it was also inhibited up to 70% by SOD. There was no inhibition by desferal, DTPA, mannitol or dimethylsulphoxide, which excluded the involvement of .OH. Our results indicate that generation of O2- by neutrophils enables these cells to enhance their production of HOCl. Furthermore, inhibition of neutrophil processes by SOD and catalase does not necessarily implicate .OH. We propose that O2- may potentiate oxidant damage at inflammatory sites by boosting the myeloperoxidase-dependent production of HOCl.

Catalase↗
PubMed1991

Ferritin, lipid peroxidation and redox-cycling xenobiotics.

A number of xenobiotics are toxic because they redox cycle and generate free radicals. Interaction with iron, either to produce reactive species such as the hydroxyl radical, or to promote lipid peroxidation, is an important factor in this toxicity. A potential biological source of iron is ferritin. The cytotoxic pyrimidines, dialuric acid, divicine and isouramil, readily release iron from ferritin and promote ferritin-dependent lipid peroxidation. Superoxide dismutase and GSH, which maintain the pyrimidines in their reduced form, enhance both iron release and lipid peroxidation. Microsomes plus NADPH can reduce a number of iron complexes, although not ferritin. Reduction of Adriamycin, paraquat or various quinones to their radicals by the microsomes enhances reduction of the iron complexes, and in some cases, enables iron release from ferritin. Adriamycin stimulates iron-dependent lipid peroxidation of the microsomes. Ferritin can provide the iron, and peroxidation is most pronounced at low pO2. Complexing agents that suppress intracellular iron reduction and lipid peroxidation may protect against the toxicity of Adriamycin.

Animals↗
PubMed1991

Tetravalent vanadium releases ferritin iron which stimulates vanadium-dependent lipid peroxidation.

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe(2+)-ferrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates, and iron release over 30 minutes, were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanadyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Ferritin alone did not promote significant levels of lipid peroxidation.

Catalase↗
PubMed1990

Ferritin-dependent lipid peroxidation by stimulated neutrophils: inhibition by myeloperoxidase-derived hypochlorous acid but not by endogenous lactoferrin.

Human neutrophils stimulated with phorbol myristate acetate or formylmethionylleucylphenylalanine caused superoxide-dependent release of iron from feritin, measured as the formation of a ferrous-ferrozine complex. The stimulated cells also caused ferritin-dependent peroxidation of phospholipid liposomes. Peroxidation was inhibited by lactoferrin, but only at concentrations considerably in excess of what could be achieved by release of endogenous lactoferrin. Peroxidation was enhanced by catalase and methionine, especially when stimulants that release myeloperoxidase were used. Peroxidation was inhibited by added myeloperoxidase. These results are explained by myeloperoxidase catalysing the formation of hypochlorous acid (HOCl) and the HOCl reacting with the lipid to inhibit peroxidation. Thus, neutrophils are able to use ferritin to promote lipid peroxidation. This may be limited under some conditions by iron binding to lactoferrin or transferrin, and more generally by reactions of the lipid with myeloperoxidase-derived HOCl. However, the latter reactions themselves may be harmful.

Ferritins↗
PubMed1990

Human neutrophil collagenase cleaves alpha 1-antitrypsin.

Inactivation of the plasma serine-proteinase inhibitor alpha 1-antitrypsin (alpha 1-AT) by neutrophil metalloproteinases has been reported [Vissers, George, Bathurst, Brennan & Winterbourn (1987) Fed. Proc. Fed. Am. Soc. Exp. Biol. 46, 1390a; (1988) J. Clin. Invest. 82, 706-711; Desrochers & Weiss (1988) J. Clin. Invest. 81, 1646-1650]. To identify the enzyme responsible, supernatant from neutrophils stimulated with phorbol 12-myristate 13-acetate was subjected to preparative SDS/PAGE, both with and without activation of latent metalloproteinases with HgCl2. The lanes were subsequently sliced into pieces, the slices incubated with equimolar amounts of type I collagen and alpha 1-AT in the presence of HgCl2, and the reaction products separated by SDS/PAGE. With the latent supernatant, the characteristic collagen-cleavage products and cleaved alpha 1-AT were present in the same slices, corresponding to an Mr of 80,000-85,000. On treatment with HgCl2 both degradative activities underwent the same molecular-mass shift to a position corresponding to Mr 60,000-65,000. Western blots of neutrophil supernatants, using a polyclonal antibody to purified collagenase, showed Mr values of 83,000 for the latent enzyme and 63,000 for the HgCl2-activated enzyme. Neutrophil collagenase was purified to homogeneity and shown also to exist in a second latent form with Mr 70,000. When activated to the Mr-63,000 form by HgCl2 and incubated with equimolar amounts of collagen and alpha 1-AT, collagenase cleaved alpha 1-AT at almost twice the rate at which collagen was cleaved. alpha 1-AT cleavage was inhibited by 1,10-phenanthroline and by high concentrations of collagen. That the purified collagenase did not contain a contaminant proteinase such as stromelysin was indicated by inability of the preparation to cleave casein. Taken together these results lead us to conclude that neutrophil collagenase is capable of degrading alpha 1-AT. Neutrophil gelatinase also cleaved alpha 1-AT, but cleavage was slow when compared with its activity against gelatin.

Caseins↗