Christine C. Winterbourn

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.

Assays for the chlorination activity of myeloperoxidase.

Publisher Summary Myeloperoxidase (donor:hydrogen-peroxide oxidoreductase) is the most abundant protein in neutrophils and is also found in monocytes. It contains two heme-prosthetic groups and is a unique peroxidase that catalyzes the conversion of hydrogen peroxide and chloride to hypochlorous acid. Hydrogen peroxide is formed from the spontaneous dismutation of superoxide, which is produced by an NADPH oxidase in the cell membrane. Hypochlorous acid is the major strong oxidant produced by neutrophils. It has powerful antimicrobial activity, and it is extremely reactive with biological molecules. It inactivates enzymes and α 1 -antitrypsin, cross-links proteins, and reacts with unsaturated fatty acids to form chlorohydrins, which may destabilize cell membranes. Given this broad spectrum of reactivity, hypochlorous acid is an obvious candidate for causing much of the damage mediated by neutrophils in inflammatory diseases. The ferric or native enzyme (MP 3+ ) reacts with hydrogen peroxide (H 2 O 2 ) to form the active redox intermediate compound I, which oxidizes chloride (CI - ) to hypochlorous acid (HOCl).

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.

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.

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.

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.

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. J. Lcukoc. Biol. 55: 147–152; 1994.

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.

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.

Selenium and Glutathione Peroxidase Levels in Premature Infants in a Low Selenium Community (Christchurch, New Zealand)

ABSTRACT: 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 <0.13 μmol/L (10 μg/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.