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The ability of granulocytes to generate superoxide anions (O2-) and hypochlorite (OCl-) during phagocytosis was investigated using peripheral blood samples from adults and cord blood samples from neonates, using the chemiluminescence probe cypiridina luciferin analog (CLA) for O2- generation and luminol (L) for OCl- generation. OCl- generation by granulocytes was also monitored by taurine chloramine formation. The chemiluminescence probe based upon CLA was highly specific for and sensitive to O2- and could be adopted to determine O2- generation in terms of xanthine oxidase units. The CLA-dependent chemiluminescence by cord blood granulocytes was significantly higher than that by normal adult granulocytes. Taurine chloramine formation was significantly correlated with the L-dependent chemiluminescence (L-CL). Thus, the L-CL is considered to be mainly involved in OCl- generated by phagocytizing granulocytes. L-CLs by cord blood granulocytes and normal adult granulocytes were essentially the same during phagocytosis.

The erythrocyte (RBC) lysis by human monocytes incubated with opsonized zymosan particles (OPZ), was inhibited by catalase, chloride-free medium, azide and hypochlorous acid (HOCl) scavengers (taurine, alanine). These findings suggest the requirement for the HOCl-generating myeloperoxidase-hydrogen peroxide-chloride system (MPO-H2O2-Cl- system). The HOCl-dependent lysis was increased by inhibiting RBC catalase with aminotriazole (AT). Conversely, the inhibition of RBC glutathione cycle with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) had no detectable effect. Moreover, the recovery of H2O2 and HOCl from OPZ-triggered monocytes was reduced by the presence of RBCs through a process almost completely preventable by pulsing RBCs with AT but not with BCNU. Thus, it appears that RBC targets protect themselves by consuming, primarily via catalase, significant amounts of monocyte-derived H2O2 with a consequent impairment of the HOCl generation. The results suggest a potential role of target cells in modulating the cytolysin production by monocytes.

We investigated the effect of antiarthritic drugs containing thiol groups, such as D-penicillamine, tiopronin (N-[2-mercaptopropionyl]glycine), sodium aurothiomalate, and aurothioglucose, on the chlorinating activity of myeloperoxidase purified from human leukocytes. Hypochlorite, the reactive product of the reaction catalyzed by myeloperoxidase, was effectively scavenged by these antiarthritic drugs, and in addition, D-penicillamine and tiopronin inhibited myeloperoxidase itself. The above-mentioned effects of these drugs were observed at concentrations that occur in the serum of rheumatoid arthritis patients treated with these agents. We suggest that the therapeutic effect of these antiarthritic drugs may be due to the protection of tissues against the reactive HOCI released by activated granulocytes at inflamed sites.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.

Urea can be oxidized electrochemically in a chloride solution to carbon dioxide, water, and nitrogen. The microkinetics of this hypochlorite-mediated urea oxidation are elucidated. Based on this kinetic information, the optimal conditions and construction principles for an electrochemical reactor are deduced. The construction of a cheap, disposable oxidation cell and necessary auxiliary equipment are described. In vitro data are reported for urea removal. A 36-L volume was used to simulate a 60-kg patient; 18 L was recirculated through a 0.12-m2 oxidation cell. Within 3 h, 35 g urea could be removed from the system. The technical and economic possibilities as well as safety requirements for hemofiltrate regeneration to a reinfusable substitution solution by anodic urea oxidation are discussed critically. Although the process does not appear to be economically practical for discontinuous hemofiltration, it might be desirable for continuous (24 h/day) treatment.

The ability of serum proteins (albumin, immunoglobulin G) and protein antioxidants (ceruloplasmin, superoxide dismutase and transferrin) to react with O2-. and OCl-, was studied. The interaction between serum proteins and OCl- was shown to be non-specific. Ceruloplasmin is the most effective OCl- trapping protein, and it reacts with O2-. with a considerable efficiency. Therefore, ceruloplasmin is supposed to be the main scavenger of toxic oxygen species generated by stimulated neutrophils.

Trypanosoma musculi are readily killed when phagocytosed by mononuclear phagocytes but the nature of the mediators of this cytotoxicity is unclear. Among the most potent mediators are oxygen-derived species. The generation of chemiluminescence (CL) by peritoneal macrophages from 12 day T. musculi-infected mice, which phagocytose and kill parasites when opsonizing antibodies are present, was recorded in the presence of antibody-coated trypanosomes. Taurine, a specific quencher of hypochlorous acid (HOCl) inhibited CL production by peritoneal macrophages, showing that HOCl is produced during phagocytosis of T. musculi. In vitro, HOCl alone exerted a powerful trypanocidal activity which was inhibited in the presence of specific quenchers. The role of HOCl generated by phagocytes in trypanosome killing was studied using granulocytes which produce more oxygen-derived species than macrophages when stimulated. Phorbol myristate acetate-triggered granulocytes can destroy T. musculi and trypanosome killing is inhibited in the presence of taurine. These data demonstrate that HOCl produced by phagocytes can effectively destroy T. musculi.

Although 5-amino-salicylic acid (5-ASA) provides effective treatment for inflammatory bowel disease, its mode of action is unestablished. 5-ASA inhibits luminol-dependent chemiluminescence triggered by activated neutrophils, hydrogen peroxide plus peroxidase or sodium hypochlorite. The concentrations required for 50% inhibition of the cells was approximately 3.6 microM. In the non-cellular system, the concentration of 5-ASA required for total inhibition being approximately equivalent to concentration of sodium hypochlorite. The reaction of 5-ASA with hypochlorite or activated neutrophils resulted in the production of a non-fluorescent product of 5-ASA. The production of this metabolite by cells was dependent upon the activity of the peroxidase and occurred with a time course which was coincident with oxygen consumption. It was concluded that by reacting with hypochlorite, 5-ASA would provide protection against the potentially damaging effects of activated neutrophils in the inflamed bowel.

The ability of major serum proteins (albumin, immunoglobulin G) and free radical scavenger proteins (ceruloplasmin, superoxide dismutase, transferrin) to interact with O2-. and OCl- was studied. The interaction between serum proteins and OCl- was shown to be nonspecific and cause protein degradation. During SDS polyacrylamide gel electrophoresis ceruloplasmin and transferrin were degraded in the highest degree. Protein damage was also recorded by fluorescence changes. It is suggested that the damaging influence of active oxygen species secreted by stimulated neutrophils into the extracellular space can be abolished only by ceruloplasmin.

Human neutrophils, incubated with phorbol myristate acetate (PMA), caused a rapid and substantial adenosine triphosphate (ATP) depletion in lymphoblastoid Daudi cells without producing lysis. Catalase (which destroys hydrogen peroxide), taurine and methionine (which scavenge hypochlorous acid), and chloride omission from the medium prevented the ATP fall. An ATP depletion comparable to that induced by neutrophils was observed by replacing neutrophils with an appropriate myeloperoxidase-H2O2-Cl- enzymatic system. Together, these data suggest that the neutrophil ATP depleting activity involves the myeloperoxidase-catalyzed transformation of H2O2 into HOCl. Moreover, the free H2O2 remaining in the neutrophil extracellular environment is ineffective. In fact, a comparable amount of enzymatically generated H2O2 did not cause Daudi cell ATP loss. A direct role for H2O2 in the neutrophil-induced Daudi cell ATP depletion was observed only under artificial conditions, that is, in the presence of the heme enzyme inhibitor azide, which prevented the HOCl production but dramatically augmented the extracellular H2O2 level. Similar levels of ATP depletion in Daudi cells were induced by amounts of reagent HOCl comparable to those generated by neutrophils. As the generated HOCl can rapidly react with a variety of neutrophil-derived nitrogenous compounds (primarily ammonia and taurine) to yield chloramines, these chlorinated oxidants might contribute to the neutrophil-mediated ATP depletion. Nevertheless, the main and well-characterized chloramines (ammonia-derived monochloramine, NH2Cl, and taurine monochloramine, TauNHCl) were devoid of ATP-depleting capacity. Thus, the results suggest that the neutrophil-induced ATP depletion in Daudi cells is HOCl-dependent, is not mediated by NH2Cl or TauNHCl, and could be promoted either by HOCl directly or by an unknown derivative oxidant.(ABSTRACT TRUNCATED AT 250 WORDS)

Allopurinol is a scavenger of the highly reactive hydroxyl radical (k2 approx. 10(9) M-1 X s-1). One product of attack of hydroxyl radical upon allopurinol is oxypurinol, which is a major metabolite of allopurinol. Oxypurinol is a better hydroxyl radical scavenger than is allopurinol (k2 approx. 4 X 10(9) M-1 X s-1) and it also reacts with the myeloperoxidase-derived oxidant hypochlorous acid. Hence the protective actions of allopurinol against reperfusion damage after hypoxia need not be entirely due to xanthine oxidase inhibition.

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H2O2) prevents polyspermy in Arbacia punctulata. We found that it is not H2O2 but probably hypochlorous acid/hypochlorite (HOCl/OCl-) derived from H2O2 that is toxic to the supernumerary sperm. The spermicidal activity of H2O2 is potentiated by at least one order of magnitude by cupric ions (Cu2+). This increased toxicity is not due to the formation of hydroxyl radicals (.OH) because .OH scavengers did not counteract the activity of Cu2+. Moreover, substitution of Cu2+ by ferrous ions (Fe2+), which are known to cause formation of .OH from H2O2, had no effect on fertilization even at 10(2)-10(3) times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), and HOCl/OCl- scavenger, totally reversed the toxic effects of Cu2+. Furthermore, we found that HOCl/OCl- is generated in solutions of H2O2 and Cu2+ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl-. HOCl/OCl- generated by Cu2+ from H2O2 and Cl-, a low concentration of exogenously added HOCl/OCl-, or increased concentrations of H2O2 has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl- from the Cl- present in sea water through reaction with H2O2 generated by the newly fertilized egg.

Stimulated neutrophils produce several potent oxidants including H2O2, O2- and HOCl. Previous studies have revealed all of these compounds to be capable of oxidizing luminol, a reagent often used to indicate, by its chemiluminescence, the oxidative burst of neutrophils. Data presented in this paper indicate that H2O2 and HOCl spontaneously react at physiologic pH to produce luminol-dependent chemiluminescence 100 times the sum of the chemiluminescence of either reagent alone. This enhancement is due to a co-oxidation by HOCl and H2O2, or to a novel oxidant generated by the interaction of HOCl and H2O2. The HOCl scavenger, taurine, inhibits the chemiluminescence. Evidence is presented against the participation of hydroxyl radical, O2- or singlet oxygen in the oxidation of luminol by HOCl and H2O2. These findings have implications for potential anti-inflammatory compounds.

Human peripheral blood monocytes (M), incubated with opsonized zymosan particles (OPZ), lysed human erythrocyte (RBC) targets, as detected by a 51Cr release method. Conversely, cells derived in vitro from M (monocyte-derived macrophages, MDM) were ineffective. When added to the M-RBC system, MDM enhanced the lysis. The lysis by M and M plus MDM was prevented by catalase, azide and amino acids (alanine, taurine), consistent with the requirement for hypochlorous acid (HOCl). Moreover, MDM per se incapable of generating HOCl augmented the HOCl recovery from the M-RBC system. The results provide evidence for a previously unrecognized form of interaction between two distinct populations of mononuclear phagocytes.

The hypochlorous acid (HOCL)-dependent lysis of human red blood cells (HRBC) targets by neutrophils, activated with opsonized zymosan particles (OPZ), was increased by inhibiting HRBC catalatic activity with aminotriazole (AT; HRBCAT). The inhibition of HRBC glutathione cycle activity with carmustine (BCNU; HRBCBCNU) had no effect. In addition, the recovery of hydrogen peroxide (H2O2) and HOCL from neutrophils, activated under conditions similar to those used for cytotoxicity assay, was reduced by the presence of HRBC and restored by replacing HRBC with HRBCAT, but not with HRBCBCNU. Linear relationships were found between the increments in the neutrophil-mediated lysis, observed by using HRBCAT instead of HRBC, and the increments in the H2O2 or HOCL recovery, detected by replacing HRBC with HRBCAT. Together these data, coupled with the results obtained by probing neutrophil cytolysis with chemical agents, suggest that the increased cytolytic efficiency displayed by neutrophils against HRBCAT, inhibited in their catalatic activity, is due to an enhanced availability of neutrophil-derived H2O2, with a consequent enhancement in the HOCL production (according to the following reaction: (formula; see text). Thus it appears that HRBC catalase restrains the neutrophil cytolytic activity, by interfering with an early step of the pathway through which neutrophils generate cytotoxins.

Oxidation of Escherichia coli by hypochlorous acid (HOCl) or chloramine (NH2Cl) gives rise to massive hydrolysis of cytosolic nucleotide phosphoanhydride bonds, although no immediate change occurs in either the nucleotide pool size or the concentrations of extracellular end products of AMP catabolism. Titrimetric curves of the extent of hydrolysis coincide with curves for loss of cell viability, e.g., reduction in the adenylate energy charge from 0.8 to 0.1-0.2 accompanies loss of 99% of the bacterial CFU. The oxidative damage caused by HOCl is irreversible within 100 ms of exposure of the organism, although nucleotide phosphate bond hydrolysis requires several minutes to reach completion. Neither HOCl nor NH2Cl reacts directly with nucleotides to hydrolyze phosphoanhydride bonds. Loss of viability is also accompanied by inhibition of induction of beta-galactosidase. The proton motive force, determined from the distribution of 14C-radiolabeled lipophilic ions, declines with incremental addition of HOCl after loss of respiratory function; severalfold more oxidant is required for the dissipation of the proton motive force than for loss of viability. These observations establish a causal link between loss of metabolic energy and cellular death and indicate that the mechanisms of oxidant-induced nucleotide phosphate bond hydrolysis are indirect and that they probably involve damage to the energy-transducing and transport proteins located in the bacterial plasma membrane.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) catalyse the formation of hypochlorite (OCl-) from chloride ions (OCl-) and hydrogen peroxide (H2O2). OCl- proved to be highly toxic for Trichinella spiralis newborn larvae (NBL) in in vitro assays. Using purified human MPO and EPO it was found that even at neutral pH both enzymes under appropriate conditions are able to kill NBL. The rate at which OCl- is produced is much lower in the EPO- than in the MPO-mediated reaction. This difference in enzymic activity may explain why in the MPO-mediated reaction half the amount of OCl- was sufficient to kill 50% of the NBL, as compared to the EPO-mediated reaction. Purified human eosinophil major basic protein showed excellent OCl- scavenging properties, resulting in a significant decrease in the EPO-mediated NBL killing. Addition of ammonium ions [(NH4)2SO4] to the EPO-mediated reaction increased the NBL killing remarkably. It was concluded that in vitro MPO is more efficient than EPO in killing NBL. Furthermore, it was suggested that although eosinophils show marked parasiticidal effects in various in vitro systems, their primary biological role might be the regulation of the inflammatory reactions.

Oxidative degradation of biological substrates by hypochlorous acid has been examined under reaction conditions similar to those found in active phagosomes. Iron sulfur proteins are bleached extremely rapidly, followed in decreasing order by beta-carotene, nucleotides, porphyrins, and heme proteins. Enzymes containing essential cysteine molecules are inactivated with an effectiveness that roughly parallels the nucleophilic reactivities of their sulfhydryl groups. Other compounds, including glucosamines, quinones, riboflavin, and, except for N-chlorination, phospholipids, are unreactive. Rapid irreversible oxidation of cytochromes, adenine nucleotides, and carotene pigments occurs when bacterial cells are exposed to exogenous hypochlorous acid; with Escherichia coli, titrimetric oxidation of cytochrome was found to coincide with loss of aerobic respiration. The occurrence of these cellular reactions implicates hypochlorous acid as a primary microbicide in myeloperoxidase-containing leukocytes; the reactivity patterns observed are consistent with the view that bactericidal action results primarily from loss of energy-linked respiration due to destruction of cellular electron transport chains and the adenine nucleotide pool.

The relative importance of several oxygen intermediates in fungicidal action against opsonized Aspergillus fumigatus conidia was investigated with monocytes from normal volunteers and patients with either chronic granulomatous disease or myeloperoxidase (MPO) deficiency. Results from experiments in which catalase, taurine, mannitol, or glucose-glucose oxidase were added to these phagocytes indicated that the MPO-hydrogen peroxide-halide system and an MPO-independent oxidative system exerted comparable conidiacidal activity. These findings offer a plausible explanation for the susceptibility of patients with chronic granulomatous disease to invasive Aspergillus infections; their phagocytes fail to generate hydrogen peroxide, a substrate necessary for both systems. Patients with MPO deficiency are not known to be predisposed to invasive aspergillosis, suggesting that an MPO-independent oxidative system may provide an alternative mechanism for the oxidative killing of Aspergillus spp.

Eosinophils are white blood cells that in humans are found in association with helminthic infections and various inflammatory disease processes. These cells contain a unique lysosomal peroxidase that oxidizes halides to generate highly reactive and toxic hypohalous acids. Although chloride is found in vivo at concentrations at least 1000-fold greater than those of other halides, human eosinophils did not preferentially oxidize chloride under physiologic conditions. Instead, eosinophils used bromide, a halide with a hitherto unknown function in humans, to generate a halogenating oxidant with characteristics similar, if not identical, to those of hypobromous acid. These results indicate that physiological concentrations of bromide arm human eosinophils with the ability to generate and release an unusual oxidant capable of destroying a wide range of prokaryotic and eukaryotic targets.

Ascorbic acid, at physiological concentrations, can scavenge the myeloperoxidase-derived oxidant hypochlorous acid at rates sufficient to protect alpha 1-antiprotease against inactivation by this molecule. The rapid depletion of ascorbic acid at sites of inflammation, as in the inflamed rheumatoid joint, may therefore facilitate proteolytic damage.

Acetaminophen binds via its acetamido side chain to purified myeloperoxidase in a pH-dependent manner and maximum binding occurred around pH 6. The H2O2-dependent myeloperoxidase-catalysed polymerization products of acetaminophen had excitation maxima at 304 nm and 334 nm in acid and alkaline solutions, respectively, and an intense blue fluorescence maximum at 426 nm. Acetaminophen can compete effectively with Cl- as myeloperoxidase substrate and thus HOCl formation is suppressed while HOCl, nevertheless present, can be scavenged by the drug. In this way the microbicidal action of the myeloperoxidase-H2O2-Cl- system can be seriously limited in the presence of high concentrations of acetaminophen. To study the effect of acetaminophen on peptide bond splitting in the myeloperoxidase antimicrobial system, thyroglobulin was used as a model peptide. Peptide bond splitting was inhibited at acetaminophen concentrations below the accepted toxic range for plasma values.

The specific thiol protease inhibitor, NCO-700, which is related to L-trans-epoxysuccinylpeptides, inhibited oxidant production by chemoattractant-stimulated rabbit polymorphonuclear leukocytes. NCO-700 could also scavenge active oxygen generated from sodium hypochlorite-hydrogen peroxide and hypoxanthine-xanthine oxidase systems.

In acidic solutions, bromide stimulates H2O2 decomposition catalyzed by chloroperoxidase or horseradish peroxidase, altogether similar to stimulation, by chloride and bromide, of H2O2 oxidation by HOCl. Low levels of an ultra-pure NaCl inhibited chloroperoxidase catalatic activity whereas very high concentrations of this salt stimulated the reaction. The stimulation reflects low bromide contamination of the ultra-pure NaCl, which is considerably less than Br levels in most AR grade chloride salts. In all of these systems, bromide functions as a catalyst which is first oxidized to Br2 by HOX (added directly or generated enzymatically), and subsequently regenerated by H2O2 reduction of Br2. These results provide strong support for a dissociable species, most likely HOX, as the first product of chloroperoxidase catalyzed oxidation of Br- and Cl- by H2O2.

Spectral-scan results obtained on the millisecond time scale are reported for reactions of chloroperoxidase with peracetic acid and chloride ion in both the presence and the absence of monochlorodimedone. A multimixing experiment is performed in which stoichiometric amounts of chloroperoxidase and peracetic acid are premixed for 0.7 s before the resultant compound I is reacted with chloride ion. The combined results show that the only detectable enzyme intermediate species is compound I (except in very late stages of the reaction), that the disappearance of compound I is accelerated by the presence of chloride ion, and that it is further accelerated if both chloride and monochlorodimedone are present. It is concluded that compound I is an obligate intermediate species in the reaction. Experiments are performed on the reaction of monochlorodimedone with hypochlorous acid in both the presence and the absence of added chloride ion, but in the absence of chloroperoxidase. The presence of chloride ion greatly accelerates the reaction rate apparently by setting off a chlorine chain reaction. This reaction would be important in the enzyme-catalyzed reaction if hypochlorous acid were liberated into the solution. A careful analysis of steady-state kinetic results shows that in the chlorination of monochlorodimedone at least, liberation of free hypochlorous acid is not important in the enzyme-catalyzed pathway. Rather the reaction proceeds from compound I to formation of iron(III)-OCl by chloride ion addition to the ferryl oxygen atom. This obligate intermediate species then chlorinates the substrate. It is well described as enzyme-activated hypochlorous acid, in which replacement of the proton in HOCl by the heme iron ion produces a Cl+ species of great potency. Thus the enzyme controls chlorination of monochlorodimedone rather than unleashing an uncontrolled chain reaction in which it would be rapidly destroyed.

Apolactoferrin and apotransferrin lost their ability to subsequently bind iron when exposed to an excess of either HOCl or myeloperoxidase plus H2O2 and Cl-. Apolactoferrin, however, was more resistant than apotransferrin. By oxidizing a mixture of the two proteins, then separating them by immunoprecipitation, the difference in susceptibility was shown to be due to the greater reactivity of transferrin iron-binding groups, rather than protective groups on the lactoferrin molecule. The iron-saturated proteins were much more resistant to oxidative modification than the apoproteins. The greater resistance of apolactoferrin should be advantageous for maintaining its iron binding capacity when co-released with myeloperoxidase and reactive oxygen species from stimulated neutrophils.

Hypochlorous acid generated by myeloperoxidase reacts with histamine to produce chloramines. At pH 7, one mole of histamine monochloramine (HisCl) was generated per mole of H2O2 provided as substrate for myeloperoxidase. At pH 5, one mole of histamine dichloramine (HisCl2) was generated per two moles of H2O2. HisCl and HisCl2 had two and four oxidizing equivalents per molecule, respectively. In vitro, 30 microM HisCl and HisCl2 induced mepyramine-sensitive guinea pig lung parenchyma contraction with 89 and 56 percent of the response of an equivalent concentration of histamine. Pretreatment of lung strips with chloramines reduced the subsequent contractile response of the tissues to methacholine. These results suggest that H-1 histamine receptors provide for targeting of histamine chloramines to pulmonary tissue which may facilitate modification of tissue responses.

Despite the critical role for neutrophils in host defenses against invasive aspergillosis, previous studies have established that neutrophils are unable to kill resting conidia of Aspergillus fumigatus. The mechanisms of resistance of the conidia were therefore investigated. Electron microscopy studies showed the fusion of phagosomes containing A. fumigatus conidia with lysosomes of the neutrophil. Resting conidia of A. fumigatus were then compared with those that had been preincubated in broth until swollen, but not germinated, as well as with blastospores of Candida albicans (two fungal forms that are killed by neutrophils) and zymosan particles. Despite comparable susceptibility to phagocytosis, resting conidia of A. fumigatus stimulated production of significantly less superoxide anion, hydrogen peroxide, and hypochlorous acid and induced less myeloperoxidase-dependent iodination by neutrophils than did the preincubated conidia of A. fumigatus, blastospores of C. albicans, or zymosan particles. In addition, resting conidia of A. fumigatus were relatively resistant to cell-free killing by oxidants presumed to be generated by neutrophils. Thus, resistance of resting conidia of A. fumigatus to neutrophil fungicidal mechanisms appears to be secondary to both failure of the conidia to stimulate an optimal respiratory burst as well as resistance of the conidia to neutrophil oxidants. However, the reversal of this resistance by preincubation of the conidia suggests that neutrophils still may form an important host defense against the conidia of A. fumigatus.

The ability of the polymorphonuclear leukocyte (PMN) oxidants, hypochlorous acid (HOC1) and hydrogen peroxide (H2O2), to oxidize proteins in rat heart and lung tissues was investigated. Cardiac myocytes, heart tissue slices, isolated perfused hearts, and lung tissue slices, were treated with HOC1 and H2O2 and the extent of methionine and cysteine oxidation was determined in the cellular proteins. Cardiac tissues were found to be highly susceptible to oxidation by physiological concentrations of HOC1. For example, in isolated hearts perfused for 60 min with 100 microM HOC1, approximately 18% of the methionine and 28% of the cysteine residues were oxidized. Lung tissues, unlike those of the heart, were resistant to physiological concentrations of HOC1, showing no oxidation of proteins. HOC1 was much more effective than H2O2 in oxidizing proteins, suggesting that HOC1 may be the most reactive oxidant produced by activated PMN. These studies show that PMN oxidants, in particular HOC1, can cause significant oxidation of proteins in target tissues, and may therefore constitute a primary cause of tissue injury at sites of inflammation. In addition, these studies show that different tissues may have varying susceptibilities to PMN oxidants.

The sulfur-centered compound dimethylthiourea (DMTU) affords antioxidant protection in animal models of acute lung injury, an effect that has been attributed to its OH. scavenging properties. Although DMTU can also react with H2O2 in certain experimental systems, the effect of DMTU on the neutrophil myeloperoxidase (MPO) pathway has not been studied. DMTU (1-10 mM) completely blocked stable oxidants and hypochlorous acid formation by phorbol myristate acetate- and zymosan-stimulated neutrophils. DMTU also provided complete inhibition when incubated with cell-free supernatants after the formation of the MPO products. DMTU prevented the oxidative inactivation of alpha 1-antitrypsin by neutrophil-stable oxidants. Evidence that DMTU was oxidized by the MPO products was obtained by titration of oxidized DMTU with reduced glutathione. Surprisingly, supernatants from cells incubated with DMTU (10 mM) consumed two- to threefold higher amounts of reduced glutathione than supernatants from cells incubated with taurine (15 mM). Metabolic studies with stimulated neutrophils and experiments with the MPO enzyme system in a cell-free system suggested that DMTU acts by scavenging the products of the MPO pathway rather than by blocking H2O2 production in the intact cell. These findings demonstrate that DMTU blocks the neutrophil MPO pathway in addition to its known ability to scavenge other reactive O2 species. The capacity of DMTU to scavenge MPO products may explain some of its protective effects in acute lung injury.

Spores of Bacillus subtilis NCTC 10073 were converted to ion-exchange (Ca, H) forms and coat-defective (urea-mercaptoethanol, urea-dithiothreitol-sodium lauryl sulphate) forms. The resistance of these to sodium hypochlorite (1000 parts/10(6) free chlorine) was compared and related to uptake from which the assumed monolayer capacities were calculated. Hypochlorite effects on spore protoplasts and cortical fragments were also examined in relation to DPA and hexosamine release. A spore lytic enzyme was extracted and examined in respect of hypochlorite activity. The results are discussed in terms of the mechanism and site of action of hypochlorite on the bacterial spore.

Myeloperoxidase-catalyzed oxidation of chloride (Cl-) to hypochlorous acid (HOCl) resulted in formation of mono- and dichloramine derivatives (RNHCl and RNCl2) of primary amines. The RNCl2 derivatives could undergo a reaction that resulted in incorporation of the R moiety into proteins. The probable mechanism was attack of RNCl2 or an intermediate formed in the decomposition of RNCl2 on histidine, tyrosine, and cystine residues and on lysine residues at high pH. Incorporation of radioactivity from labeled amines into stable, high molecular weight derivatives of proteins was measured by acid or acetone precipitation and by gel chromatography and electrophoresis. Whereas formation of RNCl2 was favored at low pH, the subsequent incorporation reaction was favored at high pH. Up to several hours were required for the maximum amount of incorporation, which was less than 10% of the label in RNCl2. For the amines tested, incorporation was in the order histamine greater than 1,2-diaminoethane greater than putrescine greater than taurine greater than lysine greater than glucosamine greater than leucine greater than methylamine. Initiation of the reaction required HOCl, and oxidized forms of bromide, iodide, or thiocyanate did not substitute. Inhibitors of incorporation fell into three classes. First, ammonia or amines competed with the labeled amine for reaction with HOCl, so that larger amounts of HOCl were required. Second, readily oxidized substances such as sulfhydryl or diketo compounds or thioethers (methionine) reduced RNCl2. Third, certain compounds competed with protein as the acceptor for the incorporation reaction. The amount required to block incorporation into protein depended on protein concentration. Among these inhibitors were imidazole compounds (histidine), phenols (tyrosine), and disulfides (glutathione disulfide, GSSG). Low yields of derivatives of histidine, tyrosine, and GSSG were detected by thin-layer chromatography. Acid-precipitable derivatives were obtained by reacting RNCl2 with polyhistidine or polytyrosine, and to a lesser extent with polylysine at high pH, but not with other poly(amino acids). Precipitable derivatives were also obtained by incubating MPO-containing extracts from leukocyte granules with hydrogen peroxide, Cl-, and labeled amines. The extracts were found to have a high content of substances with primary amino groups, which competed for incorporation. The results account for oxidative incorporation of amines into proteins in leukocytes and provide evidence that HOCl and nitrogen-chlorine (N-Cl) derivatives are formed in these cells. The characteristics of the incorporation reaction suggest that it would not contribute significantly to the antimicrobial activity of myeloperoxidase (MPO). Nevertheless, the reaction may provide a sensitive method for studying MPO action in vivo.

Neutrophils recruited to different tissues undergo respiratory burst activity at widely different PO2 levels. The present study investigated the in vitro effects of PO2 on neutrophil oxidative metabolism. When neutrophils were stimulated with either zymosan or phorbol myristate acetate (PMA) under different PO2's (0-700 Torr), hexose monophosphate shunt activity, H2O2, and hydroxyl radical (OH.) production were directly related to the level of PO2. Neutrophils functioned surprisingly well at PO2's as low as 10 Torr, where metabolic burst activity was prolonged and usually exceeded 50% of maximal values. The production of neutrophil stable oxidants and hypochlorous acid (HOCl) by zymosan-stimulated neutrophils was also directly related to PO2. In contrast, the production of stable oxidants and HOCl by PMA-stimulated neutrophils was significantly higher at 10 Torr compared with 700 Torr. The decrease in stable oxidant production by PMA-stimulated neutrophils at elevated PO2's was explained by both increased destruction of stable oxidant products and by decreased availability of the precursor HOCl. Superoxide dismutase and the OH. scavenger benzoate partially prevented the fall in stable oxidants at elevated PO2's. Measurements of stable oxidants in PMA-stimulated supernates generated at 10 and 700 Torr correlated with the ability of these supernates to decrease the elastase inhibitory capacity of the serum antiprotease alpha 1-antitrypsin. These findings suggest that different PO2's alter the magnitude and pattern of neutrophil oxidative metabolism.

The interaction of myeloperoxidase (MPO) with H2O2 and Cl- provides a potent antimicrobial/cytotoxic system for polymorphonuclear leukocytes (PMNs). MPO-related cytotoxicity may be associated with the formation of toxic oxidant MPO intermediates, HOCl, or both. MPO itself is able to oxidize drugs and cellular components. Non-steroidal anti-inflammatory drugs (NSAIDs) able to act as antioxidant free radical scavengers have recently been shown to inhibit luminol-enhanced chemiluminescence (CL) which results from the MPO-H2O2-Cl- reaction. CL is a measure of the activity of this reaction. At that time it was not clear whether the source of CL which these NSAIDs affected was HOCl or components of the initial MPO-H2O2-Cl- reaction. A NSAID antioxidant mechanism could affect MPO oxidant intermediates and HOCl. This study compares the effects of antioxidant NSAIDs, methylprednisone and free radical scavengers against MPO-based and NaOCl-based luminol-enhanced CL. Most NSAIDs which affected both MPO and NaOCl-CL appeared to share similar mechanisms, suggesting that MPO oxidant intermediates and HOCl are susceptible to NSAID effects. However, most NSAIDs were more effective against MPO-CL. The effect of these NSAIDs against MPO-CL followed the profile of NSAIDs effective in previous studies against PMN-CL. One exception to this was methylprednisone, which has no effect on PMN or MPO-CL, yet inhibited NaOCl-CL. This and other data suggest that MPO and not HOCl-related reactions are a major source of PMN-CL. Less effective NSAIDs affected NaOCl-CL better than MPO-CL. While both HOCl and MPO oxidant intermediates may be affected by NSAIDs, it appears that MPO oxidant intermediates or MPO itself are the primary target for NSAID antioxidant free radical scavenging mechanisms. These antioxidant effects impair the major killing system of the PMN and may be NSAIDs' primary anti-inflammatory mechanism. Although our data suggests the production of superoxide anion and hydroxyl radical from the MPO-H2O2-Cl- reaction, the actual presence or involvement of these free radical species is not confirmed herein.

Although salicylates have been used for centuries as treatment of inflammatory diseases, the mechanism of action of these drugs is still not clear. Aspirin (acetylsalicylic acid) and other nonsteroidal anti-inflammatory drugs (NSAID) inhibit prostaglandin biosynthesis, a property that appears to explain part of their anti-inflammatory activity. However, this mechanism does not appear to explain the anti-inflammatory properties of salicylic acid, which is a major metabolite of ASA in vivo. Results of prior studies in our laboratory have established that benzoic acid, the parent compound of the salicylate group of drugs, is decarboxylated and hydroxylated by the hydroxyl free radical (OH.) produced by stimulated granulocytes. These observations suggested that salicylates might be similarly metabolized by granulocytes. If so, the capacity of salicylates to rapidly react with OH. might relate directly to their known anti-inflammatory properties. Preliminary experiments established that salicylic acid and aspirin were decarboxylated by the hydroxyl free radical generated by the enzyme system xanthine-xanthine oxidase. We then studied the metabolism of salicylates by human granulocytes. Unstimulated granulocyte suspensions did not oxidize ASA or salicylic acid. However, suspensions stimulated by opsonized zymosan particles rapidly oxidized both substrates in pharmacological concentrations. The rate of oxidation of salicylic acid was 16-fold higher than benzoic acid, whereas the rate of oxidation of ASA was four-fold higher. The reaction was oxygen dependent and could be inhibited by known hydroxyl scavengers, particularly dimethylthiourea. The reaction could also be inhibited by superoxide dismutase and azide, indicating that O-2 and heme or an iron-dependent enzyme were required for the reaction. The reaction was not impaired by compounds known to react with the HOCL and the chloramines generated by stimulated PMN. Furthermore, salicylic acid in high concentrations did not impair the HMPS pathway, the production of O-2 or the production of H2O2 by granulocytes. These data provide evidence that salicylates are rapidly oxidized by the hydroxyl free radical produced by granulocytes and not O-2, H2O2, or HOCL. This capacity of salicylates to react rapidly and selectively react with OH. may directly relate to their anti-inflammatory properties. In addition, results of our experiments indicate that stimulated granulocytes acquire the capacity to metabolize these drugs. Therefore, several metabolites of salicylates may be produced at a site of inflammation, all of which may have altered biological activity compared with the parent compound.

Under natural conditions or because of therapy with heat or cold, neutrophils may function at times in the human body at temperatures other than 37 degrees C. Therefore, we evaluated the effects of temperature on several functions of these cells. Phagocytosis and superoxide production by stimulated neutrophils were optimal at 37 degrees C and attained at least 70% of this peak value at 42 degrees C. In contrast, production of hypochlorous acid (as measured by an assay using the chlorination of taurine) by stimulated neutrophils was optimal at temperatures less than 37 degrees C and attained only 13% to 15% of this peak value at 42 degrees C. During a 2-hour incubation, the major suppressive effects of the higher temperature occurred during the second hour. This result was not explainable by factors related to the hypochlorous acid assay system or by loss of cell viability or myeloperoxidase activity in the cell supernatants, but rather appeared to be caused by reduced generation of hydrogen peroxide at the higher temperatures. Because the extracellular release of a strong oxidant such as hypochlorous acid might result in significant tissue injury, suppression of the release of this oxidant by elevated temperatures may explain why the application of local heat sometimes benefits certain inflammatory conditions.

In the presence of Escherichia coli, myeloperoxidase-catalyzed oxidation of chloride ion resulted in formation of long-lived chloramine and/or chloramide derivatives of bacterial components. The same amount of these nitrogen-chlorine (N-Cl) derivatives was obtained with either hypochlorous acid (HOCl) or the myeloperoxidase system, indicating that myeloperoxidase catalyzed the oxidation of chloride to HOCl. Identical killing was obtained with HOCl or the myeloperoxidase system. About 30 to 50% of the oxidizing equivalents of HOCl were detected as N-Cl derivatives of peptides or peptide fragments that were released from the bacteria. The apparent molecular weight distribution of the peptides decreased with increasing amounts of HOCl, suggesting that peptides were fragmented by oxidative cleavage of chloramide derivatives of peptide bonds. The remaining 50 to 70% of the oxidizing equivalents of HOCl were rapidly consumed in peptide bond cleavage or the oxidation of other bacterial components. There was a close correspondence between the oxidation of bacterial sulfhydryls and bactericidal action. The N-Cl derivatives were lost and the oxidation of bacterial sulfhydryls increased over a period of several h at 37 degrees C. These changes were accompanied by increased killing. The increase in sulfhydryl oxidation and killing could be prevented by washing the bacteria to remove the N-Cl derivatives. Therefore, the N-Cl derivatives could oxidize bacterial components long after the myeloperoxidase-catalyzed oxidation of chloride was complete.

Alginic acid was purified from a mucoid clinical isolate of Pseudomonas aeruginosa. Luminol-dependent chemiluminescence of phorbol myristate acetate-stimulated neutrophils was inhibited by this alginate, but oxygen consumption was unaffected. Further studies indicated that this effect was due to the ability of the pseudomonal alginate to scavenge hypochlorite. A seaweed alginate was less effective and dextran T500 was ineffective in hypochlorite scavenging. It appears that the uronic acid core and the O-acetyl groups of pseudomonal alginate are involved in its hypochlorite-scavenging ability. The relevance of this phenomenon was demonstrated by the greater resistance to killing by hypochlorite of mucoid P. aeruginosa compared with a nonmucoid revertant, and the addition of purified alginate to the nonmucoid revertant protected the organism from hypochlorite. Thus, this extracellular polysaccharide may enhance the virulence of P. aeruginosa by scavenging the phagocyte-generated oxidant HOCl. This enhanced virulence may be involved in disease processes in which mucoid organisms predominate, such as cystic fibrosis.

The chemical reactions that hypochlorite undergoes in the body when chlorinated water is ingested have received very little attention. Because amino nitrogen compounds are important components of the average diet, the reactions of hypochlorite with amino compounds in the stomach were investigated. Stomach fluid was recovered from Sprague-Dawley rats that had been fasted for 48 hr and administered 4 mL deionized water. The chlorine demand of the stomach fluid was determined. An average volume-independent demand of 2.7 mg chlorine was measured. At doses below 40 mg/L chlorine reducing reactions appeared to account for reduction of all oxidizing species within 15 min as measured by the FAS-DPD titrimetric method. At least part of the chlorine demand is associated with amino acids present in the stomach fluid. Amino acids were identified and quantified in the stomach fluid by precolumn derivatization with o-phthalaldehyde and high-pressure liquid chromatography (HPLC). When stomach fluid is chlorinated to concentrations of chlorine between 200 and 1000 mg/L, organic N-chloramines are formed. After derivatization of chlorinated stomach fluid with dansyl sulfinic acid, fluorescent derivatives of chloramines were separated by HPLC. Three chloramino acid derivatives, N-chloroalanine, N-chloroglycine, and N-chlorophenylalanine, were identified by cochromatography with known standards using two chromatographic methods. The yield of a chloramine that would form in stomach fluid on administration of hypochlorite to animals was determined using tritiated piperidine and doses of 200 and 1000 mg/L chlorine. Yields of tritiated N-chloropiperidine in recovered stomach fluid were 70% and 42%, respectively, of the theoretical amount expected.

The problem of trihalomethane formation now exists with the use of chlorine (technically, a mixture of HOCl and OCl-, depending on pH) as a disinfectant in drinking water. Monochloramine (NH2Cl) may be considered as an alternative to HOCl as a disinfectant in public water supplies. This study was conducted to compare the kinetics of NH2(36)Cl with HO36Cl in rats. The peak plasma levels of 36Cl occurred at 8 and 2 h for NH2(36)Cl and and HO36Cl, respectively. The distribution of NH2(36)Cl and HO36Cl was highest in plasma, while the lowest activity was observed in the fat. The half-life for 36Cl absorption was 2.49 and 2.16 h, while the elimination from plasma was 38.8 and 44.1 h after oral NH2(36)Cl and HO36Cl treatment, respectively. Urinary excretion accounted for most of the 36Cl eliminated in both treatments.

Phagocytes generate superoxide (O2-.) and hydrogen peroxide (H2O2) and their interaction in an iron-catalyzed reaction to form hydroxyl radicals (OH.) (Haber-Weiss reaction) has been proposed. Deferoxamine chelates iron in a catalytically inactive form, and thus inhibition by deferoxamine has been employed as evidence for the involvement of OH. generated by the Haber-Weiss reaction. We report here that deferoxamine also inhibits reactions catalyzed by the peroxidases of phagocytes, i.e., myeloperoxidase (MPO) and eosinophil peroxidase (EPO). The reactions inhibited include iodination in the presence and absence of chloride and the oxidation of guaiacol. Iodination by MPO and H2O2 is stimulated by chloride due to the intermediate formation of hypochlorous acid (HOCl). Iodination by reagent HOCl also is inhibited by deferoxamine with the associated consumption of HOCl. Iron saturation of deferoxamine significantly decreased but did not abolish its inhibitory effect on iodination by MPO + H2O2 or HOCl. Deferoxamine did not affect the absorption spectrum of MPO, suggesting that it does not react with or remove the heme iron. The conversion of MPO to Compound II by H2O2 was not seen when H2O2 was added to MPO in the presence of deferoxamine, suggesting either that deferoxamine inhibited the formation of Compound II by acting as an electron donor for MPO Compound I or that deferoxamine immediately reduced the Compound II formed. Iodination by stimulated neutrophils also was inhibited by deferoxamine, suggesting an effect on peroxidase-catalyzed reactions in intact cells. Thus deferoxamine has multiple effects on the formation and activity of phagocyte-derived oxidants and therefore its inhibitory effect on oxidant-dependent damage needs to be interpreted with caution.

Cartilage is a focal point of attack by cellular and molecular elements of the inflammatory response which occurs in arthritic diseases. Neutrophils damage articular cartilage by degrading matrix components and inhibiting their synthesis. The aim of this study was to elucidate mechanisms of this damage. Human neutrophils were isolated from blood by centrifuging through Ficoll-Hypaque and granule extract prepared from them. Articular cartilage from adult humans and cattle was maintained in organ culture. Cartilage degradation (release of 35S-labelled proteoglycan) or synthesis (incorporation of 35S into proteoglycan) was determined after various treatments. Human neutrophils and neutrophil granule extract degraded proteoglycan and inhibited proteoglycan synthesis. The specific leucocyte elastase inhibitor N-methoxysuccinyl-(ala)2-pro-val-chloromethylketone (MAAPVCMK) partially reversed these effects. H2O2, a product of the neutrophil respiratory burst, when added directly at 10(-6)mol/L, or generated by glucose oxidase (GO)/glucose inhibited proteoglycan synthesis but had no effect on degradation. Hypochlorous acid (OHCl), a product of the myeloperoxidase (MPO)/H2O2/Cl system at 50 mumol/L degraded proteoglycan and inhibited its synthesis. OHCl produced by granule extract (as a source of MPO) + GO-generated H2O2 + Cl- degraded proteoglycan. The results indicate that neutrophil-mediated proteoglycan degradation and inhibition of synthesis is largely attributable to elastase and secondarily to OHCl, whereas H2O2 impairs synthesis without affecting degradation of proteoglycan.

Species such as superoxide radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (.OH), and hypochlorous acid (HOCl) can be formed in vivo, e.g., by activated phagocytic cells. Generation of .OH from H2O2 in vivo usually involves iron-dependent reactions. Good evidence exists for increased generation of oxidants in vivo in patients with active rheumatoid disease, but the contribution of these oxidants to the disease process is still uncertain. The likelihood that anti-inflammatory drugs used in the treatment of arthritis could act by scavenging oxidants or preventing their formation is discussed.

Recent investigations have indicated the presence of a fatty acid binding protein (FABP) in mammalian heart. This protein binds free fatty acids and their esters with high affinity, however, its physiological role remains unknown. Since FABP constitutes a significant amount of cystolic protein, it is likely that it would be a target for free radical attack. To test this hypothesis, FABP was examined for scavenging against free radicals such as the superoxide anion (O2-), hydroxyl radical (OH.) and hypochlorite radical (OCl.) which may be present in an ischemic reperfused heart. Our results suggest that FABP scavenges O2-, OH. and OCl. as indicated by the FABP inhibition of O2- -dependent reduction of cytochrome c, OH.-dependent hydroxybenzoic acid formation and OCl.-mediated chemiluminescence response. FABP was found to be a more potent scavenger of these free radicals compared to bovine serum albumin. Furthermore, FABP was more effective in scavenging OH. than O2-, and inhibited OH. mediated lipid peroxidation process. These results indicate that FABP can scavenge free radicals which may be present in an ischemic/reperfused heart and, thus, may play a significant physiological role in the heart during ischemia and reperfusion.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.