Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.     

Oxidation of chloride and thiocyanate by isolated leukocytes

Peroxidase-catalyzed oxidation of chloride (Cl-) and thiocyanate (SCN-) was studied using neutrophils from human blood and eosinophils and macrophages from rat peritoneal exudates. The aims were to determine whether Cl- or SCN- is preferentially oxidized and whether leukocytes oxidize SCN- to the antimicrobial oxidizing agent hypothiocyanite (OSCN-). Stimulated neutrophils produced H2O2 and secreted myeloperoxidase. Under conditions similar to those in plasma (0.14 M Cl-, 0.02-0.12 mM SCN-), myeloperoxidase catalyzed the oxidation of Cl- to hypochlorous acid (HOCl), which reacted with ammonia and amines to yield chloramines. HOCl and chloramines reacted with SCN- to yield products without oxidizing activity, so that high SCN- blocked accumulation of chloramines in the extracellular medium. Under conditions similar to those in saliva and the surface of the oral mucosa (20 mM Cl-, 0.1-3 mM SCN-), myeloperoxidase catalyzed the oxidation of SCN- to OSCN-, which accumulated in the medium to concentrations of up to 40-70 microM. Sulfonamide compounds increased the yield of stable oxidants to 0.2-0.3 mM by reacting with OSCN- to yield derivatives analogous to chloramines. Stimulated eosinophils produced H2O2 and secreted eosinophil peroxidase, which catalyzed the oxidation of SCN- to OSCN- regardless of Cl- concentration. Stimulated macrophages produced H2O2 but had low peroxidase activity. OSCN- was produced when SCN- was 0.1 mM or higher and myeloperoxidase, eosinophil peroxidase, or lactoperoxidase was added. The results indicate that SCN- rather than Cl- may be the physiologic substrate (electron donor) for eosinophil peroxidase and that OSCN- may contribute to leukocyte antimicrobial activity under conditions that favor oxidation of SCN- rather than Cl-.     

Novel products generated from 2'-deoxyguanosine by hypochlorous acid or a myeloperoxidase-H2O2-Cl- system: identification of diimino-imidazole and amino-imidazolone nucleosides

Hypochlorous acid (HOCl), generated by myeloperoxidase from H2O2 and Cl-, plays an important role in host defense and inflammatory tissue injury. We report here the identification of products generated from 2'-deoxyguanosine (dGuo) with HOCl. When 1 mM dGuo and 1 mM HOCl were reacted at pH 7.4 and 37 degrees C for 15 min and the reaction was terminated with N-acetylcysteine (N-AcCys), two products were generated in addition to 8-chloro-2'-deoxyguanosine (8-Cl-dGuo). One was identified as an amino-imidazolone nucleoside (dIz), a previously reported product of dGuo with other oxidation systems. The other was identified as a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dDiz) by spectrometric measurements. The yields were 1.4% dDiz, 0.6% dIz and 2.4% 8-Cl-dGuo, with 61.5% unreacted dGuo. Precursors of dDiz and dIz containing a chlorine atom were found in the reaction solution in the absence of termination by N-AcCys. dDiz, dIz and 8-Cl-dGuo were also formed from the reaction of dGuo with myeloperoxidase in the presence of H2O2 and Cl- under mildly acidic conditions. These results imply that dDiz and dIz are generated from dGuo via chlorination by electrophilic attack of HOCl and subsequent dechlorination by N-AcCys. These products may play a role in cytotoxic and/or genotoxic effects of HOCl.     

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.     

Molecular chlorine generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes produces 5-chlorocytosine in bacterial RNA

Myeloperoxidase, a heme enzyme secreted by activated phagocytes, uses H(2)O(2) and Cl(-) to generate the chlorinating intermediate hypochlorous acid (HOCl). This potent cytotoxic oxidant plays a critical role in host defenses against invading pathogens. In this study, we explore the possibility that myeloperoxidase-derived HOCl might oxidize nucleic acids. When we exposed 2'-deoxycytidine to the myeloperoxidase-H(2)O(2)-Cl(-) system, we obtained a single major product that was identified as 5-chloro-2'-deoxycytidine using mass spectrometry, high performance liquid chromatography, UV-visible spectroscopy, and NMR spectroscopy. 5-Chloro-2'-deoxycytidine production by myeloperoxidase required H(2)O(2) and Cl(-), suggesting that HOCl is an intermediate in the reaction. However, reagent HOCl failed to generate 5-chloro-2'-deoxycytidine in the absence of Cl(-). Moreover, chlorination of 2'-deoxycytidine was optimal under acidic conditions in the presence of Cl(-). These results implicate molecular chlorine (Cl(2)), which is in equilibrium with HOCl through a reaction requiring Cl(-) and H(+), in the generation of 5-chloro-2'-deoxycytidine. Activated human neutrophils were able to generate 5-chloro-2'-deoxycytidine. Cellular chlorination was blocked by catalase and heme poisons, consistent with a myeloperoxidase-catalyzed reaction. The myeloperoxidase-H(2)O(2)-Cl(-) system generated similar levels of 5-chlorocytosine in RNA and DNA in vitro. In striking contrast, only cell-associated RNA acquired detectable levels of 5-chlorocytosine when intact Escherichia coli was exposed to the myeloperoxidase system. This observation suggests that oxidizing intermediates generated by myeloperoxidase selectively target intracellular RNA for chlorination. Collectively, these results indicate that Cl(2) derived from HOCl generates 5-chloro-2'-deoxycytidine during the myeloperoxidase-catalyzed oxidation of 2'-deoxycytidine. Phagocytic generation of Cl(2) therefore may constitute one mechanism for oxidizing nucleic acids at sites of inflammation.     

Oxidation of NADH by chloramines and chloramides and its activation by iodide and by tertiary amines

Irreversible oxidation of reduced nicotinamide nucleotides by neutrophil-derived halogen oxidants (HOCl, chloramines, HOBr, etc.) is likely to be a highly lethal process, because of the essential role of NAD(P)H in important cell functions such as mitochondrial electron transport, and control of the cellular thiol redox state by NADPH-dependent glutathione reductase. Chloramines (chloramine-T, NH(2)Cl, etc.) and N-chloramides (N-chlorinated cyclopeptides) react with NADH to generate the same products as HOCl, i.e., pyridine chlorohydrins, as judged from characteristic changes in the NADH absorption spectrum. Compared with the fast oxidation of NADH by HOCl, k approximately 3 x 10(5) M(-1) s(-1) at pH 7.2, the oxidation by chloramines is about five orders of magnitude slower; that by chloramides is about four orders of magnitude slower. Apparent rate constants for oxidation of NADH by chloramines increase with increasing proton or buffer concentration, consistent with general acid catalysis, but oxidation by chloramides proceeds with pH-independent kinetics. In presence of iodide the oxidation of NADH by chloramines or chloramides is faster by at least two orders of magnitude; this is due to reaction of iodide with the N-halogen to give HOI/I(2), the most reactive and selective oxidant for NADH among HOX species. Quinuclidine derivatives (QN) like 3-chloroquinuclidine and quinine are capable of catalyzing the irreversible degradation of NADH by HOCl and by chloramines; QN(+)Cl, the chain carrier of the catalytic cycle, is even more reactive toward NADH than HOCl/ClO(-) at physiological pH. Oxidation of NADH by NH(2)Br proceeds by fast, but complex, biphasic kinetics. A compilation of rate constants for interactions of reactive halogen species with various substrates is presented and the concept of selective reactivity of N-halogens is discussed.     

Interaction of myeloperoxidase with vascular NAD(P)H oxidase-derived reactive oxygen species in vasculature: implications for vascular diseases

Vascular NAD(P)H oxidase-derived reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have emerged as important molecules in the pathogenesis of atherosclerosis, hypertension, and diabetic vascular complications. Additionally, myeloperoxidase (MPO), a transcytosable heme protein that is derived from leukocytes, is also believed to play important roles in the above-mentioned inflammatory vascular diseases. Previous studies have shown that MPO-induced vascular injury responses are H2O2 dependent. It is well known that MPO can use leukocyte-derived H2O2; however, it is unknown whether the vascular-bound MPO can use vascular nonleukocyte oxidase-derived H2O2 to induce vascular injury. In the present study, ANG II was used to stimulate vascular NAD(P)H oxidases and increase their H2O2 production in the vascular wall, and vascular dysfunction was used as the vascular injury parameter. We demonstrated that vascular-bound MPO has sustained activity in the vasculature. MPO could use the vascular NAD(P)H oxidase-derived H2O2 to produce hypochlorus acid (HOCl) and its chlorinating species. More importantly, MPO derived HOCl and chlorinating species amplified the H2O2-induced vascular injury by additional impairment of endothelium-dependent relaxation. HOCl-modified low-density lipoprotein protein (LDL), a specific biomarker for the MPO-HOCl-chlorinating species pathway, was expressed in LDL and MPO-bound vessels with vascular NAD(P)H oxidase-derived H2O2. MPO-vascular NAD(P)H oxidase-HOCl-chlorinating species may represent a common pathogenic pathway in vascular diseases and a new mechanism involved in exacerbation of vascular diseases under inflammatory conditions.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Peroxidase-catalyzed halide ion oxidation

The first complete mechanistic analysis of halide ion oxidation by a peroxidase was that of iodide oxidation by horseradish peroxidase. It was shown conclusively that a two-electron oxidation of iodide by compound I was occurring. This implied that oxygen atom transfer was occurring from compound I to iodide, forming hypoiodous acid, HOI. Searches were conducted for other two-electron oxidations. It was found that sulfite was oxidized by a two-electron mechanism. Nitrite and sulfoxides were not. If a competing substrate reduces some compound I to compound II by the usual one-electron route, then compound II will compete for available halide. Thus compound II oxidizes iodide to an iodine atom, I*, although at a slower rate than oxidation of I by compound I. An early hint that mammalian peroxidases were designed for halide ion oxidation was obtained in the reaction of lactoperoxidase compound II with iodide. The reaction was accelerated by excess iodide, indicating a co-operative effect. Among the heme peroxidases, only chloroperoxidase (for example from Caldariomyces fumago) and mammalian myeloperoxidase are able to oxidize chloride ion. There is not yet a consensus as to whether the chlorinating agent produced in a peroxidase-catalyzed reaction is hypochlorous acid (HOCl), enzyme-bound hypochlorous acid (either Fe-HOCl or X-HOCl where X is an amino acid residue), or molecular chlorine Cl2. A study of the nonenzymatic iodination of tyrosine showed that the iodinating reagent was either HOI or I2. It was impossible to tell which species because of the equilibria: [reaction: see text] The same considerations apply to product analysis of an enzyme-catalyzed reaction. Detection of molecular chlorine Cl2 does not prove it is the chlorinating species. If Cl2 is in equilibrium with HOCl then one cannot tell which (if either) is the chlorinating reagent. Examples will be shown of evidence that peroxidase-bound hypochlorous acid is the chlorinating agent. Also a recent clarification of the mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride along with accurate determination of the elementary rate constants will be discussed.     

1,3-Butadiene oxidation by human myeloperoxidase. Role of chloride ion in catalysis of divergent pathways.

1,3-Butadiene was oxidized by human myeloperoxidase in the absence of KCl to yield butadiene monoxide (BM) and crotonaldehyde (CA), but at KCl concentrations higher than 50 mM, 1-chloro-2-hydroxy-3-butene (CHB) was the major metabolite detected; metabolite formation was dependent on incubation time, pH, KCl, 1,3-butadiene, and H2O2 concentrations. The data are best explained by 1,3-butadiene being oxidized by myeloperoxidase by two different mechanisms. First, oxygen transfer from the hemoprotein would occur to either C-1 or C-4 of 1,3-butadiene to form an intermediate which may cyclize to form BM or undergo a hydrogen shift to form 3-butenal, an unstable precursor of CA. Further evidence for this mechanism was provided by the inability to detect methyl vinyl ketone, a possible product of an oxygen transfer reaction to C-2 or C-3 of 1,3-butadiene, and by the finding that CA was not simply a decomposition product of BM under assay conditions. In the second mechanism, however, chloride ion is oxidized by myeloperoxidase to HOCl which reacts with 1,3-butadiene to yield CHB. Further evidence for this mechanism was provided by the finding that CHB was readily formed when 1,3-butadiene was added to the filtrate of a myeloperoxidase/H2O2/KCl incubation and when 1,3-butadiene was allowed to react with authentic HOCl. In addition, CHB was not detected when BM or CA was incubated with myeloperoxidase, H2O2, and KCl for up to 60 min, or when 1,3-butadiene and KCl were incubated with chloroperoxidase and H2O2 or with mouse liver microsomes and NADPH, enzyme systems which catalyze 1,3-butadiene oxidation to BM and CA, but unlike myeloperoxidase, do not catalyze chloride ion oxidation to HOCl. These results provide clear evidence for novel olefinic oxidation reactions by myeloperoxidase.     

The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.     

Production of glutathione sulfonamide and dehydroglutathione from GSH by myeloperoxidase-derived oxidants and detection using a novel LC-MS/MS method

GSH is rapidly oxidized by HOCl (hypochlorous acid), which is produced physiologically by the neutrophil enzyme myeloperoxidase. It is converted into, mainly, oxidized glutathione. Glutathione sulfonamide is an additional product that is proposed to be covalently bonded between the cysteinyl thiol and amino group of the gamma-glutamyl residue of GSH. We have developed a sensitive liquid chromatography-tandem MS assay for the detection and quantification of glutathione sulfonamide as well as GSH and GSSG. The assay was used to determine whether glutathione sulfonamide is a major product of the reaction between GSH and HOCl, and whether it is formed by other two-electron oxidants. At sub-stoichiometric ratios of HOCl to GSH, glutathione sulfonamide accounted for up to 32% of the GSH that was oxidized. It was also formed when HOCl was generated by myeloperoxidase and its yield increased with the flux of oxidant. Of the other oxidants tested, only hypobromous acid and peroxynitrite produced substantial amounts of glutathione sulfonamide, but much less than with HOCl. Chloramines were able to generate detectable levels only when at a stoichiometric excess over GSH. We conclude that glutathione sulfonamide is sufficiently selective for HOCl to be useful as a biomarker for myeloperoxidase activity in biological systems. We have also identified a novel oxidation product of GSH with a molecular weight two mass units less than GSH, which we have consequently named dehydroglutathione. Dehydroglutathione represented a few percent of the total products and was formed with all of the oxidants except H2O2.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

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.     

The effect of D-penicillamine on myeloperoxidase: formation of compound III and inhibition of the chlorinating activity

The inhibitory effect of the anti-arthritic drug D-penicillamine on the formation of hypochlorite (HOCl) by myeloperoxidase from H2O2 and Cl- was investigated. When D-penicillamine was added to myeloperoxidase under turnover conditions, Compound III was formed, the superoxide derivative of the enzyme. Compound III was not formed when D-penicillamine was added in the presence of EDTA or in the absence of oxygen. However, when H2O2 was added to myeloperoxidase, D-penicillamine and EDTA, Compound III was formed. Therefore it is concluded that formation of Compound III is initiated by metal-catalysed oxidation of the thiol group of this anti-arthritic drug, resulting in formation of superoxide anions. Once Compound III is formed, a chain reaction is started via which the thiol groups of other D-penicillamine molecules are oxidized to disulphides. Concomitantly, Compound I of myeloperoxidase would be reduced to Compound II and superoxide anions would be generated from oxygen. This conclusion is supported by experiments which showed that formation of Compound III of myeloperoxidase by D-penicillamine depended on the chloride concentration. Thus, an enzyme intermediate which is active in chlorination (i.e. Compound I) participated in the generation of superoxide anions from the anti-arthritic drug. From the results described in this paper it is proposed that D-penicillamine may exert its therapeutic effect in the treatment of rheumatoid arthritis by scavenging HOCl and by converting myeloperoxidase to Compound III, which is inactive in the formation of HOCl.     

Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants

An important aspect of myocardial injury is the role of neutrophils in post-ischemic damage to the heart. Stimulated neutrophils initiate a series of reactions that produce toxic oxidizing agents. Superoxide rapidly dismutases to H2O2 and neutrophils contain myeloperoxidase which catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The highly reactive HOCl combines non-enzymatically with nitrogenous compounds to generate long-lived, non-radical oxidants, monochloramine and taurine N-monochloramine. We investigated the role of oxygen radicals and long-lived oxidants on cardiac sarcoplasmic reticulum function, which plays a major role in the regulation of intracellular Ca2+ and thereby in the generation of force. Incubation of sarcoplasmic reticulum with phorbol myristate acetate (PMA)-stimulated neutrophils (4 x 10(6) cells/ml) significantly decreased calcium uptake rate (0.85 +/- 0.11 to 0.11 +/- 0.06 mumol/min per mg) and Ca2+-ATPase activity (1.67 +/- 0.08 to 0.46 +/- 0.10 mumol/min per mg). Inclusion of myeloperoxidase inhibitors (cyanide, sodium azide and 3-amino-1,2,4-triazole), catalase, superoxide dismutase plus catalase, and alpha-tocopherol significantly protected (P less than 0.01) calcium uptake rates and Ca2+-ATPase activity of sarcoplasmic reticulum. Superoxide dismutase (10 microgram/ml) alone or deferoxamine (1 mM) had no protective effect in this system. The maximum inhibition of sarcoplasmic reticulum function was observed with (3-4) x 10(6) cells/ml in 4-6 min. HOCl and NH2Cl inhibited calcium uptake rate and Ca2+-ATPase activity of sarcoplasmic reticulum in a dose-dependent manner (2-20 microM), whereas H2O2 damaged sarcoplasmic reticulum at concentrations ranging from 5 to 25 mM. HOCl (20 microM) inhibited 80-90% of Ca2+-uptake rate and Ca2+-ATPase activity and L-methionine (0.1-1 mM) provided complete protection. We conclude that stimulated neutrophils damage cardiac sarcoplasmic function by generation of myeloperoxidase-catalyzed oxidants.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

p-Hydroxyphenylacetaldehyde, the major product of tyrosine oxidation by the activated myeloperoxidase system can act as an antioxidant in LDL

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. HOCl generated by the myeloperoxidase/H2O2/Cl- system of activated neutrophils may be operative in vivo making LDL atherogenic. Tyrosine has been found to be oxidized by HOCl to p-hydroxyphenylacetaldehyde (p-HA) capable of modifying phospholipid amino groups in LDL. As an amphiphatic phenolic compound, p-HA may have the potential to act as an antioxidant in the lipid phase of LDL. The present results show that (a) tyrosine exerts a protective effect on LDL modification by HOCl, (b) p-HA could act as antioxidant associated with the lipoprotein preventing cell- and transition metal ion-mediated LDL oxidation and (c) p-HA was able to scavenge free radicals.     

The oxidation of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O2 to O2- and that vanadate plus O2- then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.     

Cytochemical localization of hydrogen peroxide production in the rat uterus

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent H2O2-generating activity of the rat uterus was investigated both electron cytochemically and biochemically. We tried to cytochemically demonstrate H2O2 generation from the oxidation of reduced NADH or NADPH using the cerium method. NADPH oxidation resulted in electron-dense deposits on the apical plasma membrane covering the microvilli of the surface epithelium of the lightly fixed endometrium. In control specimens incubated in a medium from which substrate was omitted, no such deposits were observed. The reduction of ferricytochrome c due to NADH oxidation was spectrophotometrically detected in the lightly fixed uterus. Absorption at 550 nm increased with the addition of NADH, but not with that of NAD. The reaction was weakened by preheating and adversely affected by the addition of superoxide dismutase, but it was not inhibited by adding 50 mM sodium azide. These results suggest that a kind of NAD(P)H oxidase, generating H2O2 via superoxide formation, may possibly be present on the apical plasma membrane of the rat endometrial epithelium.