Effect of N-chloroamino acids on the erythrocyte

Amino acids present in blood plasma may be targets for oxidation and chlorination by HOCl/OCl^?. N-Chloroamino acids have been reported to be less reactive, but more selective than HOCl/OCl^? in their reactions; therefore, they may act as secondary mediators of HOCl/OCl^?-induced injury. This study compared the effects of five N-chloroamino acids (AlaCl, LysCl, SerCl, AspCl and PheCl) on erythrocytes with the action of HOCl/OCl^?. The N-chloroamino acids differed in stability and reactivity. They had a weaker haemolytic action than HOCl/OCl^?; HOCl/OCl^?, AlaCl and PheCl increased osmotic fragility of erythrocytes at a concentration of 1 mm. Oxidation of glutathione, formation of protein-glutathione mixed disulphides and efflux of GSSG from erythrocytes were observed for erythrocytes treated with all the employed chloroderivatives, while increased oxidation of 2′,7′-dichlorofluorescin was detected only after treatment of the cells with 1 mm HOCl/OCl^?, AlaCl and PheCl. Generally, the reactivity of at least some N-chloroamino acids may be not much lower with respect to HOCl/OCl^?.     

Collagen type II modification by hypochlorite

Oxidation of proteins is a common phenomenon in the inflammatory process mediated by highly reactive agents such as hypochlorite (HOCl/OCl(-)) produced by activated neutrophils. For instance, in rheumatoid arthritis hypochlorite plays an important role in joint destruction. One of the major targets for HOCl/OCl(-) is collagen type II (CII) - the primary cartilage protein. In our study, HOCl/OCl(-) mediated collagen II modifications were tested using various methods: circular dichroism (CD), HPLC, ELISA, dynamic light scattering (DLS), fluorimetry and spectrophotometry. It was shown that hypochlorite action causes deamination with consecutive carbonyl group formation and transformation of tyrosine residues to dichlorotyrosine. Moreover, it was shown that ammonium chloramine (NH(2)Cl) formed in the reaction mixture reacts with CII. However, in this case the yield of carbonyl groups and dichlorotyrosine is lower than that observed for HOCl/OCl(-) by 50%. CD data revealed that collagen II exists as a random coil in the samples and that chlorination is followed by CII fragmentation. In the range of low HOCl/OCl(-) concentrations (up to 1 mM) 10-90 kDa peptides are predominant whereas massive production of shorter peptides was observed for high (5 mM) hypochlorite concentration. DLS measurements showed that chlorination with HOCl/OCl(-) decreases the radius of collagen II aggregates from 30 to 6.8 nm. Taking into account the fact that chlorinated collagen is partially degraded, the DLS results suggest that smaller micelles are formed of the 10-90 kDa peptide fraction. Moreover, collagen chlorination results in epitope modification which affects CII recognition by anti-CII antibodies. Finally, since in the synovial fluid the plausible hypochlorite concentration is smaller than that used in the model the change of size of molecular aggregates seems to be the best marker of hypochlorite-mediated collagen oxidation.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

Hypochlorite action on plasma fibronectin promotes its extended conformation in complexes with antibodies

We investigated the influence of hypochlorite (HOCl/OCl-) on plasma fibronectin (Fn) aggregation and examined an affinity of Fn aggregates to Fn specific antibodies. Human plasma Fn HOCl/OCl(-)-mediated modification was monitored with differential OD method and with measurements of tryptophan fluorescence followed by acrylamide quenching of tryptophan emission. Antibody fibronectin complex formation was examined in ELISA systems with chemiluminescence (CL) detection. Results were expressed as an average of three experiments performed in triplicate. Fn aggregation/fragmentation was monitored with dynamic light scattering method. It was showed that HOCl/OCl- mediated chlorination promotes Fn aggregation/fragmentation with concomitant oxidation of tryptophan moieties and dichlorotyrosine formation. Quenching experiments revealed that in chlorinated Fn the percentage of intact tryptophan moieties buried in the hydrophobic Fn core increases as compared to unchlorinated Fn. In general, ELISA experiments showed that chlorination of plasma Fn diminished the number of available epitopes but for lower HOCl/OCl- concentrations (1-2 mM) the reverse effect is observed--the number of accessible fibronectin epitopes is increased when Fn adopts extended conformation in complex with antibody. Our results suggest that HOCl/OCl(-)-mediated plasma Fn chlorination leads to the formation of soluble aggregates and is followed by refolding processes. Fn chlorination with low doses of HOCl/OCl- promotes extended Fn conformation which in turn increases affinity toward specific antibodies and may promote Fn-IgG cluster formation. Thus it seems possible that mildly chlorinated plasma Fn promotes formation of IgG clusters which in turn may activate neutrophils.     

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.     

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.     

Protein carbonyl formation on mucosal proteins in vitro and in dextran sulfate-induced colitis.

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease, but few studies have investigated protein oxidation in the inflamed mucosa. In this study, protein carbonyl formation on colonic mucosal proteins from mice was investigated following in vitro exposure of homogenates to iron/ascorbate, hydrogen peroxide, hypochloric acid (HOCl), or nitric oxide (*NO). Total carbonyl content was measured spectrophotometrically by derivatization with dinitrophenylhydrazine (DNPH), and oxidation of component proteins within the tissue was examined by Western blotting for DNPH-derivatized proteins using anti-dinitrophenyl DNP antibodies. These results were compared with protein carbonyl formation found in the acutely inflamed mucosa from mice with colitis induced by dextran sulfate sodium (DSS) administered at 5% w/v in the drinking water for 7 d. In vitro, carbonyl formation was observed after exposure to iron/ascorbate, HOCl and *NO. Iron/ascorbate (20 microM/20 mM) exposure for 5 h increased carbonyl groups by 80%, particularly on proteins of 48, 75-100, 116, 131, and 142 kDa. Oxidation by 0.1 and 0.5 mM HOCl did not increase total carbonyl levels, but Western blotting revealed carbonyl formation on many proteins, particularly in the 49-95 kDa region. After exposure to 1-10 mM HOCl, total carbonyl levels were increased by 0.5 to 12 times control levels with extensive cross-linking and fragmentation of proteins rich in carbonyl groups observed by Western blotting. In mice with acute colitis induced by DSS, protein carbonyl content of the inflamed mucosa was not significantly different from control mucosa, (7.80 +/- 1.05 vs. 8.43 +/- 0.59 nmo/mg protein respectively, p = .16 n = 8, 10); however, Western blotting analysis indicated several proteins of molecular weight 48, 79, 95, and 131 kDa that exhibited increased carbonyl content in the inflamed mucosa. These proteins corresponded to those observed after in vitro oxidation of normal intestinal mucosa with iron/ ascorbate and HOCl, suggesting that both HOCl and metal ions may be involved in protein oxidation in DSS-induced colitis. Identification and further analysis of the mucosal proteins susceptible to carbonyl modification may lead to a better understanding of the contribution of oxidants to the colonic mucosa tissue injury in inflammatory bowel disease.     

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.     

Selective modification of apoB-100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro

Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl.The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.     

13C-NMR study of taurine and chlorotaurine in human cells

13C-NMR with 13C-enriched taurine [( 13C]taurine) has been utilized to study the formation and reactions of N-chlorotaurine in solution and in human cells. Taurine reacts instantaneously with HOCl at pH 7.0 to form N-chlorotaurine, which is stable in solution by itself. In the presence of alpha-amino acids, a chlorine transfer reaction taken place to produce N-chloroamino acids, which quickly convert to the corresponding aldehydes. [13C]Taurine was incubated with human neutrophils and with cultured human lymphoblastoid cells and 13C-NMR spectra of the whole cell mixtures were acquired in order to examine the formation of N-chlorotaurine from reaction between taurine and the endogenous HOCl produced by myeloperoxidase-catalyzed reactions (Zgliczynski, J.M., et al. (1968) Eur. J. Biochem. 4, 540; Weiss, S.J., et al. (1982) J. Clin. Invest. 70, 598). The presence of N-chlorotaurine in the cells was not detected on the 13C-NMR spectra. On the other hand, N-chloro[13C]taurine incubated with the cells was found to be converted to taurine, which must have been produced by a chlorine transfer reaction of the N-chlorotaurine to other cellular components such as amino acids, peptides or proteins. A 13C-NMR study of taurine uptake in human lymphoblastoid cells indicated that taurine is incorporated into a freely mobile intracellular pool. These results suggest that the presence of abundant taurine in a freely mobile intracellular pool may serve as a buffer in preventing oxidative damage to the cells from attacks by HOCl or other oxidants.     

Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin

After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.     

Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.     

Histamine chloramine reactivity with thiol compounds, ascorbate, and methionine and with intracellular glutathione

Histamine is stored in granules of mast cells and basophils and released by inflammatory mediators. It has the potential to intercept some of the HOCl generated by the neutrophil enzyme, myeloperoxidase, to produce histamine chloramine. We have measured rate constants for reactions of histamine chloramine with methionine, ascorbate, and GSH at pH 7.4, of 91 M(-1)s(-1), 195 M(-1)s(-1), and 721 M(-1)s(-1), respectively. With low molecular weight thiols, the reaction was with the thiolate and rates increased exponentially with decreasing thiol group pK(a). Comparing rate constants for different chloramines reacting with ascorbate or a particular thiol anion, these were higher when there was less negative charge in the vicinity of the chloramine group. Histamine chloramine was the most reactive among biologically relevant chloramines. Consumption of histamine chloramine and oxidation of intracellular GSH were examined for human fibroblasts. At nontoxic doses, GSH loss over 10 min was slightly greater than that with HOCl, but the cellular uptake of histamine chloramine was 5-10-fold less. With histamine chloramine, GSSG was a minor product and most of the GSH was converted to mixed disulfides with proteins. HOCl gave a different profile of GSH oxidation products, with significantly less GSSG and mixed disulfide formation. There was irreversible oxidation and losses to the medium, as observed with HOCl and other cell types. Thus, histamine chloramine shows high preference for thiols both in isolation and in cells, and in this respect is more selective than HOCl.     

The inhibition of bacterial growth by hypochlorous acid. Possible role in the bactericidal activity of phagocytes.

The 'respiratory burst' of phagocytes such as neutrophils generates superoxide which forms H2O2 by dismutation. H2O2 and Cl- ions serve as substrates for the enzyme myeloperoxidase to generate hypochlorous acid (HOCl). HOCl is thought to play an important role in bacterial killing, but its mechanism of action is not well characterized. Furthermore, although many studies in vitro have shown HOCl to be a damaging oxidant with little or no specificity (particularly at high concentrations), bacteria which have been ingested by phagocytes appear to experience a rapid and selective inhibition of cell division. Bacterial membrane disruption, protein degradation, and inhibition of protein synthesis, do not seem to occur in the early phases of phagocyte action. We have now found that low concentrations of HOCl exert a rapid and selective inhibition of bacterial growth and cell division, which can be blocked by taurine or amino acids. Only 20 microM-HOCl was required for 50% inhibition of bacterial growth (5 x 10(8) Escherichia coli/ml), and 50 microM-HOCl completely inhibited cell division (colony formation). These effects were apparent within 5 min of HOCl exposure, and were not reversed by extensive washings. DNA synthesis (incorporation of [3H]-thymidine) was significantly affected by even a 1 min exposure to 50 microM-HOCl, and decreased by as much as 96% after 5 min. In contrast, bacterial membrane disruption and extensive protein degradation/fragmentation (release of acid-soluble counts from [3H]leucine-labelled cells) were not observed at concentrations below 5 mM-HOCl. Protein synthesis (incorporation of [3H]leucine) was only inhibited by 10-30% following 5 min exposure to 50 microM-HOCl, although longer exposure produced more marked reductions (80% after 30 min). Neutrophils deficient in myeloperoxidase cannot convert H2O2 to HOCl, yet can kill bacteria. We have found that H2O2 is only 6% as effective as HOCl in inhibiting E. coli growth and cell division (0.34 mM-H2O2 required for 50% inhibition of colony formation), and taurine or amino acids do not block this effect. Our results are consistent with a rapid and selective inhibition of bacterial cell division by HOCl in phagocytes. H2O2 may substitute for HOCl in myeloperoxidase deficiency, but by a different mechanism and at a greater metabolic cost.     

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-.     

Fatty acid and phospholipid chlorohydrins cause cell stress and endothelial adhesion

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis, which is an inflammatory disease involving activation of phagocytic cells. Myeloperoxidase, an enzyme which is able to produce hypochlorous acid (HOCl), is released from these phagocytic cells, and has been found in an active form in atherosclerotic plaques. HOCl can oxidize both the lipid and protein moiety of LDL, and HOCl-modified LDL has been found to be pro-inflammatory, although it is not known which component is responsible for this effect. As HOCl can oxidize lipids to give chlorohydrins, we hypothesized that phospholipid chlorohydrins might have toxic and pro-inflammatory effects. We have formed chlorohydrins from fatty acids (oleic, linoleic and arachidonic acids) and from phospholipids (stearoyl-oleoyl phosphatidylcholine, stearoyl-linoleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine), and investigated various biological effects of these oxidation products. Fatty acid and phospholipid chlorohydrins were found to deplete ATP levels in U937 cells in a concentration-dependent manner, with significant effects observed at concentrations of 25 microM and above. Low concentrations (25 microM) of stearoyl-oleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine chlorohydrins were also found to increase caspase-3 activity. Finally, stearoyl-oleoyl phosphatidylcholine chlorohydrin increased leukocyte adhesion to artery segments isolated from C57Bl/6 mice. These results demonstrate potentially harmful effects of lipid chlorohydrins, and suggest that they may contribute to some of the pro-inflammatory effects that HOCl-modified low density lipoprotein has been found to induce.     

Hypochlorite-induced oxidation of amino acids, peptides and proteins

Summary. Activated phagocytes generate the potent oxidant hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is known to react with a number of biological targets including proteins, DNA, lipids and cholesterol. Proteins are likely to be major targets for reaction with HOCl within a cell due to their abundance and high reactivity with HOCl. This review summarizes information on the rate of reaction of HOCl with proteins, the nature of the intermediates formed, the mechanisms involved in protein oxidation and the products of these reactions. The predicted targets for reaction with HOCl from kinetic modeling studies and the consequences of HOCl-induced protein oxidation are also discussed.     

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.     

Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes

It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c(50%)=258+/-24 microM HOCl) and bimane-S-glutathione (B-SG, c(50%)=125+/-16 microM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH](oxidized)=4) and inhibits basal as well as 2,4-dinitrophenol- (DNP) and 2,4-dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg(2+)-ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c(50%)=478+/-36 microM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid.     

The vinyl ether linkages of plasmalogens are favored targets for myeloperoxidase-derived oxidants: a kinetic study

Plasmalogens, which contain a vinyl ether bond, are major phospholipids of the plasma membranes of endothelial and vascular smooth muscle cells and cardiac myocytes. These lipids, in contrast to other phospholipids, have been reported to be targets of HOCl/HOBr generated by myeloperoxidase, with elevated levels of the products of these reactions (alpha-chloro/alpha-bromo aldehydes and unsaturated lysophospholipids) having been detected in human atherosclerotic lesions. The reason(s) for the targeting of this lipid class, over other phospholipids, is poorly understood, and is examined here. It is shown that HOCl and HOBr react with a model vinyl ether (ethylene glycol vinyl ether) 200-300-fold faster ( k = 1.6 x 10 (3) and 3.5 x 10 (6) M (-1) s (-1), respectively) than with aliphatic alkenes (models of phospholipids). True plasmalogens react ca. 20-fold slower than the models. Chloramines and bromamines (from reaction of HOCl/HOBr with primary amines and alpha-amino groups) also react with vinyl ethers, unlike aliphatic alkenes, with k = 10 (-3)-10 (2) M (-1) s (-1) for chloramines (with the His side chain chloramine being the most reactive, k = 172 M (-1) s (-1)) and k = 10 (3)-10 (4) M (-1) s (-1) for bromamines. The bromamine rate constants are typically 10 (5)-10 (6) larger than those of the chloramines. Intermolecular vinyl ether oxidation by phospholipid headgroup bromamines can also occur. These kinetic data indicate that plasmalogens are significantly more susceptible to oxidation than the aliphatic alkenes of phospholipids, thereby rationalizing the detection of products from the former, but not the latter, in human atherosclerotic lesions.