The nitric oxide congener nitrite inhibits myeloperoxidase/H2O2/ Cl- -mediated modification of low density lipoprotein

Nitric oxide, a pivotal molecule in vascular homeostasis, is converted under aerobic conditions to nitrite. Recent studies have shown that myeloperoxidase (MPO), an abundant heme protein released by activated leukocytes, can oxidize nitrite (NO(2-)) to a radical species, most likely nitrogen dioxide. Furthermore, hypochlorous acid (HOCl), the major strong oxidant generated by MPO in the presence of physiological concentrations of chloride ions, can also react with nitrite, forming the reactive intermediate nitryl chloride. Since MPO and MPO-derived HOCl, as well as reactive nitrogen species, have been implicated in the pathogenesis of atherosclerosis through oxidative modification of low density lipoprotein (LDL), we investigated the effects of physiological concentrations of nitrite (12.5-200 microm) on MPO-mediated modification of LDL in the absence and presence of physiological chloride concentrations. Interestingly, nitrite concentrations as low as 12.5 and 25 microm significantly decreased MPO/H2O2)/Cl- -induced modification of apoB lysine residues, formation of N-chloramines, and increases in the relative electrophoretic mobility of LDL. In contrast, none of these markers of LDL atherogenic modification were affected by the MPO/H2O2/NO2-) system. Furthermore, experiments using ascorbate (12.5-200 microm) and the tyrosine analogue 4-hydroxyphenylacetic acid (12.5-200 microm), which are both substrates of MPO, indicated that nitrite inhibits MPO-mediated LDL modifications by trapping the enzyme in its inactive compound II form. These data offer a novel mechanism for a potential antiatherogenic effect of the nitric oxide congener nitrite.     

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.     

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.     

Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals

Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.     

Ibuprofen protects low density lipoproteins against oxidative modification

Oxidative modification of LDL by vascular cells has been proposed as the mechanism by which LDL become atherogenic. The effect of ibuprofen on LDL modification by copper ions, monocytes and endothelial cells was studied by measuring lipid peroxidation products. Ibuprofen inhibited LDL oxidation in a dose-dependent manner over a concentration range of 0.1 to 2.0 mM. Ibuprofen (2 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during 2 and 6 h incubation in the presence of copper ions by 52 and 28%, respectively. Weak free radical scavenging activity of ibuprofen was observed in the DPPH test. The protective effect of ibuprofen was more marked when oxidation was induced by monocytes or endothelial cells. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides generated in LDL during monocyte-mediated oxidation by 40%. HUVEC-mediated oxidation of LDL in the absence and presence of Cu2+ was reduced by 32 and 39%, respectively. More lipid peroxides appeared when endothelial cells were stimulated by IL-1beta or TNFalpha and the inhibitory effect of ibuprofen in this case was more pronounced. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during incubation of LDL with IL-1beta-stimulated HUVEC by 43%. The figures in the absence and presence of Cu2+ for HUVEC stimulated with TNFalpha were 56 and 59%, respectively. To assess the possibility that ibuprofen acts by lowering the production rate of reactive oxygen species, the intracellular concentration of H2O2 was measured. Ibuprofen (1 mM) reduced intracellular production of hydrogen peroxide in PMA-stimulated mononuclear cells by 69%. When HUVEC were stimulated by IL-1beta or TNFalpha the reduction was 62% and 66%, respectively.     

Products of the reaction of HOCl with tryptophan protect LDL from atherogenic modification

Hypochlorite (HOCl) attacks amino acid residues in LDL making the particle atherogenic. Tryptophan is prone to free radical reactions and modification by HOCl. We hypothesized, that free tryptophan may quench the HOCl attack therefore protecting LDL. Free tryptophan inhibits LDL apoprotein modification and lipid oxidation. Tryptophan-HOCl metabolites associate with LDL reducing its oxidizability initiated by endothelial cells, Cu(2+) and peroxyl radicals. One tryptophan-HOCl metabolite was identified as 4-methyl-carbostyril which showed antioxidative activity when present during Cu(2+) mediated lipid oxidation, but did not associate with LDL. Indole-3-acetaldehyde, a decomposition product of tryptophan chloramine (the product of the tryptophan-HOCl reaction) was found to associate with LDL increasing its resistance to oxidation. Myeloperoxidase treatment of LDL in the presence of chloride, H(2)O(2) and tryptophan protected the lipoprotein from subsequent cell-mediated oxidation. We conclude that, in vivo, the activated myeloperoxidase system can generate antioxidative metabolites from tryptophan by the reaction of hypochlorite with this essential amino acid.     

Salicylate promotes myeloperoxidase-initiated LDL oxidation: antagonization by its metabolite gentisic acid.

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. Tyrosyl radicals generated by myeloperoxidase (MPO) can act as prooxidants of LDL oxidation. Taking into consideration, that monophenolic compounds are able to form phenoxyl radicals in presence of peroxidases, we have tested salicylate, in its ability to act as a prooxidant in the MPO system. Measurement of conjugated dienes and lipid hydroperoxides were taken as indicators of lipid oxidation. Exposure of LDL preparations to MPO in presence of salicylate revealed that the drug could act as a catalyst of lipid oxidation in LDL. The radical scavenger ascorbic acid as well as heme poisons (cyanide, azide) and catalase were inhibitory. The main metabolite of salicylic acid, gentisic acid, showed inhibitory action in the MPO system. Even when lipid oxidation was maximally stimulated by salicylate the LDL oxidation was efficaciously counteracted in presence of gentisic acid at salicylate/gentisic acid ratios that could be reached in plasma of patients receiving aspirin medication. Gentisic acid was also able to impair the tyrosyl radical catalyzed LDL peroxidation. The results suggest that salicylate could act like tyrosine via a phenoxyl radical as a catalyst of LDL oxidative modification by MPO. But the prooxidant activity of this radical species is effectively counteracted by the salicylate metabolite gentisic acid.     

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-.     

An improved method for the sensitive monitoring of low density lipoprotein modification by myeloperoxidase

The aim of this investigation was to compare an improved fluorometric method with an UV absorbance assay for their ability to monitor low density lipoprotein (LDL) modification by myeloperoxidase (MPO) and to evaluate determining factors influencing the modification of LDL. Using absorbance at 234 nm to study the kinetics of LDL aggregation, and a native fluorescence assay for protein oxidation, we found that all components of the MPO/H2O2/Cl- system may have rate determining effects on LDL modification. While the lipoprotein modification rate correlated positively with enzyme concentration, variation of the concentration of H2O2 had a biphasic effect on the maximal rate of LDL modification with both methods. Furthermore, a positive association was found between the maximal rate of LDL modification and the acidity of the medium, with a pathophysiologically relevant optimal rate at a slightly acidic pH of 5-6, but hardly any modification above pH 6.8. In summary, both methods provide simple and useful tools for the continuous monitoring of LDL modification by the MPO/H2O2/Cl- system, but the more sensitive fluorometric method is preferable, since it allows the application of experimental conditions which are much closer to the situation in vivo.     

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.     

High plasma thiocyanate levels in smokers are a key determinant of thiol oxidation induced by myeloperoxidase

Smokers have an elevated risk of atherosclerosis but the origins of this elevated risk are incompletely defined, though evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall. We hypothesized that smokers would have a high level of thiocyanate (SCN(-)), a preferred substrate for MPO, which in turn would predispose to thiol oxidation, an established independent risk factor for atherosclerosis. In this study it is shown that on exposure to MPO/H(2)O(2), thiols on plasma proteins from nonsmokers were increasingly oxidized with increasing added SCN(-) concentrations. Plasma from smokers contained significantly higher endogenous levels of SCN(-) than that from nonsmokers (131±31 vs 40±24 μM, P<0.0001). When plasma from smokers and nonsmokers was exposed to MPO/H(2)O(2)-stimulated oxidation, a strong positive correlation (r=0.8139, P<0.0001) between the extent of thiol oxidation and the plasma SCN(-) concentrations was observed. Computational calculations indicate a changeover from HOCl to HOSCN as the major MPO-generated oxidant in plasma, with increasing SCN(-) levels. These data indicate that plasma SCN(-) levels are a key determinant of the extent of thiol oxidation on plasma proteins induced by MPO, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage to proteins in smokers.     

Hydrogen sulphide: a novel physiological inhibitor of LDL atherogenic modification by HOCl

Hypochlorite (HOCl), the product of the activated myeloperoxidase/H₂O₂/chloride (MPO/H₂O₂/Cl^- ) system is favored as a trigger of LDL modifications, which may play a pivotal role in early atherogenesis. As HOCl has been shown to react with thiol-containing compounds like glutathione and N-acetylcysteine protecting LDL from HOCl modification, we have tested the ability of hydrogen sulfide (H₂S)—which has recently been identified as an endogenous vasorelaxant—to counteract the action of HOCl on LDL. The results show that H₂S could inhibit the atherogenic modification of LDL induced by HOCl, as measured by apolipoprotein alterations. Beside its HOCl scavenging potential, H₂S was found to inhibit MPO (one may speculate that this occurs via H₂S/heme interaction) and destroy H₂O₂. Thus, H₂S may interfere with the reactants and reaction products of the activated MPO/H₂O₂/Cl^-  system. Our data add to the evidence of an anti-atherosclerotic action of this gasotransmitter taking the role of HOCl in the atherogenic modification of LDL into account.     

Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite

Oxidative modification of low-density lipoprotein (LDL) is a pivotal process in early atherogenesis and can be brought about by myeloperoxidase (MPO), which is capable of reacting with nitrite, a NO metabolite. We studied MPO-mediated formation of conjugated dienes in isolated human LDL in dependence on the concentrations of nitrite and chloride. This reaction was strongly stimulated by low concentrations (5-50 microM) of nitrite which corresponds to the reported concentration in the arterial vessel wall. Under these conditions no protein tyrosine nitration occurred; this reaction required much higher nitrite concentrations (100 microM-1 mM). Chloride neither supported lipid peroxidation alone nor was its presence mandatory for the effect of nitrite. We propose a prominent role of lipid peroxidation for the proatherogenic action of the MPO/nitrite system, whereas peroxynitrite may be competent for protein tyrosine nitration of LDL. Monomeric and oligomeric flavan-3-ols present in cocoa products effectively counteracted, at micromolar concentrations, the MPO/nitrite-mediated lipid peroxidation of LDL. Flavan-3-ols also suppressed protein tyrosine nitration induced by MPO/nitrite or peroxynitrite as well as Cu2+-mediated lipid peroxidation of LDL. This multi-site protection by (-)-epicatechin or other flavan-3-ols against proatherogenic modification of LDL may contribute to the purported beneficial effects of dietary flavan-3-ols for the cardiovascular system.     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin, transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.     

Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids

Patients with increased haemolytic haemoglobin (Hb) have 10-20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H(2)O(2) generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN(3), a peroxidase inhibitor, catalase, a H(2)O(2) decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H(2)O(2) production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN(3) and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.     

Lactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide: 15N nuclear magnetic resonance and optical spectral studies

To establish the agent(s) responsible for the activity of the lactoperoxidase (LPO)/SCN-/H2O2 system, the oxidation of thiocyanate with hydrogen peroxide, catalyzed by lactoperoxidase, has been studied by 15N NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pHs. The formation of hypothiocyanite ion (OSCN-) as one of the oxidation products correlated well with the activity of the LPO/SCN-/H2O2 system and was maximum when the concentrations of the H2O2 and SCN- were nearly the same and the pH was less than 6.0. At [H2O2]/[SCN-] = 1, OSCN- decomposed very slowly back to thiocyanate. When the ratio [H2O2]/[SCN-] was above 2, formation of CN- was observed, which was confirmed by 15N NMR and also by changes in the optical spectrum of LPO. The oxidation of thiocyanate by H2O2 in the presence of LPO does not take place at pH greater than 8.0. Since thiocyanate does not bind to LPO above this pH, the binding of thiocyanate to LPO is considered to be prerequisite for the oxidation of thiocyanate. Maximum inhibition of oxygen uptake by Streptococcus cremoris 972 bacteria was observed when hydrogen peroxide and thiocyanate were present in equimolar amounts and the pH was below 6.0.     

Thiocyanate modulates the catalytic activity of mammalian peroxidases

We investigated the potential role of the co-substrate, thiocyanate (SCN-), in modulating the catalytic activity of myeloperoxidase (MPO) and other members of the mammalian peroxidase superfamily (lactoperoxidase (LPO) and eosinophil peroxidase (EPO)). Pre-incubation of SCN- with MPO generates a more complex biological setting, because SCN- serves as either a substrate or inhibitor, causing diverse impacts on the MPO heme iron microenvironment. Consistent with this hypothesis, the relationship between the association rate constant of nitric oxide binding to MPO-Fe(III) as a function of SCN- concentration is bell-shaped, with a trough comparable with normal SCN- plasma levels. Rapid kinetic measurements indicate that MPO, EPO, and LPO Compound I formation occur at rates slower than complex decay, and its formation serves to simultaneously catalyze SCN- via 1e- and 2e- oxidation pathways. For the three enzymes, Compound II formation is a fundamental feature of catalysis and allows the enzymes to operate at a fraction of their possible maximum activities. MPO and EPO Compound II is relatively stable and decays gradually within minutes to ground state upon H2O2 exhaustion. In contrast, LPO Compound II is unstable and decays within seconds to ground state, suggesting that SCN- may serve as a substrate for Compound II. Compound II formation can be partially or completely prevented by increasing SCN- concentration, depending on the experimental conditions. Collectively, these results illustrate for the first time the potential mechanistic differences of these three enzymes. A modified kinetic model, which incorporates our current findings with the mammalian peroxidases classic cycle, is presented.     

Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation

Initiation of lipid peroxidation and the formation of bioactive eicosanoids are pivotal processes in inflammation and atherosclerosis. Currently, lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases are considered the primary enzymatic participants in these events. Myeloperoxidase (MPO), a heme protein secreted by activated leukocytes, generates reactive intermediates that promote lipid peroxidation in vitro. For example, MPO catalyzes oxidation of tyrosine and nitrite to form tyrosyl radical and nitrogen dioxide ((.)NO(2)), respectively, reactive intermediates capable of initiating oxidation of lipids in plasma. Neither the ability of MPO to initiate lipid peroxidation in vivo nor its role in generating bioactive eicosanoids during inflammation has been reported. Using a model of inflammation (peritonitis) with MPO knockout mice (MPO(-/-)), we examined the role for MPO in the formation of bioactive lipid oxidation products and promoting oxidant stress in vivo. Electrospray ionization tandem mass spectrometry was used to simultaneously quantify individual molecular species of hydroxy- and hydroperoxy-eicosatetraenoic acids (H(P)ETEs), F(2)-isoprostanes, hydroxy- and hydroperoxy-octadecadienoic acids (H(P)ODEs), and their precursors, arachidonic acid and linoleic acid. Peritonitis-triggered formation of F(2)-isoprostanes, a marker of oxidant stress in vivo, was reduced by 85% in the MPO(-/-) mice. Similarly, formation of all molecular species of H(P)ETEs and H(P)ODEs monitored were significantly reduced (by at least 50%) in the MPO(-/-) group during inflammation. Parallel analyses of peritoneal lavage proteins for protein dityrosine and nitrotyrosine, molecular markers for oxidative modification by tyrosyl radical and (.)NO(2), respectively, revealed marked reductions in the content of nitrotyrosine, but not dityrosine, in MPO(-/-) samples. Thus, MPO serves as a major enzymatic catalyst of lipid peroxidation at sites of inflammation. Moreover, MPO-dependent formation of (.)NO-derived oxidants, and not tyrosyl radical, appears to serve as a preferred pathway for initiating lipid peroxidation and promoting oxidant stress in vivo.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin,transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.