Myeloperoxidase-induced lipid peroxidation of LDL in the presence of nitrite. Protection by cocoa flavanols

Lipid peroxidation (LPO) of low-density lipoprotein (LDL) is believed to be a pivotal process rendering this plasma lipoprotein atherogenic. Several endogenous factors have been proposed to mediate LPO of LDL, among them myeloperoxidase (MPO), which is active in atherosclerotic lesions, and the plasma level of which has been proposed to be a prognostic parameter for cardiac events. Nitrite, a major oxidation product of nitric oxide, is substrate of MPO and a cofactor of MPO-mediated LPO under physiological conditions. Dietary flavonoids including (-)-epicatechin, a major flavan-3-ol in cocoa products, grapes and wine, are substrates of MPO as well as potent inhibitors of LPO in LDL at micromolar concentrations. Moreover, they strongly suppress protein tyrosine nitration of LDL by MPO/nitrite or peroxynitrite. By blunting undesirable MPO-mediated actions of nitrite, presumably via scavenging of the strong prooxidant and nitrating *NO2 radical, dietary flavonoids modulate NO metabolism in a favorable direction and thus counteract endothelial dysfunction. This article gives a survey on recent progress in this field with special reference to own recently published work.     

Betanin inhibits the myeloperoxidase/nitrite-induced oxidation of human low-density lipoproteins

Production of nitrogen dioxide by the activity of myeloperoxidase (MPO) in the presence of nitrite is now considered a key step in the pathophysiology of low-density lipoprotein (LDL) oxidation. This study shows that betanin, a phytochemical of the betalain class, inhibits the production of lipid hydroperoxides in human LDL submitted to a MPO/nitrite-induced oxidation. Kinetic measurements including time-course of particle oxidation and betanin consumption, either in the presence or in the absence of nitrite, suggest that the antioxidant effect is possibly the result of various actions. Betanin scavenges the initiator radical nitrogen dioxide and can also act as a lipoperoxyl radical-scavenger. In addition, unidentified oxidation product(s) of betanin by MPO/nitrite inhibit(s) the MPO/nitrite-induced LDL oxidation as effectively as the parent compound. In the light of betanin bioavailability and post-absorbtion distribution in humans, present findings may suggest favourable in vivo activity of this phytochemical.     

Betanin inhibits the myeloperoxidase/nitrite-induced oxidation of human low-density lipoproteins

Production of nitrogen dioxide by the activity of myeloperoxidase (MPO) in the presence of nitrite is now considered a key step in the pathophysiology of low-density lipoprotein (LDL) oxidation. This study shows that betanin, a phytochemical of the betalain class, inhibits the production of lipid hydroperoxides in human LDL submitted to a MPO/nitrite-induced oxidation. Kinetic measurements including time-course of particle oxidation and betanin consumption, either in the presence or in the absence of nitrite, suggest that the antioxidant effect is possibly the result of various actions. Betanin scavenges the initiator radical nitrogen dioxide and can also act as a lipoperoxyl radical-scavenger. In addition, unidentified oxidation product(s) of betanin by MPO/nitrite inhibit(s) the MPO/nitrite-induced LDL oxidation as effectively as the parent compound. In the light of betanin bioavailability and post-absorbtion distribution in humans, present findings may suggest favourable in vivo activity of this phytochemical.     

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.     

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.     

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.     

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.     

Thiocyanate catalyzes myeloperoxidase-initiated lipid oxidation in LDL

There is evidence that LDL oxidation may render the lipoprotein atherogenic. The myeloperoxidase-hydrogen peroxide (MPO/H2O2) system of activated phagocytes may be involved in this process. Chloride is supposed to be the major substrate for MPO, generating reactive hypochlorous acid (HOCl), modifying LDL. The pseudo-halide thiocyanate (SCN-) has been shown to be a suitable substrate for MPO, forming reactive HOSCN/SCN*. As relatively abundant levels of SCN- are found in plasma of smokers--a well-known risk group for cardiovascular disease--the ability of SCN- to act as a catalyst of LDL atherogenic modification by MPO/H2O2 was tested. Measurement of conjugated diene and lipid hydroperoxide formation in LDL preparations exposed to MPO/H2O2 revealed that SCN- catalyzed lipid oxidation in LDL. Chloride did not diminish the effect of SCN- on lipid oxidation. Surprisingly, SCN inhibited the HOCl-mediated apoprotein modification in LDL. Nitrite--recently found to be a substrate for MPO--showed some competing properties. MPO-mediated lipid oxidation was inhibited by heme poisons (azide, cyanide) and catalase. Ascorbic acid was the most effective compound in inhibiting the SCN- -catalyzed reaction. Bilirubin showed some action, whereas tocopherol was ineffective. When LDL oxidation was performed with activated human neutrophils, which employ the MPO pathway, SCN- catalyzed the cell-mediated LDL oxidation. The MPO/H2O2/SCN- system may have the potential to play a significant role in the oxidative modification of LDL--an observation further pointing to the link between the long-recognized risk factors of atherosclerosis: elevated levels of LDL and smoking.     

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.     

Antioxidant and Prooxidant Effects of Ascorbic Acid, Dehydroascorbic Acid and Flavonoids on LDL Submitted to Different Degrees of Oxidation

Although a high intake of antioxidants may decrease the risk of developing cardiovascular diseases, under certain circunstances they may promote free radical generation and lipid peroxidation. The objectives of the present study were to determine the antioxidant effects of ascorbic acid (AA), dehydroascorbic acid (DHA) and flavonoids on LDL submitted to different degrees of oxidation. LDL was submitted to oxidation with CuCl₂ (2.4 μM). Before or at different times after the propagation of the oxidation process, 28 μM (5 μg/ml) of either AA or DHA or 5 μg/mL flavonoids extract were added. Alpha-tocopherol, conjugated dienes, thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility were determined as indices of LDL oxidation. The presence of any of the three antioxidants from the onset of the incubation delayed the oxidation process. However, the addition of both DHA and flavonoids to the oxidation process when it was already initiated and alpha-tocopherol consumed, accelerated the oxidation. In contrast, AA delayed the oxidation process even when added after alpha-tocopherol was consumed. Nevertheless, it also accelerated LDL oxidation when added during the propagation phase of the oxidation process. In conclusion: although AA, DHA and flavonoids delay LDL oxidation when added before the initiation of the process, they accelerate the process if added to minimally oxidized LDL.     

Immunohistochemical evidence for the myeloperoxidase/H2O2/halide system in human atherosclerotic lesions: colocalization of myeloperoxidase and hypochlorite-modified proteins.

The 'oxidation theory' of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although the precise mechanisms of in vivo oxidation are widely unknown, increasing evidence suggests that myeloperoxidase (MPO, EC 1.11.1.7), a protein secreted by activated phagocytes, generates modified/oxidized (lipo)proteins via intermediate formation of hypochlorous acid (HOCl). In vitro generation of HOCl transforms lipoproteins into high uptake forms for macrophages giving rise to cholesterol-engorged foam cells. To identify HOCl-modified-epitopes in human plaque tissues we have raised monoclonal antibodies (directed against human HOCl-modified LDL) that do not cross-react with other LDL modifications, i.e. peroxynitrite-LDL, hemin-LDL, Cu2+-oxidized LDL, 4-hydroxynonenal-LDL, malondialdehyde-LDL, glycated-LDL, and acetylated-LDL. The antibodies recognized a specific epitope present on various proteins after treatment with OCl- added as reagent or generated by the MPO/H2O2/halide system. Immunohistochemical studies revealed pronounced staining for HOCl-modified-epitopes in fibroatheroma (type V) and complicated (type VI) lesions, while no staining was observed in aortae of lesion-prone location (type I). HOCl-oxidation-specific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control segments. Staining was shown to be inside and outside monocytes/macrophages, endothelial cells, as well as in the extracellular matrix. A similar staining pattern using immunohistochemistry could be obtained for MPO. The colocalization of immunoreactive MPO and HOCl-modified-epitopes in serial sections of human atheroma (type IV), fibroatheroma (type V) and complicated (type VI) lesions provides further convincing evidence for MPO/H2O2/halide system-mediated oxidation of (lipo)proteins under in vivo conditions. We propose that MPO could act as an important link between the development of atherosclerotic plaque in the artery wall and chronic inflammatory events.     

Kinetics of lipid peroxidation in mixtures of HDL and LDL, mutual effects.

In view of the proposed central role of LDL oxidation in atherogenesis and the established role of HDL in reducing the risk of atherosclerosis, several studies were undertaken to investigate the possible effect of HDL on LDL peroxidation. Since these investigations yielded contradictory results, we have conducted systematic kinetic studies on the oxidation in mixtures of HDL and LDL induced by different concentrations of copper, 2, 2'-azo bis (2-amidinopropane) hydrochloride (AAPH) and myeloperoxidase (MPO). These studies revealed that oxidation of LDL induced either by AAPH or MPO is inhibited by HDL under all the studied conditions, whereas copper-induced oxidation of LDL is inhibited by HDL at low copper/lipoprotein ratio but accelerated by HDL at high copper/lipoprotein ratio. The antioxidative effects of HDL are only partially due to HDL-associated enzymes, as indicated by the finding that reconstituted HDL, containing no such enzymes, inhibits peroxidation induced by low copper concentration. Reduction of the binding of copper to LDL by competitive binding to the HDL also contributes to the antioxidative effect of HDL. The acceleration of copper-induced oxidation of LDL by HDL may be attributed to the hydroperoxides formed in the "more oxidizable" HDL, which migrate to the "less oxidizable" LDL and enhance the oxidation of the LDL lipids induced by bound copper. This hypothesis is supported by the results of experiments in which native LDL was added to oxidizing lipoprotein at different time points. When the native LDL was added prior to decomposition of the hydroperoxides in the oxidizing lipoprotein, the lag preceding oxidation of the LDL was much shorter than the lag observed when the native LDL was added at latter stages, after the level of hydroperoxides became reduced due to their copper-catalyzed decomposition. The observed dependence of the interrelationship between the oxidation of HDL and LDL on the oxidative stress should be considered in future investigations regarding the oxidation of lipoprotein mixtures.     

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.     

Effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL

Myeloperoxidase, in the presence of hydrogen peroxide and nitrite, promotes the lipid peroxidation of low density lipoprotein (LDL); the modified lipoprotein is then capable of being readily endocytosed by macrophages. Since acetaminophen has been shown to inhibit the leukocyte myeloperoxidase antimicrobial system and is, under certain experimental conditions, an antioxidant, the effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL was examined. The content of LDL lipid hydroperoxides after incubation with 50 nM myeloperoxidase, 100 microM nitrite and a hydrogen peroxide generating system for 6 h was reduced by approx. 80% in the presence of 25-250 microM acetaminophen. The production of thiobarbituric acid-reactive substances was also inhibited by acetaminophen to a similar extent. Acetylsalicylic acid (25-100 microM) did not inhibit LDL lipid peroxidation mediated by the myeloperoxidase enzyme system. LDL, treated with myeloperoxidase, hydrogen peroxide and nitrite for 14 h, was metabolized by macrophages to a much greater extent than native LDL. The presence of acetaminophen prevented the modification of LDL; the lipoprotein was metabolized by macrophages to the same extent as was native LDL. These results demonstrate that acetaminophen is a potent inhibitor of the myeloperoxidase-hydrogen peroxide-nitrite mediated modification of LDL.     

Conception of myeloperoxidase inhibitors derived from flufenamic acid by computational docking and structure modification

The development of myeloperoxidase (MPO) inhibitors has been conducted using flufenamic acid as a lead compound. Computational docking of the drug and its analogs in the MPO active site was first attempted. Several molecules were then synthesized and assessed using three procedures for the measurement of their inhibiting activity: (i) the taurine assay, (ii) the accumulation of compound II, and (iii) the LDL oxidation by ELISA. Most of the synthesized molecules had an activity in the same range as flufenamic acid but none of them were able to inhibit the MPO-dependent LDL oxidation. The experiments however gave some useful indications for a rational conception of MPO inhibitors.     

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.     

Hemin/nitrite/H2O2 induces brain homogenate oxidation and nitration: effects of some flavonoids

Oxidative injury has been implicated in the pathogenesis of numerous neurodegenerative diseases. Recently, it has been found that with the existence of hydrogen peroxide and nitrite, hemin catalyzes protein nitration. We hypothesize under certain pathological conditions, hemin catalyzed protein nitration may happen in the brain. In this paper, the effects of three flavonoids, i.e. quercetin, catachin and baicalein on hemin/nitrite/H2O2 induced brain homogenate oxidation and nitration were studied. The results showed that hemin/nitrite/H2O2 system could effectively induce brain homogenate protein oxidation and nitration. Quercetin, catachin and baicalein dose-dependently inhibited hemin/nitrite/H2O2 system-induced protein nitration in a dose-dependent manner, the inhibition of protein nitration was in the order of quercetin>catachin>baicalein. These compounds also inhibited hemin/H2O2 system-induced lipid peroxidation, the inhibition order was baicalein >quercetin>catachin. However, these flavonoids showed marginal effect on hemin/nitrite/H2O2 system caused protein oxidation and thiol oxidation. The inhibition activities of flavonoids on hemin/nitrite/H2O2 system-induced protein nitration may closely relate to their radical scavenging activities, since the inhibition order of protein nitration is the same as the radical scavenging order. These results indicate hemin/nitrite/H2O2 system induces different types of oxidative assault on bio-molecules. Flavonoids could act as antioxidants inhibiting ROS and RNS caused brain damage.     

Myeloperoxidase binds to low-density lipoprotein: potential implications for atherosclerosis

Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can modify low-density lipoprotein (LDL) to a form that converts macrophages into lipid-laden or 'foam' cells, the hallmark of atherosclerotic lesions. Since MPO has been shown to bind to a number of different cell types, we investigated binding of MPO to LDL. Using the precipitation reagents phosphotungstate or isopropanol, MPO co-precipitated with LDL, retaining its catalytic activity. The association of MPO with LDL was confirmed using native gel electrophoresis. MPO was also found to co-precipitate with apolipoprotein B-100-containing lipoproteins in whole plasma. No precipitation of MPO was observed in lipoprotein-deficient plasma, and there was a dose-dependent increase in precipitation following addition of LDL to lipoprotein-deficient plasma. Binding of MPO to LDL could potentially enhance site-directed oxidation of the lipoprotein and limit scavenging of reactive oxygen species by antioxidants.     

Interaction between flavonoids and alpha-tocopherol in human low density lipoprotein

Oxidative modification of low density lipoprotein (LDL) may play an important role in the development of atherosclerosis. Alpha-tocopherol functions as a major antioxidant in human LDL. The present study was to test whether four natural flavonoids (kempferol, morin, myricetin, and quercetin) would protect or regenerate alpha-tocopherol in human LDL. The oxidation of LDL incubated in sodium phosphate buffer (pH 7.4, 10 mM) was initiated by addition of either 5.0 mM CuSO(4) at 37 degrees C or 1.0 mM of 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH) at 40 degrees C. It was found that alpha-tocopherol was completely depleted within 1 hour. Under the same experimental conditions, all four flavonoids demonstrated a dose-dependent protecting activity to alpha-tocopherol in LDL at the concentration ranging from 1 to 20microM. All flavonoids showed a varying protective activity against depletion of alpha-tocopherol in LDL, with kempherol and morin being less effective than myricetin and quercetin. The addition of flavonoids to the incubation mixture after 5 minutes demonstrated a significant regeneration of alpha-tocopherol in human LDL. The protective activity of four flavonoids to LDL is related to the number and location of hydroxyl groups in the B ring as well as the stability in sodium phosphate buffer.     

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.