The Chemiluminescence Assay of Lipid Peroxidation Products in Human Blood Plasma Lipoproteins

A chemiluminescence (CL) flash kinetics on the addition of Fe²⁺ ions into oxidized low density lipoprotein (LDL) suspension has been studied. LDL oxidation was carried out at 37°C without and in the presence of 5 or 50 μM of Cu.²⁺ It has been found that under certain experimental conditions (the addition of excess iron ions, more than 1 mM) the amplitude of CL flash depended almost linearly (1) on the concentration of oxidized LDL and (2) on the extent of LDL oxidation measured as diene conjugates (DC) and 2-thiobarbituric acid-reactive substance (TBARS) accumulation. The corresponding correlation coefficients were: for TBARS - 0.94 and for DC - 0.97, in the case of LDL autooxidation; 0.72 and 0.98, in the case of copper-induced LDL oxidation. A sensitivity of the CL method was shown to be significantly enhanced (by more than two orders) in the presence of CL sensitizer - 2, 3,5, 6-lH,4H-tetrahydro-9-(2' -benzoimidazolyl)-quinolizin-(9, 9a, 1 -gh)coumarin.     

Role of Amino Thiols in Luminol Chemiluminescence Coupled with Copper(II)-catalysed Oxidation of Cysteine and Glutathione

The Cu(II)-catalysed oxidation of the amino thiols cysteine and glutathione with oxygen was examined in the presence of luminol. Light emission was markedly suppressed when the concentration of amino thiols was greater than that of Cu(II). Cysteine caused a delay of chemiluminescence (CL) and no CL emission was observed at high concentrations of GSH. The suppression in CL could be explained in terms of the complexation of the Cu(II) catalyst by formation of a Cu(II)–dicysteine complex in case of cysteine, and of a Cu(II)–GSSG complex in case of GSH.     

Neopterin and 7,8-Dihydroneopterin Interfere With Low Density Lipoprotein Oxidation Mediated by Peroxynitrite and/or Copper

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- &#110 generate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO &#109 ). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO &#109 - as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.     

Sulfite facilitates LDL lipid oxidation by transition metal ions: a pro-oxidant in wine?

Lipid oxidation in LDL may play a role in atherogenesis. It has been shown that sulfite - a compound in the aqueous fraction of wine - could inhibit free radical (AAPH) mediated oxidation of plasma. Thus, sulfite has been proposed as an antioxidant. In contrast, the aqueous phase of wine has recently been shown to contain not fully identified compounds promoting transition metal ion (Cu(2+)) initiated LDL oxidation. As transition metal ions can catalyse the auto-oxidation of sulfite, we studied the influence of sulfite on Cu(2+) initiated LDL oxidation. The results show that sulfite at concentrations found in vivo strongly facilitated LDL oxidation by Cu(2+). The LDL-oxidase activity of ceruloplasmin was also stimulated by sulfite. ROS formation by Cu(2+)/SO(3)(2-) was not inhibited by SOD but by catalase. We propose that formation of Cu(+), sulfite radicals (SO(3)*(-)) and hydroxyl radicals (OH(*)) is a mechanism by which sulfite could act as a pro-atherogenic agent in presence of transition metal ions.     

Neopterin and 7,8-dihydroneopterin interfere with low density lipoprotein oxidation mediated by peroxynitrite and/or copper

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- γgenerate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO -). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO -- as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.     

Glucose Accelerates Copper- and Ceruloplasmin-induced Oxidation of Low-density Lipoprotein and Whole Serum

Glucose at pathophysiological concentrations was able to accelerate copper-induced oxidation of isolated low-density lipoprotein (LDL) and whole serum. The efficiency of glucose was favored under the following circumstances: (a) when LDL oxidation was induced by low copper concentration, (b) when LDL was partly oxidized, i.e. enriched with lipid peroxides. The glucose derivative methyl- &#102 - d -glucoside was ineffective on Cu 2+ -induced LDL oxidation, pointing out the essential role of the reactivity of the aldehydic carbon for the pro-oxidative effect. When LDL oxidation was induced by a peroxyl radical generator, as a model of transition metal independent oxidation, glucose was ineffective. Glucose was found to stimulate oxidation of LDL induced by ceruloplasmin, the major copper-containing protein of human plasma. Thus, glucose accelerated oxidation of LDL induced by both free and protein bound copper. Considering the requirement for catalytically active copper and for the aldehydic carbon, the pro-oxidative effect of glucose is likely to depend on the increased availability of Cu + ; this is more efficient in decomposing lipid peroxide than Cu 2+ , accounting for acceleration of LDL oxidation. The possible biological relevance of our work is supported by the finding that glucose was able to accelerate oxidation of whole serum, which was assessed by monitoring low-level chemiluminescence associated with lipid peroxidation.     

Prooxidant and antioxidant properties of Trolox C, analogue of vitamin E, in oxidation of low-density lipoprotein

Trolox C (Trolox), a water-soluble analogue of vitamin E lacking the phytyl chain, was investigated with respect to its effect on the oxidation of low-density lipoprotein (LDL). Trolox was added at different time points of LDL oxidation induced by Cu2+ and aqueous peroxyl radicals. In the case of Cu2+ -induced LDL oxidation, the effect of Trolox changed from antioxidant to prooxidant when added at later time points during oxidation; this transition occurred whenever alpha-tocopherol was just consumed in oxidizing LDL. Thus, in the case of Cu2+ -dependent LDL oxidation, the presence of lipophilic antioxidants in the LDL particle is likely to be a prerequisite for the antioxidant activity of Trolox. When oxidation was induced by peroxyl radicals, as a model of metal-independent oxidation, the effect of Trolox was always antioxidant, suggesting the importance of Cu2+ /Cu+ redox-cycling in the prooxidant mechanism of Trolox. Our data suggest that, in the absence of significant amounts of lipophilic antioxidants, LDL becomes highly susceptible to oxidation induced by transition metals in the presence of aqueous reductants.     

Resveratrol inhibits copper ion-induced and azo compound-initiated oxidative modification of human low density lipoprotein.

To investigate whether resveratrol, a polyphenolic compound in red wine, affects the oxidation of human low density lipoprotein (LDL), LDL purified from normolipidemic subjects was subjected to Cu(2+)-induce and azo compound-initiated oxidative modification, with and without the addition of varying concentrations of resveratrol. Modification of LDL was assessed by the formation of thiobarbituric acid reactive substances (TBARS) and changes in the relative electrophoretic mobility (REM) of LDL on agarose gels. Resveratrol (50 microM) reduced TBARS and REM of LDL during Cu(2+)-induced oxidation by 70.5% and 42.3%, respectively (p < 0.01), and prolonged the lag phase associated with the oxidative modification of LDL by copper ion or azo compound. These in vitro results suggest that resveratrol may afford protection of LDL against oxidative damage resulting from exposure to various environmental challenges, possibly by acting as a free radical scavenger.     

Mechanistic aspects of the relationship between low-level chemiluminescence and lipid peroxides in oxidation of low-density lipoprotein.

In this study oxidation of low-density lipoprotein (LDL) induced by different Cu2+ concentrations was investigated. Lipid peroxidation was assessed by monitoring low-level chemiluminescence (LL-CL), conjugated diene hydroperoxide (CD) and alpha-tocopherol (TocOH), the major lipophilic antioxidant in LDL. At high Cu2+ concentration, LDL oxidation was characterised by CD formation, LL-CL emission and TocOH consumption. At low Cu2+ concentration, CD formation was independent of LL-CL and occurred in the presence of TocOH. Thus, two different mechanisms lead to lipid peroxide formation in LDL. The combination of CD assay and LL-CL monitoring makes it possible to distinguish the autocatalytic mechanism of CD formation and that associated with TocOH, found at a high and a low rate of initiation, respectively.     

Glucose accelerates copper- and ceruloplasmin-induced oxidation of low-density lipoprotein and whole serum

Glucose at pathophysiological concentrations was able to accelerate copper-induced oxidation of isolated low-density lipoprotein (LDL) and whole serum. The efficiency of glucose was favored under the following circumstances: (a) when LDL oxidation was induced by low copper concentration, (b) when LDL was partly oxidized, i.e. enriched with lipid peroxides. The glucose derivative methyl-alpha-D-glucoside was ineffective on Cu2+-induced LDL oxidation, pointing out the essential role of the reactivity of the aldehydic carbon for the pro-oxidative effect. When LDL oxidation was induced by a peroxyl radical generator, as a model of transition metal independent oxidation, glucose was ineffective. Glucose was found to stimulate oxidation of LDL induced by ceruloplasmin, the major copper-containing protein of human plasma. Thus, glucose accelerated oxidation of LDL induced by both free and protein bound copper. Considering the requirement for catalytically active copper and for the aldehydic carbon, the pro-oxidative effect of glucose is likely to depend on the increased availability of Cu+; this is more efficient in decomposing lipid peroxide than Cu2+, accounting for acceleration of LDL oxidation. The possible biological relevance of our work is supported by the finding that glucose was able to accelerate oxidation of whole serum, which was assessed by monitoring low-level chemiluminescence associated with lipid peroxidation.     

LDL particle subclasses in hypercholesterolemia. Molecular determinants of reduced lipid hydroperoxide stability

Hypercholesterolemia is characterized by elevated plasma levels of LDL in which the cholesteryl ester (CE)-rich LDL subclasses of light and intermediate density (LDL1+2 and LDL3, respectively) typically predominate. The molecular mechanisms implicated in oxidation of LDL particle subclasses in hypercholesterolemia are indeterminate. Lipid hydroperoxides (LOOH), primary oxidation products in LDL, are implicated in atherogenesis. LOOH formation was evaluated in light (LDL1+2), intermediate (LDL3), and dense (LDL4+5) LDL subclasses from hypercholesterolemic (HC) subjects (n = 7) during copper-mediated oxidative stress, and compared with that in corresponding subclasses from normolipidemic subjects (n = 7). HC LDL subclasses were distinguished by lower polyunsaturated phospholipid-alpha-tocopherol ratios (P < 0.02), lower contents of phosphatidyl choline (PC)16:0-18:0/18:2 and PC16:0-18:0/20:4+22:6 (P < 0.002), and higher surface phospholipid-free cholesterol ratios (P < 0.04). The LDL3, LDL4, and LDL5 subclasses in HC subjects displayed low-core polyunsaturated CE-alpha-tocopherol ratios (P < 0.05), despite similar PUFA CE content. These physicochemical differences did not modify the oxidative susceptibility of HC LDL but underlie the marked instability of cholesterol linoleate hydroperoxides in HC LDL1+2, LDL3, and LDL4 subclasses.Elevated concentrations of large, CE-rich, light, and intermediate LDL subclasses (LDL1+2, LDL3) in hypercholesterolemia may therefore act as an abundant proatherogenic source of highly unstable LOOH in the arterial wall.     

A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin

The mechanisms by which low-density lipoprotein (LDL) particles undergo oxidative modification to an atherogenic form that is taken up by the macrophage scavenger-receptor pathway have been the subject of extensive research for almost two decades. The most common method for the initiation of LDL oxidation in vitro involves incubation with Cu(II) ions. Although various mechanisms have been proposed to explain the ability of Cu(II) to promote LDL modification, the precise reactions involved in initiating the process remain a matter of contention in the literature. This review provides a critical overview and evaluation of the current theories describing the interactions of copper with the LDL particle. Following discussion of the thermodynamics of reactions dependent upon the decomposition of preexisting lipid hydroperoxides, which are present in all crude LDL preparations, attention is turned to the more difficult (but perhaps more physiologically-relevant) system of the hydroperoxide-free LDL particle. In both systems, the key role of alpha-tocopherol is discussed. In addition to its protective, radical-scavenging action, alpha-tocopherol can also behave as a prooxidant via its reduction of Cu(II) to Cu(I). Generation of Cu(I) greatly facilitates the decomposition of lipid hydroperoxides to chain-carrying radicals, but the mechanisms by which the vitamin promotes LDL oxidation in the absence of preformed hydroperoxides remain more speculative. In addition to the so-called tocopherol-mediated peroxidation model, in which polyunsaturated fatty acid oxidation is initiated by the alpha-tocopheroxyl radical (generated during the reduction of Cu(II) by alpha-tocopherol), an evaluation of the role of the hydroxyl radical is provided. Important interactions between copper ions and thiols are also discussed, particularly in the context of cell-mediated LDL oxidation. Finally, the mechanisms by which ceruloplasmin, a copper-containing plasma protein, can bring about LDL modification are discussed. Improved understanding of the mechanisms of LDL oxidation by copper ions should facilitate the establishment of any physiological role of the metal in LDL modification. It will also assist in the interpretation of studies in which copper systems of LDL oxidation are used in vitro to evaluate potential antioxidants.     

On the chemiluminescence in the oxidation of tetravalent uranium to the uranyl ion by dimethyldioxirane

The reaction of the tetravalent uranium [U(IV)] with dimethyldioxirane (DMD) in strongly acidic water–acetone solutions is accompanied by chemiluminescence (CL) in the visible (Vis) and infra‐red (IR) regions. At least three independent reaction pathways are involved in the U(IV)–DMD oxidation: the first entails the non‐chemiluminescent oxidation of U(IV) to the uranyl ion (UO₂²⁺); the second involves the catalytic decomposition of DMD by U(IV) to afford singlet oxygen, as manifested by its characteristic IR‐CL; and in the third process, slow Vis‐CL (510–540 nm) is emitted, following DMD consumption. Copyright © 2002 John Wiley & Sons, Ltd.     

Observation of the complex formation between Cu(II) and protein by capillary electrophoretic system incorporating an UV/CL dual detector

We investigated the complex formation between Cu(II) and human serum albumin (HSA) through a biuret reaction by use of capillary electrophoretic system incorporating an ultra-violet absorption (UV) and chemiluminescence (CL) dual detector. Cu(II)-tartrate complex and Cu(II)-human serum albumin complex were detected by UV detection (282 nm) with on-capillary, followed by CL detection (luminol-hydrogen peroxide CL reaction) with end-capillary. We examined the effects of the reaction time and temperature on the UV and CL responses. On the basis of the obtained results we considered the Cu(II)-human serum albumin complex formation processes and its catalytic activity for the CL reaction. The system easily, rapidly, and simultaneously produced useful information concerning the complex formation of Cu(II) and human serum albumin due to the presence of the both detectors.     

Transient-state kinetics of the reactions of 1-methoxy-4-(methylthio)benzene with horseradish peroxidase compounds I and II

Transient-state reactions of horseradish peroxidase compounds I and II with 1-methoxy-4-(methylthio)benzene (a para-substituted thioanisole) were studied over the pH range from 3.4 to 10.5. The pH-jump technique was applied to the compound II reactions at pH values below 8.6. The reactions of both compound I and compound II with the para-substituted thioanisole consisted predominantly of an initial burst. The burst was followed by a steady-state phase that became more obvious at lower concentrations of the thioanisoles. The burst phase for both compounds I and II can be explained in terms of two independent transient-state reactions with 1-methoxy-4-(methylthio)benzene as follows: (i) a single reaction of compound I (or compound II) with the substrate and (ii) the formation of a complex between compound I or II and the substrate followed by reaction of the productive complex with another molecule of sulfide. The overall rate of reaction path ii is faster than that of path i. The preference for path i or ii is highly dependent upon the concentration of sulfide with step ii favored at higher sulfide concentrations. The experimental results obtained on the overall reaction under both pseudo-first-order and single-turnover conditions indicate that compound II reacts competitively with both the organic sulfide substrate and the sulfur cation radical produced from compound I oxidation of sulfide.     

Red wine mitigates the postprandial increase of LDL susceptibility to oxidation

The aim of the present study was to verify the extent of oxidative stress induced by a meal at plasma and LDL level, and to investigate the capacity of red wine to counteract this action. In two different sessions, six healthy men ate the same test meal consisting of "Milanese" meat and fried potatoes. The meal was taken either with 400 ml red wine or with an isocaloric hydroalcoholic solution. Oxidative stress at plasma level was estimated through the measure of ascorbic acid, alpha-tocopherol, protein SH groups, uric acid, and antioxidant capacity, measured before and 1 and 3 h after the meal. The change in the resistance of LDL to oxidative modification was taken as an index of exposure to pro-oxidants. The susceptibility to Cu(II)-catalyzed oxidation of baseline and postprandial LDL was measured as conjugated dienes formation, tryptophan residues, and relative electrophoretic mobility. The experimental meal taken with wine provoked a significant increase in the total plasma antioxidant capacity and in the plasma concentration of alpha-tocopherol and SH groups. Postprandial LDL was more susceptible to metal-catalyzed oxidation than the homologous baseline LDL after the ethanol meal. On the contrary, postprandial LDL obtained after the wine meal was as resistant or more resistant to lipid peroxidation than fasting LDL.     

The kinetics of copper-induced LDL oxidation depend upon its lipid composition and antioxidant content

Copper promotes oxidation of human low-density lipoprotein (LDL) through molecular mechanisms that are still under investigation. We employed native human LDL, phospholipid-containing delipidated LDL ghosts, or trilinolein-reconstituted, phospholipid-containing LDL to investigate both LDL oxidation and the associated process of copper reduction. Both LDL ghosts and trilinolein-reconstituted LDL were devoid of antioxidants and were extremely susceptible to AAPH-induced oxidation but, paradoxically, were rather resistant to copper-mediated oxidation. The dynamic reduction of Cu(II) to Cu(I) was quantitatively decreased in LDL ghosts and in trilinolein-reconstituted LDL, also lacking the initial rapid reduction and the subsequent inhibition phases, due to the absence of endogenous antioxidants. Conversely, the rate of copper reduction was linear and likely due to lipid peroxides, either already present or formed during copper-induced oxidation. We suggest that copper undergoes redox transitions in LDL by utilizing reducing equivalents originating from endogenous antioxidants and/or from lipid peroxides in the LDL lipid core.     

Cu2(+)-mediated oxidation of dialyzed plasma: effects on low and high density lipoproteins and cholesteryl ester transfer protein.

We previously reported that the expression of an epitope of apolipoprotein B (apoB), mapped to the C-terminus and defined by antibody Bsol7, increased during Cu2(+)-mediated oxidation of isolated low density lipoprotein (LDL). We describe now the properties of Bsol7 as a marker of LDL oxidation in whole plasma in relation to other effects of oxidative treatment of plasma, such as the distribution of apoA-I and cholesteryl ester transfer protein (CETP). In dialyzed plasma, no LDL oxidation was detected at Cu2+ concentrations (5 microM) sufficient for extensive oxidation of isolated LDL. At a higher Cu2+ concentration (50 microM), an increased expression of the Bsol7 epitope was observed; at 250 microM Cu2+, other evidence of LDL oxidation was found. The pattern of LDL response to Cu2+ observed in dialyzed plasma could be reproduced by adding 3% bovine serum albumin to isolated LDL. We demonstrate that the effect of albumin most likely results from its ability to bind copper ions. Incubation of plasma with increasing concentrations of Cu2+ resulted first in the disappearance of alpha 2-migrating HDL, the usual carrier of CEPT; free CETP and high molecular weight apoA-I-containing particles were also generated during oxidation. Addition of oxidized, but not native, LDL to plasma resulted in a transfer to LDL of some of the CETP initially associated with apoA-I. In conclusion, the increased immunoreactivity of the Bsol7 epitope was the most sensitive parameter of LDL oxidation, but other parameters, such as the presence of alpha 2-HDL and CETP-lipoprotein associations were even more sensitive evidence of lipoprotein oxidation.     

Reactions of pholasin with peroxidases and hypochlorous acid

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl(-) and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H(2)O(2) in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H(2)O(2)/Cl(-) system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.     

The protective effects of HDL and its constitutents against neutrophil respiratory burst activation by hypochlorite-oxidized LDL

Hypochlorite-oxidized low-density lipoprotein (oxLDL) possesses a substantial proinflammatory potential by modulating respiratory burst activities of polymorphonuclear neutrophils (PMN). As evaluated by luminol-amplified chemiluminescence (CL) incubation of 10(6) PMN/ml with 70 nM oxLDL was followed by substantial induction of neutrophil oxidant (ROS) generation. We evaluated the inhibitory capacity of high-density lipoprotein (HDL) and its lipid and protein constituents against the activating effects of oxLDL. At a HDL or apolipoprotein AI/LDL protein ratio of 1.0, native HDL decreased the respiratory burst activation by 64%, followed by trypsinized HDL (57%) and native apoAI (43%). The inhibitory effects of native HDL did not require prior incubation with PMN or with oxLDL suggesting an instantaneously acting protective mechanism in the minute range. OxLDL modulated ROS production not only of resting PMN but also that of activated PMN, as indicated by a 14-fold increase in FMLP-stimulated CL response and a 50% decrease in zymosan-mediated CL answer. HDL itself did not protect PMN from activation by FMLP and zymosan. However, it clearly reduced effects of oxLDL on FMLP-activation and slightly counteracted the oxLDL-mediated decrease in zymosan-induced ROS generation. Taken together, these findings may offer new insight into atheroprotective mechanisms of HDL.