High-density Lipoprotein Oxidation Research Articles

Red Wine Inhibits the Cell-Mediated Oxidation of LDL and HDL

Objective: We compared the in vitro effects of red wine, white wine and ethanol on the cell mediated oxidation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) by three frequently-used assays.Methods: LDL and HDL isolated from normolipidemic human serum were incubated with J774.A1 macrophages in DMEM with copper, with or without red wine, white wine or ethanol (equivalent to 0.2 mg ethanol/ml). Lipoprotein oxidation was assessed by conjugated diene formation as measured by changes in absorbance at 234 nm (ΔA234), thiobarbituric-acid-reactive-substance (TBARS) production and trinitrobenzene-sulfonic-acid (TNBS) reactivity.Results: Red wine (0.2 mg ethanol/mL) inhibited LDL oxidation as indicated by an 85.7% decrease in absorbance at 234 nm, a 96.5% decrease in TBARS production and complete prevention of the decrease in TNBS reactivity. White wine and ethanol did not have any significant effect at 0.2 mg/mL. White wine at 1.0 mg ethanol/mL inhibited TBARS production from LDL by 84.1%. Red wine (0.2 mg ethanol/mL) inhibited HDL oxidation as indicated by a 78.9% decrease in ΔA234, an 81.7% decrease in TBARS production and by no change in TNBS reactivity. White wine and ethanol had no effect at 0.2 mg/mL. White wine at 1.0 mg ethanol/mL inhibited TBARS production from HDL by 66.4%.Conclusions: These results indicate that red wine inhibits the cell mediated oxidation of lipoproteins, that white wine is not as effective as red wine and that the effect of the red wine is not due to its ethanol content.

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki = 40±3 nM, kobs/[I] = 6.6×105 M-1·s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0±0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki = 6.3±0.5 µM, kobs/[I] = 1.6×104 M-1·s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

Glycation of high-density lipoprotein does not increase its susceptibility to oxidation or diminish its cholesterol efflux capacity

In vitro oxidation of high-density lipoprotein (HDL) diminishes its capacity to mediate cholesterol efflux from J774 macrophages. To investigate the possible role of HDL glycation in the increased atherosclerotic risk in diabetes, we studied the effects of in vitro glycation of HDL on its susceptibility to oxidation and capacity to mediate cholesterol efflux. HDL isolated from normal volunteers was incubated with 25 mmol/L glucose for 70 hours, resulting in 6.1% additional derivatization of apoproteins as determined by trinitrobenzene sulfonic acid (TNBS) reactivity. Unmodified HDL and glycated HDL (glyHDL) were tested for susceptibility to oxidation by incubation with various concentrations of copper and three assays of lipid oxidation. GlyHDL produced 51% to 64% less lipid peroxide than HDL as determined by reaction with xylenol orange (P < .02), indicating decreased susceptibility to oxidation. However, glycation of HDL did not result in significant changes in the formation of conjugated dienes or thiobarbituric acid-reactive substances (TBARS), two other indices of oxidation. To study cholesterol efflux, J774 macrophages were labeled with 3H-cholesterol followed by incubation with the various HDL preparations. HDL and glyHDL had a similar capacity to mediate efflux. The efflux mediated by oxidized HDL (oxHDL) and oxidized glyHDL was reduced to a similar extent compared with the efflux mediated by HDL and glyHDL. These data indicate that in vitro glycation of HDL does not increase its susceptibility to oxidation and does not diminish its capacity to mediate cholesterol efflux.

Age-related increased susceptibility of high-density lipoproteins (HDL) to in vitro oxidation induced by γ-radiolysis of water

In the present study, we investigated the age-related susceptibility of high-density lipoproteins (HDL) to oxidation. HDL were obtained from healthy, normolipidemic young, middle-aged and elderly subjects. Oxidation of HDL was induced in vitro by oxygen free radicals generated by water γ-radiolysis, and followed by the decrease of endogenous vitamin E and the formation of conjugated dienes and thiobarbituric acid-reactive substances, as well as the alterations of apolipoproteins A-I/A-II. The resistance of HDL to oxidation, evaluated by the length of the lag phase, decreased with aging. This increased oxidizability of HDL with aging could have a dramatic impact on the development of atherosclerosis in the elderly population.

Enhanced Cholesterol Efflux by Tyrosyl Radical-oxidized High Density Lipoprotein Is Mediated by Apolipoprotein AI-AII Heterodimers

Myeloperoxidase secreted by phagocytes in the artery wall may be a catalyst for lipoprotein oxidation. High density lipoprotein (HDL) oxidized by peroxidase-generated tyrosyl radical has a markedly enhanced ability to deplete cultured cells of cholesterol. We have investigated the structural modifications in tyrosylated HDL responsible for this effect. Spherical reconstituted HDL (rHDL) containing the whole apolipoprotein (apo) fraction of tyrosylated HDL reproduced the ability of intact tyrosylated HDL to enhance cholesterol efflux from cholesterol-loaded human fibroblasts when reconstituted with the whole lipid fraction of either HDL or tyrosylated HDL. Free apoAI or apoAII showed no increased capacity to induce cholesterol efflux from cholesterol-loaded fibroblasts following oxidation by tyrosyl radical, either in their lipid-free forms or in rHDL. The product of oxidation of a mixture of apoAI and apoAII (1:1 molar ratio) by tyrosyl radical, however, reproduced the enhanced ability of tyrosylated HDL to induce cholesterol efflux when reconstituted with the whole lipid fraction of HDL. HDL containing only apoAI or apoAII showed no enhanced ability to promote cholesterol efflux following oxidation by tyrosyl radical, whereas HDL containing both apoAI and apoAII did. rHDL containing apoAI-apoAIImonomer and apoAI-(apoAII)2 heterodimers showed a markedly increased ability to prevent the accumulation of LDL-derived cholesterol mass by sterol-depleted fibroblasts compared with other apolipoprotein species of tyrosylated HDL. These results indicate a novel product of HDL oxidation, apoAI-apoAII heterodimers, with a markedly enhanced capacity to deplete cells of the regulatory pool of free cholesterol and total cholesterol mass. The recent observation of tyrosyl radical-oxidized LDL in vivo suggests that a similar modification of HDL would significantly enhance its ability to deplete peripheral cells of cholesterol in the first step of reverse cholesterol transport.

Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation

Increased atherosclerosis risk in hyperlipidemic patients may be a result of the enhanced oxidizability of their plasma lipoproteins. We have previously shown that hypocholesterolemic drug therapy, including the 3-hydroxy-3-methyl-glutaryl CoenzymeA (HMG-CoA) reductase inhibitors, and the hypotriglyceridemic drug bezafibrate, significantly reduced the enhanced susceptibility to oxidation of low density lipoprotein (LDL) isolated from hyperlipidemic patients. Although this antioxidative effect could not be obtained in vitro with all of these drugs, the active drug metabolites, which are formed in vivo, could affect lipoprotein oxidizability. We thus sought to analyze the effect of atorvastatin and gemfibrozil, as well as specific hydroxylated metabolites, on the susceptibility of LDL, very low density lipoprotein (VLDL), and high density lipoprotein (HDL) to oxidation. LDL oxidation induced by either copper ions (10 microM CuSO4), by the free radical generator system 2'-2'-azobis 2-amidino propane hydrochloride (5 mM AAPH), or by the J-774A.1 macrophage-like cell line, was not inhibited by the parent forms of atorvastatin or gemfibrozil, but was substantially inhibited (57-97%), in a concentration-dependent manner, by pharmacological concentrations of the o-hydroxy and the p-hydroxy metabolites of atorvastatin, as well as by the p-hydroxy metabolite (metabolite I) of gemfibrozil. On using the atorvastatin o-hydroxy metabolite and gemfibrozil metabolite I in combination an additive inhibitory effect on LDL oxidizability was found. Similar inhibitory effects (37-96%) of the above metabolites were obtained for the susceptibility of VLDL and HDL to oxidation in the oxidation systems outlined above. The inhibitory effects of these metabolites on LDL, VLDL, and HDL oxidation could be related to their free radical scavenging activity, as well as (mainly for the gemfibrozil metabolite I) to their metal ion chelation capacities. In addition, inhibition of HDL oxidation was associated with the preservation of HDL-associated paraoxonase activity. We conclude that atorvastatin hydroxy metabolites, and gemfibrozil metabolite I possess potent antioxidative potential, and as a result protect LDL, VLDL, and HDL from oxidation. We hypothesize that in addition to their beneficial lipid regulating activity, specific metabolites of both drugs may also reduce the atherogenic potential of lipoproteins through their antioxidant properties.

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.

Oxidation of High Density Lipoproteins: I. FORMATION OF METHIONINE SULFOXIDE IN APOLIPOPROTEINS AI AND AII IS AN EARLY EVENT THAT ACCOMPANIES LIPID PEROXIDATION AND CAN BE ENHANCED BY α-TOCOPHEROL

The lipids of high density lipoproteins (HDL) are initially oxidized in preference to those in low density lipoprotein when human plasma is exposed to aqueous peroxyl radicals. In this work we report on the relative susceptibility of HDL protein and lipid to oxidation and on the role HDL's alpha-tocopherol (alpha-TOH) plays in modulating protein oxidation. Exposure of isolated HDL to either low fluxes of aqueous peroxyl radicals, Cu2+ ions, or soybean lipoxygenase resulted in the oxidation of apoAI and apoAII during the earliest stages of the reaction, i.e. after consumption of ubiquinol-10 and in the presence of alpha-TOH. Hydro(pero)xides of cholesteryl esters and phospholipids initially accumulated together with specific oxidized forms of apoAI and apoAII, separated by high pressure liquid chromatography. The specific oxidized forms of apoAI were 16 and 32 mass units heavier than those of the native apolipoproteins and contained 1 and 2 methionine sulfoxide residues per protein, respectively. The third methionine residue in apoAI, as well as Trp residues, remained unoxidized during the earliest stages of HDL oxidation examined. Exposure of isolated apoAI to peroxyl radicals, Cu2+, or soybean lipoxygenase resulted in nonspecific (for peroxyl radicals) or no discernible protein oxidation (Cu2+ and soybean lipoxygenase). This indicated that the formation of the specific oxidized forms of apoAI observed with native HDL was not the result of direct reaction of these oxidants with the apolipoprotein. In vitro and in vivo enrichment of HDL with alpha-TOH resulted in a dose-dependent increase in the extent of peroxyl radical-induced formation of HDL cholesteryl ester hydroperoxides (r = 0.96) and cholesteryl ester hydroxides (r = 0. 92), as well as the loss of apoAI (r = 0.96) and apoAII (r = 0.94). alpha-TOH enrichment also enhanced HDL lipid and protein oxidation induced by Cu2+ or soybean lipoxygenase. These results indicate that the earliest stages of HDL oxidation are accompanied by the oxidation of specific methionine residues in apoAI and apoAII and that in the absence of co-antioxidants, alpha-TOH can promote this process.

Oxidation of high density lipoprotein by copper and by azobis(2-amidinopropaneïhcl): [formula omitted], Quiri Chen, Lawrence L. Rudel, and Richard Weinberg. Wake Forest Univ. School Medicine

Oxidation of high density lipoprotein by copper and by azobis(2-amidinopropaneïhcl): [formula omitted], Quiri Chen, Lawrence L. Rudel, and Richard Weinberg. Wake Forest Univ. School Medicine

動脈硬化巣における変性ＨＤＬの産生について

We obtained a monoclonal antibody 9F 5-3 a against oxidative high-density lipoprotein (HDL) modified with CuSO4. In addition, we have examined the epitope of 9F 5-3 a and the localization of oxidized HDL (oxHDL) in atheromatous plaque. The 9F 5-3 a was reacted strongly with oxHDL and to a lesser degree with oxidized low-density lipoprotein (oxLDL) and native LDL. No reactivity was found with native HDL. Furthermore, the cross-linkage of apolipoprotein A-I and/or apolipoprotein A-II was accelerated by HDL oxidation, and the crosslinked apolipoprotein forms (32, 60, 90, 120 and 200-kDas) were selectively recognized by the 9F 5-3 a antibody. OxHDL particles reacting with the 9F 5-3 a antibody were localized histochemically in the luminal side of intima of atheromatous plaques in human abdominal aortae. Using a ligand receptor assay for oxHDL, a major 130-kDa band was detected in cultured human aortic endothelial cells. These results suggested that oxHDL particles and its putative binding protein were present in atheromatous plaques and endothelial cells, respectively.

Human paraoxonase (PONA) genotype determines the susceptibility to oxidation of high-density lipoprotein (HDL) particle

Human paraoxonase (PONA) genotype determines the susceptibility to oxidation of high-density lipoprotein (HDL) particle

α-Tocopherol Enrichment of High-Density Lipoproteins: Stabilization of Hydroperoxides Produced During Copper Oxidation

In the aim to study the effect of an in vitro enrichment of high-density lipoprotein (HDL) with α-tocopherol in alcoholic solution on a copper-induced peroxidation, we monitored several markers of lipid peroxidation ( α-tocopherol consumption, formation of conjugated dienes and of fatty acid hydroperoxides, production of thiobarbituric acid-reactive substances) and the integrity of apolipoprotein A-I. High-density lipoproteins (1.063 < d < 1.21) with a mean of 0.58 α-tocopherol molecules per HDL particle were enriched whith α-tocopherol in alcoholic solution to obtain an average of 3.7 and 21 α-tocopherol molecules per HDL particle. HDL oxidation with 5 μM CuSO 4 at 37°C resulted in the total disappearance of endogenous α-tocopherol after 2 h, but after 24 h about 19% of α-tocopherol remained in the most enriched HDL. In agreement with the tocopherol-mediated peroxidation, the formation of conjugated dienes and of fatty acid hydroperoxides was very fast and increased with α-tocopherol concentration, whereas TBARS production decreased. These results showed that α-tocopherol enrichment stabilized the production of hydroperoxides in HDL and decreased the formation of secondary oxidation products. These latter products are known for deleterious effects towards apolipoproteins. This could explain why we observed that the apolipoprotein A-I of the most enriched HDL was only slightly altered after incubation with CuSO 4. Copyright © 1996 Elsevier Science Inc.

Effects of dietary vitamin C and E supplementation on the copper mediated oxidation of HDL and on HDL mediated cholesterol efflux

Copper mediated oxidative modification of high density lipoprotein (HDL) diminishes its capacity to promote cholesterol efflux from cells in culture. In the present study, HDL was isolated from eight subjects before and after a 10 day administration of the antioxidant vitamins C and E. After incubation of HDL (1.25 mg protein/ml) with 10 μM copper for 0–4 h or with 0–20 μM copper for 4 h, thiobarbituric acid reactive substances (TBARS) production was significantly decreased following vitamin administration suggesting that the vitamins decreased the susceptibility of HDL to oxidation. However, two other assays of lipoprotein oxidation, trinitrobenzene sulfonic acid reactivity and conjugated diene formation, did not show a consistent effect of vitamin administration. To study cholesterol efflux, J774 macrophages were labeled with 3H cholesterol (0.1 μCi/ml, 50 μg/ml) and incubated with HDL or oxidized HDL (100 μg protein/ml) for 24 h. HDL isolated before vitamins and oxidized in vitro was 39% less effective in mediating efflux compared to unmodified HDL, while HDL isolated after vitamins and oxidized was 22% less effective (before vs. after vitamins, P < 0.015). HDL oxidation determined by measuring TBARS production correlated with decreased cholesterol efflux ( r = −0.73, P < 0.050). These data suggest that oxidation of HDL interferes with its role in reverse cholesterol transport and that antioxidant vitamins have a protective effect.

Estradiol-17β as an antioxidant: Some distinct features when compared with common fat-soluble antioxidants

Estrogens are potent antioxidants both in vitro and in vivo. In this study the antioxidant affect of estradiol-17beta (estradiol) was compared with those of fat-soluble antioxidants (alpha-tocopherol and beta-carotene) in terms of both fatty acid (thiobarbituric acid-reactive substances and diene conjugation) and cholesterol oxidation (oxysterols). The addition of alpha-tocopherol (54 micromol/L) inhibited low-density lipoprotein (LDL) oxidation by 92.6% and high-density lipoprotein (HDL) oxidation by 76.5%. In similar experiments, estradiol (54 micromol/L) inhibited LDL oxidation by 77.5% but inhibited HDL oxidation by only 55.4%. Beta-carotene had no antioxidant effect. Lag times (diene conjugation method) for alpha-tocopherol and beta-carotene increased by 175% and 125%, respectively. Estradiol markedly reduced the maximum formation of diene conjugates as compared with results with alpha-tocopherol and beta-carotene, and it exhibited a linear curve (no change in lag time). In terms of cholesterol oxidation, estradiol was far more effective than alpha-tocopherol or beta-carotene in inhibiting oxysterol formation (microg/ml plasma) (control = 24.56 +/- 2.31, beta-carotene = 20.59 +/- 3.32, alpha-tocopherol = 20.19 +/- 1.58, estradiol = 14.38 +/- 0.70). This study shows that estradiol is as effective an antioxidant as alpha-tocopherol in terms of fatty acid peroxidation but is far more effective than alpha-tocopherol in terms of cholesterol peroxidation.

Oxidation of high density lipoproteins: characterization and effects on cholesterol efflux from J774 macrophages

Oxidative modification of high density lipoproteins (HDL) may alter their capacity to mediate cellular cholesterol efflux. We studied the kinetics of copper-mediated oxidation of HDL and cholesterol efflux mediated by unmodified and oxidized HDL (oxHDL). Oxidation was measured by increases in absorbance at 234 nm (ΔA234), production of thiobarbituric acid reactive substances (TBARS) and loss of trinitrobenzene sulfonic acid reactivity. Oxidation was dependent on copper concentration and showed a lag phase and propagation phase. Efflux of cholesterol from J774 macrophages measured by appearance of cellular [ 3H]cholesterol in the medium was lower by 16% after 4 h and 36% after 24 h with oxHDL compared to HDL. OxHDL-mediated efflux was also lower by 27% to 36% at lipoprotein concentrations of 10 to 200 μg protein/ml. Cholesterol efflux correlated negatively with TBARS production ( r= −0.97, P < 0.003) and ΔA234 ( r = −0.77, P < 0.080). There was no difference in efflux mediated by apoproteins prepared from HDL and oxHDL. Efflux measured by change in cholesterol mass in medium was 78% lower with oxHDL. Inhibition of oxidation with butylated hydroxytoluene maintained the capacity of HDL to stimulate efflux. These results suggest that oxidation of HDL may impair its protective role against atherosclerosis.

Inhibitory effects of oxidized low-density lipoprotein on the activity of plasma lecithin: cholesterol acyltransferase.

Lecithin: cholesterol acyltransferase (LCAT) is an enzyme that is important in the cholesterol reverse-transport system by converting the surface cholesterol of high-density lipoprotein (HDL) to cholesterol ester inside HDL. We have investigated changes in plasma LCAT activity induced by oxidized low-density lipoprotein (ox-LDL). (i) LCAT activity was inhibited by the addition of even a small amount of ox-LDL, (ii) This inhibitory effect was dose-dependent and was related to the degree of oxidation of LDL, (iii) As this inhibitory effect was not prevented by antioxidants, it is suggested that it does not occur via oxidation of substrate HDL. These results suggest that cholesterol reverse-transport via HDL may be affected by ox-LDL and they are noteworthy in suggesting a new physiological function for ox-LDL.

Preserved antioxidative defense of lipoproteins in renal failure and during hemodialysis

Contact to artificial surfaces during hemodialysis activates leukocytes, which then form oxidized arachidonic acid products and free radicals. This might promote the oxidative modification of low-density lipoproteins (LDL) that play a key role in the initiation of atherosclerosis. Thus, leukocyte activation could specifically contribute to the high mortality from atherosclerotic complications on long-term hemodialysis. Therefore monitored LDL and high-density lipoprotein (HDL) resistance to copper-stimulated oxidation in patients with end-stage renal disease on maintenance hemodialysis with cellulose acetate or polysulfone membranes (n = 12), in patients with chronic renal failure (n = 13) and in healthy controls (n = 12). Six of the dialysis patients were restudied during a single cuprophane dialysis. Circulating leukocytes were reversibly reduced early in hemodialysis with cellulose acetate (minimum, 83.6% ± 7.4% of baseline values at 30 minutes after dialysis start), polysulfone (minimum, 80.4% ± 10.5% at 15 minutes; P < 0.05) and cuprophane (minimum, 24.5% ± 8.5% at 60 minutes; P < 0.0001). Despite the leukocyte activation, LDL oxidation lag time was not shortened in comparison with healthy controls and was even prolonged at the end of cellulose acetate ( P < 0.05) and cuprophane ( P < 0.05) dialysis. HDL oxidation lag time increased (12.6% ± 0.9%; P < 0.0001) 15 to 60 minutes after start of hemodialysis and returned to predialysis values thereafter. In patients with chronic renal failure, the lag time of HDL oxidation was significantly prolonged (13.34 minutes ± 0.9) compared with healthy controls (10.91 ± 2.0 minutes; P < 0.01) as well as compared with the dialysis patients at baseline (9.9 minutes ± 1.4; P < 0.01). The similar pattern among these groups in LDL oxidation just missed overall significance ( P = 0.07). Therefore, neither LDL nor HDL antioxidative defenses are compromised by hemodialysis-associated leukocyte activation. The prolonged lag phase of HDL oxidation early after the start of hemodialysis may be related to heparin-induced lipoprotein lipase activity with rapid lipid changes in HDL composition. The increased antioxidative defense of lipoproteins in chronic renal failure appears to be related to the retention of reducing metabolic end products.

Interaction of oxidized HDLs with J774-A1 macrophages causes intracellular accumulation of unesterified cholesterol.

Uptake of modified lipoproteins by resident arterial monocytes/macrophages is believed to be a key event in the formation of foam cells and thus in the early phases of atherosclerosis. Low-density lipoproteins (LDLs) that undergo oxidative changes become suitable for uptake by macrophages through a specific scavenger receptor that leads to cholesteryl ester accumulation. Because the interaction of other oxidized lipoproteins with macrophages has been poorly investigated, we studied the effect of oxidatively modified high-density lipoproteins (HDLs) on the sterol metabolism of J774-A1 macrophages. Unlike native HDLs, oxidized HDLs caused a concentration-dependent accumulation of unesterified cholesterol and decreased [14C]oleate incorporation into steryl esters. Oxidized HDLs also decreased [14C]acetate incorporation into newly synthesized sterols. Cell surface binding of 125I-oxidized HDLs to the macrophages was saturable, with an apparent dissociation constant (Kd) of 0.96 nmol/mL. Both oxidized and acetylated LDLs but not native lipoproteins could compete for binding of 125I-oxidized HDL. The data support the conclusion that the effects elicited by oxidized HDLs on the sterol metabolism of macrophages are significantly different from those of native HDLs. The binding of oxidized HDLs to macrophages occurs at sites that are likely the same as those for modified LDLs. We speculate that, if occurring in vivo, HDL oxidation would generate modified lipoproteins capable of modulating the cholesterol homeostasis of macrophages.

Cross-linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu 2+-mediated oxidation

A recent study demonstrated that Cu 2+-mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457–6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross-linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane-treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross-linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.