Copper-mediated Low-density Lipoprotein Oxidation Research Articles

Soy proteins reduce progression of a focal lesion and lipoprotein oxidiability in rabbits fed a cholesterol-rich diet

The effects of different dietary proteins on the progression of a focal atheromatous lesion and on lipoprotein oxidiability were studied in male New Zealand rabbits. Focal lesions were induced on common carotid arteries by applying an electric current, using a bipolar microcoagulator. After surgery, animals were fed for 90 days two different diets, both with 1% cholesterol, 15% saturated fatty acids and 20% protein: the protein source was constituted in one group (SOY) by 16% soy protein isolate plus 4% milk whey proteins, in the other (CASEIN) by 16% casein plus 4% milk whey proteins. Lower levels of plasma cholesterol and triglycerides (−47 and −65%, respectively) ( P<0.05) were detected in the SOY versus the CASEIN group at the end of treatment. Cryosection analyses of the carotids, indicated a highly significant reduction (−39%; P<0.05) in the focal lesion progression in the SOY versus the CASEIN group. Copper-mediated oxidation of low-density lipoprotein (LDL) from rabbits fed the two different diets, performed in vitro by analysis of conjugated diene formation, showed a significantly longer lag phase in the SOY (150±5 min) versus the CASEIN animals (20±3 min) ( P<0.05). These data, while confirming the well-known lipid lowering properties of soy proteins, indicate, in this animal model, a remarkable activity on a focal atheromatous lesion, possibly also linked to a powerful antioxidant activity.

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu 2+ is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu 2+ and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 μM there is little quenching, whereas above 10 μM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue ε-aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His–X–His sequence that is located about 5 Å from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.

Seasonal Variation in Copper-mediated Low-density Lipoprotein Oxidation In Vitro is Related to Varying Plasma Concentration of Oxidised Lipids in Summer and Winter

The seasonal variation of CuCl 2 -mediated low density lipoprotein (LDL) oxidation (10 w M Cu 2+ , lag phase, rate of oxidation and maximum absorbance at 234 nm) were measured in 43 men and women on 4-6 occasions (mean 5.7 - 0.5 ) over a 12-month period. The lag phase averaged 52.7 - 0.6 min and did not differ by gender. Lag phase and rate of the rapid propagation phase of LDL oxidation showed a sinusoidal pattern over the year (increased and reduced oxidative susceptibility during January and June-July, respectively; both p <0.001 ). Changes in plasma f -tocopherol, ascorbic acid, lycopene or g -carotene concentrations did not explain seasonal differences in oxidative susceptibility of LDL in vitro. Nor did plasma lipid content of linoleic acid, the main substrate of lipid peroxidation, vary. However, the amount of hydroperoxy- plus hydroxy-fatty acids in plasma lipids varied according to season ( p <0.024) and was related to the lag phase ( r = m 0.26, p <0.001). Seasonal variation in oxidative susceptibility was not significant after adjusting for hydroperoxy- plus hydroxy-fatty acids ( p =0.506). Isolated LDL is more vulnerable to Cu 2+ -induced lipid peroxidation during the winter and this may be due to the higher amount of oxidised lipids during that period.

Contribution of superoxide to reduced antioxidant activity of glycoxidative serum albumin

Hyperglycemia increases oxidative stress in various tissues and leads to diabetic cardiovascular complication. Dyslipidemia, such as an increase in oxidized low-density lipoprotein (LDL), is well recognized in diabetic patients with hyperglycemia. However, the mechanism by which hyperglycemia causes the increased LDL oxidation remains unclear. Albumin is the most abundant protein in the circulation, and can function as an antioxidant. Therefore, we examined whether glycoxidative modification inhibits the antioxidant activity of albumin to LDL oxidation and clarified the mechanism by which this modification may suppress its antioxidant activity. Human serum albumin (HSA) was incubated in phosphate-buffered saline with and without glucose at 37 degrees C for up to 8 weeks under aerobic conditions (referred to as glycoxidation (goHSA) and oxidation (oHSA), respectively). Metal chelator-treated, nonoxidative HSA (chHSA) and freshly prepared HSA (fHSA) were used as controls. N(epsilon)-(carboxymethyl)lysine (CML), a glycoxidative product, was determined by enzyme-linked immunosorbent assay. Oxidation was estimated by measuring the thiols of the HSA molecule. Copper-mediated oxidation of LDL was conducted in the presence or absence of modified HSAs at 37 degrees C for 6 days. Malondialdehyde and negative charge of LDL were measured. To clarify the mechanism of reduced antioxidant activity of HSA, we examined firstly the binding activity of modified HSAs to copper, and secondly the effects of free radical scavengers on the formation of malondialdehyde. CML was formed in goHSA in a time- and concentration-dependent manner. Both goHSA and oHSA significantly decreased the contents of free thiol groups compared to ch- and fHSAs. The antioxidant activity of goHSA to LDL oxidation was the lowest among various modified HSAs. The oHSA showed a moderate decrease in antioxidant activity. The binding activity of go- and oHSAs to copper was lower than that of ch- and fHSAs. The formation of MDA from LDL oxidation in the presence of goHSA was completely inhibited by Tiron (1,2-dihydroxy-3,5-benzenedisulfonic acid) and superoxide dismutase. In contrast, catalase and mannitol had no effect. Our results indicate that in vitro glycoxidation of HSA induced a marked loss of antioxidant activity of this molecule to copper-mediated oxidation of LDL, which may be caused by the generation of superoxide.

Antioxidant effects of 1,5-anhydro-D-fructose, a new natural sugar, in vitro.

The antioxidant effects of 1,5-anhydro-D-fructose (1,5-AF), a unique anhydrohexulose, were studied in 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution, in human cells along with lipid peroxidation of low-density lipoprotein (LDL). We have confirmed that 1,5-AF scavenges DPPH radicals directly in solution and inhibits the formation of hydrogen peroxide and superoxide anion, typical reactive oxygen species (ROS), induced by phorbol myristate acetate (PMA) in a dose-dependent manner in THP-1 cells. We also observed the dose-dependent antioxidant effects of 1,5-AF on copper-mediated LDL oxidation. These findings suggest that 1,5-AF might play a role in reducing the risk of atherosclerosis and may help prevent coronary heart disease.

Early stages of LDL oxidation: apolipoprotein B structural changes monitored by infrared spectroscopy

Changes in the conformation of apoliprotein B-100 in the early stages of copper-mediated low density lipoprotein oxidation have been monitored by infrared spectroscopy. During the lag phase no variation in structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not modified, no changes in secondary structure are observed, but the thermal profile of the band corresponding to α-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation but not with β-sheet components. When aldehyde formation starts, a decrease of ~3% in the area of bands corresponding to α-helix and β-sheet is produced, concomitantly with an increase in β-turns and unordered structure. The two bands corresponding to β-turns vary as well under these conditions, indicating changes in these structures. Also at this stage the thermal profile shows variations in frequency for the bands corresponding to both α-helix and β-sheet.The results are consistent with the hypothesis that as soon as the polyunsaturated fatty acids from the particle core are modified, this change is reflected at the surface, in the α -helical components contacting the monolayer.

Soy and alfalfa phytoestrogen extracts become potent low-density lipoprotein antioxidants in the presence of acerola cherry extract.

Postmenopausal women have an increased risk of coronary heart disease. Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis, and the presence of modified LDL (LDL(-)) in plasma appears to represent LDL oxidation in vivo. Because previous studies have demonstrated a strong antiatherogenic effect of estrogen due to its antioxidant activity and similar antioxidant activity was found for specific isoflavones derived from soy extract, the antioxidant activity of a phytoestrogen extract derived from soy and alfalfa was studied. Copper-mediated LDL oxidation was inhibited in the presence of soy and alfalfa extracts, and this effect was further enhanced in the presence of acerola cherry extract, which is rich in ascorbic acid. Male rabbit aortic endothelial cells pretreated with soy extract were resistant to the toxic effects of high levels of LDL and LDL(-), and a lesser, but significant protection, was also afforded by alfalfa extract. Cell-mediated oxidation of LDL, measured by LDL(-) formation, was inhibited in the presence of soy extract but not alfalfa extract. However, in the presence of acerola cherry extract, both soy and alfalfa extracts potently inhibited the formation of LDL(-). These findings show that acerola cherry extract can enhance the antioxidant activity of soy and alfalfa extracts in a variety of LDL oxidation systems. The protective effect of these extracts is attributed to the presence of flavonoids in soy and alfalfa extracts and ascorbic acid in acerola cherry extract, which may act synergistically as antioxidants. It is postulated that this synergistic interaction among phytoestrogens, flavonoids, and ascorbic acid is due to the "peroxidolitic" action of ascorbic acid, which facilitates the copper-dependent decomposition of LDL peroxides to nonradical products; this synergy is complemented by a mechanism in which phytoestrogens stabilize the LDL structure and suppress the propagation of radical chain reactions. The combination of these extracts markedly lowers the concentrations of phytoestrogens required to achieve significant antioxidant activity toward LDL.

Direct Evidence for apo B-100-Mediated Copper Reduction: Studies with Purified apo B-100 and Detection of Tryptophanyl Radicals

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely α-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of α-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.

Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro.

Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The oxidation of LDL was assessed by the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS) and the loss of tryptophan fluorescence. At concentrations of 5 and 25 microM, all of the prenylchalcones tested inhibited the oxidation of LDL (50 microg protein/ml) induced by 2 microM copper sulfate. The prenylflavanones showed less antioxidant activity than the prenylchalcones, both at 5 and 25 microM. At 25 microM, the nonprenylated chalcone, chalconaringenin (CN), and the nonprenylated flavanone, naringenin (NG), exerted prooxidant effects on LDL oxidation, based on TBARS formation. Xanthohumol (XN), the major prenylchalcone in hops and beer, showed high antioxidant activity in inhibiting LDL oxidation, higher than alpha-tocopherol and the isoflavone genistein but lower than the flavonol quercetin. When combined, XN and alpha-tocopherol completely inhibited copper-mediated LDL oxidation. These findings suggest that prenylchalcones and prenylflavanones found in hops and beer protect human LDL from oxidation and that prenylation antagonizes the prooxidant effects of the chalcone, CN, and the flavanone, NG.

Protective effect of olive oil and its phenolic compounds against low density lipoprotein oxidation.

The protective effect of phenolic compounds from an olive oil extract, and of olive oils with (extra-virgin) and without (refined) phenolic components, on low density lipoprotein (LDL) oxidation was investigated. When added to isolated LDL, phenolics [0.025-0.3 mg/L caffeic acid equivalents (CAE)] increased the lag time of conjugated diene formation after copper-mediated LDL oxidation in a concentration-dependent manner. Concentrations of phenolics greater than 20 mg/L inhibited formation of thiobarbituric-acid reactive substances after AAPH-initiated LDL oxidation. LDL isolated from plasma after preincubation with phenolics (25-160 mg/L CAE) showed a concentration-dependent increase in the lag time of conjugated diene formation after copper-mediated LDL oxidation. Refined olive oil (0 mg/L CAE) and extra-virgin olive oil (0.1 and 0.3 mg/L CAE) added to isolated LDL caused an increase in the lag time of conjugated diene formation after copper-mediated LDL oxidation that was related to olive oil phenolic content. Multiple regression analysis showed that phenolics were significantly associated with the increase in lag time after adjustment for effects of other antioxidants; alpha-tocopherol also achieved a statistically significant effect. These results indicate that olive oil phenolic compounds protect LDL against peroxyl radical-dependent and metal-induced oxidation in vitro and could associate with LDL after their incubation with plasma. Both types of olive oil protect LDL from oxidation. Olive oil containing phenolics, however, shows more antioxidant effect on LDL oxidation than refined olive oil.

Aceite de oliva e inhibición de la oxidación de las lipoproteínas de baja densidad. Importancia de los compuestos fenólicos

To investigate the protective effect of several olive oils with different phenolic composition on low density lipoprotein susceptibility to oxidation. Refined olive oil (phenolic content: 0 mg/l caffeic acid equivalents [CAE]), common olive oil (0.1 and 0.3 mg/l CAE), and virgin olive oil diluted with refined olive oil (0.1 y 0.3 mg/l CAE), were added to isolated low density lipoprotein. Conjugated dienes formation was monitored after copper-mediated low density lipoprotein oxidation. An increase in the lag time of conjugated dienes formation after copper-mediated low density lipoprotein oxidation was observed linked to olive oil phenolic content (p < 0.0001, ANOVA). Multiple regression analysis showed that phenolics were the most significant antioxidants with 0.1 mg/l--increase in phenolic concentration, adjusted for alpha-tocopherol and beta-carotene, was 72 minutes (95% confidence interval [CI] 64 to 80 min) for common olive oil, and 111 min (CI 95%: 100-123 min) for virgin olive oil. In common olive oil alpha-tocopherol levels were significatively associated with the increase in the lag time (p = 0.003), reaching in virgin olive oil a borderline significant (p = 0.084). Olive oil containing phenolics showed more antioxidant effect on low density lipoprotein oxidation than refined olive in relation to its phenolic content. The nature of the phenolic content influences the antioxidant capacity of an olive oil.

Influence of low density lipoprotein (LDL) subfraction profile and LDL oxidation on endothelium-dependent and independent vasodilation in patients with type 2 diabetes.

Recent studies have suggested that hypercholesterolemia is associated with endothelial dysfunction. In patients with type 2 diabetes mellitus, dyslipidemia is mainly characterized by hypertriglyceridemia, low high density lipoprotein, and a preponderance of small dense low density lipoprotein (LDL) particles. We have examined the relationships among LDL subfractions, the susceptibility of LDL to oxidation in vitro, and endothelial function in type 2 diabetes mellitus. LDL subfractions were measured by density gradient ultracentrifugation. The susceptibility of LDL to oxidation was determined by measuring the kinetics of conjugated dienes formation during copper-mediated oxidation of LDL. Endothelium-dependent and independent vasodilation of the brachial artery were assessed by high resolution vascular ultrasound. Diabetic patients had a higher concentration of small dense LDL-III than matched controls (P < 0.01). The lag phase of conjugated dienes formation was shorter in the diabetic patients (P < 0.05), and the rate of LDL oxidation was faster (P < 0.05). Both endothelium-dependent (P < 0.01) and independent dilation of the brachial artery (P < 0.01) were impaired in the diabetic patients. On multivariate analysis, the rate of oxidation and LDL-III concentration accounted for 12% and 6%, respectively, of the variation in endothelium-dependent vasodilation (adjusted r2 = 0.18; P < 0.05), whereas LDL-III concentration and the maximum amount of conjugated dienes formed accounted for 27% and 5%, respectively, of the variation in endothelium-independent vasodilation (adjusted r2 = 0.32; P < 0.01) in the diabetic patients. In conclusion, endothelial and smooth muscle cell dysfunction in type 2 diabetes were related to abnormalities in LDL subfractions and in LDL oxidation.

Indole Derivatives, 3,3′-Di-indolylmethane and Ascorbigen, Inhibit Oxidation of Human Low-density Lipoproteins In-vitro

Oxidized low-density lipoprotein (LDL) has atherogenic properties. Antioxidants that can prevent LDL oxidation may therefore act as anti-atherogens. In this study the effect of two indole derivatives, 3,3′-di-indolylmethane and ascorbigen, which are present in cruciferous vegetables, on copper (Cu)-induced human LDL oxidation in-vitro was examined. Both compounds inhibited copper-mediated oxidation of LDL in a concentration-dependent manner by lengthening the time (lag phase) for conjugated diene formation, decreasing the propagation rate and the maximum concentration of conjugated dienes at the concentration of 1 μM or higher. 3,3′-Di-indolylmethane completely inhibited the diene formation at a concentration of 20 μM. Our data suggest that 3,3′-di-indolylmethane and ascorbigen could act as antioxidants in-vitro.

LDL isolated from Greek subjects on a typical diet or from American subjects on an oleate-supplemented diet induces less monocyte chemotaxis and adhesion when exposed to oxidative stress.

The mechanisms underlying the cardiovascular benefits of Mediterranean-style diets are not fully understood. The high content of monounsaturated fatty acids in Mediterranean-style diets derived from oleate-rich olive oil may be beneficial in reducing low density lipoprotein (LDL) oxidation and its subsequent development of atherogenic properties. This study sought to assess the proinflammatory potential of LDL isolated from subjects consuming a diet naturally rich in olive oil. LDL was isolated from 18 Greek, 18 American, and 11 Greek-Americans subjects, all of whom were living in the United States. Fatty acid composition and vitamin E levels of LDL were determined, as was the extent of copper-mediated LDL oxidation. LDL was also mildly oxidized by exposure to fibroblasts overexpressing 15-lipoxygenase and tested in vitro for bioactivity by determining its ability to stimulate monocyte chemotaxis and adhesion to endothelial cells. To confirm that dietary fatty acids influence the proinflammatory properties of mildly oxidized LDL, LDL was also isolated from 13 healthy American subjects after consumption of an 8-week liquid diet supplemented with either oleic (n=6) or linoleic (n=7) acid and tested for bioactivity in a similar fashion. There were no differences in the baseline lipid profiles among the Greeks, Americans, or Greek-Americans. Oleic acid content in LDL was 20% higher in the Greek compared with the American or Greek-American subjects (P<0.001). The extent of in vitro LDL oxidation, measured by conjugated diene formation, was lower in the Greek subjects (P<0.02), but there was no difference in the lag time. Induction of monocyte chemotaxis and adhesion by mildly oxidized LDL was decreased by 42% in the Greek group compared with the American subjects (P<0.001). There was an inverse correlation between the oleic acid content of LDL and stimulation of monocyte chemotaxis (r=-0.64, P<0.001) and a positive correlation between the polyunsaturated fatty acid content of LDL (total linoleate and arachidonic acids levels in LDL) and stimulation of monocyte chemotaxis (r=0.51, P<0.01) in the entire cohort. There were no differences in LDL vitamin E content between the groups. In the liquid-diet groups, the oleic acid-supplemented group had a 113% higher oleic acid content in LDL and a 46% lower linoleic acid content in LDL than the linoleate-supplemented group (P<0.001), whereas the vitamin E content in LDL was equal in both groups. When exposed to oxidative stress, the LDL enriched in oleic acid promoted less monocyte chemotaxis (52% lower) and reduced monocyte adhesion by 77% in comparison with linoleate-enriched LDL (P<0.001). There was a strong, negative correlation between oleic acid LDL content and monocyte adhesion (r=-0.73, P<0.001) and a strong, positive correlation between polyunsaturated fatty acid LDL content and monocyte adhesion (r=0.87, P<0.001). This study demonstrates that dietary enrichment of LDL with oleic acid is realistic and readily achieved by using diets currently in use in Mediterranean countries. In addition, these data suggest that LDL enriched with oleic acid and reduced in polyunsaturated fatty acids may be less easily converted to a proinflammatory, minimally modified LDL.

The isoflavone genistein inhibits copper and peroxyl radical mediated low density lipoprotein oxidation in vitro

Oxidation of low density lipoprotein (LDL) is implicated in the development of atherosclerosis and dietary antioxidants may provide a useful therapy in the prevention of LDL oxidation and atheroma development. The aim of these experiments was to investigate the antioxidant activity of the soybean isoflavone, genistein, in in vitro models of LDL oxidation. Genistein inhibited copper-mediated oxidation of LDL in a concentration-dependent manner by lengthening the time for conjugated diene formation (54.1±5.1 min in control LDL and 107.1±1.8 min with 5 μmol/l genistein, P<0.001) and decreasing the oxidation rate (14.4±1.9 nmol conjugated diene/mg LDL protein/min in control LDL and 7.4±1.1 nmol conjugated diene/mg LDL protein/min with 5 μmol/l genistein, P<0.001). Peroxy radical (azo-initiated) oxidation of LDL was significantly inhibited by 200 μmol/l genistein as indicated by: (i) increase in the time required for malondialdehyde (MDA) formation (7 h incubation compared to 3 h incubation with control LDL), (ii) 32, 44 and 46% decreases in MDA concentration compared to control samples following 3, 4 and 5 h incubation, respectively and (iii) decrease in relative electrophoretic mobility (REM) of LDL. Incorporation of genistein into LDL and its resultant antioxidant activity was also investigated. LDL was isolated from plasma which had been pre-incubated with 25, 50 or 100 μmol/l genistein at 37°C for 24 h. Approximately 3–4% of genistein present in plasma was incorporated into LDL, however copper-mediated oxidation of control LDL and LDL isolated from plasma pre-incubated with genistein was not significantly different.

The dynamic reduction of Cu(II) to Cu(I) and not Cu(I) availability is a sufficient trigger for low density lipoprotein oxidation

Copper (II) is one of the most widely employed experimental models to induce in vitro low density lipoprotein (LDL) oxidation. It is generally assumed that Cu(I), generated from active reduction of Cu(II), is the real trigger for the peroxidation of polyunsaturated fatty acids in LDL. We have employed isolated human LDL challenged with Cu(II) and the Cu(I) chelator bathocuproine disulfonic acid (BC) to test the validity of this hypothesis. At lower Cu(II)/LDL molar ratios, BC completely inhibited copper-mediated LDL oxidation evaluated either as thiobarbituric acid reactive substances (TBARS) production or changes in apo B100 electrophoretic mobility. On the contrary, at higher Cu(II)/LDL molar ratios, BC did not prevent LDL oxidation but rather markedly stimulated both the generation of TBARS and the increase of apo B100 electronegativity. These oxidative modifications were completely prevented by the Cu(II) chelator EDTA. Furthermore, the BC-Cu(I) complex alone was neither redox active nor active inducer of TBARS generation. These findings indicate that, under these experimental conditions, [1]Cu(I) is not necessary to promote LDL oxidation, [2]the availability of Cu(II) is a prerequisite and [3]some of the reaction(s) involved in Cu(II) reduction may play an essential role in initiating LDL oxidation.

Simulation of the induction of oxidation of low-density lipoprotein by high copper concentrations: evidence for a nonconstant rate of initiation.

Kinetic simulation can help obtain deeper insight into the molecular mechanisms of complex processes, such as lipid peroxidation (LPO) in low-density lipoprotein (LDL). We have previously set up a single-compartment model of this process, initiating with radicals generated externally at a constant rate to show the interplay of radical scavenging and chain propagation. Here we focus on the initiating events, substituting constant rate of initiation (Ri) by redox cycling of Cu2+ and Cu+. Our simulation reveals that early events in copper-mediated LDL oxidation include (1) the reduction of Cu2+ by tocopherol (TocOH) which generates tocopheroxyl radical (TocO.), (2) the fate of TocO. which either is recycled or recombines with lipid peroxyl radical (LOO.), and (3) the reoxidation of Cu+ by lipid hydroperoxide which results in alkoxyl radical (LO.) formation. So TocO., LOO., and LO. can be regarded as primordial radicals, and the sum of their formation rates is the total rate of initiation, Ri. As experimental information of these initiating events cannot be obtained experimentally, the whole model was validated experimentally by comparison of LDL oxidation in the presence and absence of bathocuproine as predicted by simulation. Simulation predicts that Ri decreases by 2 orders of magnitude during lag time. This has important consequences for the estimation of oxidation resistance in copper-mediated LDL oxidation: after consumption of tocopherol, even small amounts of antioxidants may prolong the lag phase for a considerable time.

The effects of pH on the oxidation of low-density lipoprotein by copper and metmyoglobin are different

The amplification of low-density lipoprotein (LDL) peroxidation in vitro by copper and myoglobin are well-studied biochemical approaches for investigating the oxidative modification of LDL and its role in the pathogenesis of atherosclerosis. Since the acidity of the environment is increased in inflammatory sites, the aim of this study was to investigate the effects of acidic pH on the oxidisability of LDL mediated by the haem protein myoglobin in comparison with that of copper-mediated LDL oxidation. The results show that acidic pH enhances myoglobin-mediated LDL oxidation as measured by conjugated dienes, lipid hydroperoxides and electrophoretic mobility, whilst a retardation is observed with copper as pro-oxidant; the mechanism probably relates to the effects of pH on the decomposition and formation of lipid hydroperoxides and the relative influences of copper ions and of myoglobin under these conditions.

Oxidative degradation of cholesteryl esters in low-density lipoproteins: analysis by liquid chromatography-light scattering and protection by a new synthetic antioxidant, S20478.

Cholesteryl esters in the hydrophobic core of low-density lipoprotein (LDL) particles constitute a major molecular target during copper-mediated oxidation. To facilitate the rapid analysis and quantitation of the oxidative degradation of LDL cholesteryl esters, we describe a new approach based on light scattering detection following separation by HPLC. We have applied this approach to the evaluation of the protective capacity of a new synthetic antioxidant, S20478, during oxidation of LDL in the presence of copper ions. HPLC separation of cholesterol and the four major molecular species of cholesteryl esters (C16:0, C18:1, C18:2 and C20:4) of LDL was achieved in a single run of 20 min with high sensitivity (50 ng) and low background. Time course studies of the oxidative modification of LDL (ratio LDL protein: copper, 100 micrograms/mL: 1 microM) revealed that the content of unsaturated cholesteryl esters (C20:4 and C18:2) decreased (-30% and -15%, respectively) within 90 min of copper-mediated oxidation, while only minor degradation (up to 15%) of monounsaturated (C18:1) and saturated (C16:0) esters occurred. At 24 hours of oxidation, only traces (< 5%) of the C20:4 and C18:2 esters were detectable; whereas 52% of the C18:1 ester remained (P < 0.01). Of the saturated esters, only minor proportions (35% or less) underwent oxidative modification. In addition, some 81% of free cholesterol was conserved as the native sterol. The synthetic antioxidant, S20478 (50 microM) was capable of inhibiting the initiation and the propagation of copper-mediated LDL oxidation as determined by the time- and dose-dependent inhibition of the formation of conjugated dienes and thiobarbituric acid-reactive substances, as well as the conservation of the net electrical charge of LDL; indeed S20478 conserved cholesteryl esters in their native form up to 24 hours. However, after prolonged exposure to copper ions (48 hours), only 47% of the unsaturated esters remained (C18:2, P < 0.05). Nonetheless, S20478 (10 microM) was more efficient in inhibiting copper-mediated LDL oxidation as compared to probucol at the same concentration. These findings suggest that S20478 may be of potential interest in a new antioxidant approach to therapeutic stabilisation and regression of atherosclerotic plaques. Moreover, this method should prove useful in the assessment of the integrity of native LDL, and provides a new chemical marker of the degree of LDL oxidation.

In vitro oxidised HDL exerts a cytotoxic effect on macrophages

The cytotoxic effect of native high density lipoprotein (n-HDL) and oxidised high density lipoprotein (ox-HDL) on macrophages was studied and compared with that of low density lipoprotein (LDL). Copper-mediated oxidation of HDL and LDL was conducted in vitro and assessed by the analysis of conjugated dienes (CD). The kinetics of CD production during lipoprotein oxidation showed that HDL, relative to LDL, exhibited a shorter lag phase (47.7 ± 17.8 vs. 82.9 ± 24.5 min), higher diene production (242.2 ± 23.0 vs. 210.4 ± 14.9 nmol/mg lipid) and reached maximal diene concentration in less time (100.0 ± 35.4 vs. 136.4 ± 27.9 min). The maximal rate of CD production was 5.38 ± 1.30 nmol/mg lipid/min for HDL and 4.42 ± 0.60 nmol/mg lipid/min for LDL. Vitamin E concentration was higher in HDL than in LDL (2.76 ± 0.41 vs. 2.19 ± 0.33 μg α-tocopherol equivalents/mg lipid). Ox-HDL and oxidised LDL (ox-LDL), under the same experimental conditions, were cytotoxic to macrophages in a dose-dependent manner. At the same protein, or total mass concentration, ox-HDL was less cytotoxic than ox-LDL. However, when both lipoproteins were compared at the same lipid or cholesterol concentrations, ox-HDL was equally or more cytotoxic than ox-LDL. In conclusion, HDL is more susceptible to in vitro oxidation than LDL and the resultant modification of HDL converts this lipoprotein into a cytotoxic particle.