Rate Of Low-density Lipoprotein Oxidation Research Articles

Virtual exploration of early stage atherosclerosis.

Biological mechanisms contributing to atherogenesis are multiple and complex. The early stage of atherosclerosis (AS) is characterized by the accumulation of low-density lipoprotein (LDL) droplets, leading to the creation of foam cells (FC). To address the difficulty to explore the dynamics of interactions that controls this process, this study aimed to develop a model of agents and infer on the most influential cell- and molecule-related parameters. FC started to accumulate after six to eight months of simulated hypercholesterolemia. A sensitivity analysis revealed the strong influence of LDL oxidation rate on the risk of FC creation, which was exploited to model the antioxidant effect of statins. Combined with an empirical simulation of the drug ability to decrease the level of LDL, the virtual statins treatment led to reductions of oxidized LDL levels similar to reductions measured in vivo. An Open source software was used to develop the agent-based model of early AS. Two different concentrations of LDL agents were imposed in the intima layer to simulate healthy and hypercholesterolemia groups of 'virtual patients'. The interactions programmed between molecules and cells were based on experiments and models reported in the literature. A factorial sensitivity analysis explored the respective effects of the less documented model parameters as (i) agent migration speed, (ii) LDL oxidation rate and (iii) concentration of autoantibody agents. Finally, the response of the model to known perturbations was assessed by introducing statins agents, able to reduce the oxidation rate of LDL agents and the LDL boundary concentrations. jerome.noailly@upf.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Lifestyle intervention enhances high-density lipoprotein function among patients with metabolic syndrome only at normal low-density lipoprotein cholesterol plasma levels

Metabolic syndrome (MetS) is associated with altered lipoprotein metabolism and impairment in the functionality of small, dense high-density lipoprotein (HDL) particles secondary to compositional alterations. The objective of this study was to investigate the capacity of a lifestyle program to improve the composition and antioxidative function (AOX) of small dense HDL3c in MetS. Patients with MetS (n=33) not taking lipid-lowering drugs were recruited to follow a 12-week educational program to reduce caloric intake and to increase physical activity. HDL subfractions were preparatively isolated by isopycnic density-gradient ultracentrifugation. AOX of HDL3c was assessed as its capacity to inhibit low-density lipoprotein oxidation induced by an azoinitiator. AOX of HDL3c was significantly improved (mean reduction in the propagation rate of low-density lipoprotein oxidation by HDL3c, -6.8%, P=.03) and systemic oxidative stress, assessed as plasma levels of 8-isoprostanes, tended to decrease in normocholesterolemic MetS patients (low-density lipoprotein cholesterol [LDL-C] < 130mg/dL) but not in patients with elevated LDL-C levels and in the whole study population. In both the whole study population and the normocholesterolemic subgroup, lifestyle intervention resulted in a significant degree of normalization of HDL3c composition, (enrichment in apolipoprotein A-I and cholesteryl esters, depletion in triglycerides), which was more pronounced at LDL-C < 130mg/dL. In patients with MetS, a lifestyle program improves AOX of small, dense HDL in subjects with normal LDL-C levels. Correction of HDL composition, involving partial normalization of apoA-I content and core lipid composition, 2 central features of the lipid hydroperoxide-inactivating capacity of HDL, may account for this effect.

Kinetic modeling of low density lipoprotein oxidation in arterial wall and its application in atherosclerotic lesions prediction

Oxidation of low-density lipoprotein (LDL) is one of the major factors in atherogenic process. Trapped oxidized LDL (Ox-LDL) in the subendothelial matrix is taken up by macrophage and leads to foam cell generation creating the first step in atherosclerosis development. Many researchers have studied LDL oxidation using in vitro cell-induced LDL oxidation model. The present study provides a kinetic model for LDL oxidation in intima layer that can be used in modeling of atherosclerotic lesions development. This is accomplished by considering lipid peroxidation kinetic in LDL through a system of elementary reactions. In comparison, characteristics of our proposed kinetic model are consistent with the results of previous experimental models from other researches. Furthermore, our proposed LDL oxidation model is added to the mass transfer equation in order to predict the LDL concentration distribution in intima layer which is usually difficult to measure experimentally. According to the results, LDL oxidation kinetic constant is an important parameter that affects LDL concentration in intima layer so that existence of antioxidants that is responsible for the reduction of initiating rates and prevention of radical formations, have increased the concentration of LDL in intima by reducing the LDL oxidation rate.

Human LDL core cholesterol ester packing: three-dimensional image reconstruction and SAXS simulation studies

Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a "disrupted shell"-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition.

Plasma asymmetric dimethylarginine (ADMA), nitrate and the indices of low-density lipoprotein oxidation

Oxidative modification of low-density lipoprotein (LDL) is an important contributor to atherosclerosis. Also, oxidized LDL is suspected to cause accumulation of asymmetric dimethylarginine (ADMA), an endogenous competitive nitric oxide synthase inhibitor, which is suggested to be an independent risk factor for atherosclerosis. This study was performed to evaluate how plasma ADMA is related to plasma nitric oxide production, oxidized LDL and ex vivo susceptibility of LDL to oxidation in mildly hypercholesterolemic otherwise healthy subjects. Plasma ADMA was determined using high performance liquid chromatography tandem mass spectrometry. LDL oxidation was estimated by the lag time and rate of copper-induced LDL oxidation. The nitric oxide production in plasma was estimated based on nitrate (NO(3)(-)) determination and plasma oxidized LDL was determined by a capture ELISA. Low ADMA was a significant determinant for high LDL oxidation rate and concentration of plasma ADMA was associated with nitrate levels. There may be an interplay between LDL fatty acid oxidation rate and plasma ADMA and nitrate. We hypothesize that plasma ADMA has a bivalent role: high ADMA may have a protective role in decelerating LDL fatty acid oxidation and also a risk factor for endothelial dysfunction by decreasing availability of nitric oxide.

Effect of bicarbonate on iron-mediated oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) may play an important role in atherosclerosis. We studied the effects of bicarbonate/CO2 and phosphate buffer systems on metal ion-catalyzed oxidation of LDL to malondialdehyde (MDA) and to protein carbonyl and MetO derivatives. Our results revealed that LDL oxidation in mixtures containing free iron or heme derivatives was much greater in bicarbonate/CO2 compared with phosphate buffer. However, when copper was substituted for iron in these mixtures, the rate of LDL oxidation in both buffers was similar. Iron-catalyzed oxidation of LDL was highly sensitive to inhibition by phosphate. Presence of 0.3-0.5 mM phosphate, characteristic of human serum, led to 30-40% inhibition of LDL oxidation in bicarbonate/CO2 buffer. Iron-catalyzed oxidation of LDL to MDA in phosphate buffer was inhibited by increasing concentrations of albumin (10-200 microM), whereas MDA formation in bicarbonate/CO2 buffer was stimulated by 10-50 microM albumin but inhibited by higher concentrations. However, albumin stimulated the oxidation of LDL proteins to carbonyl derivatives at all concentrations examined in both buffers. Conversion of LDL to MDA in bicarbonate/CO2 buffer was greatly stimulated by ADP, ATP, and EDTA but only when EDTA was added at a concentration equal to that of iron. At higher than stoichiometric concentrations, EDTA prevented oxidation of LDL. Results of these studies suggest that interactions between bicarbonate and iron or heme derivatives leads to complexes with redox potentials that favor the generation of reactive oxygen species and/or to the generation of highly reactive CO2 anion or bicarbonate radical that facilitates LDL oxidation.

α-tocopherol is important to inhibit low-density lipoprotein oxidation in smokers

Abstract Smoking is recognized as an independent risk factor for atherosclerosis. This study was designed to study the effect of smoking on plasma and low-density lipoprotein (LDL) antioxidant concentrations and LDL oxidation susceptibility. A total of 89 subjects, consisting of 27 smokers and 62 nonsmokers, were recruited. Biochemical values and plasma ascorbic acid, α-tocopherol, and LDL α-tocopherol levels were assessed. LDL lag time, oxidation rate, and correlation between LDL α-tocopherol and LDL oxidation rate were determined. No differences were demonstrated for ascorbic acid and α-tocopherol concentrations in plasma between smokers and nonsmokers; however, LDL α-tocopherol was significantly higher for nonsmokers ( P = 0.012). Furthermore, the lag time for LDL oxidation was significantly longer for nonsmokers ( P = 0.019), whereas the LDL oxidation rate was significantly greater for smokers ( P = 0.018). Additionally, a highly significant negative correlation was demonstrated for LDL α-tocopherol concentration and LDL oxidation rate in smokers ( r = −0.440, P = 0.026), but not in nonsmokers. The results of this study suggest that LDL α-tocopherol is more susceptible to oxidative stress caused by smoking than ascorbic acid and α-tocopherol in plasma. For smokers, the presence of α-tocopherol in LDL effectively protects LDL from oxidation.

Efficacy of a complex multivitamin supplement

Multivitamin supplements are often sold to consumers with the claim that supplements modify risk factors associated with disease. Because few products are validated scientifically, we examined the effects of a 24-ingredient multivitamin formula in an open-label pilot investigation. We examined 150 subjects for specific endpoints including blood concentrations of selected vitamins, homocysteine, lipids, and low-density lipoprotein (LDL) oxidation indices at baseline and at 12 and 24 wk. One hundred forty-one subjects were successfully assayed for and showed significant time effects for homocysteine and vitamin B6 (as pyridoxal-5'-phosphate), B12, and folic acid concentrations during treatment (P < 0.0001). Vitamin B6, B12, and folic acid concentrations were significantly elevated at weeks 12 and 24 (P < 0.05). Homocysteine concentration decreased significantly during the same periods (7.9 +/- 2.4 versus 6.7 +/- 1.7 versus 6.7 +/- 1.9 mM/mL; P < 0.05). There were correlations relating homocysteine to vitamins B6 (P = 0.001, r(2) = 0.03), B12 (P < 0.001, r(2) = 0.09), and folic acid (P = 0.001, r(2) = 0.10). Significant time effects were noted for 121 subjects successfully assayed for vitamin C, E, beta-carotene, LDL oxidation rate, and LDL lag time (P < 0.0001). Post hoc assessment showed elevations in vitamin C, E, and beta-carotene concentrations at 12 and 24 wk (P < 0.05). LDL oxidation lag time at baseline (57.5 +/- 13.9 min) increased by 12 wk (63.5 +/- 19.0 min; P < 0.05) and 24 wk (63.8 +/- 16.3 min; P < 0.05). LDL oxidation rate at baseline (9.7 +/- 3.0 microM x min(-1). g(-1)) was reduced at 12 wk (7.1 +/- 2.5 microM x min(-1) x g(-1); P < 0.05) and 24 wk (6.0 +/- 2.0 microM x min(-1) x g(-1); P < 0.05). Only vitamin C was significantly correlated with LDL oxidation rate (P = 0.05, r(2) = 0.003). A multi-ingredient vitamin formula with antioxidant properties has measurable effects on homocysteine and LDL oxidation indices.

Evidence for inhibition of low density lipoprotein oxidation and cholesterol accumulation by apolipoprotein H (β 2-glycoprotein I)

Low density lipoprotein (LDL) oxidation and lipid accumulation are thought to enhance the progression of atherosclerosis. Apolipoprotein H (apoH) has been implicated in the development of human atherosclerosis. However, the roles of apoH in the oxidative modification of LDL and cellular accumulation of lipid constituents remained uncharacterized. In this study, the level of plasma apoH was found to be significantly associated with the oxidative susceptibility of LDL in human subjects. Plasma levels of apoH were positively correlated with the lag time but negatively correlated with LDL oxidation rate in conjugated diene formation. By using a J774 A.1 macrophage culture system, we found that apoH could not only inhibit the formation of conjugated diene and thiobarbituric acid-reactive substances, but also reduce the electrophoretic mobility of oxidized LDL. Furthermore, apoH decreased cellular accumulation of cholesterol via a reduction in cholesterol influx and an increase in cholesterol efflux. This is the first demonstration that apoH appears to have "antioxidant"-like effects on LDL oxidation. The results also suggest that apoH can inhibit the translocation of cholesterol from extracellular pools to macrophages, suggesting that apoH may play an important role in the prevention of atherosclerosis.

Fluvastatin, an HMG-CoA reductase inhibitor, protects LDL from oxidative modification in hypercholesterolemic rabbits.

The antioxidative effect of fluvastatin sodium (fluvastatin) on low-density lipoprotein (LDL) was evaluated in vivo and in vitro. Since ex vivo measurement of the LDL oxidizability is reported to reflect the response of the atherosclerotic process, LDL isolated from rabbits fed a high cholesterol diet for 4 weeks with or without fluvastatin, pravastatin or alpha-tocopherol administration was oxidized by copper ions to estimate conjugated diene formation. Fluvastatin but not pravastatin significantly prolonged the lag time of LDL oxidized by copper ions ex vivo without affecting plasma cholesterol levels at a dose of 3 mg/kg after four weeks of treatment. Alpha-tocopherol-treated rabbits showed dramatically elongated LDL oxidation lag time at a dose of 150 mg/kg. In order to assess the mechanism, the content of alpha-tocopherol, a major endogenous antioxidant in LDL was measured, and we found that only LDL isolated from alpha-tocopherol-treated rabbits contained a significantly larger amount of alpha-tocopherol than that from high cholesterol control rabbits. To elucidate the mechanism further, the effect of fluvastatin on conjugated diene formation during copper-induced LDL oxidation in vitro was studied. Fluvastatin not only prolonged lag time, but also suppressed the rate of LDL oxidation, both in a dose dependent manner above 1 microM, while pravastatin showed no effect. These results suggest the direct antioxidative effect of fluvastatin on LDL oxidation in vivo. Since oxidation of LDL is an important step in the initiation and progression of atherosclerosis, fluvastatin may reduce the risk of this condition not only by lowering plasma cholesterol but also by protecting LDL from oxidation.

Relative Effects of alpha- and gamma-Tocopherol on Low-Density Lipoprotein Oxidation and Superoxide Dismutase and Nitric Oxide Synthase Activity and Protein Expression in Rats.

BACKGROUND: Increasing evidence suggests that vitamin E prevents the progression of atherosclerosis by inhibiting platelet aggregation, monocyte adhesion, and improving plaque stability and vasomotor function. Recently, controversy has arisen as to the relative effects of alpha- and gamma-tocopherol in modulating some mediators of atherosclerosis. METHODS AND RESULTS: We examined the effects of alpha- and gamma-tocopherol on constitutive nitric oxide synthase (cNOS) and superoxide dismutase (SOD) activity and protein expression in rats. Sprague-Dawley rats were fed regular chow or chow mixed with alpha- or gamma-tocopherol (100 mg/kg/day) for 7 to 10 days. Plasma alpha- and gamma-tocopherol levels, low-density lipoprotein (LDL) oxidation, and cNOS and SOD activity and protein expression were measured. Plasma alpha-tocopherol levels were significantly increased (eP <.01 vs control), but gamma-tocopherol levels fell (P <.01 vs control) in rats fed alpha-tocopherol. Plasma gamma-tocopherol levels were increased (P <.01 vs control), and alpha-tocopherol levels did not change in rats fed gamma-tocopherol. Both alpha- and gamma-tocopherol feeding decreased the rate of LDL oxidation induced by phorbol 12-myristate 13-acetate (PMA)-stimulated leukocytes (P <.01 vs control). Both alpha- and gamma-tocopherol increased SOD activity in plasma and arterial tissues as well as Mn SOD and Cu/Zn SOD protein expression in arterial tissues (all P <.01 vs control). gamma-Tocopherol was more potent than alpha-tocopherol in all these effects (P <.05). Both a- and gamma-tocopherol increased NO generation and cNOS activity (all P <.05 vs control). However, only gamma-tocopherol increased cNOS protein expression. CONCLUSIONS: These observations indicate that whereas both alpha- and gamma-tocopherol exert important effects on determinants of oxidationand vasomotor function, effects of dietary gamma-tocopherol supplementation in vivo are less pronounced than those of gamma-tocopherol supplementation.

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.

Prooxidant and antioxidant activities of macrophages in metal-mediated LDL oxidation: the importance of metal sequestration.

Murine macrophages incubated in metal-supplemented RPMI could block or promote oxidation of low-density lipoprotein (LDL) depending on the degree of metal supplementation. Only at high concentrations of Cu (1 micromol/L) and Fe (30 micromol/L) were cells prooxidant, leading to an accelerated rate of LDL oxidation over that measured in comparable cell-free media. At lower concentrations of Cu and Fe in RPMI, LDL oxidation in the presence of macrophages was inhibited relative to the cell-free condition. This appeared to be dependent on a stable modification of the culture medium, because preconditioning of media by incubation with macrophages could also decrease their capacity to sustain subsequent cell-free LDL oxidation. This was due, in part, to a removal of metal from the media during preconditioning. However, resupplementation of media with metals did not fully restore oxidative capacity, indicating that other cell-dependent antioxidant modifications occurred. This did not involve significant alterations to the thiol content of the media. This study highlights the complexity of the role that cells such as macrophages have with regards to LDL oxidation in vitro and demonstrate that there are both antioxidative and prooxidative components.

Microsomal cytochromes P450 catalyze the oxidation of low density lipoprotein

Low density lipoprotein (LDL) oxidation is a major contributor to foam cell formation during early atherogenesis. Several oxygenases have been implicated in the process of LDL oxidation in the arterial wall, where the environment is relatively low in antioxidants, but the exact mechanism for LDL oxidation in vivo is not known. In the present study we sought to determine the ability of cytochrome P450 2E1 (P450 2E1) and other P450s, located in the liver and in other tissues, to oxidize LDL. Upon incubation of LDL (0.1 mg of protein/ml) with purified, reconstituted rabbit P450 2E1 in the presence of NADPH and the NADPH-cytochrome P450 reductase, time- and P450 2E1 concentration-dependent LDL oxidation was observed, as analyzed by determining the formation of peroxides, thiobarbituric acid reactive substances (TBARS), and conjugated dienes. Within 1 h of initiating the reaction, almost maximal oxidation was observed. NADPH, and active P450 2E1 enzyme were required for LDL oxidation to occur. The rate of P450 2E1-induced LDL oxidation was also dependent on the lipoprotein concentration. P450 2E1 could also oxidize pure phospholipids and cholesteryl ester, the major lipids in LDL. In the presence of catalase or superoxide dismutase (SOD), LDL oxidation was completely blocked, suggesting that hydrogen peroxide and superoxide are involved in P450 2E1-induced LDL oxidation. The ability of P450 2E1 to oxidize LDL was not unique to this enzyme, and could be observed with some other purified, cytochromes P450 in the reconstituted system such as rat P450 2B1 and human P450 3A4. Finally, microsomal membranes obtained from rats that were induced to express high levels of P450s 2B1, 2E1, and 1A1/2 were able to oxidize LDL, whereas little oxidation was seen with microsomes that were induced to express 3A2. We thus conclude that LDL can be oxidized by some cytochrome P450s and, as some of these enzymes are present in liver and in arterial wall, they may have a physio/pathological relevance to LDL oxidation and atherogenesis.

A sensitive chemiluminescence method to measure the lipoxygenase catalyzed oxygenation of complex substrates

Oxidative modification of low-density lipoprotein (LDL) has been implicated as a patho-physiological process in early atherogenesis and 15-lipoxygenases (15-LOX) may be involved. While studying the in vitro kinetics of the 15-LOX/LDL interaction, we found that the conventional spectrophotometric assays failed in the range of substrate saturation owing to the high optical density of concentrated LDL solutions. Therefore, we developed a much more sensitive assay system which was based on peroxide induced isoluminol enhanced chemiluminescence. With this method reliable kinetic data were obtained at LDL concentrations of up to 1 mg/ml. To validate this luminometric method the kinetic parameters of 15-LOX catalyzed oxygenation of linoleic acid (Km=3.7 microM, kcat=17 s-1) were determined and we observed a good agreement with previously published data obtained with a spectrophotometric assay. Moreover, we found that the kinetic constants of 15-LOX catalyzed LDL oxidation (Km=0.64 microM, kcat=0.15 s-1) are quite different from those of free fatty acid oxygenation and that the cholesterol esters are preferentially oxidized during 15-LOX/LDL interaction. Vitamin E depletion does not reduce the rate of LDL oxidation and analysis of the structure of the oxygenation products suggests that the majority of the products were formed via direct LOX catalyzed oxidation of LDL ester lipids. The luminometric method described here is not restricted to the measurement of LOX catalyzed LDL oxidation, but may also be used to determine kinetic constants for the oxidation of other complex substrates such as biomembranes or liposomes.

Oxidative Changes Associated with β-Carotene and α-Tocopherol Enrichment of Human Low-Density Lipoproteins

Objective: To determine what effects enrichment of human low-density lipoprotein (LDL) with combinations of α-tocopherol and β-carotene would exert on LDL oxidation and attempt to define the nature of the effects.Methods: Human plasma was pooled and α-tocopherol and β-carotene was added in a four-by-four design resulting in the enrichment of LDL with α-tocopherol and β-carotene in varying concentrations. Enriched and control LDL was oxidized in Cu2+ mediated oxidation system and resistance of LDL to oxidation was determined by lag time, thiobarbituric acid reactive substances (TBARS) activity, and rate of oxidation.Results: Increasing LDL α-tocopherol concentration had a linear relationship with lag time and a negative correlation with rate of oxidation. LDL β-carotene concentration was linearly correlated with the rate of LDL oxidation and β-carotene loss, and exponentially related to TBARS concentration.Conclusions: These results support earlier findings for the protective effect of α-tocopherol against LDL oxidation, and suggest that β-carotene participates as a prooxidant in the oxidative degradation of LDL under these conditions. Since high levels of α-tocopherol did not mitigate the prooxidative effect of β-carotene, these result indicate that increased LDL β-carotene may cancel the protective qualities of α-tocopherol.

A Comparison of the Kinetics of Low-Density Lipoprotein Oxidation Initiated by Copper or by Azobis(2-Amidinopropane)

This article describes the kinetics of low density lipoprotein (LDL) oxidation catalyzed by azobis (2-amidinopropane) dihydrochloride, ABAP, or by copper. The LDLs were isolated from nonhuman primates fed diets enriched in one of three types of fatty acids: saturated fatty acids, monounsaturated fatty acids, predominantly, oleic acid, or polyunsaturated fatty acids, predominantly linoleic acid. Oxidation was followed by monitoring the formation of conjugated diene hydroperoxides from polyunsaturated fatty acids (PUFA). For both copper and ABAP-initiated oxidation, the rate of LDL oxidation depended on the concentrations of initiator, PUFA, and LDL. Except for the dependence on PUFA concentration the rate of LDL oxidation was not directly influenced by the fatty acid composition of the LDL particle. The two initiators had very different dependence on initiator concentration. Because LDL particles are essentially small, lipid-rich droplets, the kinetic descriptions of LDL oxidation assumed: (1), that there was only one chain per particle, and (2) that the radical chain was terminated when a second radical either entered or was formed in the particle. When two LDL samples having very different lag times were mixed, the oxidation profile was bimodal. This finding demonstrated that the oxidation of native LDL particles was independent of the oxidation state of the other native LDL particles in solution, i.e., LDL particles do not rapidly exchange radicals, for example, hydroperoxyl radicals. Oxidation initiated by ABAP was proportional to [ABAP]0.5, suggesting that hydroperoxyl radical recombination between the lipid hydroperoxyl radical and the ABAP-hydroperoxyl radical was the chain-terminating step. The reciprocal of the rate of copper oxidation was linearly related to the reciprocal copper concentration, demonstrating that the binding of copper to LDL was necessary to initiate oxidation. This binding constant showed considerable variability among LDL samples. The kinetic descriptions of LDL oxidation reflect the differences in the mechanisms of initiation and termination.

Properties of low density lipoproteins relevant to oxidative modifications change paradoxically during aging

Atherosclerosis is a common problem among the elderly. Because lipid peroxidation is considered a contributor to the development of atherosclerosis, we compared oxidative properties of lipoproteins in an otherwise healthy (SENIEUR-classified) aged population (65-74 years) with young controls (18-30 years). Relative amounts of oxidatively altered low density lipoprotein (LDL), estimated by means of an antibody against LDL modified by 4-hydroxynonenal, a product of lipid peroxidation, were increased marginally in serum from the elderly (9.8 vs. 7.4%, P = 0.07). In contrast, isolated LDL from the elderly revealed a decreased susceptibility to in vitro oxidation: the lag time was increased (2.34 vs. 2.10 h, P < 0.01), and the maximal rate of LDL oxidation decreased (0.88 vs. 1.01 O.D./h, P = 0.001). However, there were no age-related changes in lipid composition of native LDL and consumption of fatty acids during in vitro oxidation. The serum concentrations of ascorbic acid and most lipophilic anti-oxidants (the latter expressed per g serum lipids) were significantly decreased in the elderly except tocopherols which tended to be higher. In conclusion, our data reveal paradox age-related alterations of LDL as to its behaviour in oxidation in vivo vs. in vitro.

Ceruloplasmin Enhances Smooth Muscle Cell- and Endothelial Cell-mediated Low Density Lipoprotein Oxidation by a Superoxide-dependent Mechanism

Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro. We now show that ceruloplasmin also increases LDL oxidation by vascular cells. In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively. The maximal response was at 100-300 microg ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration. Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity. Three lines of evidence indicated a critical role for cellular superoxide (O2.) in ceruloplasmin-stimulated oxidation. First, the rate of production of O2. by cells correlated with their rates of LDL oxidation. Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types. Finally, O2. production by SMC quantitatively accounted for the observed rate of LDL oxidation. To show this, the course of O2. production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell-free conditions. Neither ceruloplasmin nor O2. alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC. These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O2. accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.

Dietary Supplementation With Orange and Carrot Juice in Cigarette Smokers Lowers Oxidation Products in Copper–Oxidized Low–Density Lipoproteins

Our objective was to evaluate the effect of daily supplementation with foods high in vitamin C and beta carotene on plasma vitamin levels and oxidation of low-density lipoprotein (LDL) in cigarette smokers. Fifteen normolipidemic male cigarette smokers who did not usually take vitamin supplements were recruited into the study. Throughout the study, subjects consumed a diet rich in polyunsaturated fatty acids, which provided 36% of energy as fat: 18% from meat, dairy products, vegetable oils, and fat spreads and 18% from walnuts (68 g/day). Subjects consumed a vitamin-free drink daily for 3 weeks; then for 3 weeks they consumed daily supplements of orange juice (145 mg vitamin C) and carrot juice (16 mg beta carotene). Vitamin-rich food supplements raised plasma levels of ascorbic acid (1.6-fold; P < .01) and beta carotene (2.6-fold; P < .01). Malondialdehyde, one end product of oxidation, was lower in copper-oxidized LDL after vitamin supplementation (mean +/- standard error = 65.7 +/- 2.0 and 57.5 +/- 2.9 mumol/g LDL protein before and after supplementation, respectively; P < .01). Rate of LDL oxidation and lag time before the onset of LDL oxidation were not affected by antioxidant supplementation. In habitual cigarette smokers, antioxidant vitamins, which can be feasibly provided from food, partly protected LDL from oxidation despite a diet rich in polyunsaturated fatty acids.