Very-low-density Lipoprotein Oxidation Research Articles

Eicosapentaenoic Acid Inhibits Oxidation of ApoB-containing Lipoprotein Particles of Different Size In Vitro When Administered Alone or in Combination With Atorvastatin Active Metabolite Compared With Other Triglyceride-lowering Agents

:Eicosapentaenoic acid (EPA) is a triglyceride-lowering agent that reduces circulating levels of the apolipoprotein B (apoB)-containing lipoprotein particles small dense low-density lipoprotein (sdLDL), very–low-density lipoprotein (VLDL), and oxidized low-density lipoprotein (LDL). These benefits may result from the direct antioxidant effects of EPA. To investigate this potential mechanism, these particles were isolated from human plasma, preincubated with EPA in the absence or presence of atorvastatin (active) metabolite, and subjected to copper-initiated oxidation. Lipid oxidation was measured as a function of thiobarbituric acid reactive substances formation. EPA inhibited sdLDL (IC50 ∼2.0 μM) and LDL oxidation (IC50 ∼2.5 μM) in a dose-dependent manner. Greater antioxidant potency was observed for EPA in VLDL. EPA inhibition was enhanced when combined with atorvastatin metabolite at low equimolar concentrations. Other triglyceride-lowering agents (fenofibrate, niacin, and gemfibrozil) and vitamin E did not significantly affect sdLDL, LDL, or VLDL oxidation compared with vehicle-treated controls. Docosahexaenoic acid was also found to inhibit oxidation in these particles but over a shorter time period than EPA. These data support recent clinical findings and suggest that EPA has direct antioxidant benefits in various apoB-containing subfractions that are more pronounced than those of other triglyceride-lowering agents and docosahexaenoic acid.

Separation of Lipids on Human Very Low‐density Lipoproteins by Micellar Electrokinetic Chromatography

AbstractLiquid‐phase and solid‐phase extractions (SPE) in combination with a simple micellar electrokinetic chromatography (MEKC) method were used to investigate human very low‐density lipoprotein (VLDL) lipids for two healthy donors. At absorbance 200 nm, the effective mobilities and peak areas of the MEKC profiles showed good reproducibility and precisions. A major peak and several minor peaks appeared for the total lipids of native VLDL, but both the peak numbers and areas reduced for the in vitro oxidized VLDL. Two chloroform and two methanol fractions were obtained from SPE of VLDL total lipids. Significant differences were observed for the first methanol fraction between native and in vitro oxidized VLDL lipids. The first methanol fraction showed a major peak and several minor peaks for native VLDL, but both the peak numbers and areas reduced for oxidized VLDL. Oxidation of VLDL caused decomposition of lipids, and thus the reduction of peak numbers and areas.

The two faces of α- and γ-tocopherols: an in vitro and ex vivo investigation into VLDL, LDL and HDL oxidation

BackgroundVitamin E and its derivatives, namely, the tocopherols, are known antioxidants, and numerous clinical trials have investigated their role in preventing cardiovascular disease; however, evidence to date remains inconclusive. Much of the in vitro research has focused on tocopherol's effects during low-density lipoprotein (LDL) oxidation, with little attention being paid to very LDL (VLDL) and high-density lipoprotein (HDL). Also, it is now becoming apparent that γ-tocopherol may potentially be more beneficial in relation to cardiovascular health. ObjectivesDo α- and γ-tocopherols become incorporated into VLDL, LDL and HDL and influence their oxidation potential in an in vitro and ex vivo situation? DesignFollowing (i) an in vitro investigation, where plasma was preincubated with increasing concentrations of either α- or γ-tocopherol and (ii) an in vivo 4-week placebo-controlled intervention with α- or γ-tocopherol. Tocopherol incorporation into VLDL, LDL and HDL was measured via high-pressure liquid chromatography, followed by an assessment of their oxidation potential by monitoring conjugated diene formation. ResultsIn vitro: Both tocopherols became incorporated into VLDL, LDL and HDL, which protected VLDL and LDL against oxidation. However and surprisingly, the incorporation into HDL demonstrated pro-oxidant properties. Ex vivo: Both tocopherols were incorporated into all three lipoproteins, protecting VLDL and LDL against oxidation; however, they enhanced the oxidation of HDL. ConclusionsThese results suggest that α- and γ-tocopherols display conflicting oxidant activities dependent on the lipoprotein being oxidized. Their pro-oxidant activity toward HDL may go some way to explain why supplementation studies with vitamin E have not been able to display cardioprotective effects.

Open tubular CE for in vitro oxidation studies of human very‐low‐density lipoprotein particles

Human very-low-density lipoprotein (VLDL) particles were immobilised on the inner wall of electrochromatographic fused-silica capillaries, and the applicability of these capillary columns in oxidation studies was investigated. Capillaries coated with radiolabelled VLDL particles showed a coating efficiency of 97%, and allowed estimation of the amount of VLDL present in a capillary. Radioactivity measurements and atomic force microscopy with tapping mode confirmed the presence of VLDL particles as a monolayer. The pI determined for the VLDL was 4.7-4.8 varying with the human source. The effects of VLDL concentration, coating time and pH on the coating stability were clarified, and the stability was examined in terms of the repeatability of EOF and retention factors of selected steroids. The repeatability of run-to-run and the coating-to-coating reproducibility ranged from 2.6 to 4.9% and 3.2 to 6.6%, respectively. The lifetime of a coating was at least 7 days or 84 consecutive runs. The in situ copper-mediated VLDL oxidation carried out in the capillary with optimised VLDL coating showed that, during the oxidation of VLDL particles, the negative charges of the particles are increased, leading to enhanced EOF mobilities. Several oxidation parameters, including copper sulfate concentration, amount of EDTA needed to stop the reaction, pH and the oxidation procedure, were examined. Effect of the oxidation process on the stability of the coating in one capillary, and in five different capillaries ranged between 0.4-4.1% and 0.8-6.6%, respectively. The in situ oxidation of VLDL particles was compared with that of low-density lipoproteins.

Folate: In Vitro and in Vivo Effects on VLDL and LDL Oxidation

Raised total homocysteine (tHcy) levels may be involved in the etiology of cardiovascular disease and can lead to damage of vascular endothelial cells and arterial wall matrix. Folic acid supplementation can help negate these detrimental effects by reducing tHcy. Recent evidence has suggested an additional anti-atherogenic property of folate in protecting lipoproteins against oxidation. This study utilized both an in vitro and in vivo approach. In vitro: Very-low-density lipoprotein (VLDL) and low density lipoprotein (LDL) were isolated by rapid ultracentrifugation and then oxidized in the presence of increasing concentrations (0-->10 micromol/L) of either folic acid or 5-methyltetrahydrofolate (5-MTHF). In vivo: Twelve female subjects were supplemented with folic acid (1 mg/day), and the pre- and post-VLDL and LDL isolates subjected to oxidation. In vitro: 5-MTHF, but not folic acid, significantly increased the resistance of VLDL and LDL to oxidation. In vivo: Following folic acid supplementation, tHcy decreased, serum folate increased, and both VLDL and LDL displayed a significant increase in their resistance to oxidation. These results indicated that in vitro, only the active form of folate, 5-MTHF, had antioxidant properties. In vivo results demonstrated that folic acid supplementation reduced tHcy and protected both VLDL and LDL against oxidation. These findings provide further support for the use of folic acid supplements to aid in the prevention of atherosclerosis.

Formation of N.EPSILON.-(Hexanonyl)lysine in Oxidized Human Very-low-density Lipoprotein.

A mechanism of oxidative modification of apolipoproteins (apo) in human very-low-density lipoprotein (VLDL) was investigated in vitro. Lipid peroxidation was promoted by cupric ion in VLDL. Modification of apoE and apoB-100 was observed in the VLDL oxidation. Ne-(Hexanonyl)lysine, one of the lipid hydroperoxide-modified lysine residue, was detected in VLDL oxidized for 18 hours by immunoblot analysis and enzyme-linked immunosorbent assay. The results indicate that lysine residues of apoE and apoB-100 were modified by lipid hydroperoxides. The heparin-binding activity of apoE and apoB-100 which seems to reflect their low-density lipoprotein receptor (LDLr)-binding activity decreased in the VLDL oxidation. This demonstrates that the heparin-binding site of apoE and apoB-100 which includes lysine residues was modified in the VLDL oxidation. Our data suggest that lysine residues of the LDLr-binding site of apoE and apoB-100 might be damaged by lipid hydroperoxides produced in the VLDL oxidation.

Effect of L-ascorbic acid on the oxidative modification of apolipoprotein E in human very-low-density lipoprotein.

Antioxidant activity of L-ascorbic acid (AsA) against oxidative modification of apolipoprotein (apo) E in human very-low-density lipoprotein (VLDL) was investigated. The VLDL oxidation induced by peroxyl radicals and peroxynitrite led to lipid peroxidation and oxidative modification of apoE. The binding activity of apoE to heparin was decreased by the oxidative modification. AsA (200, 500, and 1,000 microm) inhibited both the lipid peroxidation and the oxidative modification of apoE. These results suggest that AsA prevents the biological function of apoE in VLDL from the oxidation by free radicals.

Effect of alpha-tocopherol on the oxidative modification of apolipoprotein E in human very-low-density lipoprotein.

The oxidative modification of apolipoprotein (apo) E and lipid peroxidation in human very-low-density lipoprotein (VLDL) induced by peroxynitrite and cupric ions in vitro were strongly suppressed by enrichment with alpha-tocopherol (alpha-Toc; 170 microM). Alpha-Toc also suppressed the decrease in the heparin-binding activity of apoE in the VLDL oxidation. These results suggest that alpha-Toc protected apoE in VLDL from oxidative stress.

Oxidative Modification of Apolipoprotein E in Human Very-Low-Density Lipoprotein and Its Inhibition by Glycosaminoglycans

The mechanism of metal ion-catalyzed oxidative modification of apolipoprotein E (apoE) in human very-low-density lipoprotein (VLDL) and its inhibition by glycosaminoglycan (GAG) was investigated in vitro. The VLDL oxidation catalyzed by Cu2+ led to the lipid peroxidation, the formation of aggregates, and covalent modification of apoE. The modified apoE lost heparin-binding activity. These results suggest that the lipid peroxidation of VLDL and modification of apoE cause impairment of lipid uptake by cells and deposit the oxidized lipids in the tissues. The lipid peroxidation and oxidative modification of apoE in VLDL mediated by Cu2+ and an aqueous radical generator were suppressed by GAG, heparan sulfate, heparin, and chondroitin sulfate A, even though GAGs demonstrated no ability to scavenge α,α-diphenyl-β-picrylhydrazyl radical. There were no relationships between inhibitory activity of GAGs in the VLDL oxidation and their number of sulfate groups which possess chelating activity of metal ion. Therefore, it can be considered that the inhibition of VLDL oxidation by GAGs is possibly due to the interaction between GAG and VLDL which bring about the steric hindrance, interference with the reaction between VLDL particle and the reactive oxygen species. These studies suggest that GAGs preserve the biological functions of apoE from oxidative stress.

Susceptibility to oxidation of copper-induced plasma lipoproteins from Japanese eel: protective effect of vitellogenin on the oxidation of very low density lipoprotein

The susceptibility to oxidation of copper-induced plasma lipoproteins from Japanese eel Anguilla japonica was examined with the guidance of thiobarbituric acid-reactive substances (TBARS). The TBARS values of copper-induced plasma lipoproteins increased with increasing the lipid-to-apolipoprotein ratios and very low density lipoprotein (VLDL) exhibited the highest TBARS value. On the other hand, vitellogenin, estrogen-induced precursor of egg yolk proteins, was resistant to copper-induced oxidation and seemed to chelate low concentrations of copper ion. Vitellogenin also protected the copper-induced oxidation of VLDL because of its antioxidant function. Vitellogenin seemed to serve as transition metals-binding lipoprotein by which free-radical reactions in the oocytes were extensively depressed.

A simple method for assessing copper-mediated oxidation of very-low-density lipoprotein isolated by rapid ultracentrifugation.

The association of very-low-density lipoprotein (VLDL) with atherosclerosis remains controversial. However, studies have shown that oxidative modification of VLDL can promote foam cell formation, leading to the development of atherosclerosis. A rapid method is described which will allow the significance of VLDL oxidation to be assessed in clinical studies. VLDL was isolated from heparinized plasma by a 1-h, single spin ultracentrifugation. Total protein was standardized to 25 mg/L. Oxidation was promoted by the addition of copper ions (17.5 mumol/L, final concentration) incubated at 37 degrees C. Conjugated diene production was followed at 234 nm. Total assay preparation time was 2 h. Urate greatly inhibited the oxidation of VLDL and was successfully removed by size exclusion chromatography. VLDL isolated from frozen plasma (-70 degrees C) was stable for 15 weeks. This simple, rapid method for the isolation of VLDL may be applied to assess the significance of VLDL oxidation in disease.

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.

Expression of monocyte chemoattractant protein-1 in monocytes and effects of native and oxidized very low density lipoproteins.

Monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant, is thought to play an important role in migration of monocytes into atherosclerotic lesions. The present study was designed to investigate the capacity of human peripheral blood monocytes to express MCP-1 and effects of native very low density lipoprotein (VLDL) and oxidized VLDL (OX-VLDL) on the expression. The total RNA was extracted from cultured monocytes, which were exposed to VLDL and OX-VLDL, and the media conditioned by monocytes were collected. MCP-1 mRNA expression was examined by Northern blot analysis. MCP-1 protein in conditioned media was determined by using sandwich ELISA. The results showed that monocytes can express MCP-1 after a 24 h incubation at 37 degrees C, and the expression was markedly increased by a exposure to OX-VLDL, whereas the expression was slightly increased when exposed to VLDL. It suggests that the capacity of monocytes to produce MCP-1 that recruits and activates circulating monocytes may be of considerable importance in atherogenesis, and oxidation of VLDL enhances its potential to promote atherogenesis.

Increased oxidation resistance of atherogenic plasma lipoproteins at high vitamin E levels in non-vitamin E supplemented men

The oxidative modification of human low density lipoprotein (LDL) has been widely investigated. However, there are no data concerning the oxidation susceptibility of combined very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein fraction, although all of them are atherogenic and contain antioxidants such as α-tocopherol. We investigated the oxidation susceptibility and oxidation resistance of VLDL + LDL (including IDL) fraction by induction with CuCl 2 and its relation to plasma α-tocopherol concentration and lipid standardised α-tocopherol concentration in 406 non-vitamin E-supplemented men from eastern Finland. Even thought we did not give oral vitamin E or any other antioxidant supplementation to our study participants, we observed a significant, consistent relationship between measurements of oxidation resistance and plasma content of vitamin E. In the multivariate regression model, a high plasma content of vitamin E or lipid standardised vitamin E concentration were the most important determinants of lag time to maximal oxidation rate (standardised regression coefficient = 0.244, P < 0.0001 for vitamin E and 0.211, P < 0.0001 for lipid standardised vitamin E). After statistical adjustment for age, use of cigarettes, hypolipidemic medication (yes vs. no), month of the measurements, plasma concentrations of total ascorbic acid (ascorbic acid + dehydroascorbic acid), β-carotene and phospholipids, serum concentrations of LDL cholesterol and triglycerides and dietary intake of linoleic acid, the lag time to maximal oxidation rate was 10% (95% C.I. 6.0–13.5%) longer in men in the highest fifth than in the lowest fifth of plasma vitamin E content ( P < 0.0001 for trend). When the fifths of lipid standardised vitamin E were compared, the lag time to maximal oxidation rate was 6% (95% C.I. 1.8–10.1%) longer in men in the highest than in the lowest fifth ( P < 0.0001 for trend). Our data suggest that α-tocopherol is an important antioxidant preventing the in vitro oxidation of VLDL + LDL fraction even in non-supplemented subjects.

Hypertriglyceridemic serum, very low density lipoprotein, and iron enhance Mycobacterium avium replication in human macrophages.

The growth of Mycobacterium avium 7497, serovar 4, in cultured human macrophages is enhanced by Fe3+ and serum lipids over 7 days. Iron (1-80 micrograms/mL) added to macrophages cultured in normal serum resulted in 10-fold increases in growth. If iron-supplemented macrophages were cultured in serum from hypertriglyceridemic donors after infection, M. avium growth increased 10(3)- to 10(4)-fold. Without macrophages, differences in bacterial growth between sera were not seen. Removal of very low density lipoprotein (VLDL) eliminated the differences between sera. Isolated VLDL from hyperlipidemic serum resulted in 10(5)-fold increases in growth over that seen with VLDL from normal sera. Accelerated M. avium growth in macrophages cultured with hyperlipidemic serum was partly inhibited by the addition of superoxide dismutase (1000 IU/mL). Results suggest that iron stimulates O2-induced oxidation of VLDL and its subsequent accumulation in macrophages. The resultant iron- and lipid-laden cells become excellent hosts for mycobacterial growth.

Radical-mediated oxidation of isolated human very-low-density lipoprotein.

Oxidative modification of human low-density lipoprotein (LDL) has received much attention because of its suggested involvement in the early events of atherogenesis. In contrast, little data exist concerning the oxidation of human very-low-density lipoprotein (VLDL), although such modification promotes foam cell formation by these lipoproteins. We therefore investigated the radical-mediated oxidation of VLDL by using controlled oxidizing conditions and sensitive and specific methods to assess lipoprotein lipid oxidation and antioxidation. We observed that the ratio of alpha-tocopherol to coenzyme Q10 in VLDL was close to that of LDL, suggesting that these lipoproteins may transport some coenzyme Q10 to extrahepatic tissues, as they do tocopherol. Most of the coenzyme Q10 associated with VLDL was present in its reduced, antioxidant active form, ubiquinol-10. The small amounts of ubiquinol-10 in VLDL provided the lipoprotein lipids with a highly efficient antioxidant protection. Also, the kinetics of radical-mediated lipid peroxidation in VLDL resembled that in LDL and therefore also probably proceeded via the recently described tocopherol-mediated peroxidation mechanism. Oxidation competition experiments using aqueous radicals and physiological concentrations and molar ratios of LDL and VLDL indicated that in contrast to the situation with high-density lipoproteins, lipid peroxidation was initiated and detected simultaneously in the former two lipoprotein particles. However, once initiated, peroxidation propagated at an approximately twofold higher rate in VLDL than LDL. Our studies suggest that radical-mediated lipid (per)oxidation proceeds via similar mechanisms in isolated LDL and VLDL. We conclude that efficient LDL antioxidants are also likely to be effective protective agents for VLDL.

A rapid and simple method for measuring the susceptibility of low-density-lipoprotein and very-low-density-lipoprotein to copper-catalyzed oxidation

Much evidence has accumulated to suggest a role for the oxidation of low-density-lipoprotein (LDL) and very low-density-lipoprotein (VLDL) in the pathogenesis of atherosclerosis. The susceptibility of lipoprotein to copper-catalyzed oxidation is often used to evaluate its oxidizability. A method was developed which isolates the non-high-density lipoprotein (non-HDL) fraction and removes EDTA by a dextran-magnesium precipitation method. The oxidizability of this fraction is evaluated by monitoring the fluorescence and measuring thiobarbituric acid reactive substances (TBARS) at different intervals of incubation. Those parameters reflect apolipoprotein B (apo B) modification and lipid degradation during LDL and VLDL oxidation. Our assay is sensitive enough to study factors which can influence the oxidizability of LDL and VLDL. The method is simple, rapid and can be easily conducted in a routine laboratory.

Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity.

Increased lipid peroxidation products were detected in a lipoprotein fraction containing very low density lipoprotein (VLDL) and low density lipoprotein (LDL) obtained from rats made diabetic by streptozotocin injection. The enhanced oxidation in the diabetic VLDL plus LDL fraction correlated with the in vitro toxicity of this lipoprotein fraction to proliferating fibroblasts. In contrast, high density lipoprotein (HDL) was not cytotoxic. That the increased oxidation and development of cytotoxic activity in the diabetic VLDL + LDL was related to the diabetes was shown by the fact that insulin treatment of diabetic animals inhibited both oxidation and cytotoxicity of VLDL + LDL. In contrast, treatment of diabetic rats with the antioxidants vitamin E or probucol after diabetes was established also inhibited both the in vivo oxidation and in vitro cytotoxicity of diabetic VLDL + LDL, but without altering hyperglycemia. Vitamin E or probucol treatment thus allowed separation of the oxidation process from the hyperglycemia occurring in experimental diabetes. The mechanisms by which diabetes in humans or experimental animals leads to the various manifestations of tissue damage are unknown; however, these studies demonstrate for the first time that a relationship exists between the in vivo oxidation of lipoproteins in diabetes and the potential for tissue damage as monitored by in vitro cytotoxicity. Furthermore, these results suggest that the mechanism for certain aspects of tissue damage accompanying experimental diabetes may be mediated by lipid peroxidation products.

Effect of sepsis on VLDL kinetics: responses in basal state and during glucose infusion.

The effect of gram-negative sepsis on the kinetics and oxidation of very low-density lipoprotein (VLDL) fatty acids was assessed in conscious dogs in the normal state and 24 h after infusion of live Escherichia coli. VLDL, labeled with [2-3H]glycerol and [1-14C]palmitic acid, was used to trace VLDL kinetics and oxidation, and [1-13C]palmitic acid bound to albumin was infused simultaneously to quantify kinetics and oxidation of free fatty acid (FFA) in plasma. Sepsis caused a fivefold increase in the rate of VLDL production (RaVLDL). In the control dogs, the direct oxidation of VLDL-fatty acids was not an important contributor to their overall energy metabolism, but in dogs with sepsis, 17% of the total rate of CO2 production could be accounted for by VLDL-fatty acid oxidation. When glucose was infused into dogs with insulin and glucagon levels clamped at basal levels (by means of infusion of somatostatin and replacement of the hormones), RaVLDL increased significantly in the control dogs, but it did not increase further in dogs with sepsis. We conclude that the increase in triglyceride concentration in fasting dogs with gram-negative sepsis is the result of an increase in VLDL production and that the fatty acids in VLDL can serve as an important source of energy in sepsis.