Oxidative cross-linking of ApoB100 and hemoglobin results in low density lipoprotein modification in blood. Relevance to atherogenesis caused by hemodialysis.

Human blood contains a form of minimally modified low density lipoprotein (LDL), termed LDL-, whose origin remains unknown. Exploring the mechanism of formation, we found that LDL- can be produced in plasma in the absence of oxygen following LDL incubation with oxidized hemoglobin species. A high degree of apolipoprotein B100 modification results from covalent association of hemoglobin with LDL involving dityrosine formation but not due to the malonaldehyde epitope formation. This was evidenced by the cross-reactivity of oxidized LDL with antibodies against hemoglobin that was accompanied by a 60-fold increase in dityrosine levels. In this study we found significantly higher LDL- levels in the blood of hemodialysis patients, perhaps contributing to their greatly increased risk of atherosclerosis. The mechanism of LDL- formation was studied during ex vivo blood circulation using a model system resembling clinical hemodialysis in terms of the induction of inflammatory responses. This circulation increased free hemoglobin and LDL- levels compared with non-circulated blood without appreciable lipid peroxidation. Pronounced increases in LDL- were found also during circulation of plasma supplemented with nanomolar hemoglobin levels. The increase in dityrosine content and presence of heme in LDL after blood circulation suggest that LDL is modified, in part, by hemoglobin-LDL conjugates containing heme. Thus, hemoglobin-mediated reactions leading to LDL oxidation in plasma can account for high LDL- levels in hemodialysis patients.     

Red wine mitigates the postprandial increase of LDL susceptibility to oxidation

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

Comparison of two methods for radioiodination on the oxidizability properties of low density lipoprotein

Radiolabeling of low density lipoprotein (LDL) apoB100 with 125I, an oxidative process, is commonly used in lipoprotein investigation. Since 1) LDL is unstable and oxidation-prone, 2) the modification of apoB100 by oxidation increases the negative charge of particles and leads to the uptake of modified LDL through the scavenger receptor pathway, and 3) oxidized LDL is cytotoxic, it is relevant to investigate whether the oxidative stability of LDL is influenced by its labeling with 125I. The aim of this study was to investigate and compare lipid and protein oxidation markers in human LDL after labeling with 125I by two widely adopted methods that use ICl or the chloramide 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril as the oxidizing agent. Native LDL served as a common control and sham-iodinated LDL as a handling control for each procedure. The resistance against copper-induced oxidation of 125I-LDL labeled with ICl was similar to that of controls with regard to the lag time and maximal amount of conjugated diene formed, as there were levels of initial conjugated diene, alpha-tocopherol, and tryptophan. However, radioiodination with the chloramide accelerated the onset of the rapid phase of LDL oxidation due to a drastic depletion of alpha-tocopherol and increased conjugated diene content. Measurements of copper-induced LDL oxidizability showed enhanced indices of lipid oxidation. The lag time and the time to maximal diene production were 65% and 30% shorter than controls. This was accompanied by a 50% reduced tryptophan fluorescence. The anionic surface charge of the LDL particle increased moderately with both labeling procedures. The results indicate that labeling of LDL with 125I may oxidize lipids and apoB100 to a variable extent, depending on the nature of the iodinating agent. This is why assessment of the oxidizability properties of 125I-labeled LDL is recommended for reliable biological studies.     

Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor.

The low density lipoproteins (LDL) of human plasma consist of a series of discrete particle subspecies of distinct physicochemical, immunological, and hydrodynamic properties. Such structural differences are intimately linked to the metabolic heterogeneity of circulating LDL in vivo. The current studies were designed to evaluate and compare the interaction of discrete LDL subspecies from normolipidemic subjects with the LDL receptor. Plasma LDL of d 1.019-1.063 g/ml from healthy males were fractionated into 15 subspecies of defined physicochemical characteristics by isopycnic density gradient ultracentrifugation as described earlier (Chapman et al., J. Lipid Res. 1988. 29: 442-458). The major LDL subspecies, LDL-5 to LDL-10, exhibited an overall range in density from 1.0244 to 1.0435 g/ml; individual subspecies increased in density by increments of 0.027 (LDL-5), 0.026 (LDL-6), 0.030 (LDL-7), 0.031 (LDL-8), 0.035 (LDL-9), and 0.042 g/ml (LDL-10), respectively. Taken together, these subspecies accounted for approximately 70% of the total mass of LDL of d 1.019-1.063 g/ml; their cholesterol: protein ratios decreased from 1.70 to 1.12 and particle size from 275 to 260 A with increase in density. ApoB-100 was the unique protein component in subspecies 5-8, with trace amounts (less than 0.2% of apoLDL) of both apoA-I and apoE in subspecies 9 and 10. The interaction of individual LDL subspecies with the LDL receptor on cultured human U-937 monocyte-like cells was compared by determining receptor binding affinities at 4 degrees C. Scatchard analysis of specific binding curves demonstrated a single class of binding site for each subspecies. The lowest dissociation constants were displayed by LDL subspecies 6 (Kd 5.71 nM), 7 (Kd 5.24 nM) and 8 (Kd 4.67 nM), while subspecies 5, 9, and 10 displayed significantly higher Kd values (8.35, 7.20, and 6.87 nM, respectively). Competitive displacement studies at 4 degrees C, in which unlabeled subspecies from the same gradient series competed for binding with 125I-labeled LDL subspecies, confirmed the relative binding affinities of these subspecies. As the hydrophobic lipid core of LDL undergoes a thermotropic transition at approximately 37 degrees C, which may in turn influence the surface structure of the particle, internalization and degradation studies were performed at 37 degrees C. No effect of temperature was detectable; again, LDL subspecies at each extreme of the density distribution (LDL-5 and LDL-10) displayed significantly lower binding affinities for the LDL receptor than that from the peak region (LDL-7).(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycated phosphatidylethanolamine promotes macrophage uptake of low density lipoprotein and accumulation of cholesteryl esters and triacylglycerols.

Non-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 micrograms/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 +/- 1.54 micrograms/mg cell protein) and TG (285.32 +/- 4.38 micrograms/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 +/- 0.95, p < 0.01 and TG, 185.57 +/- 3.58 micrograms/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 +/- 1.35 and TG, 280.78 +/- 3.98 micrograms/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5-6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia.     

Modified phosphatidylethanolamine as the active component of oxidized low density lipoprotein promoting platelet prothrombinase activity

We analyzed the influence of the atherogenic oxidized low density lipoproteins (LDL) on the activity of the platelet prothrombinase complex, a major contributor to overall thrombin formation in vivo. Platelet dependent thrombin generation was found to be strongly stimulated by in vitro oxidized LDL. The enhancement was additive to that observed with the platelet agonist thrombin. Oxidized LDL increased the platelet binding of annexin-V, suggesting that the augmented surface exposure of aminophospholipids promoted the prothrombinase activity. All of the stimulatory activity of the oxidized LDL could be recovered in the microemulsions prepared from the lipid portion of the modified particles. Phospholipid vesicles were prepared containing the total lipids of the oxidized LDL but lacking specifically in one lipid component. Following the selective removal of the ethanolamine phospholipids (PE) from the LDL lipids, the platelet-dependent thrombin formation was markedly reduced. Vesicles enriched with the isolated PE fraction alone enhanced the thrombin generation. Analyses with autoxidized phospholipids indicated that oxidation products of unsaturated diacyl-PE were mainly responsible for the increased prothrombinase activity. Oxidized LDL and its PE fraction lost their stimulatory activity after treatment with NaCNBH(3), a chemical reductant of Schiff base adducts. Phospholipid vesicles supplemented with synthetic aldehyde-PE adducts largely reproduced the stimulation of the thrombin generation. We conclude that the oxidized LDL particles elicit a pronounced prothrombotic response by increasing the activity of the platelet prothrombinase complex. Specific oxidative modifications of the LDL-associated ethanolamine phospholipids are mainly responsible for this stimulation.     

Effect of consumption of phenols from olives and extra virgin olive oil on LDL oxidizability in healthy humans

A high intake of olive oil has been proposed as an explanation for the low incidence of coronary heart disease in Mediterranean countries, but it is unclear whether olive oil offers specific benefits beyond a low content of saturated fat. Some types of extra virgin olive oil are rich in non-polar phenols, which might be taken up by plasma LDL particles and protect these from becoming atherogenic by oxidative modification. In a pilot study we found that consumption of 47 g fortified olive oil containing 31 mg phenols significantly increased the lag time of LDL oxidation from 112 ± 5 min before to 130 ± 7 min 2h after the meal. However, this study was not controlled, and in the current study we therefore investigated whether olive oil phenols increase the lag time of LDL oxidation in postprandial samples when compared with a control group.

Twelve healthy men and women consumed four different olive oil supplements with a meal on four separate occasions: one similar to the supplement in the pilot study (positive control); one containing mainly non-polar olive oil phenols; one containing mainly polar olive oil phenols; and one without phenols (placebo). Lag time significantly increased 2 h after the meals with the positive control (8 ± 2 min), the polar phenols (8 ± 2 min), and the placebo (8 ± 2 min), but not after the non-polar phenols (-0.4 ± 3 min). Increases were not statistically different between supplements.

These results indicate that the lag time of LDL-oxidation is increased after consumption of a meal. This increase is probably due to non-specific meal or time effects and not to phenols from olives or olive oil. Furthermore, these findings stress the need for adequate controlled studies to avoid misinterpretations of the data.     

Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products

Modification of low density lipoproteins (LDL) by oxidation has been shown to permit recognition by the acetyl-LDL receptor of macrophages. The extensive oxidation of LDL that is required before interaction occurs with this receptor produces major alterations in both the lipid and protein components of LDL. Several chemical modifications of LDL also lead to recognition by this receptor; all of these involve derivatization of lysine residues of apolipoprotein B by adducts that neutralize the positively charged epsilon-amino group. The present studies show that oxidation also results in derivatization of LDL lysine residues. Analysis of amino acid composition indicated that 32% of lysine residues were modified after oxidation of LDL by exposure to 5 microM CuSO4 for 20 h. About one-half of the derivatized lysines were labile under the conditions of acid hydrolysis. Fluorescence of LDL protein was greatly increased by oxidation, with excitation maximum at 350 nm and emission maximum at 433 nm. When LDL containing phosphatidylcholine with isotopically labeled arachidonic acid in the sn-2 position was oxidized, there was a 5-fold increase in radioactivity bound to protein compared to nonoxidized LDL or oxidized LDL labeled with 2-[1-14C]palmitoyl phosphatidylcholine. Prior methylation of LDL prevented the rapid uptake and degradation by macrophages that normally accompanies oxidation. These findings suggest that oxidation of LDL is accompanied by derivatization of lysine epsilon-amino groups by lipid products and that these adducts may be important in the interaction of oxidized LDL with the acetyl-LDL receptor.     

Effect of consumption of phenols from olives and extra virgin olive oil on LDL oxidizability in healthy humans

A high intake of olive oil has been proposed as an explanation for the low incidence of coronary heart disease in Mediterranean countries, but it is unclear whether olive oil offers specific benefits beyond a low content of saturated fat. Some types of extra virgin olive oil are rich in non-polar phenols, which might be taken up by plasma LDL particles and protect these from becoming atherogenic by oxidative modification. In a pilot study we found that consumption of 47 g fortified olive oil containing 31 mg phenols significantly increased the lag time of LDL oxidation from 112 ± 5 min before to 130 ± 7 min 2h after the meal. However, this study was not controlled, and in the current study we therefore investigated whether olive oil phenols increase the lag time of LDL oxidation in postprandial samples when compared with a control group.

Twelve healthy men and women consumed four different olive oil supplements with a meal on four separate occasions: one similar to the supplement in the pilot study (positive control); one containing mainly non-polar olive oil phenols; one containing mainly polar olive oil phenols; and one without phenols (placebo). Lag time significantly increased 2 h after the meals with the positive control (8 ± 2 min), the polar phenols (8 ± 2 min), and the placebo (8 ± 2 min), but not after the non-polar phenols (-0.4 ± 3 min). Increases were not statistically different between supplements.

These results indicate that the lag time of LDL-oxidation is increased after consumption of a meal. This increase is probably due to non-specific meal or time effects and not to phenols from olives or olive oil. Furthermore, these findings stress the need for adequate controlled studies to avoid misinterpretations of the data.     

Atherosclerosis: another protein misfolding disease?

The secondary structure and conformation of apo-B 100 in low-density lipoproteins (LDL) are imposed by lipid-protein interactions and dynamics, and affected by the introduction or removal of lipids during the course of lipoprotein metabolism. Following an alteration of the water-lipid interface as a result of, for example, oxidation of lipids, the supramolecular structure becomes destabilized and apoB can misfold. These events have been observed in LDL(-), a fraction of oxidatively modified LDL isolated in vivo. This modified lipoprotein possesses several atherogenic properties and represents an in vivo counterpart of in vitro modified LDL that is implicated in atherosclerosis. The misfolding of apoB, its aggregation, resistance to proteolysis, and cytotoxicity are common motifs shared by LDL(-) and amyloidogenic proteins. Based on these analogies, we propose that atherogenesis could be considered as a disease produced by the accumulation of cytotoxic and pro-inflammatory misfolded lipoproteins.     

Intracellular generation of MDA-LYS epitope in foam cells.

Oxidative stress plays a central role in atherogenesis. Antioxidants, such as probucol, inhibit oxidation of LDL, retard secretion of interleukin-1, growth factors and chemoattractants, and thus inhibit progression of atherosclerosis. Other antioxidants with an ability to inhibit LDL oxidation, however, could not prevent progression of atherosclerosis. The inconsistency between antioxidant potencies indicated oxidative events might have occurred at locations other than LDL. MDA-lysine epitope (MDA-lys) is closely associated with atherogenesis and was recognized as marker for oxidation. We traced formation of MDA-lys during oxidation of LDL and formation of foam cells. The results indicated that thiobarbituric acid reactive substance (TBARS) was primarily present in lipid fraction of ox-LDL not associated with protein fraction after Cu2+ oxidation in vitro. Oxidized LDL did not increase significant immunoreactivity of MDA-lys epitope under our experimental conditions. Foam cells, however, showed the presence of MDA-lys epitope suggesting that intracellular oxidation events occurred to internalized lipids. The uptake of non-oxidatively modified LDL (acetylated LDL) was sufficient to generate MDA-lys epitope in foam cells, consistent with the hypothesis that atherosclerosis is associated with oxidative events in addition to LDL oxidation. We hypothesized that MDA-lys may be generated through intracellular lipid metabolism during the formation of foam cells.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Emulsified lipids increase endotoxemia: possible role in early postprandial low-grade inflammation

Low-grade inflammation is a risk factor for the onset of atherosclerosis. Little is known about the involvement of endotoxin absorption from the gut during the digestion of lipids. In the present study, we first investigated in humans the impact of a mixed meal containing dispersed lipids on postprandial endotoxemia and inflammation. We then investigated the effect of (i) oil emulsification in vivo in rats and (ii) fatty acid amounts in vitro using Caco-2 cells on postprandial endotoxemia. In humans, postprandial endotoxemia increased early after the meal. Moreover, we evidenced that the endotoxin receptor sCD14 increased during digestion and that chylomicrons could contribute to absorbed endotoxin transport. This could explain the significant peak of inflammatory cytokine IL-6 that we observed 2 h after the mixed meal. Interestingly, in rats, the emulsion led to both higher endotoxemia and hypertriglyceridemia than oil and compared to a control saline load. In vitro, incubation of Caco-2 cells with increasing fatty acid concentrations enhanced epithelial absorption of endotoxin. To our knowledge, this is the first study evidencing in healthy humans that, following a mixed meal containing lipids, increased endotoxemia is associated with raised sCD14 and a peak of IL-6. On a repeated basis, this may thus be a triggering cascade for the onset of atherosclerosis. In this respect, optimizing both dietary fat amount and structure could be a possible strategy to limit such low-grade endotoxemia and inflammation by the control of postprandial lipemia.     

The effect of histidine modification on copper-dependent lipid peroxidation in human low-density lipoprotein

Summary

Lipid peroxidation and subsequent oxidative modification of low-density lipoprotein (LDL) have been implicated as causal events in atherosclerosis. Cu²⁺ may play an important role in LDL oxidation by binding to histidine residues of apolipoprotein B-100 (apo B) and initiating and propagating lipid peroxidation. To investigate the role of histidine residues, we used diethylpyrocarbonate (DEPC), a lipid-soluble histidine-specific modifying reagent. When LDL (0.1 mg protein/ml, or 0.2 µM) was incubated with DEPC (1 mM), at least 76 ± 7% of the histidine residues in apo B were modified. Treatment of LDL with DEPC led to an increase in the rate of Cu²⁺-induced initiation of lipid peroxidation (R_i), but a significant decrease in the rate of propagation. These changes resulted in an overall increased resistance of LDL to oxidation, with a significantly increased lag phase preceding the propagation phase of lipid peroxidation. In contrast to DEPC, ascorbate completely prevented the initiation of LDL oxidation (R_i = 0). Our data indicate that there are two types of copper/histidine binding sites on apo B: those facing the lipid core of the LDL particle, which mediate the propagation of lipid peroxidation and are modified by DEPC; and those found on the surface of the LDL particle exposed to the aqueous environment, which are responsible for mediating the initiation of lipid peroxidation and are modifiable by ascorbate in the presence of Cu²⁺.     

Concentration-dependent antioxidant activity of probucol in low density lipoproteins in vitro: probucol degradation precedes lipoprotein oxidation

The ability of probucol, a lipid-lowering drug with antioxidant properties, to prevent the Cu2+-induced oxidation of human plasma low density lipoproteins (LDL) was examined as a function of the concentration of probucol in LDL. In the absence of probucol, 3 microM Cu2+ induced half-maximal LDL lipid oxidation, as determined by the formation of thiobarbituric acid reactive substances (TBARS). Oxidation was associated with a loss of apolipoprotein B-100 and the appearance of higher molecular weight forms of the protein. In the presence of 0.6 mol% probucol (relative to phospholipid) and with 3 microM Cu2+, the time required to obtain half-maximal LDL lipid oxidation increased from 130 to 270 min and was explained by an increase in the lag time prior to LDL lipid oxidation. Once rapid oxidation of LDL had begun, the rate of TBARS formation was similar to that for LDL containing no probucol. At a probucol concentration of 4.2 mol%, the antioxidant prevented the oxidation of LDL-lipids. The delay in Cu2+-induced LDL oxidation with probucol corresponded to the time required for free radical-mediated processes to convert probucol to a spiroquinone and a diphenoquinone. These in vitro findings suggest that the potent antioxidant property of probucol is directly related to the amount of drug in the LDL particle and may have relevance to its antiatherosclerotic effects observed in vivo.     

Structural changes of low density lipoproteins with Cu2+ and glucose induced oxidation

The compositional and structural changes of lipids and apolipoproteins during in vitro oxidation of low density lipoprotein (LDL) are investigated in this study by IR spectroscopy. For comparison, LDL samples containing either copper or glucose at physiological or pathological concentrations are considered in order to know the separate affects of these chemical factors on LDL oxidation. The results show that the initial steps of lipid oxidation proceed through hydrogen atom loss from methylene groups, as well as loss of cholesteryl ester molecules, and later a recovering of carbonyl compounds resulting from aldehyde formation that generally occurs in autooxidation processes. Lipid oxidation is induced by copper ions, and glucose enhances metal ion induced LDL oxidation as determined by conjugated diene formation. As to the protein conformational changes, IR spectroscopy reveals for the first time that LDL oxidation involves formation of beta-sheet structures, these being more abundant in LDL samples with pathological concentrations of glucose or copper. Consequently, the LDL structural changes may contribute to the recognition of oxidized LDL particles by scavenger receptors.     

Mechanisms of oxidative modification of low density lipoproteins under conditions of oxidative and carbonyl stress

Low-molecular-weight aldehydes (glyoxal, methylglyoxal, 3-deoxyglucosone) generated on autooxidation of glucose under conditions of carbonyl stress react much more actively with amino groups of L-lysine and epsilon-amino groups of lysine residues of apoprotein B-100 in human blood plasma low density lipoproteins (LDL) than their structural analogs (malonic dialdehyde (MDA), 4-hydroxynonenal) resulting on free radical oxidation of lipids under conditions of oxidative stress. Glyoxal-modified LDL aggregate in the incubation medium with a significantly higher rate than LDL modified by MDA, and MDA-modified LDL are markedly more poorly absorbed by cultured human macrophages and significantly more slowly eliminated from the rat bloodstream upon intravenous injection. Studies on kinetics of free radical oxidation of rat liver membrane phospholipids have shown that ubiquinol Q(10) is the most active lipid-soluble natural antioxidant, and suppression of ubiquinol Q(10) biosynthesis by beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors (statins) is accompanied by intensification of lipid peroxidation in rat liver biomembranes and in LDL of human blood plasma. Injection of ubiquinone Q(10) protects the human blood plasma LDL against oxidation and prevents oxidative stress-induced damages to rat myocardium. A unified molecular mechanism of atherogenic action of carbonyl-modified LDL in disorders of lipid and carbohydrate metabolism is discussed.     

Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble alpha-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

Modification of the lipid-protein interaction in human low-density lipoprotein destabilizes ApoB-100 and decreases oxidizability

The interactions of the lipid and protein moiety of human low-density lipoprotein (LDL) and their influence on the oxidation behavior of LDL were modified using an amphipathic peptide, melittin, as a probe. The interaction of melittin with the LDL phospholipid surface resulted in a destabilization of apolipoprotein B-100 (apoB-100) as monitored by differential scanning calorimetry, while the characteristics of lipid core melting remained nearly unchanged. Binding of melittin caused a restriction of lipid chain mobility near the glycerol backbone, but not in the middle or near the methyl terminus of the fatty acyl chains as observed by electron paramagnetic resonance. Also, upon melittin addition, the level of copper binding to apoB-100 and the oxidizability of LDL by Cu2+ ions were greatly reduced, as indicated by abolished tryptophan fluorescence quenching upon Cu2+ binding and, during oxidation, prolongation of the lag phase of oxidation, attenuated consumption of alpha-tocopherol, and a lowered maximal rate of conjugated diene formation. This reduction of oxidizability could not be reversed by increasing the Cu2+ concentration. It is deduced that interaction of Cu2+ and alpha-tocopherol is required for reductive activation of the metal. It can be abolished by interfering with the interactions between apoB-100 and the lipid moiety of LDL which modifies the conformation of LDL and, as a consequence, hinders copper binding to apoB-100.     

Dietary supplementation with coenzyme Q10 results in increased levels of ubiquinol-10 within circulating lipoproteins and increased resistance of human low-density lipoprotein to the initiation of lipid peroxidation.

Ubiquinol-10 (CoQH2, the reduced form of coenzyme Q10) is a potent antioxidant present in human low-density lipoprotein (LDL). Supplementation of humans with ubiquinone-10 (CoQ, the oxidized coenzyme) increased the concentrations of CoQH2 in plasma and in all of its lipoproteins. Intake of a single oral dose of 100 or 200 mg CoQ increased the total plasma coenzyme content by 80 or 150%, respectively, within 6 h. Long-term supplementation (three times 100 mg CoQ/day) resulted in 4-fold enrichment of CoQH2 in plasma and LDL with the latter containing 2.8 CoQH2 molecules per LDL particle (on day 11). Approx. 80% of the coenzyme was present as CoQH2 and the CoQH2/CoQ ratio was unaffected by supplementation, indicating that the redox state of coenzyme Q10 is tightly controlled in the blood. Oxidation of LDL containing various [CoQH2] by a mild, steady flux of aqueous peroxyl radicals resulted immediately in very slow formation of lipid hydroperoxides. However, in each case the rate of lipid oxidation increased markedly with the disappearance of 80-90% CoQH2. Moreover, the cumulative radical dose required to reach this 'break point' in lipid oxidation was proportional to the amount of CoQH2 incorporated in vivo into the LDL. Thus, oral supplementation with CoQ increases CoQH2 in the plasma and all lipoproteins thereby increasing the resistance of LDL to radical oxidation.