Probabilistic kinetic model of slow oxidation of low-density lipoprotein: I. Theory

The microscopic probabilistic model has been introduced to explain the kinetics of very slow oxidation of low-density lipoprotein (LDL) from human plasma. The LDL oxidation, carried out in very unfavorable conditions, is assumed to be initiated by the traces of the transition-metal ions associated with the lipoprotein. The substrates for the metal-ion attack are alpha-tocopherol and the pre-formed lipid hydroperoxide. The theory assumes oscillation of the metal ions and alpha-tocopherol from the oxidized to the reduced states. In this model alpha-tocopherol acts as a pro-oxidant. The entire oxidation process consists of rare bursts of events in individual LDL particles. The reactions within the particles are treated in terms of probabilities of individual active species to participate in a specified reaction. The circular flow of the radical reactions could be visualized as circular flow of microscopic probabilities. The empirical, macroscopic quantities are quantitatively related with the microscopic probabilities, determined by a set of five adjustable parameters. The differential equations describing the initial radical generation rate and the rates of change of concentration of oxygen, hydroperoxide, co-antioxidant and trapped radicals in an LDL system are numerically solved in a finite difference approach.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

A comparison of the kinetics of low-density lipoprotein oxidation induced by copper or by gamma-rays: influence of radiation dose-rate and copper concentration

The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis. The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or by oxygen free radicals generated by 60Co gamma-rays. The effects of copper concentration and irradiation dose-rate on LDL peroxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene, hydroperoxides, and thiobarbituric acid reactive substance formation as well as alpha-tocopherol disappearance. In the case of gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiation dose-rate. The radiation chemical rates (nu) increased with increasing dose-rate. Copper-induced LDL peroxidation followed two kinetic patterns: a slow kinetic for copper concentrations between 5-20 microM, and a fast kinetic for a copper concentration of 40 microM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copper binding site on apo-B. When compared with gamma-rays, copper ions act as drastic and powerful oxidants only at higher concentrations (> or = 40 microM).     

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: A beneficial “side effect” of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic–phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.     

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: a beneficial "side effect" of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic-phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.     

Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.     

Protective Effect of Dehydroepiandrosterone Against Copper-Induced Lipid Peroxidation in the Rat

This study investigates the effectiveness and multitargeted activity of dehydroepiandrosterone (DHEA) as antioxidant in vivo. A single dose of DHEA was given IP to male rats. Liver and brain microsomes, and plasma low density lipoprotein (LDL), were isolated from rats sacrified 17 h later. Liver and brain microsomes were challenged with CuSO₄ and, as index of lipid peroxidation, the production of thiobarbituric acid reactive substances (TBARS) was measaured. Also, plasma low-density lipoprotein (LDL) were challenged with copper and the time course of lipid peroxidation was evaluated following the formation of conjugated dienes. The onset of TBARS generation induced by copper was marked delayed in both liver and brain microsomes from DHEA-treated animals. Also, the resistance of LDL to oxidation, expressed by the duration of the lag-phase of the kinetic curve, was significantly enhanced in DHEA-treated rats. Results indicate that in vivo DHEA supplementation makes subcellular fractions isolated from different tissues and plasma constituents (LDL) more resistant to lipid peroxidation triggered by copper. The antioxidant effect on plasma LDL might be of special relevance to the proposed antiatherogenic activity of DHEA. Moreover, multitargeted antioxidant activity of DHEA might protect tissues from oxygen radicals damage. © 1997 Elsevier Science Inc.     

Sulfite facilitates LDL lipid oxidation by transition metal ions: a pro-oxidant in wine?

Lipid oxidation in LDL may play a role in atherogenesis. It has been shown that sulfite - a compound in the aqueous fraction of wine - could inhibit free radical (AAPH) mediated oxidation of plasma. Thus, sulfite has been proposed as an antioxidant. In contrast, the aqueous phase of wine has recently been shown to contain not fully identified compounds promoting transition metal ion (Cu(2+)) initiated LDL oxidation. As transition metal ions can catalyse the auto-oxidation of sulfite, we studied the influence of sulfite on Cu(2+) initiated LDL oxidation. The results show that sulfite at concentrations found in vivo strongly facilitated LDL oxidation by Cu(2+). The LDL-oxidase activity of ceruloplasmin was also stimulated by sulfite. ROS formation by Cu(2+)/SO(3)(2-) was not inhibited by SOD but by catalase. We propose that formation of Cu(+), sulfite radicals (SO(3)*(-)) and hydroxyl radicals (OH(*)) is a mechanism by which sulfite could act as a pro-atherogenic agent in presence of transition metal ions.     

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or -tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and -tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.     

Protective effect of dietary capsaicin on induced oxidation of low-density lipoprotein in rats

An animal study was carried out to examine the beneficial influence of the known hypocholesterolemic spice principle-capsaicin on the susceptibility of low-density lipoprotein to oxidation in normal and hypercholesterolemic condition. In rats rendered hypercholeterolemic by maintaining them on a cholesterol-enriched diet for eight weeks, inclusion of capsaicin (0.015%) in the diet, produced significant hypocholesterolemic effect. Oxidation of low-density lipoprotein was induced either by copper ion in vitro after its isolation, or by ferrous ion in vivo in experimental rats under either normal or hypercholesterolemic situation and the beneficial effect of dietary capsaicin on the same was evaluated. LDL oxidation was measured by the thiobarbituric acid reactive substances (TBARS) formed and relative electrophoretic mobility of oxidized LDL. Dietary capsaicin was found to be protective to the LDL oxidation in vitro in the case of normal rats as indicated by reduction in TBARS by more than 40%. In the case of LDL isolated from hypercholesterolemic rats the extent of copper induced LDL oxidation was significantly lower than that of LDL isolated from normal rats. Dietary capsaicin did not make any difference in the extent of LDL oxidation in vitro in hypercholesterolemic rats. Ferrous ion induced in vivo oxidation of LDL was 71% lower in capsaicin fed normal rats. In high cholesterol feeding, Fe-induced in vivo oxidation of LDL was 73% lower, while the same was still marginally lower in capsaicin fed hypercholesterolemic rats. Hepatic lipid peroxidation was significantly decreased by dietary capsaicin in normal rats. While a significantly decreased level of lipid peroxidation was observed in hypercholesterolemic rats compared to normal rats, the same was not significantly altered by dietary capsaicin. Results suggest that dietary spice principle capsaicin is protective to LDL oxidation both in vivo and in vitro under normal situation, while in hypercholesterolemic situation where the extent of LDL oxidation is already lowered, capsaicin does not offer any further reduction.     

Modulation of LDL oxidation by 7,8-dihydroneopterin

Human macrophages stimulated with interferon-γ generate neopterin and 7,8-dihydroneopterin which interfere with reactive species involved in LDL oxidation. While neopterin was found to have pro-oxidative effects on copper-mediated LDL oxidation, the influence of 7,8-dihydroneopterin is more complex. This study provides detailed information that 7,8-dihydroneopterin reveals both pro-oxidative and anti-oxidative effects on copper mediated LDL oxidation. 7,8-dihydroneopterin inhibited the oxidation of native LDL effectively monitored by (i) formation of conjugated dienes, (ii) relative electrophoretic mobility (EM) and (iii) specific oxidized epitopes. Using minimally oxidized LDL (mi-LDL) or moderately oxidized LDL (mo-LDL) 7,8-dihydroneopterin changed its antioxidative behavior to a strongly pro-oxidative. Incubation of 7,8-dihydroneopterin with native LDL, mi-LDL or mo-LDL in the absence of copper ions showed that formation of conjugated dienes was more increased in mo-LDL than in mi-LDL while no diene formation was observed with native LDL.

We suggest that 7,8-dihydroneopterin is a modulator for LDL oxidation in the presence of copper ions depending on the “oxidative status” of this lipoprotein.     

The Chemiluminescence Assay of Lipid Peroxidation Products in Human Blood Plasma Lipoproteins

A chemiluminescence (CL) flash kinetics on the addition of Fe²⁺ ions into oxidized low density lipoprotein (LDL) suspension has been studied. LDL oxidation was carried out at 37°C without and in the presence of 5 or 50 μM of Cu.²⁺ It has been found that under certain experimental conditions (the addition of excess iron ions, more than 1 mM) the amplitude of CL flash depended almost linearly (1) on the concentration of oxidized LDL and (2) on the extent of LDL oxidation measured as diene conjugates (DC) and 2-thiobarbituric acid-reactive substance (TBARS) accumulation. The corresponding correlation coefficients were: for TBARS - 0.94 and for DC - 0.97, in the case of LDL autooxidation; 0.72 and 0.98, in the case of copper-induced LDL oxidation. A sensitivity of the CL method was shown to be significantly enhanced (by more than two orders) in the presence of CL sensitizer - 2, 3,5, 6-lH,4H-tetrahydro-9-(2' -benzoimidazolyl)-quinolizin-(9, 9a, 1 -gh)coumarin.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin, transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.     

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 (K_m=3.7 μM, k_cat=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 (K_m=0.64 μM, k_cat=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.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin,transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.     

Slow oxidation of high density lipoproteins as studied by EPR spectroscopy

There is relatively little information on the role of high density lipoprotein (HDL) oxidation in atherogenesis although there are indications that oxidation might affect atheroprotective activities of HDL. Recently we reported the study on LDL oxidation initiated and sustained by traces of the transition metal ions under conditions, which favor slow oxidation. Here we report the results of the analogous study on the oxidation of the two HDL subclasses. The oxidation process was monitored by measuring the time dependence of oxygen consumption and concentration of the spin-trapped free radicals using EPR spectroscopy. In both HDL2 and HDL3 subclasses, the dependence of the oxidation process on the copper/lipoprotein molar ratio is different from that in LDL dispersions. Comparison of the kinetic profiles of HDL2 and HDL3 oxidation revealed that under all studied experimental conditions HDL2 was more susceptible to copper-induced oxidation than HDL3.     

A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin

The mechanisms by which low-density lipoprotein (LDL) particles undergo oxidative modification to an atherogenic form that is taken up by the macrophage scavenger-receptor pathway have been the subject of extensive research for almost two decades. The most common method for the initiation of LDL oxidation in vitro involves incubation with Cu(II) ions. Although various mechanisms have been proposed to explain the ability of Cu(II) to promote LDL modification, the precise reactions involved in initiating the process remain a matter of contention in the literature. This review provides a critical overview and evaluation of the current theories describing the interactions of copper with the LDL particle. Following discussion of the thermodynamics of reactions dependent upon the decomposition of preexisting lipid hydroperoxides, which are present in all crude LDL preparations, attention is turned to the more difficult (but perhaps more physiologically-relevant) system of the hydroperoxide-free LDL particle. In both systems, the key role of alpha-tocopherol is discussed. In addition to its protective, radical-scavenging action, alpha-tocopherol can also behave as a prooxidant via its reduction of Cu(II) to Cu(I). Generation of Cu(I) greatly facilitates the decomposition of lipid hydroperoxides to chain-carrying radicals, but the mechanisms by which the vitamin promotes LDL oxidation in the absence of preformed hydroperoxides remain more speculative. In addition to the so-called tocopherol-mediated peroxidation model, in which polyunsaturated fatty acid oxidation is initiated by the alpha-tocopheroxyl radical (generated during the reduction of Cu(II) by alpha-tocopherol), an evaluation of the role of the hydroxyl radical is provided. Important interactions between copper ions and thiols are also discussed, particularly in the context of cell-mediated LDL oxidation. Finally, the mechanisms by which ceruloplasmin, a copper-containing plasma protein, can bring about LDL modification are discussed. Improved understanding of the mechanisms of LDL oxidation by copper ions should facilitate the establishment of any physiological role of the metal in LDL modification. It will also assist in the interpretation of studies in which copper systems of LDL oxidation are used in vitro to evaluate potential antioxidants.     

Detection of apolipoprotein B100 early conformational changes during oxidation

Conformational changes of human plasma apolipoprotein B100 (apoB) during oxidative modification of low-density lipoproteins (LDL) have been investigated. Emphasis has been put on the early stages of LDL oxidation and the modification of apoB. We have applied two different modes of LDL oxidation initiation in order to approach the problem from different perspectives. To study conformational changes of the protein and the phospholipids surface monolayer, we have applied attenuated total reflection infrared as well as fluorescence spectroscopy. We have found for the first time that conformational changes of apoB occur even in the earliest stages of oxidation process and that those are located predominantly in the β-sheet regions. The dynamics of changes has also been described and related to different stages of oxidation. After initial increase in particle surface accessibility and mobility, by entering into the propagation phase of oxidation process, LDL surface accessibility and mobility are decreased. Finally, in the decomposition phase of LDL oxidation, as the particle faces large chemical and physical changes, surface mobility and accessibility is increased again. These observations provide new insights into the modifications of LDL particles upon oxidation.     

Oxidation of Low Density Lipoprotein Upon Sequential Exposure to Copper Ions

Copper-induced LDL oxidation is characterized by an 'induction phase' (lag phase) during which the endogenous antioxidants are consumed, followed by a 'propagation phase' in which the LDL-associated polyunsaturated fatty acids are oxidized. Oxidation products may play an important role in the propagation of the oxidative process in the arterial intima as they increase the permeability of the damaged endothelium to various plasma components, including LDL. We therefore found it of interest to investigate the kinetics of LDL oxidation in vitro under conditions where LDL is sequentially exposed to Cu²⁺-induced oxidation.

The results of our studies demonstrate that when native LDL is exposed to copper oxidation in a medium containing oxidized LDL, oxidation of the added LDL may be almost instantaneous. Furthermore, even when native LDL is added to 'oxidizing LDL' towards the end of the lag phase or during the propagation phase it becomes oxidized after a very short lag. This oxidation process, occurring in spite of the possible protective effect of the antioxidants present in the newly added LDL, indicates that although antioxidants prolong the latency period by preventing the formation of active free radicals, when such radicals are present in the system, oxidation propagates. These results lend strong support to the generally accepted paradigm regarding the mechanism of propagation of lipid oxidation.

In view of the effect of oxidation products on the permeability of the endothelium, the observed shortening of the lag period may result in a vicious cycle, independent of the LDL-associated antioxidants, leading to continuing oxidation and foam cell formation.