Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals

Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.     

Glucose enhancement of LDL oxidation is strictly metal ion dependent

Recent evidence suggests that lipoprotein oxidation is increased in diabetes, however, the mechanism(s) for such observations are not clear. We examined the effect of glucose on low-density lipoprotein (LDL) oxidation using metal ion-dependent and -independent oxidation systems. Pathophysiological concentrations of glucose (25 mM) enhanced copper-induced LDL oxidation as determined by conjugated diene formation or relative electrophoretic mobility (REM) on agarose gels. Similarly, iron-induced LDL oxidation was stimulated by glucose resulting in 4- to 6-fold greater REM than control incubations without glucose. In contrast, glucose had no effect on metal ion-independent LDL oxidation by aqueous peroxyl radicals. The effect of glucose on metal ion-dependent LDL oxidation was associated with enhanced reduction of metal ions, and in the case of iron-induced LDL oxidation, was completely inhibited by superoxide dismutase. The effect of glucose was mimicked by other reducing sugars, such as fructose and mannose, and the extent to which each sugar enhanced LDL oxidation was closely linked to its metal ion-reducing activity. Thus, promotion of LDL oxidation by glucose is specific for metal ion-dependent oxidation and involves increased metal ion reduction. These results provide one potential mechanism for enhanced LDL oxidation in diabetes.     

p-Hydroxyphenylacetaldehyde, the major product of tyrosine oxidation by the activated myeloperoxidase system can act as an antioxidant in LDL

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. HOCl generated by the myeloperoxidase/H2O2/Cl- system of activated neutrophils may be operative in vivo making LDL atherogenic. Tyrosine has been found to be oxidized by HOCl to p-hydroxyphenylacetaldehyde (p-HA) capable of modifying phospholipid amino groups in LDL. As an amphiphatic phenolic compound, p-HA may have the potential to act as an antioxidant in the lipid phase of LDL. The present results show that (a) tyrosine exerts a protective effect on LDL modification by HOCl, (b) p-HA could act as antioxidant associated with the lipoprotein preventing cell- and transition metal ion-mediated LDL oxidation and (c) p-HA was able to scavenge free radicals.     

Dual effect of glucose on LDL oxidation: dependence on vitamin E

The aim of the present study was to determine the direct effect of glucose on LDL oxidation, a key step in the development of atherosclerosis. Purified human LDL were incubated with glucose (500 mg/dl) and LDL oxidation was started by adding CuCl(2) to the media. Glucose delayed the vitamin E consumption, but accelerated the formation of conjugated dienes and increased both the formation of thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility. When LDL were incubated with increasing concentrations of glucose and submitted to oxidation, the formation of conjugated dienes, TBARS, and the electrophoretic mobility increased in a concentration-dependent manner. When LDL was enriched with vitamin E, it showed a delay in the formation of conjugated dienes, even in the presence of glucose. To determine whether glucose had any effect on LDL oxidation, once the process was started and vitamin E consumed, LDL were submitted to oxidation and, at different times thereafter, glucose was added into the media. Under these conditions glucose also accelerated the LDL oxidation. In summary, present results show that in LDL submitted to oxidation, glucose delays the early phases of the oxidation, slowing the vitamin E consumption, but it accelerates the rate of LDL oxidation once LDL vitamin E has been consumed; the effect being concentration-dependent.     

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.     

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.     

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.     

Glucose-Modified Low Density Lipoprotein Enhances Human Monocyte Chemotaxis

In diabetes mellitus the progression of atherosclerosis is accelerated. The interaction of glucose with athero-genic lipoproteins may be relevant to the mechanisms responsible for this vascular damage. The aim of this study was to examine the effect of glucose-modified low density lipoprotein (LDL) on human monocyte chemotaxis and to investigate the roles of oxidation and glycation in the generation of chemotactic LDL. Cu(II)-mediated LDL oxidation was potentiated by glucose in a dose-dependent manner and increased its chemotactic activity. Incubation with glucose alone, under conditions where very little oxidation was observed, also increased the chemotactic property of LDL. Neither diethylenetriamine pentaacetic acid (DETAPAC) nor aminoguanidine, which both inhibited LDL oxidation, completely inhibited the chemotactic activity of glycated oxidised LDL. The results suggest that both oxidation and glycation contribute to increased chemotactic activity.     

Products of the reaction of HOCl with tryptophan protect LDL from atherogenic modification

Hypochlorite (HOCl) attacks amino acid residues in LDL making the particle atherogenic. Tryptophan is prone to free radical reactions and modification by HOCl. We hypothesized, that free tryptophan may quench the HOCl attack therefore protecting LDL. Free tryptophan inhibits LDL apoprotein modification and lipid oxidation. Tryptophan-HOCl metabolites associate with LDL reducing its oxidizability initiated by endothelial cells, Cu(2+) and peroxyl radicals. One tryptophan-HOCl metabolite was identified as 4-methyl-carbostyril which showed antioxidative activity when present during Cu(2+) mediated lipid oxidation, but did not associate with LDL. Indole-3-acetaldehyde, a decomposition product of tryptophan chloramine (the product of the tryptophan-HOCl reaction) was found to associate with LDL increasing its resistance to oxidation. Myeloperoxidase treatment of LDL in the presence of chloride, H(2)O(2) and tryptophan protected the lipoprotein from subsequent cell-mediated oxidation. We conclude that, in vivo, the activated myeloperoxidase system can generate antioxidative metabolites from tryptophan by the reaction of hypochlorite with this essential amino acid.     

Prooxidant and antioxidant properties of Trolox C, analogue of vitamin E, in oxidation of low-density lipoprotein

Trolox C (Trolox), a water-soluble analogue of vitamin E lacking the phytyl chain, was investigated with respect to its effect on the oxidation of low-density lipoprotein (LDL). Trolox was added at different time points of LDL oxidation induced by Cu2+ and aqueous peroxyl radicals. In the case of Cu2+ -induced LDL oxidation, the effect of Trolox changed from antioxidant to prooxidant when added at later time points during oxidation; this transition occurred whenever alpha-tocopherol was just consumed in oxidizing LDL. Thus, in the case of Cu2+ -dependent LDL oxidation, the presence of lipophilic antioxidants in the LDL particle is likely to be a prerequisite for the antioxidant activity of Trolox. When oxidation was induced by peroxyl radicals, as a model of metal-independent oxidation, the effect of Trolox was always antioxidant, suggesting the importance of Cu2+ /Cu+ redox-cycling in the prooxidant mechanism of Trolox. Our data suggest that, in the absence of significant amounts of lipophilic antioxidants, LDL becomes highly susceptible to oxidation induced by transition metals in the presence of aqueous reductants.     

The initiation of free radical peroxidation of low-density lipoproteins by glucose and its metabolite methylglyoxal: a common molecular mechanism of vascular wall injure in atherosclerosis and diabetes

It was found that glucose in the range of concentrations 12.5–100 mM stimulated Cu²⁺–mediated free radical peroxidation of low-density lipoproteins (LDL) from human blood plasma. Considering the kinetic parameters of LDL peroxidation we proposed that intensification of this process may be caused by formation of free radical intermediates of glucose auto-oxidation. Addition of SOD to the medium inhibited LDL oxidation, indicating the formation of superoxide anion-radicals under autoxidation of glucose. Similarly, SOD inhibited free radical peroxidation of liposomes from egg lecithin in the presence of glucose that confirms the generation of superoxide radicals under co-oxidation of unsaturated lipids and glucose. Normalization of glucose level in the blood of patients with type 2 diabetes mellitus during therapy was accompanied by a significant decrease in LDL oxidation in vivo (the decrease in primary and secondary lipoperoxidation products). The formation of superoxide anion-radicals was observed during interaction of aminoacid l-lysine with a product of glucose oxidative metabolism–methylglyoxal, but not with a product of lipoperoxidation malonyldialdehyde. In accordance with the foregoing the administration of sugar-lowering drug metformin, which binds and utilizes methylglyoxal, caused a stronger inhibition of LDL peroxidation in the blood of patients with diabetes mellitus, probably due to decrease in methylglyoxal-dependent generation of superoxide anion-radicals. Based on the results we set out the hypothesis about autocatalytic mechanism of free radical reactions involving natural dicarbonyls and suppose the common molecular mechanism of vascular wall injury in atherosclerosis and diabetes.     

Morin hydrate inhibits azo-initiator induced oxidation of human low density lipoprotein

We demonstrated that the flavonoid morin hydrate at 75–100 μM protects against the oxidation of low density lipoprotein (LDL) by free radicals produced by 2,2′-azo-bis(2-amidinopropane) dihydrochloride. Morin hydrate reduces the relative electrophoretic mobility, malondialdehyde equivalents and lipid peroxide level of oxidized LDL. On the other hand, Trolox (an analogue of vitamin E) showed less protective effect in the present system. Since free radical mediated oxidation of LDL is implicated to be a cause of atherogenesis, morin hydrate may be a candidate chemotherapeutic agent herein.     

Human apo-lipoprotein B from normal plasma contains oxidised peptides

Oxidation of low density lipoprotein (LDL) may be atherogenic, but radical-initiated oxidation of its apoprotein B-100 (apoB) has been little studied. Transition metal ions iron and copper are candidates for mediating radical oxidation of LDL in vivo. Therefore, we studied the copper-ion-induced oxidation of apoB in human LDL. Using HPLC methods developed in our recent work, we studied the destruction of native and the generation of six oxidised amino acids; we also assessed the release of peptides from the LDL particle by FPLC. We observed time-dependent losses of apoB histidine, lysine and glycine. Long-lived reactive species, the reductant DOPA, and the oxidant hydroperoxides of valine and leucine (measured as hydroxides after reduction), were generated. Their relative abundance (mol/mol of parent amino acid) was DOPA>o- and m-tyrosine>dityrosine, valine-hydroxides, leucine hydroxides. Low molecular weight fragments were also released from the LDL in a time-dependent manner, contained hydroperoxides sensitive to GSH peroxidase, and generated radicals on reaction with iron–EDTA. The fragments contained peptides active in the quinone redox cycling procedure, comprising 0.25% of the supplied LDL amino acids. Characteristic peptides were present in each FPLC fraction containing the fragments, as judged by further HPLC fractionation. Some fragments were present in the unoxidised LDL preparations, and when these were largely removed by FPLC, copper oxidation could still generate fragments, suggesting that those present in the starting material might indicate prior oxidation. Concordantly, we found that fresh plasma LDL apoB contained 3% of total plasma protein-bound oxidised amino acids, and with the same relative abundance. We conclude that plasma proteins including apoB are subject to physiological oxidation, similar to that inflicted by copper ions; the latter may contribute to intimal LDL oxidation, which could be the source of oxidised plasma apoB.     

Estimating Hydroxyl Radical Content in Rat Brain Using Systemic and Intraventricular Salicylate: Impact of Methamphetamine

Abstract: Free radicals have been implicated in the etiology of many neurodegenerative conditions. Yet, because these species are highly reactive and thus short-lived it has been difficult to test these hypotheses. We adapted a method in which hydroxyl radicals are trapped by salicylate in vivo, resulting in the stable and quantifiable products, 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. After systemic (100 mg/kg i.p.) or intraventricular (4 µmol) administration of salicylate, the amount of DHBA in striatal tissue correlated with tissue levels of salicylate. After systemic salicylate, the ratio of total DHBA to salicylate in neostriatum was at least 10-fold higher than that observed after central salicylate. In addition, systemic salicylate resulted in considerably higher concentrations of 2,3- and 2,5-DHBA in plasma than in brain. Therefore, a large portion of the DHBA present in brain after systemic salicylate may have been formed in the periphery. A neurotoxic regimen of methamphetamine increased the concentration of DHBA in neostriatum after either central or systemic administration of salicylate. The increase in 2,3-DHBA after the central administration of salicylate was significant at 2 h, but not at 4 h, after the last dose of methamphetamine. These results suggest that (1) when assessing specific events in brain, it is preferable to administer salicylate centrally, and (2) neurotoxic doses of methamphetamine increase the hydroxyl radical content in brain in a time-dependent manner.     

The influence of glucose on free radical peroxidation of low density lipoproteins in vitro and in vivo

In the range of concentrations 12.5–100 mM glucose stimulated Cu-mediated free radical peroxidation of low density lipoproteins (LDL) from human blood plasma. Based on analysis of kinetic parameters of the LDL peroxidation it was found that intensification of this process is caused by formation of free radical intermediates of glucose autooxidation during generation of reactive oxygen species in the presence of transition metal ions. Normalization of blood glucose in patients with type 2 diabetes during therapy was accompanied by a significant decrease of LDL oxidation. Therapy with the sugar-lowering drug metformin, which utilizes methylglyoxal, caused much higher inhibition of the in vivo LDL peroxidation in blood of patients with diabetes mellitus probably due to the decrease of methylglyoxal-dependent generation of superoxide anion radicals shown by us earlier [Biochemistry (Moscow) 2009, vol. 74, pp. 568–574].     

Salicylate promotes myeloperoxidase-initiated LDL oxidation: antagonization by its metabolite gentisic acid.

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. Tyrosyl radicals generated by myeloperoxidase (MPO) can act as prooxidants of LDL oxidation. Taking into consideration, that monophenolic compounds are able to form phenoxyl radicals in presence of peroxidases, we have tested salicylate, in its ability to act as a prooxidant in the MPO system. Measurement of conjugated dienes and lipid hydroperoxides were taken as indicators of lipid oxidation. Exposure of LDL preparations to MPO in presence of salicylate revealed that the drug could act as a catalyst of lipid oxidation in LDL. The radical scavenger ascorbic acid as well as heme poisons (cyanide, azide) and catalase were inhibitory. The main metabolite of salicylic acid, gentisic acid, showed inhibitory action in the MPO system. Even when lipid oxidation was maximally stimulated by salicylate the LDL oxidation was efficaciously counteracted in presence of gentisic acid at salicylate/gentisic acid ratios that could be reached in plasma of patients receiving aspirin medication. Gentisic acid was also able to impair the tyrosyl radical catalyzed LDL peroxidation. The results suggest that salicylate could act like tyrosine via a phenoxyl radical as a catalyst of LDL oxidative modification by MPO. But the prooxidant activity of this radical species is effectively counteracted by the salicylate metabolite gentisic acid.     

Glucose accelerates copper- and ceruloplasmin-induced oxidation of low-density lipoprotein and whole serum

Glucose at pathophysiological concentrations was able to accelerate copper-induced oxidation of isolated low-density lipoprotein (LDL) and whole serum. The efficiency of glucose was favored under the following circumstances: (a) when LDL oxidation was induced by low copper concentration, (b) when LDL was partly oxidized, i.e. enriched with lipid peroxides. The glucose derivative methyl-alpha-D-glucoside was ineffective on Cu2+-induced LDL oxidation, pointing out the essential role of the reactivity of the aldehydic carbon for the pro-oxidative effect. When LDL oxidation was induced by a peroxyl radical generator, as a model of transition metal independent oxidation, glucose was ineffective. Glucose was found to stimulate oxidation of LDL induced by ceruloplasmin, the major copper-containing protein of human plasma. Thus, glucose accelerated oxidation of LDL induced by both free and protein bound copper. Considering the requirement for catalytically active copper and for the aldehydic carbon, the pro-oxidative effect of glucose is likely to depend on the increased availability of Cu+; this is more efficient in decomposing lipid peroxide than Cu2+, accounting for acceleration of LDL oxidation. The possible biological relevance of our work is supported by the finding that glucose was able to accelerate oxidation of whole serum, which was assessed by monitoring low-level chemiluminescence associated with lipid peroxidation.     

Recycling of vitamin E in human low density lipoproteins.

Oxidative modification of low density lipoproteins (LDL) and their unrestricted scavenger receptor-dependent uptake is believed to account for cholesterol deposition in macrophage-derived foam cells. It has been suggested that vitamin E that is transported by LDL plays a critical role in protecting against LDL oxidation. We hypothesize that the maintenance of sufficiently high vitamin E concentrations in LDL can be achieved by reducing its chromanoxyl radicals, i.e., by vitamin E recycling. In this study we demonstrate that: i) chromanoxyl radicals of endogenous vitamin E and of exogenously added alpha-tocotrienol, alpha-tocopherol or its synthetic homologue with a 6-carbon side-chain, chromanol-alpha-C6, can be directly generated in human LDL by ultraviolet (UV) light, or by interaction with peroxyl radicals produced either by an enzymic oxidation system (lipoxygenase + linolenic acid) or by an azo-initiator, 2,2'-azo-bis(2,4-dimethylvaleronitrile) (AMVN; ii) ascorbate can recycle endogenous vitamin E and exogenously added chromanols by direct reduction of chromanoxyl radicals in LDL; iii) dihydrolipoic acid is not efficient in direct reduction of chromanoxyl radicals but recycles vitamin E by synergistically interacting with ascorbate (reduces dehydroascorbate thus maintaining the steady-state concentration of ascorbate); and iv) beta-carotene is not active in vitamin E recycling but may itself be protected against oxidative destruction by the reductants of chromanoxyl radicals. We suggest that the recycling of vitamin E and other phenolic antioxidants by plasma reductants may be an important mechanism for the enhanced antioxidant protection of LDL.     

Paradoxical protective effect of aminoguanidine toward low-density lipoprotein oxidation: inhibition of apolipoprotein B fragmentation without preventing its carbonylation. Mechanism of action of aminoguanidine

Oxidative modification of low-density lipoproteins (LDLs) is an important feature in the initiation and progression of atherosclerosis. Aminoguanidine (AMG), classically described as an inhibitor of advanced glycation end products, turned out to be also efficient in animal models as an antioxidant against lipid peroxidation. The originality of the present study was based on the simultaneous assessment of the oxidation of LDL lipid and protein moieties in order to characterize the molecular sites of AMG protection. Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes (CD) and hydroperoxide molecular species from cholesteryl esters (CEOOH) and phosphatidylcholines (PCOOH). LDL protein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. The LDL oxidation was mediated by water gamma radiolysis, which has the advantage of being quantitative and highly selective with regard to the free radicals produced. Here, we reported that AMG resulted in a protection of LDLs against lipid peroxidation (both in the lag phase and in the propagation phase) and against apoB fragmentation in a concentration-dependent manner, due to the scavenging effect of AMG toward lipid peroxyl radicals. Paradoxically, AMG was poorly efficient against apoB carbonylation that began during the lag phase. We hypothesize that, even in the presence of AMG, a nonnegligible proportion of (*)OH radicals remained able to initiate oxidation of the LDL protein moiety, leading to apoB carbonylation.     

Why is glycated LDL more sensitive to oxidation than native LDL? A comparative study

It is well established that oxidative modification of low-density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. To examine the influence of different agents which may influence LDL-glycation and oxidation, experiments including glycation with glucose, glucose 6-phosphate, metal chelators (EDTA) and antioxidants (BHT) were performed. The influence of time dependence on the glycation process and the alteration of the electrophoretic mobility of LDL under diverse glycation and/or oxidation conditions was also investigated. The formation of conjugated dienes and levels of lipid peroxides in these different LDL-modifications were estimated. The copper-induced oxidation of LDL in vitro was determined by measurement of thiobarbituric acid reactive substances (TBARS) and expressed as nmol MDA/mg of LDL protein. We found that glycated LDL is more prone to oxidation than native LDL. Using native LDL, the maximal oxidation effect was found to reach a value of 49.72 nmol MDA/mg protein after 8 h. The maximum oxidation of the 31 days, glycated LDL with glucose was 71.76 nmol MDA/mg protein amounting to 144.33% of the value found for native LDL. In the case of glucose 6-phosphate glycation, the maximum oxidation under the same conditions amounted to 173.77% of the value found for native LDL. To measure the extent of glycation, fluorescence of advanced glycation end products (AGEs) was determined (370 nm excitation and 440 nm emission). The most potent glycation agent was glucose 6-phosphate leading to the formation of very high amounts of AGEs. This process was promoted in the absence of EDTA, which prevents the oxidative cleavage of modified Amadori products (ketoamines) to AGEs. We therefore conclude that both processes, glycation and oxidation, result in the modification of LDL. The lower the glycation-rate (+/- EDTA) as measured by relative fluorescence units RFU (generation of AGEs), the lower the additional oxidation rate after glycation as measured by TBARS (generation of MDA equivalents). Glycation and/or oxidation change the electrophoretic mobility of LDL.