An immunochemical marker of low density lipoprotein oxidation

Using monoclonal antibodies against apolipoprotein B (apoB) we studied changes in apoB immunoreactivity during copper ion-mediated oxidation of human low density lipoprotein (LDL). The radioimmunoassay experiments demonstrated the decrease of immunoreactivity of three different epitopes of apoB located in different parts of the protein; at the same time the immunoreactivity of another epitope, previously mapped to the C-terminal 20 amino acids of apoB increased markedly during the first 6 h of LDL oxidation and diminished gradually upon prolonged incubation with copper ions. The fate of LDL during oxidation was also monitored using electrophoretic techniques combined with immunodetection. These experiments showed a rapid fragmentation and disappearance of immunoreactive apoB. They also indicated that the diminishing LDL immunoreactivity detectable during oxidation is associated with apoB fragments still attached to the lipid core. The changes in apoB immunoreactivity during Cu2+ treatment of LDL are similar to those observed upon LDL aging. Therefore, it appears that the enhancement of immunoreactivity of the C-terminus of apoB is a general phenomenon associated with various kinds of oxidative modifications of LDL.     

Tetravalent vanadium mediated oxidation of low density lipoprotein

1. Tetravalent vanadium causes oxidation of low density lipoprotein (LDL) as manifest by protein degradation and lipid peroxidation. 2. Oxidative modification of the apolipoprotein B-100 is paralleled by the formation of thiobarbituric acid reactive substance and fluorescent chromolipid production. 3. The metal chelators ethylenediamine tetracetic acid and desferrioxamine, and the alcohols, ethanol and isopropanol inhibit the oxidation of LDL by tetravalent vanadium. No inhibition is observed with superoxide dismutase, catalase or mannitol. 4. The data suggest that aldehydes formed during the process of lipid peroxidation induced by tetravalent vanadium react with the proteins in LDL to form fluorescent chromolipids and that the oxidative process originates within the hydrophobic domain of LDL.     

Inhibition of human low density lipoprotein oxidation by flavonols and their glycosides

Antioxidative effects of the flavonols and their glycosides, i.e., quercetin (Q), quercetin galactopyranoside (QG), quercetin rhamnolpyranoside (QR), rutin (R), morin (MO), myrecetin (MY), kaempferol (K) and kaempferol glucoside (KG), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either by a water-soluble initiator 2,2'-azobis(2-amidino propane hydrochloride) (AAPH), or by cupric ion (Cu2+). The reaction kinetics were monitored either by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL, or by the formation of thiobarbituric acid reactive substances (TBARS). Kinetic analysis of the antioxidation process demonstrates that these flavonols and their glycosides are effective antioxidants against AAPH- and Cu(2+)-initiated LDL peroxidation, the flavonols bearing ortho-dihydroxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities, and the glycosides are less active than their parent aglycones.     

Inhibition of human low density lipoprotein oxidation by active principles from spices

Spice components and their active principles are potential antioxidants. In this study we examined the effect of phenolic and non-phenolic active principles of common spices on copper ion-induced lipid peroxidation of human low density lipoprotein (LDL) by measuring the formation of thiobarbituric acid reactive substance (TBARS) and relative electrophoretic mobility (REM) of LDL on agarose gel. Curcurriin, capsaicin, quercetin, piperine, eugenol and allyl sulfide inhibited the formation of TBARS effectively through out the incubation period of 12 h and decreased the REM of LDL. Spice phenolic active principles viz. curcumin, quercetin and capsaicin at 10 M produced 40–85% inhibition of LDL oxidation at different time intervals while non-phenolic antioxidant allyl sulfide was less potent in inhibiting oxidation of LDL. However, allyl sulfide, eugenol and ascorbic acid showed pro-oxidant activity at lower concentrations (10 M) and antioxidant activity at higher concentrations (50 M) only. Among the spice principles tested quercetin and curcumin showed the highest inhibitory activity while piperine showed least antioxidant activity at equimolar concentration during initiation phase of oxidation of LDL. The inhibitory effect of curcumin, quercetin and capsaicin was comparable to that of BHA, but relatively more potent than ascorbic acid. Further, the effect of curcurnin, quercetin, capsaicin and BHA on initiation and propagation phases of LDL oxidation showed that curcurnin significantly inhibited both initiation and propagation phases of LDL oxidation, while quercetin was found to be ineffective at propagation phase. These data suggest that the above spice active principles, which constitute about 1–4% of above spices, are effective antioxidants and offer protection against oxidation of human LDL.     

Tanshinone II-A inhibits low density lipoprotein oxidation in vitro

Tanshinone II-A (TSII-A) is a major component of Salvia miltorrhiza Bunge which has long been used for preventing and ameliorating anginal pain in China. However the effect of TSII-A on low density lipoprotein (LDL) oxidation has not been studied. The present study was performed to investigate the effects of TSII-A on LDL oxidation using four oxidizing systems, including copper-, peroxyl radical- and peroxynitriteinitiated and macrophage-mediated LDL oxidation. LDL oxidation was measured in terms of formation of thiobarbituric acid-reactive substances (TBARS), relative electrophoretic mobility (REM) on agarose gel and lag time. In all four systems, TSII-A has apparent antioxidative effects against LDL oxidation, as evidenced by its dose-dependent inhibition of TBARS formation, prolongation of lag time and suppression of increased REM.

Regarding the mechanism underlying its antioxidative effect, TSII-A neither scavenged superoxide nor peroxynitrite. It also did not chelate copper. But it has mild peroxyl radical scavenging activity. The direct binding to LDL particles and conformational change of LDL structure by TSII-A were suggested, because it increased negative charge of LDL which was shown by increased REM on agarose gel. In conclusion, TSII-A is an effective antioxidant against LDL oxidation in vitro. The underlying mechanism appears to be related to its peroxyl radical scavenging and LDL binding activity.     

Tanshinone II-A inhibits low density lipoprotein oxidation in vitro

Tanshinone II-A (TSII-A) is a major component of Salvia miltorrhiza Bunge which has long been used for preventing and ameliorating anginal pain in China. However the effect of TSII-A on low density lipoprotein (LDL) oxidation has not been studied. The present study was performed to investigate the effects of TSII-A on LDL oxidation using four oxidizing systems, including copper-, peroxyl radical- and peroxynitriteinitiated and macrophage-mediated LDL oxidation. LDL oxidation was measured in terms of formation of thiobarbituric acid-reactive substances (TBARS), relative electrophoretic mobility (REM) on agarose gel and lag time. In all four systems, TSII-A has apparent antioxidative effects against LDL oxidation, as evidenced by its dose-dependent inhibition of TBARS formation, prolongation of lag time and suppression of increased REM.

Regarding the mechanism underlying its antioxidative effect, TSII-A neither scavenged superoxide nor peroxynitrite. It also did not chelate copper. But it has mild peroxyl radical scavenging activity. The direct binding to LDL particles and conformational change of LDL structure by TSII-A were suggested, because it increased negative charge of LDL which was shown by increased REM on agarose gel. In conclusion, TSII-A is an effective antioxidant against LDL oxidation in vitro. The underlying mechanism appears to be related to its peroxyl radical scavenging and LDL binding activity.     

Low density lipoprotein receptor-related protein is required for macrophage-mediated oxidation of low density lipoprotein by 12/15-lipoxygenase

The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-deficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL receptor family, also suppressed the cell-mediated LDL oxidation in a dose-dependent manner. The cell-mediated LDL oxidation was dose-dependently blocked by an anti-LRP antibody but not by an anti-LDL receptor antibody. Furthermore, antisense oligodeoxyribonucleotides against LRP reduced the cell-mediated LDL oxidation under the conditions in which the expression of LRP was decreased. The results taken together indicate that LRP was involved essentially for the cell-mediated LDL oxidation by 12/15-lipoxygenase expressed in J774A.1 cells, suggesting an important pathophysiological role of this receptor-enzyme system as the initial trigger of the progression of atherosclerosis.     

Apple juice inhibits human low density lipoprotein oxidation.

Dietary phenolic compounds, ubiquitous in vegetables and fruits and their juices possess antioxidant activity that may have beneficial effects on human health. The phenolic composition of six commercial apple juices, and of the peel (RP), flesh (RF) and whole fresh Red Delicious apples (RW), was determined by high performance liquid chromatography (HPLC), and total phenols were determined by the Folin-Ciocalteau method. HPLC analysis identified and quantified several classes of phenolic compounds: cinnamates, anthocyanins, flavan-3-ols and flavonols. Phloridzin and hydroxy methyl furfural were also identified. The profile of phenolic compounds varied among the juices. The range of concentrations as a percentage of total phenolic concentration was: hydroxy methyl furfural, 4-30%; phloridzin, 22-36%; cinnamates, 25-36%; anthocyanins, n.d.; flavan-3-ols, 8-27%; flavonols, 2-10%. The phenolic profile of the Red Delicious apple extracts differed from those of the juices. The range of concentrations of phenolic classes in fresh apple extracts was: hydroxy methyl furfural, n.d.; phloridzin, 11-17%; cinnamates, 3-27%; anthocyanins, n.d.-42%; flavan-3-ols, 31-54%; flavonols, 1-10%. The ability of compounds in apple juices and extracts from fresh apple to protect LDL was assessed using an in vitro copper catalyzed human LDL oxidation system. The extent of LDL oxidation was determined as hexanal production using static headspace gas chromatography. The apple juices and extracts, tested at 5 microM gallic acid equivalents (GAE), all inhibited LDL oxidation. The inhibition by the juices ranged from 9 to 34%, and inhibition by RF, RW and RP was 21, 34 and 38%, respectively. Regression analyses revealed no significant correlation between antioxidant activity and either total phenolic concentration or any specific class of phenolics. Although the specific components in the apple juices and extracts that contributed to antioxidant activity have yet to be identified, this study found that both fresh apple and commercial apple juices inhibited copper-catalyzed LDL oxidation. The in vitro antioxidant activity of apples support the inclusion of this fruit and its juice in a healthy human diet.     

Mechanistic aspects of the relationship between low-level chemiluminescence and lipid peroxides in oxidation of low-density lipoprotein.

In this study oxidation of low-density lipoprotein (LDL) induced by different Cu2+ concentrations was investigated. Lipid peroxidation was assessed by monitoring low-level chemiluminescence (LL-CL), conjugated diene hydroperoxide (CD) and alpha-tocopherol (TocOH), the major lipophilic antioxidant in LDL. At high Cu2+ concentration, LDL oxidation was characterised by CD formation, LL-CL emission and TocOH consumption. At low Cu2+ concentration, CD formation was independent of LL-CL and occurred in the presence of TocOH. Thus, two different mechanisms lead to lipid peroxide formation in LDL. The combination of CD assay and LL-CL monitoring makes it possible to distinguish the autocatalytic mechanism of CD formation and that associated with TocOH, found at a high and a low rate of initiation, respectively.     

Effects of phosphatidylserine on the oxidation of low density lipoprotein

• 1.1. LDL was incubated in the presence of 1 μ M CuSO₄ for 18 hr at 37°C. The content of lipoperoxides was found to be approx. 40 nmol MDA equivalents/mg LDL protein. The addition of 50 μM phosphatidylserine (PS) reduced the content of lipoperoxides to 15% of control values.
• 2.2. The electrophoretic mobility observed for LDL oxidized in the presence of PS approximated the mobility observed for native LDL.
• 3.3. The formation of conjugated dienes was strongly inhibited when LDL was oxidized in the presence of PS.
• 4.4. The addition of 50 μM phosphatidylcholine, phosphatidylglycerol and cardiolipin did not alter the extent of LDL oxidation.
• 5.5. PS did not inhibit the oxidation of LDL mediated by J774 macrophages in the presence of Ham's F-10 culture medium. Under these conditions, PS was found to be an excellent substrate for oxidation.

     

Dipicolinic acid prevents the copper-dependent oxidation of low density lipoprotein

Effect of dipicolinic acid (pyridine 2,6-dicarboxylic acid) and pyridine compounds on the copper-dependent oxidation of human low density lipoprotein was analyzed in relation to the inhibition of copper reduction. Dipicolinic acid inhibited copper-dependent LDL oxidation completely, but the LDL oxidation was slightly inhibited by pyridine compounds with one carboxyl group at 2 or 6-position. Reduction of copper by LDL itself and ascorbate was inhibited completely by dipicolinic acid, but only partially by picolinic acid, quinolinic acid and isocinchomeronic acid with 2- or 6-carboxylic group. Pyridine compounds without 2- or 6-carboxyl group did not show any inhibitory effect on the LDL oxidation and the copper reduction. Protective effect of dipicolinic acid on the LDL oxidation was closely correlated with the copper-reducing activity. Dipicolinic acid shows an antioxidant action by the formation of a chelation complex with copper. This may have implications in understanding mechanisms of preventing LDL oxidation during the early phase of atherosclerosis.     

Modification of copper-catalyzed oxidation of low density lipoprotein by proteoglycans and glycosaminoglycans.

Chondroitin sulfate proteoglycans (CSPG) appear to contribute to retention of low density lipoproteins (LDL) in atherosclerotic lesions. In vitro, CSPG and glycosaminoglycans (GAG) modify LDL structure and increase its uptake by macrophages. This latter effect appears related to increased exposure of arginine- and lysine-rich segments of apoB-100. We explored whether alterations of LDL induced by human arterial CSPG and purified GAG alter the lipoprotein susceptibility to transition metals-catalyzed oxidation. Human LDL was complexed with human arterial CSPG and dissociated by raising the ionic strength. The nonaggregated, CSPG- and GAG-treated LDL was subjected to oxidation by micromolar amounts of Cu+, Cu2+, Fe2+, and Fe3+. This treatment increased LDL susceptibility to Cu2+ oxidation 3- to 5-times, as indicated by the degradation rate of phospholipids and cholesteryl esters and formation rates of dienes and thiobarbituric acid-reacting substances (TBARS). Also, human macrophages degraded the CSPG-treated, Cu2+-oxidized LDL 3- to 6-times faster than native LDL similarly treated. No enhancement of oxidation was observed with Fe2+, Fe3+, and Cu+. Quenching of the LDL intrinsic fluorescence by Cu2+ showed that heparin, CSPG, and chondroitin-6-SO4 pretreatment increased the access of Cu2+ to hydrophobic chromophores, probably tryptophan, 6- to 7-, 3- to 4-, and 2- to 3-fold, respectively. Also, the affinity constant (Ka) of LDL for Cu2+ was increased from 0.12 microM to 0.20 microM by the treatment with CSPG and GAG. These results and evaluation of the fraction of surface-accessible LDL chromophores to acrylamide quenching suggest that the increased susceptibility to oxidation may be associated with an increase in the access of Cu2+ to hydrophobic regions in LDL caused by treatment with CSPG and GAG. This effect was not detected with Cu+, Fe2+, or Fe3+. The phenomenon may contribute to acceleration of the oxidative modifications of LDL in cell culture models and in vivo.     

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.     

Inhibitory effect of ebselen on the oxidation of low density lipoprotein

The inhibitory effect of a synthesized glutathione peroxidase (GSHPX) mimic- ebselen, and its cofactor glutathione (GSH), on the oxidation of low density lipoprotein (LDL) induced by Cu²⁺ was studied by determination of hydroperoxides and thiobarbituric acid reactive substances (TBARS). Ebselen alone had a strong inhibitory effect on the oxidation of LDL. The lag time of LDL oxidation was prolonged with an increase in the concentration of ebselen. The inhibitory effect of 5 μM ebselen was equivalent to that of 50 μM α-tocopherol. When GSH was present, ebselen exhibited stronger inhibitory effect than when present alone. With 50 μM GSH, ebselen at a concentration as low as 5 μM could inhibit oxidation of LDL induced by 5 μM Cu²⁺ completely for at least 24 h. Ebselen at high concentrations (100 μM) decomposed hydroperoxides in pre-oxidized LDL and effectively prevented its further oxidation, but not in the present of EDTA. Low concentration of ebselen (5 μM) plus GSH (50 μM) decomposed hydroperoxides in pre-oxidized LDL whether EDTA was added or not.     

Evaluation of low density lipoprotein oxidation in a course of hypothyroidism

INTRODUCTION: The aim of this study was to evaluate the influence of hypothyroidism on oxidative modification of low density lipoprotein (LDL). MATERIAL AND METHODS: 24 patients with overt hypothyroidism and 10 patients with mild hypothyroidism were enrolled to the study. The control group consisted of 24 healthy subjects with normal serum TSH. Plasma level of oxidized LDL (oxLDL) and serum level of antibodies against oxidized LDL (anti-oxLDL) determined lipoprotein oxidation. RESULTS: Significantly increased plasma oxLDL levels were found in patients with overt hypothyroidism in comparison to patients with mild hypothyroidism and control group. Anti-oxLDL levels in patients with overt or mild hypothyroidism and in the control group showed no significant differences. OxLDL plasma levels in patients with hypothyroidism inversely correlated with FT(4) levels and positively correlated with TSH, total cholesterol, LDL cholesterol and triglycerides levels. CONCLUSIONS: The presented study indicates increased lipoprotein oxidation in patients with hypothyroidism which depends on the degree of hypothyroidism and changes in lipid profile. Elevated cholesterol and triglycerides levels are the factors increasing lipoprotein oxidation. Plasma oxLDL levels may constitute a useful marker indicating the risk for atherosclerosis in hypothyroidism.     

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.     

Low density lipoprotein receptor-related protein-mediated membrane translocation of 12/15-lipoxygenase is required for oxidation of low density lipoprotein by macrophages

Oxidation of low density lipoprotein (LDL) is the key step for the development of atherosclerosis. The 12/15-lipoxygenase expressed in macrophages is capable of oxygenating linoleic acid esterified to cholesterol in the LDL particle, and thus this enzyme is presumed to initiate LDL oxidation. We recently reported that LDL receptor-related protein (LRP) was required for the enzyme-mediated LDL oxidation by macrophages and suggested the selective uptake of cholesterol ester from LDL to the plasma membrane (Xu, W., Takahashi, Y., Sakashita, T., Iwasaki, T., Hattori, H., and Yoshimoto. T. (2001) J. Biol. Chem. 276, 36454-36459). To elucidate precise mechanisms of lipoxygenase-mediated LDL oxidation, we investigated the intracellular localization of 12/15-lipoxygenase. The 12/15-lipoxygenase was predominantly detected in cytosol of resting peritoneal macrophages and of macrophage-like J774A.1 cells permanently transfected with the cDNA for the enzyme. When the cells were treated with LDL and subjected to subcellular fractionation, the 12/15-lipoxygenase was detected in the membranes with a concomitant decrease in cytosol as shown by Western blot analysis. The levels of the enzyme associated with the membrane reached maximum in 15 min after LDL addition and then decreased. However, the enzymatic activity of 12/15-lipoxygenase in the membrane fraction was very weak even after LDL treatment. This fact supports the suicide inactivation of the enzyme by the oxygenation of cholesterol ester transferred from the LDL particle to the plasma membrane. Immunohistochemical analysis using an antibody against 12/15-lipoxygenase revealed that the plasma membrane was the major site of the enzyme translocation by the LDL treatment. LDL-dependent 12/15-lipoxygenase translocation was inhibited by a blocking antibody against LRP. Furthermore, an enzyme translocation inhibitor, L655238, inhibited the LDL oxidation caused by the 12/15-lipoxygenase. We propose that cholesterol ester selectively transferred from the LDL particle to the plasma membrane via LRP is oxygenated by 12/15-lipoxygenase translocated to this membrane.     

Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I

We have shown previously that low density lipoprotein (LDL) subjected to vortexing forms self-aggregates that are avidly phagocytosed by macrophages. That phagocytic uptake is mediated by the LDL receptor. We now show that LDL self-aggregation is strongly inhibited (80-95%) by the presence of high density lipoprotein (HDL) or apolipoprotein (apo) A-I. Another type of LDL aggregation, namely that induced by incubation of LDL with phospholipase C, was also markedly inhibited by HDL or apoA-I. The aggregation of LDL induced by vortexing was not inhibited by 2.5 M NaCl, and apoA-I was still able to block LDL aggregation at this high salt concentration, strongly suggesting hydrophobic interactions as the basis for the effect of apoA-I. The fact that apoA-I protected against LDL aggregation induced by two apparently quite different procedures suggests that the aggregation in these two cases has common features. We propose that these forms of LDL aggregation result from the exposure of hydrophobic domains normally masked in LDL and that the LDL-LDL association occurs when these domains interact. ApoA-I, because of its amphipathic character, is able to interact with the exposed hydrophobic domains of LDL and thus block the intermolecular interactions that cause aggregation.