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.     

Decrease in the particle size of low-density lipoprotein (LDL) by oxidation

A radical reaction of low-density lipoprotein (LDL) causes fragmentation and cross-link of apolipoprotein B-100 (apoB). LDL (50 microg/ml) was subjected to the well-studied oxidation with Cu(2+) (1.67 microM). The concentration of alpha-tocopherol decreased to 10% of the initial level during the first 30 min. After this lag time, the conjugated diene content, as measured by absorption at 234 nm, started increasing and the residual apoB at 512 kDa determined by immunoblot after SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) was also decreased. The particle size of LDL determined by nondenaturing gradient gel electrophoresis decreased steadily during the initial 120 min, when residual native apoB was only 30% of the initial level. Plasma was also oxidized with Cu(2+) (400 microM). Under this condition, a clear lag time was not observed and alpha-tocopherol content, apoB, and the LDL particle size were decreased simultaneously. Based on these experiments, we propose that an oxidation reaction is involved in the formation of small dense LDL.     

Comparison of LDL trap assay to other tests of antioxidant capacity; effect of vitamin E and lovastatin treatment

Oxidized low density lipoprotein (LDL) has a major impact in the development of atherosclerosis. Risk for oxidative modification of LDL is usually determined indirectly by measuring the capability of LDL to resist radical insult. We compared three different methods quantifying the antioxidative capacity of LDL ex vivo in dyslipidemic patients with coronary heart disease. Plasma samples were obtained from two double-blinded cross-over trials. The duration of all interventions (placebo, lovastatin 60 mg/day, RRR-alpha-tocopherol 300 mg/day and lovastatin + RRR-alpha-tocopherol combined) was 6 weeks. The total radical capturing capacity of LDL (TRAP) in plasma was determined using 2,2-azo-bis(2,4-dimethyl-valeronitrile) (AMVN) -induced oxidation, and measuring the extinction time of chemiluminescence. TRAP was compared to the variables characterizing formation of conjugated dienes in copper-induced oxidation. Also the initial concentrations and consumption times of reduced alpha-tocopherol (alpha-TOH) and ubiquinol in AMVN-induced oxidation were determined. Repeatability of TRAP was comparable to that of the lag time in conjugated diene formation. Coefficient of variation within TRAP assay was 4.4% and between TRAP assays 5.9%. Tocopherol supplementation produced statistically significant changes in all antioxidant variables except those related to LDL ubiquinol. TRAP increased by 57%, the lag time in conjugated diene formation by 34% and consumption time of alpha-TOH by 88%. When data of all interventions were included in the analyses, TRAP correlated with the lag time (r = 0.75, p < 10(-6)), with LDL alpha-TOH (r = 0.50, p < 0.001) and with the consumption time of alpha-TOH (r = 0.58, p < 0.0001). In the baseline data, the associations between different antioxidant variables were weaker. TRAP correlated with the lag time (r = 0.55, p < 0.001) and alpha-TOH consumption time (r = 0.48, p < 0.05), and inversely with apolipoprotein Al (r = -0.51, p < 0.05). Lag time at the baseline did not correlate with ubiquinol or tocopherol parameters, or with any plasma lipid or lipoprotein levels analyzed. Lovastatin treatment did not significantly affect the antioxidant capacity of LDL. In conclusion, TRAP reflects slightly different properties of LDL compared to the lag time. Thus, LDL TRAP assay may complement the other methods used to quantify the antioxidant capacity of LDL. However, TRAP and the lag time react similarly to vitamin E supplementation.     

Antiatherogenic activity of extracts of Argania spinosa L. pericarp: beneficial effects on lipid peroxidation and cholesterol homeostasis

Prevention of lipoprotein oxidation by natural compounds may prevent atherosclerosis via reducing early atherogenesis. In this study, we investigated for the first time the beneficial properties of methanolic extract of argania pericarp (MEAP) towards atherogenesis by protecting human low-density lipoprotein (LDL) against oxidation while promoting high-density lipoprotein (HDL)-mediated cholesterol efflux. By measuring the formation of malondialdehyde (MDA) and conjugated diene as well as the lag phase and the progression rate of lipid peroxidation, the MEAP was found to possess an inhibitory effect. In addition, MEAP reduced the rate of disappearance of alpha-tocopherol as well as the apoB electrophoretic mobility in a dose-dependent manner. These effects are related to the free radical scavenging and copper-chelating effects of MEAP. In terms of cell viability, MEAP has shown a cytotoxic effect (0-40 microg/mL). Incubation of 3H-cholesterol-loaded J774 macrophages with HDL in the presence of increasing concentrations of MEAP enhanced HDL-mediated cholesterol efflux independently of ABCA1 receptor pathways. Our findings suggest that argania seed pericarp provides a source of natural antioxidants that inhibit LDL oxidation and enhance cholesterol efflux and thus can prevent development of cardiovascular diseases.     

The protective effect on Cu2+- and AAPH-mediated oxidation of human low-density lipoproteins depends on glycosaminoglycan structure

The effect of various glycosaminoglycans on Cu(2+)- and AAPH-induced oxidation of human low-density lipoprotein (LDL) was studied by monitoring conjugated diene formation. Heparin (Hep) increased the lag phase (t(lag)) of LDL oxidation, and fast moving and slow moving Hep species modified the kinetics of LDL oxidation to the same extent. Beef spleen heparan sulfate (HS) sample produced a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL whilst beef kidney HS species modified LDL oxidation kinetics to a lower extent. Dermatan sulfate (DS) from different sources caused a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL. Hyaluronic acid had no effect. Chondroitin sulfate (CS) from beef trachea produced a very strong protective antioxidant effect evaluated by increasing of the t(lag) and decreasing of the conjugated diene formation. Hep was completely ineffective in protecting LDL from 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation, whilst DS was moderately effective. Beef trachea CS showed a very strong ability to protect LDL oxidation induced by 1 mM AAPH. The different protective effect on Cu(2+)- and AAPH-induced LDL oxidation by glycosaminoglycans is discussed considering their various structures and properties, and their capacity to interact to different extents with hydrophobic regions of LDL protein is confirmed by measuring the LDL-tryptophan fluorescence kinetics.     

Serum ex vivo lipoprotein oxidizability in patients with ischemic heart disease supplemented with vitamin E

The decreased oxidizability of plasma lipoproteins is related to the increased vitamin E intake and its association with a relatively lower incidence of coronary heart disease has been proposed. We investigated the effect of the in vivo vitamin E supplementation on the oxidizability of serum lipids in patients with ischemic heart disease and a moderate hypercholesterolemia. Thirty-two patients (16 males and 16 postmenopausal women) participated in this placebo-controlled, randomized trial. They were treated with 400 mg vitamin E/day for 6 weeks. The copper-induced serum lipid oxidizability ex vivo was assessed by measuring conjugated diene formation at 245 nm. We also measured vitamin E, malondialdehyde (MDA) and uric acid concentrations in the plasma. Because of observed significant differences in parameters of serum lipid oxidizability (lag time and maximal rate of oxidation), plasma alpha-tocopherol and MDA levels between male patients and postmenopausal women supplemented with vitamin E, the results were compared between both genders. Six weeks of vitamin E supplementation significantly increased plasma vitamin E levels (by 87 %) in male patients but in postmenopausal women only by 34 %. Concomitantly with increased plasma levels of vitamin E the decrease in plasma MDA levels was observed in male patients (decrease by 20 %; p=0.008), but in postmenopausal women the decrease did not attain statistical significance. Plasma uric acid levels were not apparently changed in placebo or vitamin E supplemented groups of patients. The changes in ex vivo serum lipid oxidizability after vitamin E, supplementation have shown a significantly prolonged lag time (by 11 %; p=0.048) and lowered rate of lipid oxidation (by 21 %; p=0.004) in male patients in comparison with postmenopausal women. Linear regression analysis revealed a significant correlation between plasma vitamin E levels and the lag time (r=0.77; p=0.03) and the maximal rate of serum lipid oxidation (r=-0.70; p=0.05) in male patients. However, in postmenopausal women the correlations were not significant. We conclude that 400 mg vitamin E/day supplementation in patients with ischemic heart disease and a moderate hypercholesterolemia influenced favorably ex vivo serum lipid oxidation of male patients when compared with postmenopausal women. The observed differences between both genders could be useful in the selection of the effective vitamin E doses in the prevention of coronary heart disease.     

Effects of some flavonoids on the susceptibility of low-density lipoprotein to oxidative modification

The effects of six flavonoids viz., apigenin, genistein, morin, naringin, pelargonidin and quercetin on the susceptibility of low-density lipoprotein (LDL) to oxidative modification were investigated. Flavonoids were added to plasma and incubated for 3 hr at 37 degrees C, and the LDL fraction was separated by ultracentrifugation. Oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS), after cupric sulfate solution was added. Quercetin and morin significantly (P<0.01 by ANOVA) prolonged the lag time before initiation of oxidation reaction in dose-dependent manner. They also suppressed the formation of lipid peroxides and TBARS more markedly than other flavonoids. The ability to prolong lag time and suppression of lipid peroxides and TBARS formation was in the following order: quercetin >morin >pelargonidin >genistein >naringin >apigenin. LDL exposed to flavonoids reduced oxidizability. These findings suggest that flavonoids may have a role in ameliorating atherosclerosis.     

Effects of flavonoids on the susceptibility of low-density lipoprotein to oxidative modification

Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds on the susceptibility of low-density lipoprotein (LDL) to oxidative modification. In this study, six flavonoids, "apigenin, genistein, morin, naringin, pelargonidin and quercetin", were added to plasma and incubated for 3h at 37 degrees C. Then, the LDL fraction was separated by ultracentrifugation. The oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS) after cupric sulfate solution was added. We showed that among flavonoids used, quercetin and morin significantly (P<0.01 by ANOVA) and dose-dependently prolonged the lag time before initiation of oxidation reaction. Also, these two flavonoids suppressed the formation of lipid peroxides and TBARS more markedly than others. Their ability to prolong lag time and suppression of lipid peroxides and TBARS formation resulted to be in the following order: quercetin>morin>pelargonidin>genistein>naringin>apigenin. LDL exposed to flavonoids in vitro reduced oxidizability. These findings show that flavonoids may have a role in ameliorating atherosclerosis.     

The monoterpene terpinolene from the oil of Pinus mugo L. in concert with alpha-tocopherol and beta-carotene effectively prevents oxidation of LDL.

Antioxidants from several nutrients, e.g. vitamin E, beta-carotene, or flavonoids, inhibit the oxidative modification of low-density lipoproteins. This protective effect could possibly retard atherogenesis and in consequence avoid coronary heart diseases. Some studies have shown a positive effect of those antioxidants on cardiovascular disease. Another class of naturally occurring antioxidants are terpenoids, which are found in essential oils. The essential oil of Pinus mugo and the contained monoterpene terpinolene effectively prevent low-density lipoprotein (LDL)-oxidation. In order to test the mechanism by which terpinolene protects LDL from oxidation, LDL from human blood plasma enriched in terpinolene was isolated. In this preparation not only the lipid part of LDL is protected against copper-induced oxidation--as proven by following the formation of conjugated dienes, but also the oxidation of the protein part is inhibited, since loss of tryptophan fluorescence is strongly delayed. This inhibition is due to a retarded oxidation of intrinsic carotenoids of LDL, and not, as in the case of some flavonoids, attributable to a protection of intrinsic alpha-tocopherol. These results are in agreement with our previous results, which showed the same effects for a monoterpene from lemon oil, i.e. gamma-terpinene.     

Comparison of LDL TRAP assay to other tests of antioxidant capacity; Effect of vitamin E and lovastatin treatment

Oxidized low density lipoprotein (LDL) has a major impact in the development of atherosclerosis. Risk for oxidative modification of LDL is usually determined indirectly by measuring the capability of LDL to resist radical insult. We compared three different methods quantifying the antioxidative capacity of LDL ex vivo in dyslipidemic patients with coronary heart disease. Plasma samples were obtained from two double-blinded cross-over trials. The duration of all interventions (placebo, lovastatin 60 mg/day, RRR-α-tocopherol 300 mg/day and lovastatin + RRR-α-tocopherol combined) was 6 weeks. The total radical capturing capacity of LDL (TRAP) in plasma was determined using 2,2-azobis(2,4-dimethyl-valeronitrile) (AMVN)-induced oxidation, and measuring the extinction time of chemiluminescence. TRAP was compared to the variables characterizing formation of conjugated dienes in copper-induced oxidation. Also the initial concentrations and consumption times of reduced α-tocopherol (α-TOH) and ubiquinol in AMVN-induced oxidation were determined.

Repeatability of TRAP was comparable to that of the lag time in conjugated diene formation. Coefficient of variation within TRAP assay was 4.4% and between TRAP assays 5.9%. Tocopherol supplementation produced statistically significant changes in all antioxidant variables except those related to LDL ubiquinol. TRAP increased by 57%, the lag time in conjugated diene formation by 34% and consumption time of α-TOH by 88%. When data of all interventions were included in the analyses, TRAP correlated with the lag time (r = 0.75, p < 10^-6), with LDL α -TOH (r = 0.50, p < 0.001) and with the consumption time of α-TOH (r = 0.58, p < 0.0001). In the baseline data, the associations between different antioxidant variables were weaker. TRAP correlated with the lag time (r = 0.55, p < 0.001) and α-TOH consumption time (r = 0.48, p < 0.05), and inversely with apolipoprotein Al (r = -0.51, p < 0.05). Lag time at the baseline did not correlate with ubiquinol or tocopherol parameters, or with any plasma lipid or lipoprotein levels analyzed. Lovastatin treatment did not significantly affect the antioxidant capacity of LDL. In conclusion, TRAP reflects slightly different properties of LDL compared to the lag time. Thus, LDL TRAP assay may complement the other methods used to quantify the antioxidant capacity of LDL. However, TRAP and the lag time react similarly to vitamin E supplementation.     

Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein.

Much data has accumulated supporting a proatherogenic role for oxidized low density lipoprotein (Ox-LDL). Micronutrient antioxidants such as alpha-tocopherol, the principal lipid-soluble antioxidant, assume potential significance because levels can be manipulated by dietary measures without resulting in side effects. Co-incubation of LDL in vitro with alpha-tocopherol inhibits its oxidative modification. Hence the effect of dietary supplementation with alpha-tocopherol on the time course of copper-catalyzed oxidation of LDL was tested in a randomized placebo-controlled single-blind study. Two groups of 12 male subjects were given either placebo or alpha-tocopherol (800 IU/day) for a period of 12 weeks. Alpha-tocopherol therapy did not result in any side effects or exert an adverse effect on the plasma lipid and lipoprotein profile. While the lipid standardized alpha-tocopherol levels were similar at baseline, the supplemented group had 3.3-fold and 4.4-fold higher levels compared to placebo at 6 and 12 weeks, respectively. In the 15 subjects in whom both plasma and LDL alpha-tocopherol levels were quantitated, there was a significant correlation (r = 0.79, P less than 0.0001). At baseline there were no significant differences in the time course curves of thiobarbituric acid-reacting substances (TBARS) activity or conjugated diene formation between the alpha-tocopherol and placebo groups. However, at both 6 and 12 weeks the mean levels of TBARS activity and conjugated diene formation were lower in the alpha-tocopherol group; the most significant differences were manifest at the 3-h time point. Also at both 6 and 12 weeks the mean rate of oxidation was lower in the alpha-tocopherol group.2+_     

Comparison of the effect of alpha-lipoic acid and alpha-tocopherol supplementation on measures of oxidative stress

In vitro studies have shown that alpha-lipoic acid (LA) is an antioxidant. There is a paucity of studies on LA supplementation in humans. Therefore, the aim of this study was to assess the effect of oral supplementation with LA alone and in combination with alpha-tocopherol (AT) on measures of oxidative stress. A total of 31 healthy adults were supplemented for 2 months either with LA (600 mg/d, n = 16), or with AT (400 IU/d, n = 15) alone, and then with the combination of both for 2 additional months. At baseline, after 2 and 4 months of supplementation, urine for F2-isoprostanes, plasma for protein carbonyl measurement and low-density lipoprotein (LDL) oxidative susceptibility was collected. Plasma oxidizability was assessed after incubation with 100 mM 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) for 4 h at 37 degrees C. LDL was subjected to copper- and AAPH-catalyzed oxidation at 37 degrees C over 5 h and the lag time was computed. LA significantly increased the lag time of LDL lipid peroxide formation for both copper-catalyzed and AAPH-induced LDL oxidalion (p < .05), decreased urinary F2-isoprostanes levels (p < .05), and plasma carbonyl levels after AAPH oxidation (p < .001). AT prolonged LDL lag time of lipid peroxide formation (p < .01 ) and conjugated dienes (p < .01) after copper-catalyzed LDL oxidation, decreased urinary F2-isoprostanes (p < .001), but had no effect on plasma carbonyls. The addition of LA to AT did not produce an additional significant improvement in the measures of oxidative stress. In conclusion, LA supplementation functions as an antioxidant, because it decreases plasma- and LDL-oxidation and urinary isoprostanes.     

Effects of isoflavones containing soy protein isolate compared with fish protein on serum lipids and susceptibility of low density lipoprotein and liver lipids to in vitro oxidation in hamsters

The effects of dietary soy protein isolate (SPI), ethanol-extracted SPI (E-SPI) low in isoflavones, and fish protein (FP) on the concentration of blood lipids and the susceptibility of low density lipoprotein (LDL) to copper-induced oxidation were compared in male golden Syrian hamsters fed a moderate hypercholesterolemic semi-purified diet for 10 weeks. SPI, E-SPI, and FP were incorporated into the isonitrogenous experimental diets as protein sources. The SPI group exhibited significantly lower serum total cholesterol concentration compared with the E-SPI group (P < 0.05) and the FP group (P < 0.01). Both the SPI and E-SPI groups showed lower LDL cholesterol (P < 0.001 and P < 0.05, respectively) and less LDL apolipoprotein B (P < 0.01) compared with the FP group. The distribution pattern of serum lipoprotein cholesterol fractions of the SPI and E-SPI groups were similar to each other, but different from that of the FP group. The lysine/arginine ratio of the three diets was significantly correlated with serum total cholesterol concentration (r = 0.462, P = 0.023). The resistance of LDL to copper-induced oxidation was greater in the SPI group than in the E-SPI and FP groups as assessed by the lower concentrations of thiobarbituric acid-reactive substances (TBARS) and the longer lag time required for the formation of conjugated dienes (P < 0.01). Livers of hamsters fed the FP diet had a higher amount of TBARS than those of hamsters fed SPI (P < 0.01) and E-SPI (P < 0.05) diets. The SPI diet showed sparing effects on alpha-tocopherol contents in both serum and liver. It seems likely that soy isoflavones protect the circulating and membrane lipids by sparing alpha-tocopherol and endogenous antioxidants.     

Kinetic analysis of copper-induced peroxidation of HDL, autoaccelerated and tocopherol-mediated peroxidation

Comparison of the kinetic profiles of copper-induced peroxidation of HDL and LDL at different copper concentrations reveals that under all the studied experimental conditions HDL is more susceptible to oxidation than LDL. The mechanism responsible for HDL oxidation is a complex function of the copper/HDL ratio and of the tocopherol content of the HDL. At high copper concentrations, the kinetic profiles were similar to those observed for LDL oxidation, namely, relatively rapid accumulation of oxidation products, via an autoaccelerated, noninhibited mechanism, was preceded by an initial "lag phase." Under these conditions, the maximal peroxidation rate (V(max)) of HDL and LDL depended similarly on the molar ratio of bound copper/lipoprotein. Analysis of this dependency in terms of the binding characteristics of copper to lipoprotein, yielded similar dissociation constant (K = 10(-6) M) but different maximal binding capacities for the two lipoproteins (8 Cu(+2)/HDL as compared to 17 Cu(+2)/LDL). Given the size difference between HDL and LDL, these results imply that the maximal surface density of bound copper is at least 2-fold higher for HDL than for LDL. This difference may be responsible for the higher susceptibility of HDL to copper-induced oxidation in the presence of high copper concentrations. At relatively low copper concentrations, the kinetic profile of HDL oxidation was biphasic, similar to but more pronounced than the biphasic kinetics observed for the oxidation of LDL lipids at the same concentration of copper. Our results are consistent with the hypothesis that the first phase of rapid oxidation occurs via a tocopherol-mediated-peroxidation (TMP) mechanism. Accordingly, enrichment of HDL with tocopherol resulted in enhanced accumulation of hydroperoxides during the first phase of copper-induced oxidation. Notably, the maximal accumulation during the first phase decreased upon increasing the ratio of bound copper/HDL. This behavior can be predicted theoretically for peroxidation via a TMP mechanism, in opposition to autoaccelerated peroxidation. The possible pathophysiological significance of these findings is discussed.     

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.     

Implications of Lag Time Concept in the Oxidation of LDL

Oxidation of low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. The most common technique for measuring the oxidation of lipoproteins is the continuos measurement of the formation of conjugated diene at OD 234 nm. The concept of “lag time”, derived from such measurements, has been used to test the efficacy of various antioxidants for their ability to inhibit the oxidation of LDL. This review will elaborate on some of the factors that might affect the lag time.     

Antioxidant activities of a new lignan and a neolignan from Saururus chinensis

A new diarylbutane lignan, 2'-hydroxy dihydroguaiaretic acid (4), and a known 8-O-4'-type neolignan, machilin D (5), were isolated from the ethyl acetate extracts of the underground parts of Saururus chinensis. Compounds 4 and 5 exhibited low-density lipoprotein (LDL)-antioxidant activity in the thiobarbituric acid-reactive substances (TBARS) assay (4: IC(50)=3.3 microM and 5: IC(50)=3.8 microM), the lag time of conjugated diene production, the relative electrophoretic mobility (REM) of ox-LDL, the apoB-100 fragmentation on copper-mediated LDL oxidation and the macrophage-mediated LDL oxidation, and radical DPPH scavenging activity.     

Effect of gangliosides on the copper-induced oxidation of human low-density lipoproteins

The role of gangliosides in the copper-induced oxidative modification of human low-density lipoprotein (LDL) was studied focusing on the early stage of LDL oxidation in which the concentration of conjugated dienes increases only weakly. The changes in the protein and lipid component were followed using fluorescence spectroscopy. The results indicate that binding of gangliosides to LDL causes slower destruction of tryptophan fluorescence and suppresses cross-linking between the reactive groups of the protein and the products of lipid peroxidation. The protective role of gangliosides could be assigned to their interference with the lipid-protein interaction in the LDL particle, which might be important for the maintenance of the native plasma antioxidant status in vivo.     

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.     

Comparative time-courses of copper-ion-mediated protein and lipid oxidation in low-density lipoprotein

Free radicals damage both lipids and proteins and evidence has accumulated for the presence of both oxidised lipids and proteins in aged tissue samples as well as those from a variety of pathologies including atherosclerosis, diabetes, and Parkinson's disease. Oxidation of the protein and lipid moieties of low-density lipoprotein is of particular interest due to its potential role in the unregulated uptake of lipids and cholesterol by macrophages; this may contribute to the initial stage of foam cell formation in atherosclerosis. In the study reported here, we examined the comparative time-courses of lipid and protein oxidation during copper-ion-mediated oxidation of low-density lipoprotein. We show that there is an early, lipid-mediated loss of 40-50% of the Trp residues of the apoB100 protein. There is no comparable loss over an identical period during the copper-ion-mediated oxidation of lipid-free BSA. Concomitant with Trp loss, the antioxidant alpha-tocopherol is consumed with subsequent extensive lipid peroxidation. Further changes to the protein, including the copper-ion-dependent 3.5-fold increase in 3,4-dihydroxyphenylalanine and the copper-ion-independent 3-5-fold increase in o-tyrosine, oxidation products of Tyr and Phe, respectively, only occur after maximal lipid peroxidation. Long incubation periods result in depletion of 3,4-dihydroxyphenylalanine, presumably reflecting further oxidative changes. Overall, copper-ion-mediated oxidation of LDL appears to proceed initially by lipid radical-dependent processes, even though some of the earliest detectable changes occur on the apoB100 protein. This is followed by extensive lipid peroxidation and subsequent additional oxidation of aromatic residues on apoB100, though it is not yet clear whether this late protein oxidation is lipid-dependent or occurs as a result of direct radical attack.