Copper-mediated Low-density Lipoprotein Oxidation Research Articles

Effect of lycopene on the copper-induced oxidation of low-density lipoprotein in broiler chickens.

The present study was undertaken to investigate whether dietary lycopene (LP) could prevent the copper-mediated oxidation of low density lipoprotein (LDL), and affect growth performance, relative organ weights, plasma and meat lipid profiles, and LP contents in plasma and tissues in broiler chickens. A total of 160 day-old male broiler chicks were randomly allotted into 16 pens with rice husk as a bedding material. Each experiment had 4 replicates, 10 chicks per replicate (n = 4 per treatment). A corn-soybean meal base diet was used as a control diet (CONT). To formulate the experimental diets, the base diet was added with LP at the levels of 10 (LP10) or 20 mg/kg (LP20), or 17 g/kg of tomato paste (TP17) which was equivalent to 5 ppm of LP. The experiment lasted 4 weeks. Growth performance and relative organ weights were not affected (p > 0.05) by any of dietary treatments. Dietary LP significantly lowered (p < 0.05) triglyceride and LDL cholesterol at 2 weeks of age, but did not affect them at 4 weeks of age. Total cholesterol in thigh meats was not altered by dietary treatments. LP was not detected in leg meats in all chicks, nor in liver or plasma of the CONT group. LP was found in liver and plasma, being the former greater in the concentration, of the chicks fed diets containing LP10, LP20, or TP17. At 2 and 4 weeks, the copper-mediated oxidation of LDL was delayed (p < 0.05) in either LP- or TP-fed chickens compared with the CONT group. In conclusion, LP lowers triglyceride and LDL cholesterol, is deposited into serum and liver, and prevents the LDL oxidation in broiler chickens, confirming the role of LP in the lipid-lowering and antioxidant properties in broiler chickens.

English

The modified low-density lipoprotein (LDL; glycated and oxidized) is more atherogenic and are poorly recognized by LDL receptor. In the present study, an attempt has been made to evaluate the effects of Triphala on LDL modification in experimentally treated rats and in human LDL. The effect of ethanolic extract of Triphala on LDL oxidation susceptibility in high fat diet fed and STZ induced rats was evaluated. The inhibitory effect of Triphala on LDL oxidation and glycation was investigated using copper and glucose induced human LDL, respectively in vitro. The co-presence of Triphala extract significantly increased lag phase and minimized the conjugated dienes formation in dose dependent manner. Copper mediated LDL oxidation was characterized by elevated indices of thiobarbituric acid reactive substance (TBARS), whereas co-presence of Triphala extract significantly minimized the production of TBARS. The results of this investigation shows that Triphala probably with their antioxidant properties inhibited LDL glycation and oxidation and provide scientific reasons regarding the possible medical benefits of using Triphala to prevent diabetic and cardiovascular complications. &nbsp; Key words: Triphala, low-density lipoprotein (LDL) oxidation, LDL glycation, high fat diet, streptozotocin (STZ).

Inhibitory Activities of Compounds from the Twigs of Garcinia hombroniana Pierre on Human Low‐density Lipoprotein (LDL) Oxidation and Platelet Aggregation

The methanol extract of the twigs of Garcinia hombroniana, which showed strong LDL antioxidation and antiplatelet aggregation activities, was subjected to column chromatography to obtain 3,5,3',5'-tetrahydroxy-4-methoxybenzophenone, 1,7-dihydroxyxanthone and eight triterpenoids, garcihombronane B, D, E and F, friedelin, glutin-5-en-3β-ol, stigmasterol and lupeol. The structures of the compounds were elucidated by spectroscopic methods. The compounds were evaluated for their ability to inhibit copper-mediated LDL oxidation and arachidonic acid (AA)-, adenosine diphosphate (ADP)-, collagen-induced platelet aggregation in vitro. Among the compounds tested, 3,5,3',5'-tetrahydroxy-4-methoxybenzophenone and 1,7-dihydroxyxanthone showed strong inhibitory activity on LDL oxidation with half-maximal inhibitory concentration (IC(50)) values of 6.6 and 1.7 µM, respectively. 3,5,3',5'-Tetrahydroxy-4-methoxybenzophenone exhibited strong activity on AA-, ADP- and collagen-induced platelet aggregation with IC(50) values of 53.6, 125.7 and 178.6 µM, respectively, while 1,7 dihydroxyxanthone showed significant and selective inhibitory activity against ADP-induced aggregation with IC(50) value of 5.7 µM. Of the triterpenoids tested, garcihombronane B showed moderate activity against LDL oxidation and garcihombronane D and F showed selective inhibition on ADP-induced platelet aggregation.

Correlation between Chemical Composition ofCurcuma domesticaandCurcuma xanthorrhizaand Their Antioxidant Effect on Human Low-Density Lipoprotein Oxidation

The antioxidant activity of the curcuminoids of Curcuma domestica L. and C. xanthorrhiza Roxb. and eight compounds which are prevalent constituents of their rhizome oils were investigated in an effort to correlate human low-density lipoprotein (LDL) antioxidant activity with the effect of the herbs and their components. The antioxidant activity was examined using thiobarbituric acid reactive substances (TBARSs) assay with human LDL as the oxidation substrate. The methanol extracts and rhizome oils of C. xanthorrhiza and C. domestica showed strong inhibitory activity on copper-mediated oxidation of LDL. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin, isolated from the methanol extracts of both plants, exhibited stronger activity than probucol (IC50 value 0.57 μmol/L) as reference, with IC50 values ranging from 0.15 to 0.33 μmol/L. Xanthorrhizol, the most abundant component (31.9%) of the oil of C. xanthorrhiza, showed relatively strong activity with an IC50 value of 1.93 μmol/L. The major components of C. domestica, ar-turmerone (45.8%) and zerumbone (3.5%), exhibited IC50 values of 10.18 and 24.90 μmol/L, respectively. The high levels of curcuminoids in the methanol extracts and xanthorrhizol, ar-turmerone and zerumbone in the oils, and in combination with the minor components were responsible for the high LDL antioxidant activity of the herbs.

Synthesis and pharmacological evaluation of antioxidant chalcone derivatives of 2(3H)-benzoxazolones

Chalcones featuring an analgesic/anti-inflammatory pharmacophore, i.e., the 2(3H)-benzoxazolone heterocycle, on the one hand, and a radical scavenger moiety, i.e., 2,6-di-t-butylphenol, on the other hand were synthesized by condensation of a ketone 2(3H)-benzoxazolone precursor with 3,5-di-t-butyl-4-hydroxybenzaldehyde. Among the various methods explored (acid homogenous or heterogenous catalysis, base catalysis), heterogenous catalysis conditions using KSF Montmorillonite were found to be the most convenient. The E-geometry of the so-obtained chalcones was ascertained both by 1H and 13C-nuclear magnetic resonance (NMR) spectroscopy as well as B3LYP/6-31G** quantum mechanics calculations. Chalcones 1–8 were pharmacologically evaluated in vitro for their ability to prevent human low-density lipoprotein (LDL) copper-induced oxidation using Cu2+ as oxidizing agent. Compound 4 emerged as the most promising agent as it was able to inhibit copper-mediated human LDL oxidation with an activity ten times greater than that of Probucol, a reference antioxidant drug.

Protective Effect of Magnoflorine Isolated from Coptidis Rhizoma on Cu2+-Induced Oxidation of Human Low Density Lipoprotein

The aim of this study was to investigate the antioxidant activity of magnoflorine, an alkaloid isolated from Coptidis Rhizoma, against the oxidation of native low density lipoprotein (LDL) and modified LDL. Magnoflorine was found to inhibit the copper-mediated (Cu2+) oxidation of LDL, as well as of glycated and glycoxidated LDL by increasing the lag time of conjugated diene formation and preventing the generation of thiobarbituric acid reactive substances (TBARS). In addition, the results from the fluorescence emission spectra of tryptophan (Trp) supported that the antioxidant activity of magnoflorine could be associated with the protective effect on the structural modification of apolipoprotein B (apoB) required for LDL oxidation. These results suggest that magnoflorine may be useful for preventing the oxidation of various LDL forms.

C-reactive protein inhibits in vitro oxidation of low-density lipoprotein

C-reactive protein (CRP) is elevated in cardiovascular disease and binds to oxidized phosphatidylcholine (oxPtC) in the low-density lipoprotein (LDL) surface. In the present study, we tested if CRP influences the susceptibility of LDL to oxidation. At physiological concentrations of 1–7 μg/ml, CRP strongly inhibited copper-mediated oxidation of LDL and phospholipid liposomes in a concentration-dependent manner. Similar concentrations of different monoclonal antibodies or albumin did not influence LDL oxidation. Antioxidant activity of CRP was inhibited by phosphocholine (PC), indicating that the observed activity involves binding of CRP to oxPtC. These results suggest that CRP may limit atherogenic oxidation of LDL in vivo.

Antioxidant Activities of Abietane Diterpenoids Isolated from Torreya nucifera Leaves

Investigation on antioxidant compounds from the ethanolic extracts of Torreya nucifera leaves resulted in the isolation of abietane diterpenoids, a known 18-methylesterferruginol (1) and a new 18-dimethoxyferruginol (2). The structures of compounds 1 and 2 were elucidated on the basis of their spectroscopic analyses. Compounds 1 and 2 inhibited the Cu2+-mediated, 2,2'-azobis(2-amidinopropane)hydrochloride-mediated and 3-morpholinosydnonimine-1-mediated low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substances assay as well as the macrophage-mediated LDL oxidation. Compounds 1 and 2 exhibited the potent antioxidant activities in the conjugated diene production, relative electrophoretic mobility, and apoB-100 fragmentation on copper-mediated LDL oxidation. Compound 1 also suppressed nitric oxide production and inducible nitric oxide synthase expression in lipopolysaccharide-stimulated RAW264.7 cells.

Antioxidant Properties of Flavone C-Glycosides from Atractylodes japonica Leaves in Human Low-density Lipoprotein Oxidation

ABSTRACT: Three antioxidant flavone C-glucosides, isoorientin, vitexin, and isovitexin, were identified for the 1st time from Atractylodes japonica leaves by an activity-guided fractionation with various analytical techniques including column chromatography, high-performance liquid chromatography (HPLC), fast atom bombardment mass spectrometry (FAB-MS), and nuclear magnetic resonance (NMR). Hydroxyl and superoxide anion radicals scavenging activities of these compounds were examined using electron spin resonance (ESR). Inhibitory activities of these compounds on human low-density lipoprotein (LDL) oxidation were evaluated by malondialdehyde (MDA) and thiobarbituric acid-reactive substances (TBARS), both representing intermediates of lipid peroxidation. These flavone C-glucosides displayed about 50% of scavenging activity against hydroxyl radicals at the concentrations below 10 μM. The superoxide dismutase (SOD)-equivalent activities of isoorientin, vitexin, and isovitexin at 1 mM were 31.37, 2.71, and 2.63 unit/mL, respectively. Isoorientin at 1 μM exhibited over 60% inhibition of MDA formed during copper-mediated human LDL oxidation. Amounts of free MDA in LDL treated with isoorientin, vitexin, isovitexin, and control were 20.06, 40.73, 34.08, and 48.03 nM/mg protein, respectively. These compounds also prolonged the lag phase time of the conjugated diene formation. There was a positive correlation between the free radical scavenging activities and the inhibitory effects on the LDL oxidation of these compounds. These results suggest that the flavone C-glucosides isolated from the leaves of A japonica possess beneficial antioxidant properties against free radicals as well as LDL oxidation.

In Vitro Antioxidant Activity and Antigenotoxicity of 5-n-Alkylresorcinols

Incubation with 5-n-alkylresorcinols (chain lengths C15:0, C17:0, C19:0, C21:0, and C23:0) increased the self-protection capacity of HT29 human colon cancer cells against DNA damage induced by hydrogen peroxide and genotoxic fecal water samples using comet assay (single-cell gel electrophoresis assay). The alkylresorcinols did not exert potent antioxidant activity in the FRAP (ferric reduction ability of plasma) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assays. However they were able to significantly inhibit copper-mediated oxidation of human LDL (low-density lipoprotein) in vitro, and pentadecylresorcinol at 25 micromol/L increased lag time by 65 min. The results show that alkylresorcinols have antigenotoxic and antioxidant activity under in vitro conditions.

Troglitazone Inhibits Long-Term Glycation and Oxidation of Low-Density Lipoprotein

Troglitazone (T) is a member of a new class of antidiabetic drugs termed thiazolidinediones (TZDs), which has previously been used as an anti-diabetic agent. In this study we investigated the influence of T, a ligand for PPAR-gamma receptor, on copper-catalyzed or cell-mediated oxidation of native, glycated, and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition of copper-mediated low-density lipoprotein-oxidation, as monitored by the formation of oxidation-specific fluorescence, was observed for both native and glycated low-density lipoprotein. At the concentration of 20 microg/mL the inhibition amounted from 14.7% to 64.7% by all low-density lipoprotein forms. For glycated low-density lipoprotein we obtained the highest oxidation rate, but the most pronounced inhibition by T was found for glycoxidated low-density lipoprotein (goLDL). Inhibitory effects of T were also investigated by measurement of relative electrophoretic mobility (REM) in the concentration range of 0 to 20 microg/mL. The inhibition of 4h oxidation of native low-density lipoprotein was found in the entire concentration range, but significance was seen at 10 microg/mL. The long-term glycation and glycoxidation of low-density lipoprotein as measured by 5-hydroxymethyl-2-furaldehyde (5-HMF) formation and binding of fructosamine was found to be inhibited by T. In endothelial cell-mediated oxidation of low-density lipoprotein cytotoxicity of T in the concentration range of 0 to 160 microg/mL during 2 to 24 h oxidation was investigated. In the non-cytotoxic concentration range of 5 to 20 microg/mL, a significantly reduced liberation of isoprostane 8-epi-PGF2alpha during 24 h cell-mediated oxidation of low-density lipoprotein and its modifications was found. This inhibitory action of T was most significant in the case of goLDL and amounted to approximately 20% to 60% inhibition at 5 to 20 microg/mL T, respectively. In the concentration range of 40 to 160 microg/mL, however, T showed an increasing cytotoxic action, as evidenced by loss of cell adhesion, loss of cellular protein, morphological changes, and cell disintegration as well as by strongly enhanced troglitazone-mediated isoprostane 8-IP liberation (fivefold to sixfold). T may be used as a model to explore the thiazolidinediones' mechanism on oxidation in a more general aspect for treatment for T2DM, because T is not clinically available.

Antioxidant Activities of a New Lignan and a Neolignan from Saururus chinensis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Association between reduced low density lipoprotein oxidation and inhibition of monocyte chemoattractant protein-1 production in statin-treated subjects

Monocyte chemoattractant protein-1 (MCP-1) is essential in atherogenesis. Oxidized lipids regulate MCP-1 expression and release from mononuclear cells. In this study we investigated (1) whether statin therapy reduces lipopolysaccharide (LPS)-stimulated MCP-1 production in human whole-blood samples and (2) the relationships between in vitro low-density lipoprotein (LDL) oxidation and MCP-1 production. Fasting blood samples were obtained from 55 healthy nonsmoking adults with moderate hypercholesterolemia who were participating in a randomized double-blind 8-week trial comparing the effects of statin therapy with those of placebo on cytokine production. Samples were analyzed for resistance to copper-mediated LDL oxidation (lag time in minutes), as well as MCP-1- and interleukin-8 (IL-8)-stimulated production. Statin therapy reduced MCP-1 production (mean +/- SD) -161 +/- 399 pg/mL/mm 3 white cells) compared with 267 +/- 985 pg/mL/mm 3 in the placebo group, but changes were not different between active and placebo groups ( P = .13). Statin therapy also increased lag times (median [interquartile range]; 20.5 [7.0-51.2] minutes vs -17.0 [-5.3-16.5] minutes; P = .067 for group difference). Inhibition of MCP-1 production correlated with prolongation of lag time ( r = .46, P = .0056) in statin-treated subjects. Statin therapy reduced MCP-1 production in the whole blood of human subjects and these changes were correlated with improvement in LDL oxidative resistance.

Antioxidant properties of two novel 2-biphenylmorpholine compounds (EP2306 and EP2302) in vitro and in vivo

The oxidation of low-density lipoprotein (LDL) is an important event in the development of atherosclerosis. In the present study, the antioxidant properties of two novel 2-biphenylmorpholine compounds (EP2306 and EP2302) were studied. Both compounds inhibited dose-dependently the in vitro oxidation of LDL induced by copper ions. EP2306 and EP2302 increased significantly the lag phase of the oxidation reaction at 0.1 and 10 μM, respectively, whereas they reduced the rate of the reaction at 1 and 10 μM, respectively. This inhibitory effect was not due to a free radical scavenging or copper-chelating activity of EP2300 compounds. Moreover, EP2306 and EP2302 inhibited 12-lipoxygenase activity dose-dependently with IC 50 values of 454 and 318 μM, respectively, but had no effect on 15-lipoxygenase activity. In hyperlipidaemic rabbits treated with EP2306 for 4 weeks, there was a decrease in thiobarbituric acid-reactive substance (TBARS) levels and a significant increase in total peroxyl radical-trapping potential (TRAP) levels as compared to control animals. The present data suggest that EP2300 compounds are effective inhibitors of copper-mediated LDL oxidation in vitro. Moreover, EP2306 acts as an antioxidant in hyperlipidaemic rabbits, a property which could be beneficial in reducing atherosclerosis.

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 μM and 5: IC 50 = 3.8 μM), 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.

Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108

The reactive oxygen species (ROS) and free radical-initiated reactions are ascertained to play multiple roles in degenerative or pathological events such as aging, cancer, heart dysfunction and Alzheimer’s disease. EPS2 with a mean molecular weight of 1.3 × 10 5 was characterized as an antioxidant exopolysaccharide from the broth of a marine filamentous fungus Keissleriella sp. YS 4108. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The radical eliminating and antioxidant actions of the glycan was assessed in different in vitro systems showing that EPS2 exhibited profound scavenging activities in superoxide radical. As a reinforcement of the action, similar radical scavenging effects of EPS2 were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. Moreover, EPS2 effectively blocked as well the non site-specific strand-breaking of DNA induced by the Fenton reaction at concentrations of 0.1 and 1 mg/mL. Further investigation of the effect of EPS2 on human low density lipoprotein (LDL) system demonstrated that it significantly inhibited copper-mediated oxidation of LDL in a dose-dependent manner. These results suggest that EPS2, possessing pronounced free radical scavenging and antioxidant activities, could be of considerable preventive and therapeutic significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer’s disease which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules.

Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108

The reactive oxygen species (ROS) and free radical-initiated reactions are ascertained to play multiple roles in degenerative or pathological events such as aging, cancer, heart dysfunction and Alzheimer’s disease. EPS2 with a mean molecular weight of 1.3 × 105 was characterized as an antioxidant exopolysaccharide from the broth of a marine filamentous fungus Keissleriella sp. YS 4108. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The radical eliminating and antioxidant actions of the glycan was assessed in different in vitro systems showing that EPS2 exhibited profound scavenging activities in superoxide radical. As a reinforcement of the action, similar radical scavenging effects of EPS2 were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. Moreover, EPS2 effectively blocked as well the non site-specific strand-breaking of DNA induced by the Fenton reaction at concentrations of 0.1 and 1 mg/mL. Further investigation of the effect of EPS2 on human low density lipoprotein (LDL) system demonstrated that it significantly inhibited copper-mediated oxidation of LDL in a dose-dependent manner. These results suggest that EPS2, possessing pronounced free radical scavenging and antioxidant activities, could be of considerable preventive and therapeutic significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer’s disease which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules.

Nitric oxide inhibits prooxidant actions of uric acid during copper-mediated LDL oxidation

Interactions between uric acid and physiologically relevant fluxes of nitric oxide ( NO) during copper-mediated low-density lipoprotein (LDL) oxidation were evaluated. In the absence of NO, a dual pro- and antioxidant action of uric acid was evident: low concentrations of uric acid enhanced lipid oxidation and α-tocopherol consumption, while its protective role was observed at higher concentrations. The prooxidant effects of uric acid were mostly related to its copper-reducing ability to form Cu +, an initiator of lipid oxidation processes. While the prooxidant action of uric acid was completely inhibited by NO, the antioxidant action of NO was slightly counterbalanced by uric acid. Enhancement of α-tocopherol consumption by uric acid was inhibited in the presence of NO while additive antioxidant effects between NO and uric acid were observed in conditions where uric acid spared α-tocopherol. Altogether, these results suggest that in the artery wall, the NO/uric acid pair may exert antioxidant actions on LDL, even if increased amounts of redox active copper were available at conditions favoring prooxidant activities of uric acid.

Synergistic inhibition of low-density lipoprotein oxidation by rutin, gamma-terpinene, and ascorbic acid.

Low-density lipoprotein (LDL) oxidation may play a significant role in atherogenesis. Flavonoids are well-known for their excellent antioxidative capacity in various model systems, therefore we examined the behaviour of rutin, a quercetin-3-rutinosid, in the copper-mediated LDL oxidation. Rutin alone has been shown to protect LDL against oxidation. Furthermore we investigated the combination of rutin with a hydrophilic (ascorbate) and a lipophilic antioxidant (gamma-terpinene) in copper-mediated LDL oxidation. In both cases we found a synergistic effect on lag phase prolongation. To elucidate whether this effect mainly depends on the copper chelating ability of rutin we examined its reaction in more detail. Although inhibiting the oxidation of alpha-linolenic acid in the "rose bengal system" no direct influence of a copper-rutin-complex was determined. We conclude that a redox active copper-rutin-complex is still able to initiate the LDL oxidation but may prevent copper from a reaction at the binding sites of apoB-100. The synergistic effect in preventing LDL oxidation is due to this trapping of copper in a complex in the case of ascorbate. The synergistic action of rutin and gamma-terpinene can be explained by different distribution of rutin and gamma-terpinene in, and around the LDL-particle, respectively.

Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 microM) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.