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 1-7mug/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.     

Inhibition of THP-1 cell-mediated low-density lipoprotein oxidation by the macrophage-synthesised pterin, 7,8-dihydroneopterin

Human macrophages release the pterin, 7,8-dihydroneopterin when exposed to the immune stimulant gamma-interferon (IFN-gamma). Previous in vitro studies have shown 7,8-dihydroneopterin is a potent antioxidant, which inhibits copper- and peroxyl-radical mediated low-density lipoprotein (LDL) oxidation. Using THP-1 cells, a human derived monocyte-like cell line, we have found that low micromolar concentrations of 7,8-dihydroneopterin inhibit cell mediated oxidation of LDL, as measured by electrophoretic mobility, alpha-tocopherol loss, and lipid oxidation. Stimulation of the THP-1 cells with IFN-gamma caused a significant reduction in the cells' ability to oxidise LDL. The extracellular pterin concentration increased from 0 to 16 nM with IFN-gamma stimulation, while the intracellular concentration increased from 0.21 to 1.69 nmol/mg cell protein.     

Myricitrin degraded by simulated digestion inhibits oxidation of human low-density lipoprotein

The inhibitory effects of myricitrin on the oxidation of human low-density lipoprotein were investigated before and after its degradation by simulated digestion. Myricitrin strongly inhibited the low-density lipoprotein oxidation induced by either 2,2'-azobis (2-amidinopropane) dihydrochloride or CuSO4 in a concentration-dependent manner. Myricitrin was very stable under an acidic condition (pH 1.8) corresponding to the gastric environment, but it was easily degraded under an alkaline condition (pH 8.5) corresponding to the intestinal environment. However, degraded myricitrin also had a strong inhibitory effect on the oxidative degradation of alpha-tocopherol, cholesterol and apolipoprotein B-100 in low-density lipoprotein. Our study revealed that myricitrin was degraded into many components under a mildly alkaline condition, but the degraded myricitrin still retained the free radical-scavenging and copper-chelating activities toward low-density lipoprotein.     

High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein

Oxidatively modified low-density lipoprotein (LDL), generated as a result of incubation of LDL with specific cells (e.g., endothelial cells, EC) or redox metals like copper, has been suggested to be an atherogenic form of LDL. Epidemiological evidence suggests that higher concentrations of plasma high-density lipoprotein (HDL) are protective against the disease. The effect of HDL on the generation of the oxidatively modified LDL is described in the current study. Incubation of HDL with endothelial cells, or with copper, produced much lower amounts of thiobarbituric acid-reactive products (TBARS) as compared to incubations that contained LDL at equal protein concentrations. Such incubations also did not result in an enhanced degradation of the incubated HDL by macrophages in contrast to similarly incubated LDL. On the other hand, inclusion of HDL in the incubations that contained labeled LDL had a profound inhibitory effect on the subsequent degradation of the incubated LDL by the macrophages while having no effect on the generation of TBARS or the formation of conjugated dienes. This inhibition was not due to the modification of HDL as suggested by the following findings. (A) There was no enhanced macrophage degradation of the HDL incubated with EC or copper alone, together with LDL, despite an increased generation of TBARS. (B) HDL with the lysine groups blocked (acetyl HDL, malondialdehyde (MDA) HDL) was still able to prevent the modification of LDL and (C) acetyl HDL and MDA-HDL competed poorly for the degradation of oxidatively modified LDL. It is suggested that HDL may play a protective role in atherogenesis by preventing the generation of an oxidatively modified LDL. The mechanism of action of HDL may involve exchange of lipid peroxidation products between the lipoproteins.     

Resonance Raman spectra of beta-carotene in native and modified low-density lipoprotein

Low-density lipoproteins isolated between density 1.02 and 1.063 g/cm3 from normal fasting human plasma, show strong resonance Raman spectra due to the presence of beta-carotene. Three intense bands, at 1010, 1160 and 1530 cm-1, are assigned to the stretching vibrations of -C-CH3, = C-C = and -C = C- bonds, respectively, of beta-carotene. High-resolution spectra of the 1500-1600 cm-1 region reveal multiple features, suggesting the coexistence of several structural populations of beta-carotene. The modifications of lipoproteins with pH and temperature (30 degrees-42 degrees) change the resonance Raman spectra of beta-carotene. The specific binding of LDL at pH 7.0 by fibroblast cells is suppressed. Our experiments thus suggest that physical and chemical perturbations of plasma lipoproteins modify the lipid-protein interactions and thereby alter the configurational distribution of beta-carotene molecules within these particles.     

Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low-density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoprotein.

4-Hydroxynonenal (HNE) is a major aldehydic propagation product formed during peroxidation of unsaturated fatty acids. The aldehyde was used to modify freshly prepared human low-density lipoprotein (LDL). A polyclonal antiserum was raised in the rabbit and absorbed with freshly prepared LDL. The antiserum did not react with human LDL, but reacted with CuCl2-oxidized LDL and in a dose-dependent manner with LDL, modified with 1, 2 and 3 mM-HNE, in the double-diffusion analysis. LDL treated with 4 mM of hexanal or hepta-2,4-dienal or 4-hydroxyhexenal or malonaldehyde (4 or 20 mM) did not react with the antiserum. However, LDL modified with 4 mM-4-hydroxyoctenal showed a very weak reaction. Lipoprotein (a) and very-low-density lipoprotein were revealed for the first time to undergo oxidative modification initiated by CuCl2. This was evidenced by the generation of lipid hydroperoxides and thiobarbituric acid-reactive substances, as well as by a marked increase in the electrophoretic mobility. After oxidation these two lipoproteins also reacted positively with the antiserum against HNE-modified LDL.     

beta-Carotene inhibits the oxidative modification of low-density lipoprotein

Several lines of evidence indicate that oxidized LDL (Ox-LDL) may promote atherogenesis. Hence, the role of antioxidants in the prevention of LDL oxidation needs to be determined. beta-Carotene, in addition to being an efficient quencher of singlet oxygen, can also function as a radical-trapping antioxidant. Since previous studies have failed to show that beta-carotene inhibits LDL oxidation, we re-examined its effect on the oxidative modification of LDL. For these studies, LDL was oxidized in both a cell-free (2.5 microM Cu2+ in PBS) and a cellular system (human monocyte macrophages in Ham's F-10 medium). beta-Carotene inhibited the oxidative modification of LDL in both systems as evidenced by a decrease in the lipid peroxide content (thiobarbituric-acid-reacting substances activity), the negative charge of LDL (electrophoretic mobility) and the formation of conjugated dienes. By inhibiting LDL oxidation, beta-carotene substantially decreased its degradation by macrophages. beta-Carotene (2 microM) was more potent than alpha-tocopherol (40 microM) in inhibiting LDL oxidation. Thus, beta-carotene, like ascorbate and alpha-tocopherol, inhibits LDL oxidation and might have an important role in the prevention of atherosclerosis.     

Mechanism of low-density lipoprotein oxidation by hemoglobin-derived iron

Excellular hemoglobin is an extremely active oxidant of low-density lipoproteins (LDL), a phenomenon explained so far by different mechanisms. In this study, we analyzed the mechanism of met-hemoglobin oxidability by comparing its mode of operation with other hemoproteins, met-myoglobin and horseradish peroxidase (HRP) or with free hemin. The kinetics of met-hemoglobin activity toward LDL lipids and protein differed from that of met-myoglobin and HRP, both quantitatively and qualitatively. Those differences were further clarified by analyzing heme transfer from the above-mentioned hemoproteins to LDL. It appeared that met-hemoglobin transferred most of its hemin to LDL, and the presence of H(2)O(2) accelerated the process. In contrast, met-myoglobin partially released hemin, but only in the presence of H(2)O(2), while HRP could not transfer heme at all. The minor amount of hemin transferred from met-myoglobin to LDL sufficed to trigger ApoB oxidation, forming covalent aggregates via inter-bityrosines. This indicated that heme bound to high affinity site(s) is responsible for oxidation. LDL components providing the sites were analyzed by binding heme-CO monomers to LDL. Soret spectra revealed that the high affinity site of monomeric hemin is located on the LDL protein, ApoB. The complex heme-CO-ApoB underwent instantaneous oxidation to hemin-ApoB, and the bound hemin then slowly disintegrated in conjunction with LDL oxidation. Hemopexin prevented LDL oxidation by trapping hemoprotein transferable heme. We concluded that met-hemoglobin exerts its oxidative activity on LDL via transfer of heme, which serves as a vehicle for iron insertion into the LDL protein, leading to formation of atherogenic LDL aggregates.     

Iron-catalyzed oxidation of Trp residues in low-density lipoprotein

The mechanisms of oxidation of low-density lipoproteins (LDLs) are not well defined, but epidemiological and experimental studies suggest that iron-catalyzed processes may contribute to atherogenesis. The aim of this study was to test the hypothesis that iron-catalyzed oxidations of LDLs in vitro produce diagnostic biomarkers of oxidation of the apolipoprotein that could be applied to studies in vivo. LDLs were oxidized in the presence of Fe2+, EDTA, and ascorbic acid for up to 40 h. Following delipidation and trypsin digestion, the peptides were separated by HPLC, with four peaks detected at 365 nm, whereas none were observed in peptides from unoxidized LDLs. The peptides were identified by MALDI-QTOF mass spectrometry as IVQILP(W+4) EQNEQVK, IYSL(W+4)EHSTK, FEGLQE(W+4)EGK, and YH(W+4)EHTGLTLR, with (W+4) rather than the W residues of the unoxidized protein. The mass gains (+4 increase in m/z in tryptophan, W) and absorbance at 365 nm indicate kynurenines, which were trypsin-releasable peptides that are on the surface of LDL particles. All four peptides thus characterized share the sequence of WE. The preferential oxidation of W residues in WE sequences suggest contributions from the C-proximate glutamate residues in chelation of the iron species, thereby influencing site selectivities of oxidation. These kynurenine-containing peptides might serve as biomarkers of iron-mediated oxidations in vivo.     

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.     

Dietary supplementation with docosahexaenoic acid,but not with eicosapentaenoic acid,reduces host resistance to fungal infection in mice

The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated. Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA. Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound. This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil. These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance. DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver. However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation. Greater infection-induced increases in spleen and serum interferon-gamma concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.     

Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis

Cells underoxidative stress induced by peroxides undergo functional andmorphological changes, which often resemble those observed duringapoptosis. Peroxides, however, also cause the oxidation ofintracellular reduced glutathione (GSH). We investigated the relationbetween these peroxide-induced effects by using human umbilical veinendothelial cells (HUVEC) and two HUVEC-derived cell lines, ECRF24 andECV304. With HUVEC, tert-butylhydroperoxide (tBH) or hydrogenperoxide application in the presence of serum induced, in adose-dependent way, reorganization of the actin cytoskeleton, membraneblebbing, and nuclear condensation. These processes were accompanied bytransient oxidation of GSH. With ECRF24 cells, this treatment resultedin less blebbing and a shorter period of GSH oxidation. However,repeated tBH addition increased thenumber of blebbing cells and prolonged the period of GSH oxidation. ECV304 cells were even more resistant to peroxide-induced bleb formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVECand ECRF24 cells, but not in ECV304 cells. Neither membrane blebbingnor nuclear condensation in any of these cell types was due toapoptosis, as evidenced by the absence of surface expression ofphosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH, although hightBH concentrations lead to nonapoptotic death. We conclude that, in endothelial cells,peroxide-induced cytoskeletal reorganization and bleb formationcorrelate with the degree of GSH oxidation but do not represent anearly stage of the apoptotic process.

     

Effects of oolonghomobisflavan A on oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been suggested to be involved in the onset of atherosclerosis. Oolong tea contains unique polyphenols including oolonghomobisflavan A (OFA). In this study, the effects of OFA on LDL oxidation by ROS and RNS were investigated in vitro. OFA suppressed formation of cholesterol ester hydroperoxides in LDL oxidized by peroxyl radical and peroxynitrite, and formation of thiobarbituric acid reactive substances in LDL oxidized by Cu²⁺. In addition, OFA inhibited fragmentation, carbonylation, and nitration of apolipoprotein B-100 (apo B-100) in the oxidized LDL, in which heparin-binding activity of apo B-100 was protected by OFA. Our results suggest that OFA exhibits antioxidant activity against both lipid peroxidation and oxidative modification of apo B-100 in LDL oxidized by ROS and RNS. Polyphenols in oolong tea may prevent atherosclerosis by reducing oxidative stress.     

Castanospermine inhibits the function of the low-density lipoprotein receptor

Castanospermine, a plant alkaloid that inhibits the glycoprotein processing enzyme glucosidase I, has been used to inhibit N-linked oligosaccharide modification, resulting in the production of glycoproteins having Glc3Man7-9(GlcNAc)2 oligosaccharides. This alkaloid caused a significant inhibition of LDL endocytosis in cultured primate smooth muscle cells and human skin fibroblasts. At an optimum concentration of 250 micrograms/mL, castanospermine caused a 40% decrease in cell surface receptor-mediated LDL binding at 4 degrees C, with no apparent change in affinity. Further, the inhibitor had no direct effect on LDL metabolism. This inhibition of LDL receptor expression and function occurred only when the drug was present during de novo receptor synthesis, i.e., during up-regulation. Although the number of cell surface LDL receptors was significantly reduced in the presence of castanospermine, the total number of receptors in the cell was only slightly reduced, indicating that castanospermine induced a redistribution rather than a reduction in the number of receptors. Similarly, subcellular fractionation studies confirmed that castanospermine treatment of fibroblasts results in an altered distribution of receptor activity compared with controls. These findings are consistent with the conclusion that the decrease in specific LDL binding to cells grown in the presence of castanospermine is due to intracellular redistribution of the LDL receptor so that more receptor remains in internal compartments as a result of a diminished rate of transport.     

Inhibition by serum components of oxidation and collagen-binding of low-density lipoprotein.

Low-density lipoprotein (LDL) is oxidized by cellular and noncellular mechanisms, both leading to an increased binding to collagen. We have investigated the effect of serum on lipid peroxidation, apoprotein oxidation and the binding of oxidized apoprotein to collagen. During noncellular oxidation, lipoprotein-deficient serum strongly inhibited all three processes. The serum fraction of M(r) > 100,000 was equally inhibitory; this effect was not due to alpha 1 or gamma globulins, alpha 2 macroglobulins, haptoglobins or ceruloplasmin. The serum fraction of M(r) 30,000-100,000 stimulated the binding of oxidized apoprotein but the albumin in this fraction inhibited lipid peroxidation and apoprotein oxidation. Serum ultrafiltrate (M(r) < 1000) inhibited lipid and protein oxidation, and binding; the inhibitory effect was abolished by deionization which removed histidine. The effects of lipoprotein-deficient serum and its fractions on cellular oxidation were similar but weaker than those on noncellular oxidation, HDL inhibited noncellular oxidation as well as binding of oxidized apoprotein. VLDL also inhibited oxidation; this could not be accounted for by its content of apo B. If present in vivo, these inhibitory effects would completely suppress both cellular and noncellular oxidation of LDL and its subsequent binding to collagen.     

Macrophage enrichment with the isoflavan glabridin inhibits NADPH oxidase-induced cell-mediated oxidation of low density lipoprotein. A possible role for protein kinase C.

Macrophage-mediated oxidation of low density lipoprotein (LDL) is considered to be of major importance in early atherogenesis; therefore, intervention means to inhibit this process are being extensively studied. In the present study, we questioned the ability of the isoflavan glabridin (from licorice) to accumulate in macrophages and to affect cell-mediated oxidation of LDL. We first performed in vitro studies, using mouse peritoneal macrophages (MPMs) and the J-774 A.1 macrophage-like cell line. Both cells accumulated up to 1.5 micrograms of glabridin/mg of cell protein after 2 h of incubation, and this process was time- and glabridin dose-dependent. In parallel, in glabridin-enriched cells, macrophage-mediated oxidation of LDL was inhibited by up to 80% in comparison with control cells. Glabridin inhibited superoxide release from MPMs in response to phorbol 12-myristate 13-acetate, or to LDL when added together with copper ions, by up to 60%. Translocation of P-47, a cytosolic component of NADPH oxidase to the plasma membrane was substantially inhibited. In glabridin-enriched macrophages, protein kinase C activity reduced by approximately 70%. All of the above effects of glabridin required the presence of the two hydroxyl groups on the flavonoid's B phenol ring. In order to assess the physiological significance of these results, we next performed in vivo studies, using the atherosclerotic apolipoprotein E-deficient (E0) mice. MPMs harvested from glabridin-treated E0 mice (20 micrograms/mouse/day for a period of 6 weeks) demonstrated reduced capability to oxidize LDL by 80% in comparison with placebo-treated mice. This latter phenomenon was associated with a reduction in the lesion oxysterols and a 50% reduction in the aortic lesion size. We thus conclude that glabridin accumulation in macrophages is associated with reduced cell-mediated oxidation of LDL and decreased activation of the NADPH oxidase system. These phenomena could be responsible for the attenuation of atherosclerosis in E0 mice, induced by glabridin.     

Plasma thiols inhibit hemin-dependent oxidation of human low-density lipoprotein

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies demonstrated that plasma thiols promote oxidation of LDL by free ferric iron (Fe3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a physiological Fe3+-protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo. In contrast to free Fe3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant, hemin readily promoted LDL oxidation. During incubation of LDL (0.2 mg of protein/ml) with hemin (10 microM) at 37 degrees C for 6 h, thiobarbituric acid-reactive substances (TBARS), a marker of lipid oxidation, increased from 0.3 (+/-0.1) nmol/mg of LDL protein to a maximal concentration of 45.8 (+/-5.2) nmol/mg of LDL protein. Under the same experimental conditions, lipid-conjugated dienes, another marker of lipid oxidation, increased from non-detectable to near-maximal levels of 78-187 nmol/mg of LDL protein, and lipoprotein polyunsaturated fatty acyl-containing cholesteryl ester content decreased to 15-36% of that present in native (i.e. unoxidized) LDL. Continued incubation of LDL with hemin for up to 24 h resulted in no further significant alterations in lipoprotein levels of TBARS, lipid-conjugated dienes, and cholesteryl esters. In addition to these chemical modifications indicative of lipoprotein oxidation, agarose gel electrophoretic analysis indicated that exposure of LDL to hemin resulted in conversion of the lipoprotein to an atherogenic form as evidenced by its increased anodic electrophoretic mobility. Addition of physiological concentrations of plasma thiols (either cysteine, homocysteine or reduced glutathione; 1-100 microM, each) inhibited hemin-mediated oxidation of LDL. Thus, whereas the maximal TBARS concentration was achieved following 6 h of incubation of LDL with hemin alone, addition of thiol extended the time required to attain maximal TBARS concentration to > or = 12 h. Similar antioxidant effects of thiols on formation of lipid-conjugated dienes, loss of cholesteryl esters, and lipoprotein anodic electrophoretic mobility were also observed. However, all thiols were not equally effective at inhibiting hemin-dependent LDL oxidation. Thus, whereas reduced glutathione was most effective at inhibiting hemin-dependent LDL oxidation, an intermediate effect was observed for homocysteine, and cysteine was least effective. The inhibition of hemin-mediated LDL oxidation by plasma thiols reported here confirms a previous observation that, under certain conditions, thiols can function as antioxidants, but contrasts with the previously documented pro-oxidant effect of the same thiols on oxidation of LDL by free Fe3+. These contrasting effects of plasma thiols on hemin- and free Fe3+-mediated LDL oxidation indicate that, in vivo, the ability of thiols to function as either anti- or pro-oxidants during LDL oxidation may, at least in part, be determined by the type of oxidant stress to which the lipoprotein is exposed.     

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.