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.     

Inhibitory effect of isoflavones on peroxynitrite-mediated low-density lipoprotein oxidation

Peroxynitrite, a potent oxidant formed in vivo from the reaction of nitric oxide with superoxide, can mediate low-density liprotein (LDL) oxidation which is thought to increase the risk of atherosclerosis. This study investigates the inhibitory effect of the isoflavones, genistein and daidzein, together with their glycosidic forms, genistin and daidzin, on the peroxynitrite-mediated LDL oxidation and nitration of tyrosine. Genistein and daidzein were observed to dose-dependently inhibit peroxynitrite-mediated LDL oxidation, while their glucoside conjugates showed less activity. Moreover, all the isoflavones used in this study were found to be potent peroxynitrite scavengers, preventing the nitration of tyrosine. The ability of the isoflavones at 50 microM to decrease the tyrosine nitration induced by peroxynitrite (1 mM) was in the ratios of genistein (49%), daidzein (40%), daidzin (41%) and genistin (42%) when compared to the control (tyrosine incubated only with peroxynitrite). These results suggest that an intake of isoflavones could contribute to protecting against cardiovascular diseases and chronic inflammatory diseases.     

Selenoprotein P protects low-density lipoprotein against oxidation

Selenoprotein P (SeP) is an extracellular glycoprotein with 8-10 selenocysteines per molecule, containing approximately 50% of total selenium in human serum. An antioxidant function of SeP has been postulated. In the present study, we show that SeP protects low-density lipoproteins (LDL) against oxidation in a cell-free in-vitro system. LDL were isolated from human blood plasma and oxidized with CuCl₂, 2,2'-azobis(2-amidinopropane) (AAPH) or peroxynitrite in the presence or absence of SeP, using the formation of conjugated dienes as parameter for lipid peroxidation. SeP delayed the CuCl₂- and AAPH-induced LDL oxidation significantly and more efficiently than bovine serum albumin used as control. In contrast, SeP was not capable of inhibiting peroxynitrite-induced LDL oxidation. The protection of LDL against CuCl₂- and AAPH-induced oxidation provides evidence for the antioxidant capacity of SeP. Because SeP associates with endothelial membranes, it may act in vivo as a protective factor inhibiting the oxidation of LDL by reactive oxygen species.     

Effects of dietary factors on oxidation of low-density lipoprotein particles

Oxidized low-density lipoproteins (ox-LDLs) appear to play a significant role in atherogenesis. In fact, circulating ox-LDL concentrations have been recognized as a risk factor for cardiovascular disease (CVD). A higher intake of some nutrients and specific food compounds such as monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) and flavonoids have also been associated with a lower risk of CVD. These dietary factors could be associated to a lower risk of CVD through a reduction of the atherogenicity of LDL particles through limited oxidation. Therefore, the purpose of this article is to review human clinical studies that evaluated effects of dietary antioxidant vitamins, fatty acids (MUFA, PUFA) and specific flavonoid-rich foods on LDL particle oxidation and describe potential mechanisms by which dietary factors may prevent oxidation of LDL particles. Antioxidant vitamin supplements such as alpha-tocopherol and ascorbic acid as well as beta-carotene and fish-oil supplements have not been clearly demonstrated to prevent oxidation of LDL particles. Moreover, inconsistent documented effects of flavonoid-rich food such as olive oil, tea, red wine and soy on LDL particle oxidizability may be explained by difference in variety and quantity of flavonoid compounds used among studies. However, a healthy food pattern such as the Mediterranean diet, which includes a combination of antioxidant compounds and flavonoid-rich foods, appears effective to decrease LDL particle oxidizability, which may give some insight of the cardiovascular benefits associated with the Mediterranean diet.     

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.     

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.     

Superoxide anion participation in human monocyte-mediated oxidation of low-density lipoprotein and conversion of low-density lipoprotein to a cytotoxin

Human monocytes, upon activation with opsonized zymosan, altered low-density lipoprotein (LDL) during a 24-h co-incubation, resulting in its oxidation and acquisition of cytotoxic activity against target fibroblast cell lines. Both the oxidation of LDL and its conversion to a cytotoxin were enhanced with time of incubation, with the most substantial changes occurring after 6 h of culture of LDL with activated monocytes. Unactivated monocytes did not mediate either alteration. Superoxide anion (O2-) participated in both the oxidation of LDL and its conversion to a cytotoxin since addition of superoxide dismutase (SOD) at the beginning of the co-incubation inhibited, in a concentration dependent fashion, both the monocyte-mediated oxidation and the monocyte-mediated conversion of LDL to a cytotoxin. As expected, the rate of superoxide anion release was greatest during the respiratory burst, very early in the 24-h incubation (0 to 2 h); however, exposure of LDL to monocytes during the respiratory burst was not required for LDL oxidation. The lower levels of O2- released by the cells hours after the respiratory burst had subsided were sufficient to lead to the initiation of LDL oxidation. Three results indicated that the oxidative modification of LDL into a cytotoxin required O2(-)-independent free radical propagation after O2(-)-dependent initiation. First, oxidation of LDL exposed to the activated, superoxide anion-releasing monocytes for 6 h could be almost completely blocked by the addition at 6 h of the general free radical scavenger butylated hydroxytoluene, but not by SOD. Second, LDL oxidation proceeded even after removal of LDL from the superoxide anion-producing, activated cells after various durations of exposure. Third, the development of substantial levels of lipid peroxidation products and the development of greater cytotoxicity occurred after 6 h of exposure of LDL to activated cells, long after peak O2- release had subsided. These results lead us to conclude that monocyte-mediated oxidation of LDL, leading to its transformation into a cytotoxin, requires release of O2- occurring as a result of activation but not necessarily during the respiratory burst, and also requires O2(-)-independent free radical propagation. The modification of LDL into a potent toxin by activated monocytes may explain the tissue damage in atherosclerotic lesions and other pathologic sites in which inflammatory cells congregate.     

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.     

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.     

Vitamin C protects low-density lipoprotein from homocysteine-mediated oxidation

Homocysteine, an atherogenic amino acid, promotes iron-dependent oxidation of low-density lipoprotein (LDL). We investigated whether vitamin C, a physiological antioxidant, could protect LDL from homocysteine-mediated oxidation. LDL (0.2 mg of protein/ml) was incubated at 37 degrees C with homocysteine (1000 microM) and ferric iron (10-100 microM) in either the absence (control) or presence of vitamin C (5-250 microM). Under these conditions, vitamin C protected LDL from oxidation as evidenced by an increased lag time preceding lipid diene formation (> or = 5 vs. 2.5 h for control), decreased thiobarbituric acid-reactive substances accumulation (< or = 19 +/- 1 nmol/mg when vitamin C > or = 10 microM vs. 32 +/- 3 nmol/mg for control, p <.01), and decreased lipoprotein anodic electrophoretic mobility. Near-maximal protection was observed at vitamin C concentrations similar to those in human blood (50-100 microM); also, some protection was observed even at low concentrations (5-10 microM). This effect resulted neither from altered iron redox chemistry nor enhanced recycling of vitamin E in LDL. Instead, similar to previous reports for copper-dependent LDL oxidation, we found that vitamin C protected LDL from homocysteine-mediated oxidation through covalent lipoprotein modification involving dehydroascorbic acid. Protection of LDL from homocysteine-mediated oxidation by vitamin C may have implications for the prevention of cardiovascular disease.     

Effects of oxidation on the structure and stability of human low-density lipoprotein

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.     

Urate attenuates oxidation of native low-density lipoprotein by hypochlorite and the subsequent lipoprotein-induced respiratory burst activities of polymorphonuclear leukocytes

Oxidation converts native low-density lipoprotein (LDL) into a signal molecule promoting inflammatory processes during atherogenesis. The exact contribution of different antioxidants in prevention of LDL oxidation is not known. Uric acid efficiently scavenges oxidants including hypochlorite. We investigated the effect of different urate concentrations (25-500 mol/l) on the oxidation of isolated native LDL by sodium hypochlorite (1000 mol/l). While relative electrophoretic mobility declined continuously with increasing urate concentrations in the oxidation medium, lipid peroxidation as measured by TBARS was blunted only at high molar urate/NaOCl ratios. By decreasing oxidative modifications, urate dose-dependently (beginning with a urate/NaOCl ratio of 1:40) diminished stimulatory effects of oxidized LDL on the respiratory burst of resting polymorphonuclear leukocytes (PMNL). Protecting effects of urate against the proinflammatory action of oxidized LDL on activated cells were evident only at a molar urate/NaOCl ratio of 1:2 suggesting different sensitivities of PMNL to LDL oxidation state in dependence on their activity state.     

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.     

In vitro oxidized and glycated human low-density lipoprotein particles characterized by capillary zone electrophoresis

A simple capillary zone electrophoresis (CZE) method was used to determine native, in vitro Cu(2+) and glucose modified low-density lipoprotein (LDL) particles for four healthy subjects. The LDL electropherograms are highly reproducible with good precisions of effective mobility and peak area. The native LDL capillary electrophoresis (CE) profile shows a major peak with lower mobility and two minor peaks with higher mobilities. For three-hour Cu(2+) oxidation, one major peak with mobility close to that of the native major peak, and one minor peak with mobility extending to -47 x 10(-5)cm(2)V(-1)s(-1) appear. For eighteen-hour Cu(2+) oxidation, one major peak with mobility much higher than that of the native major peak appears. As the reaction time for LDL and Cu(2+) increases from 0 to 24h, effective mobility of the LDL major peak increases, suggesting that LDL particles become more negatively charged and oxidized as the time increases. The in vitro glycated LDL particles are characterized by a major peak and two minor peaks. Mobility of the major peak is close to that of native major peak, but the second minor peak is much more negatively charged with mobility extending to -53 x 10(-5)cm(2)V(-1)s(-1). Native, oxidized and glycated LDL particles show distinctive differences in their CZE profiles. Agarose electrophoresis shows that the charge to mass ratios of native, three-hour Cu(2+) and glucose modified LDL particles are similar, but that of eighteen-hour Cu(2+) oxidized LDL particles is higher.     

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.     

Serum non-transferrin-bound iron and low-density lipoprotein oxidation in heterozygous hemochromatosis

Non-transferrin-bound iron (NTBI) is implicated in lipid peroxidation but the relation with oxidative modification of low-density lipoprotein (LDL) is not known. We assessed variables reflecting in vitro and in vivo LDL oxidation in two age- and sex-matched groups (n=23) of hereditary hemochromatosis heterozygotes (C282Y), characterized by a clear difference in mean serum NTBI (1.55+/-0.57 micromol/L vs 3.70+/-0.96 micromol/L). Plasma level of oxidized LDL (absolute and relative to plasma apolipoprotein B), and IgG and IgM antibodies to oxidized LDL, markers of in vivo LDL oxidation, did not differ between the groups with low and high serum NTBI. Mean lag-phase of in vitro LDL oxidation was also not significantly different between both study groups. Conclusion: these findings do not support the hypothesis that NTBI promotes oxidative modification of plasma LDL.     

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.