Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu²⁺ or the soybean lipoxidase (LPOx)/phospholipase A₂ (PLA₂). With low concentrations of DMPO (1 mM), Cu²⁺ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu²⁺ showed maximum yields after 30 min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA₂ followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50 mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu²⁺. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA₂. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu²⁺ or LPOx/PLA₂ for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu²⁺, when we used the oxidizing system LPOx/PLA₂, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.     

The effect of histidine modification on copper-dependent lipid peroxidation in human low-density lipoprotein

Summary

Lipid peroxidation and subsequent oxidative modification of low-density lipoprotein (LDL) have been implicated as causal events in atherosclerosis. Cu²⁺ may play an important role in LDL oxidation by binding to histidine residues of apolipoprotein B-100 (apo B) and initiating and propagating lipid peroxidation. To investigate the role of histidine residues, we used diethylpyrocarbonate (DEPC), a lipid-soluble histidine-specific modifying reagent. When LDL (0.1 mg protein/ml, or 0.2 µM) was incubated with DEPC (1 mM), at least 76 ± 7% of the histidine residues in apo B were modified. Treatment of LDL with DEPC led to an increase in the rate of Cu²⁺-induced initiation of lipid peroxidation (R_i), but a significant decrease in the rate of propagation. These changes resulted in an overall increased resistance of LDL to oxidation, with a significantly increased lag phase preceding the propagation phase of lipid peroxidation. In contrast to DEPC, ascorbate completely prevented the initiation of LDL oxidation (R_i = 0). Our data indicate that there are two types of copper/histidine binding sites on apo B: those facing the lipid core of the LDL particle, which mediate the propagation of lipid peroxidation and are modified by DEPC; and those found on the surface of the LDL particle exposed to the aqueous environment, which are responsible for mediating the initiation of lipid peroxidation and are modifiable by ascorbate in the presence of Cu²⁺.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

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.     

Photoredox reactivity of copper(II)-3,5-diisopropylsalicylate induced by ligand-to-metal charge transfer excitation of the copper-phenolate chromophore

The electronic spectrum of Cu^II(dps)₂ in CH₃CN with dps=3,5-diisopropylsalicylate shows a ligand field absorption at λ_max=711 nm (ε=140 M⁻¹ cm⁻¹), and a phenolate to Cu(II) ligand-to-metal charge transfer (LMCT) band at λ_max=428 nm (ε=950). LMCT excitation of Cu^II(dps)₂ leads to the reduction of Cu(II) to Cu(I). Copper(II) disappears with φ=2.8×10⁻³ at λ_irr=436 nm.     

Effect of 3,4-dihydroxyphenylalanine on Cu2+-induced Inactivation of HDL-associated Paraoxonase1 and Oxidation of HDL; Inactivation of Paraoxonase1 Activity Independent of HDL Lipid Oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Protective activity of plicatin B against human LDL oxidation induced in metal ion-dependent and -independent processes. Experimental and theoretical studies

Oxidation of low-density lipoproteins (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Natural antioxidants have been shown to protect LDL from oxidation and to inhibit atherogenic developments in animals. Structurally related prenylated pterocarpans, erybraedin C and bitucarpin A, and the prenylchalcone plicatin B were examined for their ability to inhibit LDL oxidation in vitro. The kinetic profile of peroxidation is characterized by the lag time of oxidation (t_lag), the maximal rate of oxidation (V_max) and the maximal accumulation of oxidation products (OD_max). Specific variation of the set of kinetic parameters by antioxidants may provide important information about the mechanism of inhibitory action of a given compound. At equimolar concentrations (1 μM) the prenylated derivatives tested were found to inhibit 1 μM copper sulphate-induced oxidation of LDL (50 μg protein/ml) in accordance with the following order of activity: plicatin B>erybraedin Cbitucarpin A. Structural aspects, such as hydrogen-donating substituents, their number and arrangement in the aromatic ring moieties, and the prenyl and methoxy substituents, were investigated in order to explain the findings obtained. It is well known that the antioxidant activity of flavonoids is believed to be caused by a combination of transition metal chelation and free-radical-scavenging activities. To investigate these differences we comparatively studied the protective mechanism of plicatin B in copper-dependent or -independent LDL oxidation. The latter was mediated by 2,2’-azo-bis-(2-amidinopropane) dihydrochloride (ABAP). We measured the formation of conjugated dienes (OD_234 nm). Plicatin B (0.2-1.5 μM) delayed the Cu²⁺ (1 μM) promoted oxidation as conjugate diene formation (t_lag) of the LDL by 45.2-123.5 min and reduced V_max by 0.46-0.29 μM/min. In the ABAP (0.2 mM) promoted LDL oxidation t_lag increased by 67.2-110.2 min through plicatin B (0.5-2.5 μM). In experiments in which Cu²⁺ concentrations increased (0.5 - 3 μM) and the amount of plicatin B (1 μM) was maintained constant, a significant decrease in t_lag and an increase in V_max was observed. In this study plicatin B appeared to exhibit a mixed mechanism, interfering with the formation of the radicals by chelating copper involved in the initiation/propagation reaction, but also by scavenging free hydroperoxyl radicals resulting from ABAP thermolysis. In addition, theoretical analysis indicated that plicatin B preferentially established the chelating complex with Cu²⁺, because its affinity value is notably higher (by a factor of 5) than that for Cu⁺.     

Modeling by assembly and molecular dynamics simulations of the low Cu2+ occupancy form of the mammalian prion protein octarepeat region: gaining insight into Cu2+-mediated beta-cleavage

The prion protein has garnered considerable interest because of its involvement in prion disease as well as its unresolved cellular function. The octarepeat region in the flexible N-domain is capable of binding copper through multiple coordination modes. Under conditions of low pH and low Cu²⁺ concentration, the four octarepeats (ORs) cooperatively coordinate a single copper ion. Based on the average structure of the PHGG and GWGQ portions of a copper-free OR₂ model from molecular dynamics simulations, the starting structures of the OR₄ complex could be constructed by assembling the repeating structure of PHGG and GWGQ fragments. The resulting model contains a preformed site suitable for Cu²⁺ coordination. Molecular dynamics simulations of Cu²⁺ bound to the assembled OR₄ model (Cu:OR₄) reveal a close association of specific Trp and Gly residues with the Cu²⁺ center. This low Cu²⁺-occupancy form of prion protein is redox-active and can readily initiate cleavage of the OR region, mediated by reactive oxygen species generated by Cu⁺. The OR region is known to be required for β-cleavage, as are the Trp residues within the OR region. The β-cleaved form of the prion protein accumulates in amyloid fibrils. Hence, the close approach of Trp and Gly residues to the Cu²⁺ coordination site in the low Cu²⁺-occupancy form of the OR region may signal an important interaction for the initiation of prion disease.     

Tryptophan fluorescence reveals the presence of long-range interactions in the denatured state of ribonuclease Sa

Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues. We made five variants of RNase Sa by adding Trp residues at locations where they are found in other members of the microbial ribonuclease family. To better understand the protein denatured state, we also studied the fluorescence properties of the following peptides: N-acetyl-Trp-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and the five pentapeptides with the same sequence as the Trp substitution sites in RNase Sa. The major conclusions are: 1), the wavelength of maximum fluorescence intensity, λ_max, does not differ significantly for the peptides and the denatured proteins; 2), the fluorescence intensity at λ_max, I_F, differs significantly for the five Trp containing variants of RNase Sa; 3), the I_F differences for the denatured proteins are mirrored in the peptides, showing that the short-range effects giving rise to the I_F differences in the peptides are also present in the proteins; 4) the I_F values for the denatured proteins are more than 30% greater than for the peptides, showing the presence of long-range effects in the proteins; 5), fluorescence quenching of Trp by acrylamide and iodide is more than 50% greater in the peptides than in the denatured proteins, showing that long-range effects limit the accessibility of the quenchers to the Trp side chains in the proteins; and 6), these results show that nonlocal effects in the denatured states of proteins influence Trp fluorescence and accessibility significantly.     

The two faces of α- and γ-tocopherols: an in vitro and ex vivo investigation into VLDL, LDL and HDL oxidation

BackgroundVitamin E and its derivatives, namely, the tocopherols, are known antioxidants, and numerous clinical trials have investigated their role in preventing cardiovascular disease; however, evidence to date remains inconclusive. Much of the in vitro research has focused on tocopherol's effects during low-density lipoprotein (LDL) oxidation, with little attention being paid to very LDL (VLDL) and high-density lipoprotein (HDL). Also, it is now becoming apparent that γ-tocopherol may potentially be more beneficial in relation to cardiovascular health.ObjectivesDo α- and γ-tocopherols become incorporated into VLDL, LDL and HDL and influence their oxidation potential in an in vitro and ex vivo situation?DesignFollowing (i) an in vitro investigation, where plasma was preincubated with increasing concentrations of either α- or γ-tocopherol and (ii) an in vivo 4-week placebo-controlled intervention with α- or γ-tocopherol. Tocopherol incorporation into VLDL, LDL and HDL was measured via high-pressure liquid chromatography, followed by an assessment of their oxidation potential by monitoring conjugated diene formation.ResultsIn vitro: Both tocopherols became incorporated into VLDL, LDL and HDL, which protected VLDL and LDL against oxidation. However and surprisingly, the incorporation into HDL demonstrated pro-oxidant properties. Ex vivo: Both tocopherols were incorporated into all three lipoproteins, protecting VLDL and LDL against oxidation; however, they enhanced the oxidation of HDL.ConclusionsThese results suggest that α- and γ-tocopherols display conflicting oxidant activities dependent on the lipoprotein being oxidized. Their pro-oxidant activity toward HDL may go some way to explain why supplementation studies with vitamin E have not been able to display cardioprotective effects.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin,transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.     

Methemoglobin formation in mutant hemoglobin α chains: electron transfer parameters and rates

Hemoglobin-mediated transport of dioxygen (O₂) critically depends on the stability of the reduced (Fe²⁺) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe³⁺) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe³⁺) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ_solv 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ_prot) accounts for 27–30% except for the Miyagi and J-Buda variants (λ_prot ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe²⁺ state. Semiclassical Marcus ET theory-based calculations predict experimental k_ET for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.     

Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

Background

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.

Methodology/Principal Findings

As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn₂-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu²⁺ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu²⁺: a moderate-affinity site (10⁶ M^-1) and a high-affinity site (10⁸ M^-1). Furthermore, Cu²⁺ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.

Conclusions/Significance

We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu²⁺ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.     

Dalcetrapib and anacetrapib increase apolipoprotein E-containing HDL in rabbits and humans

The large HDL particles generated by administration of cholesteryl ester transfer protein inhibitors (CETPi) remain poorly characterized, despite their potential importance in the routing of cholesterol to the liver for excretion, which is the last step of the reverse cholesterol transport. Thus, the effects of the CETPi dalcetrapib and anacetrapib on HDL particle composition were studied in rabbits and humans. The association of rabbit HDL to the LDL receptor (LDLr) in vitro was also evaluated. New Zealand White rabbits receiving atorvastatin were treated with dalcetrapib or anacetrapib. A subset of patients from the dal-PLAQUE-2 study treated with dalcetrapib or placebo were also studied. In rabbits, dalcetrapib and anacetrapib increased HDL-C by more than 58% (P < 0.01) and in turn raised large apo E-containing HDL by 66% (P < 0.001) and 59% (P < 0.01), respectively. Additionally, HDL from CETPi-treated rabbits competed with human LDL for binding to the LDLr on HepG2 cells more than control HDL (P < 0.01). In humans, dalcetrapib increased concentrations of large HDL particles (+69%, P < 0.001) and apo B-depleted plasma apo E (+24%, P < 0.001), leading to the formation of apo E-containing HDL (+47%, P < 0.001) devoid of apo A-I. Overall, in rabbits and humans, CETPi increased large apo E-containing HDL particle concentration, which can interact with hepatic LDLr. The catabolism of these particles may depend on an adequate level of LDLr to contribute to reverse cholesterol transport.     

Oxidized low density lipoprotein inhibits the hemolytic activity of Asp-hemolysin from Aspergillus fumigatus

We have examined the effect of chemically modified human low density lipoproteins (LDLs) , acetylated LDL and oxidized LDL, on the hemolytic activity of Asp-hemolysin. Oxidized LDL, but not acetylated LDL, inhibited the hemolytic activity of this toxin. The inhibitory effects of oxidized LDL increased with the time of Cu²⁺-induced LDL oxidation. Similar inhibition was observed in the filtrate which was separated from the incubation mixture of Asp-hemolysin with oxidized LDL (for 2 h of oxidation) following ultrafiltration through a membrane with a molecular mass cutoff of 100 000. However, at longer LDL oxidation times, the inhibition by the filtrates was less than the control mixture without ultrafiltration. We suggest that the inhibition by oxidized LDL was due to the binding of oxidized LDL to Asp-hemolysin at shorter LDL oxidation times .     

Effect of dioxygen on copper(II) binding to α-synuclein

Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W α-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (K_d(app)) changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-α-syn site, MDV(F/W) and M^∗DV(F/W), where M^∗ = methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F→W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a Cu^II-Trp/Phe (cation-π) interaction.     

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu²⁺-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R^−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.     

Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome

We have found that hydrogen (dihydrogen; H₂) has beneficial lipid-lowering effects in high-fat diet-fed Syrian golden hamsters. The objective of this study was to characterize the effects of H₂-rich water (0.9–1.0 l/day) on the content, composition, and biological activities of serum lipoproteins on 20 patients with potential metabolic syndrome. Serum analysis showed that consumption of H₂-rich water for 10 weeks resulted in decreased serum total-cholesterol (TC) and LDL-cholesterol (LDL-C) levels. Western blot analysis revealed a marked decrease of apolipoprotein (apo)B100 and apoE in serum. In addition, we found H₂ significantly improved HDL functionality assessed in four independent ways, namely, i) protection against LDL oxidation, ii) inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to endothelial cells, iii) stimulation of cholesterol efflux from macrophage foam cells, and iv) protection of endothelial cells from TNF-α-induced apoptosis. Further, we found consumption of H₂-rich water resulted in an increase in antioxidant enzyme superoxide dismutase and a decrease in thiobarbituric acid-reactive substances in whole serum and LDL. In conclusion, supplementation with H₂-rich water seems to decrease serum LDL-C and apoB levels, improve dyslipidemia-injured HDL functions, and reduce oxidative stress, and it may have a beneficial role in prevention of potential metabolic syndrome.     

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.