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

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

Thyroid hormone (T3) and its acetic derivative (TA3) protect low-density lipoproteins from oxidation by different mechanisms

Triiodothyronine (T3) and triiodothyroacetic acid (TA3) are thyroid compounds that similarly protect low-density lipoprotein (LDL) against oxidation induced by the free radical generator 2,2'-azobis-[2-amidinopropane] dihydrochloride (AAPH). However, TA3 is more antioxidant than T3 on LDL oxidation induced by copper ions (Cu2+), suggesting that these compounds act by different mechanisms. Here we measured conjugated diene production kinetics during in vitro human LDL (50 mg LDL-protein per l) oxidation induced by various Cu2+ (0.5-4 microM) or AAPH (0.25-2 mM) concentrations in the presence of T3, TA3, butylated hydroxytoluene (BHT) (a free radical scavenger) or ethylenediaminetetracetic acid (EDTA) (a metal chelator). From the kinetics were estimated: length of the lag phase (Tlag), maximum velocity of conjugated diene production (Vmax), and maximum amount of generated dienes (Dmax). Thyroid compound effects on these oxidation parameters were compared to those of the controls BHT and EDTA. In addition we measured by atomic absorption spectrometry copper remaining in LDL after a 30 min incubation of LDL with Cu2+ and the compounds followed by extensive dialysis, i.e. copper bound to LDL. As expected, LDL-copper was decreased by EDTA in a concentration-dependent manner, whereas it was not affected by BHT. T3 increased LDL-copper whereas TA3 slightly decreased it. The whole data suggest that T3 and TA3 are free radical scavengers that also differently disturb LDL-copper binding, an essential step for LDL lipid peroxidation. The most likely mechanisms are that T3 induces new copper binding sites inside the LDL particle, increasing the LDL-copper amount but in a redox-inactive form, whereas TA3 blocks some redox-active copper binding sites highly implicated in the initiation and the propagation of lipid peroxidation. Alternatively, we also found that a little amount of copper is tightly bound in LDL, which may be essential for the propagation of lipid peroxidation induced by free radical generators.     

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.     

The effect of albumin on copper-induced LDL oxidation

In an attempt to gain deeper understanding of the mechanism or mechanisms responsible for the protective effect of serum albumin against Cu²⁺-induced peroxidation of low density lipoprotein (LDL), we have examined the influence of the concentrations of bovine serum albumin (BSA), Cu²⁺ and LDL on the kinetics of peroxidation. Since the common method of monitoring the oxidation by continuous recording of the absorbance of conjugated dienes at 234 nm cannot be used at high BSA-concentrations because of the intensive absorption of BSA, we have monitored the time-dependent increase of absorbance at 245 nm. At this wavelength, conjugated dienes absorb intensely, whereas the background absorbance of BSA is low. Using this method, as well as the TBARS assay for determination of malondialdehyde, over a large range of BSA concentrations, we show that in many cases the influence of BSA on the kinetics of oxidation can be compensated for by increasing the concentration of copper. This reconciles the apparent contradiction between previously published data. Detailed studies of the kinetic profiles obtained under different conditions indicate that binding of Cu²⁺ to albumin plays the major role in its protective effect while other mechanisms contribute much less than copper binding. This conclusion is consistent with the less pronounced effect of BSA on the oxidation induced by the free radical generator AAPH. It is also shown that the copper-albumin complex is capable of inducing LDL oxidation, although the kinetics of the latter process is very different from that of copper-induced oxidation. Nevertheless, when compared to copper induced oxidation at similar concentration of the oxidation-promotor, the kinetics of oxidation induced by copper-albumin complex is very different and is consistent with a tocopherol mediated peroxidation, characteristic under low radical flux. Similar kinetics was observed for copper-induced oxidation only at much lower copper concentrations.     

Role of lipoprotein-copper complex in copper catalyzed-peroxidation of low-density lipoprotein.

The oxidative modification of low-density lipoprotein (LDL) is suggested to play an important role in the pathogenesis of atherosclerosis. The present study examined the role of the formation of LDL-copper (Cu) complex in the peroxidation of LDL. The amount of copper bound to LDL increased during incubation performed with increasing concentrations of CuSO4. More than 80% of the copper bound to the LDL particle was observed in the protein phase of LDL, suggesting that most of the copper ions formed complexes with the ligand-binding sites of apoprotein. The addition of histidine (1 mM), known to form a high affinity complex with copper, and EDTA (1 mM), a metal chelator, during the incubation of LDL with CuSO4 prevented the formation of both thiobarbituric acid-reactive substances (TBARS) and LDL-Cu complexes. EDTA inhibited the copper-catalyzed ascorbate oxidation whereas histidine had no effect, suggesting that the copper within the complex with histidine is available to catalyze the reaction, in contrast to EDTA. These observations indicate that the preventive effect of histidine on the copper-catalyzed peroxidation of LDL is not simply mediated by chelating free copper ions in aqueous phase. Evidence that copper bound to LDL particle still has a redox potential was provided by the observed increase in TBARS content during incubation of LDL-Cu complexes in the absence of free copper ions. The addition of either histidine or EDTA to LDL-Cu complexes inhibited the formation of TBARS by removing copper ions from the LDL forming the corresponding complexes. However, there still remained small amounts of copper in the LDL particles following the treatment of LDL-Cu complexes with histidine or EDTA. The copper ions remaining in the LDL particle lacked the ability to catalyze LDL peroxidation, suggesting that there may be two types of copper binding sites in LDL: tight-binding sites, from which the copper ions are not removed by chelation, and weak-binding sites, from which copper ions are easily removed by chelators. The formation of TBARS in the LDL preparation during incubation with CuSO4 was comparable to the incubation with FeSO4. In contrast, the formation of TBARS in the LDL-lipid micelles by CuSO4 was nearly eliminated even in the presence of ascorbate to promote metal-catalyzed lipid peroxidation, although a marked increase in TBARS content was observed in the LDL-lipid micelles with FeSO4, and with FeCl3 in the presence of ascorbate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Probabilistic kinetic model of slow oxidation of low-density lipoprotein: II. Experiments

Theoretical probabilistic kinetic model has been applied to describe the measurements of several oxidation markers as a function of time, during slow oxidation of low-density lipoprotein (LDL). It has been demonstrated that such a process could be described as tocopherol-mediated peroxidation (TMP), initiated and sustained by the action of copper ions, present in LDL in trace amounts. In that process concentration of alpha-tocopherol remains essentially unaltered. Tocopherol and copper ions act as catalysts, oscillating between the oxidized and reduced states. The fitting of the theoretical model to the experimental data resulted in determination of the numerical values for the kinetic parameters. It has been found that the parameter values used for the fitting of the data collected for a number of samples from various donors differ rather little. The kinetic chain length of 1.3 (in presence of co-antioxidants) and 2.9 (in the absence of co-antioxidants) is shorter than found by others. The difference probably comes from the much lower concentration of copper ions in our systems (about 0.1 ion per LDL particle).     

Extent of copper LDL oxidation depends on oxidation time and copper/LDL ratio: chemical characterization

The aim of our study was to determine, as a function of [Cu(2+)]/[LDL] ratios (0.5 and 0.05) and of oxidation phases, the extent of LDL oxidation by assessing the lipid and apo B oxidation products. The main results showed that: (i) kinetics of conjugated diene formation presented four phases for Cu(2+)/LDL ratio of 0.5 and two phases for [Cu(2+)]/[LDL] ratio of 0.05; (ii) oxidation product formation (cholesteryl ester and phosphatidylcholine hydroperoxides, apo B carbonyl groups) occurred early in the presence of endogenous antioxidants, under both copper oxidation conditions; (iii) apo B carbonylated fragments appeared when antioxidants were totally consumed at [Cu(2+)]/[LDL] ratio of 0.5; and (iv) antioxidant concentrations were stable, oxysterol formation was negligible, and no carbonylated fragment was detected at [Cu(2+)]/[LDL] ratio of 0.05. Depending on the copper/LDL ratio, oxidized LDL differ greatly in the nature of lipid peroxidation product and the degree of apo B fragmentation.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

The dose-dependent effect of copper-chelating agents on the kinetics of peroxidation of low-density lipoprotein (LDL)

Copper-induced peroxidation of lipoproteins involves continuous production of free radicals via a redox cycle of copper. Formation of Cu(I) during Cu(II)-induced peroxidation of LDL was previously demonstrated by accumulation of the colored complexes of Cu(I) in the presence of one of the Cu(I)-specific chelators bathocuproine (BC) or neocuproine (NC). All the studies conducted thus far employed high concentrations of these chelators (chelator/Cu(II) > 10). Under these conditions, at low copper concentrations the chelators prolonged the lag preceding oxidation, whereas at high copper concentrations the chelators shortened the lag. In an attempt to gain understanding of these non-monotonic effects, we have studied systematically the peroxidation of LDL (0.1 microM, 50 microg protein/mL) at varying concentrations of NC or BC over a wide range of concentrations of the chelators and copper. These studies revealed that: (i) At copper concentrations of 5 microM and below, NC prolonged the lag in a monotonic, dose-dependent fashion typical for other complexing agents. However, unlike with other chelators, the maximal rate of oxidation was only slightly reduced (if at all). (ii) At copper concentrations of 15 microM and above, the addition of about 20 microM NC or BC resulted in prolongation of the lag, but this effect became smaller at higher concentrations of the chelators, and at yet higher concentrations the lag became much shorter than that observed in the absence of chelators. Throughout the whole range of NC concentrations, the maximal rate of peroxidation increased monotonically upon increasing the NC concentration. (iii) Unlike in the absence of chelators, the prooxidative effect of copper did not exhibit saturation with respect to copper, up to copper concentrations of 30 microM. Based on these results we conclude that the copper-chelates can partition into the hydrophobic core of LDL particles and induce peroxidation by forming free radicals within the core. This may be significant with respect to the understanding of the possible mechanisms of peroxidation by chelated transition metals in vivo.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

Lipophilic beta-blockers inhibit monocyte and endothelial cell-mediated modification of low density lipoproteins.

The effects of propranolol, pindolol and metoprolol on the modification of low density lipoprotein (LDL) by U937 monocyte-like cells, endothelial cells and copper ions were studied by determination of the lipid peroxidation product content and measurement of the relative electrophoretic mobility of the particle. Propranolol and pindolol inhibited LDL oxidation by U937 cells in a dose-dependent manner from 10 to 100 microM, whereas metoprolol had no effect. In the case of LDL modification by endothelial cells, all the three beta-blockers were efficient within the same range of concentrations, and the order of potency was propranolol greater than pindolol greater than metoprolol. In vitro oxidation of LDL in the presence of copper ions was also inhibited by propranolol; pindolol and metoprolol had no significant protective effect in this system. These results concerning the inhibitory action of beta-blockers were confirmed by testing the degradation of modified LDL by J774 macrophages. Although the concentrations of the drugs utilized in this study are relatively high, in long-term treatment beta-blockers might accumulate in target tissues, and the protective effect of propranolol against LDL oxidation might be involved in its inhibitory action on atherosclerosis previously reported in animal models.     

Direct evidence for apo B-100-mediated copper reduction: studies with purified apo B-100 and detection of tryptophanyl radicals

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.     

1 Acyl-2 acetyl-sn-glycero-3 phosphocholine decreases the susceptibility of low-density lipoprotein to oxidative modification by copper ions,monocytes or endothelial cells

The effects of platelet-activating factor (PAF) and its analogue, 1 acyl-2 acetyl-sn-glycero-3 phosphocholine (1 acyl-2 acetyl-GPC), were investigated on the oxidative modification of low-density lipoprotein (LDL) by copper ions, U937 monocyte-like cells or endothelial cells, by determination of the lipid peroxidation end products (TBARS) content and measurement of the electrophoretic mobility of the particle. 1 Acyl-2 acetyl-GPC, in the concentration range 1–5 μg/ml, inhibited LDL oxidation in a dose-dependent manner in the three systems, whereas PAF had no effect. The protective effect of 1 acyl-2 acetyl-GPC was markedly more important when oxidative modification was performed with endothelial cells, leading to total inhibition at 5 μg/ml. At the same concentration, the TBARS production was inhibited by 60% and 20% with monocytes and copper ions, respectively. The degradation by J774 macrophage-like cells of LDL modified by copper ions, U937 monocyte-like cells or endothelial cells was also inhibited when modification was performed in the presence of 1 acyl-2 acetyl-GPC. Furthermore, preincubation of the LDL particle with 1 acyl-2 acetyl-GPC before modification protected the lipoprotein against oxidation, whereas preincubation of the cultured cells with the phospholipid had no effect. Thus 1 acyl-2 acetyl-GPC decreases the susceptibility of the LDL particle to oxidative modification, possibly by intercalation within the lipid phase of the particle. Since LDL oxidation is believed to play an important role in the initiation and progression of atherosclerosis, this inhibitory effect of 1 acyl-2 acetyl-GPC might be of importance in view of the fact that this phospholipid is produced concomitantly with PAF in some inflammatory cells.     

Kinetics of lipid peroxidation in mixtures of HDL and LDL, mutual effects.

In view of the proposed central role of LDL oxidation in atherogenesis and the established role of HDL in reducing the risk of atherosclerosis, several studies were undertaken to investigate the possible effect of HDL on LDL peroxidation. Since these investigations yielded contradictory results, we have conducted systematic kinetic studies on the oxidation in mixtures of HDL and LDL induced by different concentrations of copper, 2, 2'-azo bis (2-amidinopropane) hydrochloride (AAPH) and myeloperoxidase (MPO). These studies revealed that oxidation of LDL induced either by AAPH or MPO is inhibited by HDL under all the studied conditions, whereas copper-induced oxidation of LDL is inhibited by HDL at low copper/lipoprotein ratio but accelerated by HDL at high copper/lipoprotein ratio. The antioxidative effects of HDL are only partially due to HDL-associated enzymes, as indicated by the finding that reconstituted HDL, containing no such enzymes, inhibits peroxidation induced by low copper concentration. Reduction of the binding of copper to LDL by competitive binding to the HDL also contributes to the antioxidative effect of HDL. The acceleration of copper-induced oxidation of LDL by HDL may be attributed to the hydroperoxides formed in the "more oxidizable" HDL, which migrate to the "less oxidizable" LDL and enhance the oxidation of the LDL lipids induced by bound copper. This hypothesis is supported by the results of experiments in which native LDL was added to oxidizing lipoprotein at different time points. When the native LDL was added prior to decomposition of the hydroperoxides in the oxidizing lipoprotein, the lag preceding oxidation of the LDL was much shorter than the lag observed when the native LDL was added at latter stages, after the level of hydroperoxides became reduced due to their copper-catalyzed decomposition. The observed dependence of the interrelationship between the oxidation of HDL and LDL on the oxidative stress should be considered in future investigations regarding the oxidation of lipoprotein mixtures.     

Cu2(+)-mediated oxidation of dialyzed plasma: effects on low and high density lipoproteins and cholesteryl ester transfer protein.

We previously reported that the expression of an epitope of apolipoprotein B (apoB), mapped to the C-terminus and defined by antibody Bsol7, increased during Cu2(+)-mediated oxidation of isolated low density lipoprotein (LDL). We describe now the properties of Bsol7 as a marker of LDL oxidation in whole plasma in relation to other effects of oxidative treatment of plasma, such as the distribution of apoA-I and cholesteryl ester transfer protein (CETP). In dialyzed plasma, no LDL oxidation was detected at Cu2+ concentrations (5 microM) sufficient for extensive oxidation of isolated LDL. At a higher Cu2+ concentration (50 microM), an increased expression of the Bsol7 epitope was observed; at 250 microM Cu2+, other evidence of LDL oxidation was found. The pattern of LDL response to Cu2+ observed in dialyzed plasma could be reproduced by adding 3% bovine serum albumin to isolated LDL. We demonstrate that the effect of albumin most likely results from its ability to bind copper ions. Incubation of plasma with increasing concentrations of Cu2+ resulted first in the disappearance of alpha 2-migrating HDL, the usual carrier of CEPT; free CETP and high molecular weight apoA-I-containing particles were also generated during oxidation. Addition of oxidized, but not native, LDL to plasma resulted in a transfer to LDL of some of the CETP initially associated with apoA-I. In conclusion, the increased immunoreactivity of the Bsol7 epitope was the most sensitive parameter of LDL oxidation, but other parameters, such as the presence of alpha 2-HDL and CETP-lipoprotein associations were even more sensitive evidence of lipoprotein oxidation.     

Effect of probucol on the physical properties of low-density lipoproteins oxidized by copper

Human plasma low-density lipoproteins (LDL) were incubated with 10 microM probucol for 1 h at 37 degrees C. Probucol incorporation into the LDL was complete as judged by filtration through a 0.2-micron filter, ultracentrifugation, and gel filtration. LDL with and without probucol were incubated for up to 24 h with 5 microM Cu2+ at 37 degrees C. Copper oxidation increased the content of random structure in the LDL protein from 30% to 36% at the expense of beta-structure (which decreased from 22% to 16%) without a change in alpha-helical content as measured by circular dichroism spectroscopy. This loss of beta-structure was prevented by the presence of probucol in the LDL during the copper incubation. Probucol reduced the rate of increase of fluorescence during copper oxidation at 37 degrees C. After 6 h, the fluorescence intensity at 360-nm excitation and 430-nm emission was 30% less in probucol-containing samples. Probucol had no effect on the circular dichroic spectrum of LDL and only minimal effects (less than 5%) on the fluorescence emission spectrum at wavelengths below 500 nm. Two fluorescence peaks, with emission at 420 nm and excitation at 340 and 360 nm, are resolved in three-dimensional fluorescence spectra of oxidized LDL. Probucol reduces the intensity of both peaks equally. The binding of a highly reactive heparin (HRH) fraction to LDL was measured by titration of LDL with HRH in the presence of fluoresceinamine-labeled HRH. The decrease in fluorescence anisotropy of the labeled HRH is proportional to the concentration of bound HRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble alpha-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.     

Cellular prion protein acquires resistance to proteolytic degradation following copper ion binding

The conversion of cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)) conveys an increase in hydrophobicity and induces a partial resistance to proteinase K (PK). Interestingly, co-incubation with high copper ion concentrations also modifies the solubility of PrP(c) and induces a partial PK resistance which was reminiscent of PrP(Sc). However, concerns were raised whether this effect was not due to a copper-induced inhibition of the PK itself. We have therefore analyzed the kinetics of the formation of PK-resistant PrP(C) and excluded possible interference effects by removing unbound copper ions prior to the addition of PK by methanol precipitation or immobilization of PrP(C) followed by washing steps. We found that preincubation of PrPc with copper ions at concentrations as low as 50 microM indeed rendered these proteins completely PK resistant, while control substrates were proteolyzed. No other divalent cations induced a similar effect. However, in addition to this specific stabilizing effect on PrP(C), higher copper ion concentrations in solution (>200 microM) directly blocked the enzymatic activity of PK, possibly by replacing the Ca2+ ions in the active center of the enzyme. Therefore, as a result of this inhibition the proteolytic degradation of PrP(C) as well as PrP(Sc) molecules was suppressed.