Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation.

We reported earlier that urate may behave as a pro-oxidant in Cu2+-induced oxidation of diluted plasma. Thus, its effect on Cu2+-induced oxidation of isolated low-density lipoprotein (LDL) was investigated by monitoring the formation of malondialdehyde and conjugated dienes and the consumption of urate and carotenoids. We show that urate is antioxidant at high concentration but pro-oxidant at low concentration. Depending on Cu2+ concentration, the switch between the pro- and antioxidant behavior of urate occurs at different urate concentrations. At high Cu2+ concentration, in the presence of urate, superoxide dismutase and ferricytochrome c protect LDL from oxidation but no protection is observed at low Cu2+ concentration. The use of Cu2+ or Cu+ chelators demonstrates that both copper redox states are required. We suggest that two mechanisms occur depending on the Cu2+ concentration. Urate may reduce Cu2+ to Cu+, which in turn contributes to formation. The Cu2+ reduction is likely to produce the urate radical (UH.-). It is proposed that at high Cu2+ concentration, the reaction of UH.- radical with generates products or intermediates, which trigger LDL oxidation. At low Cu2+ concentration, we suggest that the Cu+ ions formed reduce lipid hydroperoxides to alkoxyl radicals, thereby facilitating the peroxidizing chain reaction. It is anticipated that these two mechanisms are the consequence of complex LDL-urate-Cu2+ interactions. It is also shown that urate is pro-oxidant towards slightly preoxidized LDL, whatever its concentration. We reiterate the conclusion that the use of antioxidants may be a two-edged sword.     

α-Tocopherol mediated peroxidation in the copper (II) and met myoglobininduced oxidation of human low density lipoprotein: The influence of lipid hydroperoxides

The principal antioxidant in human LDL, α-tocopherol, is converted to the α-tocopheroxyl radical after reaction with peroxyl radicals or Cu²⁺, and, if it does not terminate with peroxyl radicals, could initiate lipid peroxidation; a phenomenon called ‘tocopherol mediated peroxidation’. Only in the presence of Cu²⁺ and low levels of lipid hydroperoxides was an α-tocopherol dependent decrease in the resistance of LDL to oxidation detected. This suggests that tocopherol mediated peroxidation will probably not contribute significantly as a pro-oxidant process in those individuals most at risk of developing atherosclerosis through an oxidative mechanism.     

Human apo-lipoprotein B from normal plasma contains oxidised peptides

Oxidation of low density lipoprotein (LDL) may be atherogenic, but radical-initiated oxidation of its apoprotein B-100 (apoB) has been little studied. Transition metal ions iron and copper are candidates for mediating radical oxidation of LDL in vivo. Therefore, we studied the copper-ion-induced oxidation of apoB in human LDL. Using HPLC methods developed in our recent work, we studied the destruction of native and the generation of six oxidised amino acids; we also assessed the release of peptides from the LDL particle by FPLC. We observed time-dependent losses of apoB histidine, lysine and glycine. Long-lived reactive species, the reductant DOPA, and the oxidant hydroperoxides of valine and leucine (measured as hydroxides after reduction), were generated. Their relative abundance (mol/mol of parent amino acid) was DOPA>o- and m-tyrosine>dityrosine, valine-hydroxides, leucine hydroxides. Low molecular weight fragments were also released from the LDL in a time-dependent manner, contained hydroperoxides sensitive to GSH peroxidase, and generated radicals on reaction with iron–EDTA. The fragments contained peptides active in the quinone redox cycling procedure, comprising 0.25% of the supplied LDL amino acids. Characteristic peptides were present in each FPLC fraction containing the fragments, as judged by further HPLC fractionation. Some fragments were present in the unoxidised LDL preparations, and when these were largely removed by FPLC, copper oxidation could still generate fragments, suggesting that those present in the starting material might indicate prior oxidation. Concordantly, we found that fresh plasma LDL apoB contained 3% of total plasma protein-bound oxidised amino acids, and with the same relative abundance. We conclude that plasma proteins including apoB are subject to physiological oxidation, similar to that inflicted by copper ions; the latter may contribute to intimal LDL oxidation, which could be the source of oxidised plasma apoB.     

A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin

The mechanisms by which low-density lipoprotein (LDL) particles undergo oxidative modification to an atherogenic form that is taken up by the macrophage scavenger-receptor pathway have been the subject of extensive research for almost two decades. The most common method for the initiation of LDL oxidation in vitro involves incubation with Cu(II) ions. Although various mechanisms have been proposed to explain the ability of Cu(II) to promote LDL modification, the precise reactions involved in initiating the process remain a matter of contention in the literature. This review provides a critical overview and evaluation of the current theories describing the interactions of copper with the LDL particle. Following discussion of the thermodynamics of reactions dependent upon the decomposition of preexisting lipid hydroperoxides, which are present in all crude LDL preparations, attention is turned to the more difficult (but perhaps more physiologically-relevant) system of the hydroperoxide-free LDL particle. In both systems, the key role of alpha-tocopherol is discussed. In addition to its protective, radical-scavenging action, alpha-tocopherol can also behave as a prooxidant via its reduction of Cu(II) to Cu(I). Generation of Cu(I) greatly facilitates the decomposition of lipid hydroperoxides to chain-carrying radicals, but the mechanisms by which the vitamin promotes LDL oxidation in the absence of preformed hydroperoxides remain more speculative. In addition to the so-called tocopherol-mediated peroxidation model, in which polyunsaturated fatty acid oxidation is initiated by the alpha-tocopheroxyl radical (generated during the reduction of Cu(II) by alpha-tocopherol), an evaluation of the role of the hydroxyl radical is provided. Important interactions between copper ions and thiols are also discussed, particularly in the context of cell-mediated LDL oxidation. Finally, the mechanisms by which ceruloplasmin, a copper-containing plasma protein, can bring about LDL modification are discussed. Improved understanding of the mechanisms of LDL oxidation by copper ions should facilitate the establishment of any physiological role of the metal in LDL modification. It will also assist in the interpretation of studies in which copper systems of LDL oxidation are used in vitro to evaluate potential antioxidants.     

Protective effect of the oligomeric acylphloroglucinols from Myrtus communis on cholesterol and human low density lipoprotein oxidation

Myrtle (Myrtus communis L.), a culinary spice and flavouring agent for alcoholic beverages widespread in the Mediterranean area and especially in Sardinia, contains the structurally unique oligomeric non-prenylated acylphloroglucinols, semimyrtucommulone and myrtucommulone A, whose antioxidant activity was investigated during the oxidative modification of lipid molecules implicated in the onset of cardiovascular diseases. Both acylphloroglucinols showed powerful antioxidant properties during the thermal (140 degrees C), solvent-free degradation of cholesterol. Moreover, the pre-treatment with semimyrtucommulone and myrtucommulone A significantly preserved LDL from oxidative damage induced by Cu(2+) ions at 2h of oxidation, and showed remarkable protective effect on the reduction of polyunsaturated fatty acids and cholesterol, inhibiting the increase of their oxidative products (conjugated dienes fatty acids hydroperoxides, 7beta-hydroxycholesterol, and 7-ketocholesterol). Taking into account the widespread culinary use of myrtle leaves, the results of the present work qualify the natural compounds semimyrtucommulone and myrtucommulone A as interesting dietary antioxidants with potential antiatherogenicity.     

Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging

Oxidation of lipids has been implicated in the pathophysiology of atherosclerosis. It has been suggested that scavenging of lipid peroxyl radicals contribute to the antiatherosclerotic effects of naturally occurring compounds such as the isoflavones. This group of polyphenolics includes genistein and is present in relatively high concentrations in food products containing soy. Soy isoflavones are capable of inhibiting lipoprotein oxidation in vitro and suppressing formation of plasma lipid oxidation products in vivo. However, key aspects of the antioxidant mechanisms remain unknown. In this study the antioxidant effects of genistein and other soy isoflavones on lipid peroxidation initiated by mechanistically diverse oxidants was investigated. Although isoflavones inhibited lipid peroxidation stimulated by both metal-dependent and independent processes, the concentration required for these effects were relatively high compared to those found in vivo. Interestingly, however, isoflavones were not consumed and remained in the native state over the time during which inhibition of lipid peroxidation was observed. This was also the case under conditions where synergistic inhibition of LDL oxidation was observed with ascorbate. Furthermore, in an oxidation system driven solely by peroxyl radicals, isoflavones were found to be relatively poor peroxyl radical scavengers. Consistent with the apparent lack of reactivity with lipid-derived oxidants, isoflavones were also relatively resistant to oxidation mediated by the potent oxidant peroxynitrite. The potential antioxidant mechanisms of isoflavones are discussed in the context of possible reactivities of isoflavone-derived phenoxyl radicals.     

Anti- and pro-oxidant effects of quercetin in copper-induced low density lipoprotein oxidation. Quercetin as an effective antioxidant against pro-oxidant effects of urate.

We recently reported that, depending on its concentration, urate is either a pro- or an antioxidant in Cu(2+)-induced low-density lipoprotein (LDL) oxidation. We also previously demonstrated an antioxidant synergy between urate and some flavonoids in the Cu(2+)-induced oxidation of diluted serum. As a result, the effect of the flavonoid quercetin on the Cu(2+)-induced oxidation of isolated LDL has been studied either in the presence or absence of urate. We demonstrate that, like urate, quercetin alone, at low concentration, exhibits a pro-oxidant activity. The pro-oxidant behavior depends on the Cu(2+) concentration but it is not observed at high Cu(2+) concentration. When compared with urate, the switch between the pro- and the antioxidant activities occurs at much lower quercetin concentrations. As for urate, the pro-oxidant character of quercetin is related to its ability to reduce Cu(2+) with the formation of semioxidized quercetin and Cu(+) with an expected yield larger than that obtained with urate owing to a more favorable redox potential. It is also shown that the pro-oxidant activity of urate can be inhibited by quercetin. An electron transfer between quercetin and semioxidized urate leading to the repair of urate could account for this observation as suggested by recently published pulse radiolysis data. It is anticipated that the interactions between quercetin-Cu(2+)-LDL and urate, which are tightly controlled by their respective concentration, determine the balance between the pro- and antioxidant behaviors. Moreover, as already observed with other antioxidants, it is demonstrated that quercetin alone behaves as a pro-oxidant towards preoxidized LDL.     

Sulfite facilitates LDL lipid oxidation by transition metal ions: a pro-oxidant in wine?

Lipid oxidation in LDL may play a role in atherogenesis. It has been shown that sulfite - a compound in the aqueous fraction of wine - could inhibit free radical (AAPH) mediated oxidation of plasma. Thus, sulfite has been proposed as an antioxidant. In contrast, the aqueous phase of wine has recently been shown to contain not fully identified compounds promoting transition metal ion (Cu(2+)) initiated LDL oxidation. As transition metal ions can catalyse the auto-oxidation of sulfite, we studied the influence of sulfite on Cu(2+) initiated LDL oxidation. The results show that sulfite at concentrations found in vivo strongly facilitated LDL oxidation by Cu(2+). The LDL-oxidase activity of ceruloplasmin was also stimulated by sulfite. ROS formation by Cu(2+)/SO(3)(2-) was not inhibited by SOD but by catalase. We propose that formation of Cu(+), sulfite radicals (SO(3)*(-)) and hydroxyl radicals (OH(*)) is a mechanism by which sulfite could act as a pro-atherogenic agent in presence of transition metal ions.     

Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid

Oxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of Mr below 500 was prepared as a model for the low Mr components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low Mr serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL. These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants.     

Lipid peroxidation and 4-hydroxy-2-nonenal formation by copper ion bound to amyloid-beta peptide

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is proposed to be a toxic factor in the pathogenesis of Alzheimer disease. The primary products of lipid peroxidation are phospholipid hydroperoxides, and degraded reactive aldehydes, such as HNE, are considered secondary peroxidation products. In this study, we investigated the role of amyloid-beta peptide (A beta) in the formation of phospholipid hydroperoxides and HNE by copper ion bound to A beta. The A beta1-42-Cu2+ (1:1 molar ratio) complex showed an activity to form phospholipid hydroperoxides from a phospholipid, 1-palmitoyl-2-linoleoyl phosphatidylcholine, through Cu2+ reduction in the presence of ascorbic acid. The phospholipid hydroperoxides were considered to be a racemic mixture of 9-hydroperoxide and 13-hydroperoxide of the linoleoyl residue. When Cu2+ was bound to 2 molar equivalents of A beta(1-42) (2 A beta1-42-Cu2+), lipid peroxidation was inhibited. HNE was generated from one of the phospholipid hydroperoxides, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH), by free Cu2+ in the presence of ascorbic acid through Cu2+ reduction and degradation of PLPC-OOH. HNE generation was markedly inhibited by equimolar concentrations of A beta(1-40) (92%) and A beta(1-42) (92%). However, A beta(1-42) binding 2 or 3 molar equivalents of Cu2+ (A beta1-42-2Cu2+, A beta1-42-3Cu2+) acted as a pro-oxidant to form HNE from PLPC-OOH. These findings suggest that, at moderate concentrations of copper, A beta acts primarily as an antioxidant to prevent Cu2+-catalyzed oxidation of biomolecules, but that, in the presence of excess copper, pro-oxidant complexes of A beta with Cu2+ are formed.     

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.     

Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide

The combination of ascorbate, transition metal ions, and hydrogen peroxide (H(2)O(2)) is an efficient hydroxyl radical generating system called "the Udenfriend system." Although the pro-oxidant role of ascorbate in this system has been well characterized in vitro, it is uncertain whether ascorbate also acts as a pro-oxidant under physiological conditions. To address this question, human plasma, used as a representative biological fluid, was either depleted of endogenous ascorbate with ascorbate oxidase, left untreated, or supplemented with 25 microM-1 mM ascorbate. Subsequently, the plasma samples were incubated at 37 degrees C with 50 microM-1 mM iron (from ferrous ammonium sulfate), 60 or 100 microM copper (from cupric sulfate), and/or 200 microM or 1 mM H(2)O(2). Although endogenous and added ascorbate was depleted rapidly in the presence of transition metal ions and H(2)O(2), no cholesterol ester hydroperoxides or malondialdehyde were formed, i.e., ascorbate protected against, rather than promoted, lipid peroxidation. Conversely, depletion of endogenous ascorbate was sufficient to cause lipid peroxidation, the rate and extent of which were enhanced by the addition of metal ions but not H(2)O(2). Ascorbate also did not enhance protein oxidation in plasma exposed to metal ions and H(2)O(2), as assessed by protein carbonyl formation and depletion of reduced thiols. Interestingly, neither the rate nor the extent of endogenous alpha-tocopherol oxidation in plasma was affected by any of the treatments. Our data show that even in the presence of redox-active iron or copper and H(2)O(2), ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in human plasma in vitro.     

Products of the reaction of HOCl with tryptophan protect LDL from atherogenic modification

Hypochlorite (HOCl) attacks amino acid residues in LDL making the particle atherogenic. Tryptophan is prone to free radical reactions and modification by HOCl. We hypothesized, that free tryptophan may quench the HOCl attack therefore protecting LDL. Free tryptophan inhibits LDL apoprotein modification and lipid oxidation. Tryptophan-HOCl metabolites associate with LDL reducing its oxidizability initiated by endothelial cells, Cu(2+) and peroxyl radicals. One tryptophan-HOCl metabolite was identified as 4-methyl-carbostyril which showed antioxidative activity when present during Cu(2+) mediated lipid oxidation, but did not associate with LDL. Indole-3-acetaldehyde, a decomposition product of tryptophan chloramine (the product of the tryptophan-HOCl reaction) was found to associate with LDL increasing its resistance to oxidation. Myeloperoxidase treatment of LDL in the presence of chloride, H(2)O(2) and tryptophan protected the lipoprotein from subsequent cell-mediated oxidation. We conclude that, in vivo, the activated myeloperoxidase system can generate antioxidative metabolites from tryptophan by the reaction of hypochlorite with this essential amino acid.     

Aggregation of LDL with chondroitin-4-sulfate makes LDL oxidizable in the presence of water-soluble antioxidants

The content of plasma and arterial interstitial fluid in water-soluble antioxidants makes it unlikely for low-density lipoprotein (LDL) to oxidize by the oxidation mechanisms most frequently discussed. By aggregation of LDL in the presence of chondroitin-4-sulfate (C-4-S), but not with chondroitin-6-sulfate or sphingomyelinase, a complex arises which can oxidize in the presence of 20 microM ascorbate and 300 microM urate. This oxidation sensitivity even persists after the gel-filtration of an LDL/C-4-S/Cu(2+) complex, indicating entrapment of Cu(2+) within. This corresponds well to the known ability of C-4-S to bind copper ions and is a potential mechanism by which LDL oxidation in the arterial intima is facilitated after prolonged retention by the extracellular matrix.     

The Effect of the Phenolic Antioxidant Ferulic Acid on the Oxidation of Low Density Lipoprotein Depends on the Pro-oxidant Used

The action of ferulic acid during the oxidation of LDL has been investigated using both copper ions and the haem protein metmyoglobin as pro-oxidants. The results demonstrate the ability of ferulic acid to act as a pro-oxidant when LDL oxidation is induced by copper at concentrations of the phenolic acid which are protective when the LDL oxidation is mediated by metmyoglobin. The suggested mechanism involves the reduction of Cu²⁺ to Cu⁺ by ferulic acid resulting in the production of the ferulic phenoxyl radical.     

Homocysteine promotes the LDL oxidase activity of ceruloplasmin

Ceruloplasmin (CP) oxidises low density lipoprotein (LDL). The oxidising potential depends on the formation of Cu(+)-CP which is redox-cycled during oxidation. Homocysteine (HCY) reduces free Cu(2+), potentiating its cell-damaging property. We show that HCY enhanced LDL oxidation by CP, but did not activate the LDL oxidising potential of Cu(2+)-diamine oxidase. Selective removal of the redox-active Cu(2+) abolished the LDL oxidase activity of CP. However, HCY partially restored the LDL oxidase activity of redox-copper depleted CP, indicating that the remaining six copper atoms in CP may also be involved in the process. Spectroscopic and oxidation inhibition studies using the Cu(+)-reagent bathocuproine revealed that HCY induced Cu(+)-CP formation, thus promoting its LDL oxidase activity.     

Inhibition of lipid peroxidation by S-nitrosoglutathione and copper

The antioxidant properties of S -nitrosoglutathione, a nitric oxide-derived product were studied in different experimental systems. By using the crocin bleaching test, S -nitrosoglutathione, in the presence of copper ions, shows an antioxidant capacity about six times higher than that of Trolox c and referable to the interception of peroxyl radicals by nitric oxide. Copper alone shows a modest inhibitory action, which is about seven times lower than that of Trolox c. S -nitrosoglutathione prevents lipid peroxidation induced by the well-known Fe 2+ /ascorbate system (IC 50 =450 μM) and the inhibitory effect is strongly reinforced by the presence of copper ions (IC 50 =6.5 μM). In addition, cumene hydroperoxide-induced lipid peroxidation is markedly decreased by S -nitrosoglutathione, provided that copper ions, maintained reduced by ascorbate, are present. Decomposition of S -nitrosoglutathione through metal catalysis and/or the presence of reducing agents and the consequent release of nitric oxide are of crucial importance for eliciting the antioxidant power. In this way, copper ions and/or reducing species with low antioxidant potency are able to promote the formation of an extremely strong antioxidant species such as nitric oxide.     

Inhibition of Lipid Peroxidation by S -nitrosoglutathione and Copper

The antioxidant properties of S -nitrosoglutathione, a nitric oxide-derived product were studied in different experimental systems. By using the crocin bleaching test, S -nitrosoglutathione, in the presence of copper ions, shows an antioxidant capacity about six times higher than that of Trolox c and referable to the interception of peroxyl radicals by nitric oxide. Copper alone shows a modest inhibitory action, which is about seven times lower than that of Trolox c. S -nitrosoglutathione prevents lipid peroxidation induced by the well-known Fe 2+ /ascorbate system (IC 50 =450 &#119 M) and the inhibitory effect is strongly reinforced by the presence of copper ions (IC 50 =6.5 &#119 M). In addition, cumene hydroperoxide-induced lipid peroxidation is markedly decreased by S -nitrosoglutathione, provided that copper ions, maintained reduced by ascorbate, are present. Decomposition of S -nitrosoglutathione through metal catalysis and/or the presence of reducing agents and the consequent release of nitric oxide are of crucial importance for eliciting the antioxidant power. In this way, copper ions and/or reducing species with low antioxidant potency are able to promote the formation of an extremely strong antioxidant species such as nitric oxide.     

Amyloid beta-Cu2+ complexes in both monomeric and fibrillar forms do not generate H2O2 catalytically but quench hydroxyl radicals

Oxidative stress plays a key role in Alzheimer's disease (AD). In addition, the abnormally high Cu(2+) ion concentrations present in senile plaques has provoked a substantial interest in the relationship between the amyloid beta peptide (Abeta) found within plaques and redox-active copper ions. There have been a number of studies monitoring reactive oxygen species (ROS) generation by copper and ascorbate that suggest that Abeta acts as a prooxidant producing H2O2. However, others have indicated Abeta acts as an antioxidant, but to date most cell-free studies directly monitoring ROS have not supported this hypothesis. We therefore chose to look again at ROS generation by both monomeric and fibrillar forms of Abeta under aerobic conditions in the presence of Cu(2+) with/without the biological reductant ascorbate in a cell-free system. We used a variety of fluorescence and absorption based assays to monitor the production of ROS, as well as Cu(2+) reduction. In contrast to previous studies, we show here that Abeta does not generate any more ROS than controls of Cu(2+) and ascorbate. Abeta does not silence the redox activity of Cu(2+/+) via chelation, but rather hydroxyl radicals produced as a result of Fenton-Haber Weiss reactions of ascorbate and Cu(2+) rapidly react with Abeta; thus the potentially harmful radicals are quenched. In support of this, chemical modification of the Abeta peptide was examined using (1)H NMR, and specific oxidation sites within the peptide were identified at the histidine and methionine residues. Our studies add significant weight to a modified amyloid cascade hypothesis in which sporadic AD is the result of Abeta being upregulated as a response to oxidative stress. However, our results do not preclude the possibility that Abeta in an oligomeric form may concentrate the redox-active copper at neuronal membranes and so cause lipid peroxidation.     

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.