Interactions of copper with glycated proteins: possible involvement in the etiology of diabetic neuropathy

Humans and animals with diabetes frequently develop peripheral vascular dysfunction and peripheral neuropathies. There is accumulating evidence that impaired peripheral nerve function may derive from diminished endoneural blood flow. The decrements in nerve blood flow may, in turn, be due to diminished endothelium-dependent vasodilation. Although a number of possible causes of this defective vasodilation have been suggested, none has been definitely proven. Regardless of the precise cause, the impaired vasodilatory activity may reflect diminished availability of endothelium-derived relaxing factor (EDRF), variously thought to be nitric oxide or thiol adducts of nitric oxide. Other investigators have reported that administration of transition metal chelators to diabetic rats corrects EDRF-mediated arterial relaxation and restores both neural blood flow and nerve conduction velocity, suggesting the involvement of transition metals. Our investigations center about the hypothesis that glycated proteins bind transition metals such as copper and iron, and that such 'glycochelates' accumulate within the vasculature in diabetes and catalytically inactivate EDRF. In partial support of this hypothesis: (1) Glycated albumin binds approximately 3-fold greater amounts of both copper and iron. (2) Copper bound to glycated albumin remains redox active (e.g. capable of supporting the oxidation of ascorbic acid). (3) Copper and copper-containing glycochelates cause the rapid decomposition of one putative form of EDRF, nitrosocysteine. (4) The amount of exchangeable (i.e. chelatable) copper in the plasma of diabetic rats is approximately twice that in normal rat plasma. (5) Similarly, tail tendons of diabetic animals have about twice as much bound copper as do tendons of normal rats. (6) Implants bearing adsorbed glycated albumin placed in the peritonea of normal mice for 48 h accumulate approximately 5 times as much bound copper as do implants coated with control albumin. Overall, these observations support--but do not conclusively prove - the hypothesis that transition metals such as copper, bound to glycated proteins, may blunt normal EDRF-dependent relaxation of diabetic arteries and provide a rationale for the use of transition metal chelators in the therapy of diabetic vasculopathy and neuropathy.     

Glycochelates and the etiology of diabetic peripheral neuropathy

People with diabetes are prone to develop peripheral vascular and nerve abnormalities which, in extreme cases, can lead to limb amputations. Although numerous theories have been advanced for these complications, no firm explanation is yet available. Recently, evidence has appeared suggesting that these vascular and nerve abnormalities may involve transition metals; administration of chelators such as desferrioxamine has been shown to prevent or actually reverse slowed peripheral nerve conduction and neuronal blood flow, as well as impaired endothelium-dependent arterial relaxation. Here, we argue that (i) the heavily glycated proteins known to accumulate in people with diabetes gain an increased affinity for transition metals such as iron and copper, (ii) as a result, proteins such as elastin and collagen within the arterial wall-which are known to be particularly heavily glycosylated in diabetes-may accumulate bound metal, especially copper, (iii) the bound metal causes the catalytic destruction of endothelium derived relaxing factor (nitric oxide or a derivative thereof), thereby engendering a state of chronic vasoconstriction. The resulting impairment of blood flow to peripheral nerves restricts the delivery of oxygen and nutrients and, in extremis, nerve death eventuates. If this hypothesis is proved correct, there are important implications for the development of novel pharmaceuticals for the treatment of diabetic peripheral neuropathy.     

Characterization of a 160 kD Photosystem II reaction center complex isolated from photoinhibited Dunaliella salina thylakoids

Photoinhibition in the green alga Dunaliella salina is accompanied by the formation of inactive Photosystem II reaction centers. In SDS-PAGE analysis, the latter appear as 160 kD complexes. These complexes are structurally stable, enough to withstand re-electrophoresis of excised gel slices from the 160 kD region. Western blot analyses with specific polyclonal antibodies raised against the D1 or D2 reaction center proteins provided evidence for the presence of both of these polypeptides in the re-electrophoresed 160 kD complex. Incubation of excised gel slices from the 160 kD region, under aerobic conditions at 4°C for a prolonged period of time, caused a break-up of the 160 kD complex into a 52 kD D1-containing and 80 and 26 kD D2-containing pieces. Western blot analysis with polyclonal antibodies raised against the apoproteins of CPI (reaction center proteins of PS I) did not show cross-reaction either with the 160 kD complex or with the 52, 80 and 26 kD pieces. The results show the presence of both D1 and D2 in the 160 kD complex and strengthen the notion of a higher molecular weight D1- and D2-containing complex that forms upon disassembly of photodamaged PS II units.Abbreviations Chl chlorophyll - PS II Photosystem II - D1 the 32 kD reaction center protein of PS II, encoded by the chloroplast psbA gene - D2 the 34 kD reaction center protein of PS II, encoded by the chloroplast psbD gene - CPI the 82 and 83 kD reaction center proteins of PS I, encoded by the chloroplast psaA and psaB genes - HL high light - LL low light This publication is dedicated to the memory of the late Professor Daniel Arnon, whom the first author will fondly remember for his many accounts of past scientific discovery and debate.     

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities

Background

Albumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties.

Methods

Most studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin.

Results

Both in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells.

Conclusions and general significance

Our results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.     

Synthesis of TPEN variants to improve cancer cells selective killing capacity

TPEN is an amino chelator of transition metals that is effective at the cellular and whole organism levels. Although TPEN of often used as a selective zinc chelators, it has affinity for copper and iron and has been shown to chelate both biologically. We have previously shown that TPEN selectively kills colon cancer cells based on its ability to chelate copper, which is highly enriched in colon cancer cells. The TPEN-copper complex is redox active thus allowing for increased ROS production in cancer cells and as such cellular toxicity. Here we generate TPEN derivatives with the goal of increasing its selectivity for copper while minimizing zinc chelation to reduce potential side effects. We show that one of these derivatives, TPEEN despite the fact that it exhibits reduced affinity for transition metals, is effective at inducing cell death in breast cancer cells, and exhibits less toxicity to normal breast cells. The toxicity effect of the both chelators coupled to the metal content of the different cell types reveals that they exhibit their toxicity through chelating redox active metals (iron and copper). As such TPEEN is an important novel chelators that can be exploited in anti-cancer therapies.     

Activation of protein-bound copper ions during early glycation: study on two proteins

This study proposes several possible pathways by which hyperglycemia could make protein-bound metal ions more redox active. These mechanisms were tested on bovine serum albumin and calf lens protein. Proteins rich in early glycation products were less capable of competing for copper ions in the presence of other ligands (e.g., glycine and calcein), suggesting that glycated proteins might have diminished stability constants of their copper chelates compared to control counterparts. When protein-copper complexes were tested for their ability to cause the oxidation of ascorbic acid, as well as the reduction of molecular oxygen to hydrogen peroxide, glycated and control proteins differed considerably in their redox abilities. Oxidative damage on proteins documented by protein carbonyl content and amino acid analysis indicates the involvement of Fenton chemistry upon metal chelation. The possible biological consequences of the observed activation of metal ions bound to early glycated proteins are discussed.     

Chelation and intercalation: complementary properties in a compound for the treatment of Alzheimer's disease

Selective application of metal chelators to homogenates of human Alzheimer's disease (AD) brain has led us to propose that the architecture of aggregated beta-amyloid peptide, whether in the form of plaques or soluble oligomers, is determined at least in part by high-affinity binding of transition metals, especially copper and zinc. Of the two metals, copper is implicated in reactive oxygen species generating reactions, while zinc appears to be associated with conformational and antioxidant activity. We tested the copper chelators trientine, penicillamine, and bathophenanthroline for their ability to mobilize brain Abeta as measured against our benchmark compound bathocuproine (BC). All of these agents were effective in solubilizing brain Abeta, although BC was the most consistent across the range of AD brain tissue samples tested. Similarly, all of the copper chelators depleted copper in the high-speed supernatants. BC alone had no significant effect upon zinc levels in the soluble fraction. BC extraction of brain tissue from C100 transgenic mice (which express human Abeta but do not develop amyloid) revealed SDS-resistant dimers as Abeta was mobilized from the sedimentable to the soluble fraction. NMR analysis showed that, in addition to its copper chelating properties, BC interacts with Abeta to form a complex independent of the presence of copper. Such hybrid copper chelating and "chain breaking" properties may form the basis of a rational design for a therapy for Alzheimer's disease.     

Inhibition of methyl jasmonate-promoted senescence in rice leaves by a metal chelator, 2,2′-bipyridine

The possible mediatory role of transition metals in methyl jasmonate- (MJ-)induced senescence of rice leaves was investigated. Metal chelators(2,2-bipyridine, 8-hydroxylquinoline and 1,10-phenanthroline) reducedMJ-promoted senescence of rice leaves. The reduction of MJ-promoted senescenceby 2,2-bipyridine(BP) is closely associated with the decrease in lipidperoxidation and increase in activity of superoxide dismutase (SOD). Our resultssuggest that iron or copper plays a major role in MJ-promoted senescence ofdetached rice leaves. BP-reduced senescence of detached rice leaves induced byMJ was reversed by adding Fe²⁺ or Cu²⁺, but notby Mn²⁺ or Mg²⁺. Reduction of MJ-promotedsenescence of detached rice leaves by BP is most likely mediated throughchelation of iron or copper and an increase in SOD activity.     

Protein aging by carboxymethylation of lysines generates sites for divalent metal and redox active copper binding: relevance to diseases of glycoxidative stress.

Aging and age-related diseases are associated with the production of reactive oxygen species which modify lipids, proteins and DNA. Here we hypothesized the glyco- and lipoxidation product N(epsilon)-(carboxymethyl)lysine (CML) in proteins should bind divalent and redox active transition metal binding. CML-rich poly-L-lysine and bovine serum albumin (BSA) were chemically prepared and found to bind non-dialyzable Cu(2+), Zn(2+) and Ca(2+). CML-BSA-copper complexes oxidized ascorbate and depolymerized protein in the presence of H(2)O(2). CML-rich tail tendons implanted for 25 days into the peritoneal cavity of diabetic rats had a 150% increase in copper content and oxidized ascorbate three times faster than controls. CML-rich proteins immunoprecipitated from serum of uremic patients oxidized four times more ascorbate than control and generated spin adducts of DMPO in the presence of H(2)O(2). The chelator DTPA suppressed ascorbate oxidation thereby implicating transition metals in the process. In aging and disease, CML accumulation may result in a deleterious vicious cycle since CML formation itself is catalyzed by lipoxidation and glycoxidation.     

Thermodynamic analysis of human serum albumin interactions with glucose: insights into the diabetic range of glucose concentration

The interaction of proteins with glucose results in their non-enzymatic glycation and influences their structural and functional properties. Human serum albumin (HSA) interacts with glucose forming glycated HSA. However, the glucose binding sites and the thermodynamic characteristics of the glycated HSA require further delineation. Here, the binding properties of HSA and glucose were studied utilizing fluorescent techniques. HSA was incubated with glucose in the 0-300mM range at 27 or 37 degrees C. The interaction of HSA with glucose showed two sets of binding sites. The first set consists of two sites with positive cooperativity and the second set consists of nine identical non-cooperative sites. The percentage of glycated HSA (gly%) and the moles of glucose bound to moles of HSA (r) were utilized to obtain binding constants and thermodynamic parameters based on the Wyman binding potential. The enthalpy of binding, obtained by van't Hoff relation, presented exothermicity up to 7mM glucose (126mg/dl, normal range) and endothermic propensity at higher glucose concentrations (>7mM, diabetic range). The start of endothermic propensity was consistent with the diabetic range of glucose concentration and indicates unfolding of HSA. The Gibbs free energy and entropy of binding further supports the unfolding of HSA. Therefore, glucose interacts with multiple sites on HSA affecting its biochemical and biophysical properties. This may interfere with HSA normal function contributing to diabetic complications.     

Insulin resistance and glycemic abnormalities are associated with deterioration of left ventricular diastolic function: a cross-sectional study

Background

The purpose of this study was to test the hypothesis that sidestream tobacco smoke extracts would inhibit the culture of endothelial cells and enhance platelet aggregation under diabetic vascular conditions. Sidestream tobacco smoke and advanced glycation end products are known cardiovascular risk factors and we aimed to determine the combined interaction between these two risk factors to promote cardiovascular diseases associated with diabetes.

Methods

Human umbilical vein endothelial cells were cultured in the presence of sidestream tobacco smoke extracts (SHS) or nicotine and glycated albumin (AGE) or non-glycated albumin. After 3 days, endothelial cell viability and density were investigated. Platelets were also incubated with these compounds for up to 6 hours. Platelet aggregation and the surface expression of CD41 and CD62P were examined. In some experiments, platelets were added to the endothelial cell culture to determine if an interaction between platelets and endothelial cells occurs that can alter the responses to SHS or AGE.

Results

In general, the endothelial cell culture conditions were reduced in the presence of AGE and SHS. Nicotine, did not play a role in this reduction. Platelet aggregation proceeded faster in the presence of AGE and SHS. Interestingly, with the combined culture of endothelial cells and platelets, the endothelial cell culture conditions were improved and the platelet functional changes were diminished in the presence of SHS and AGE, as compared with the individual incubations.

Conclusions

Our data suggests that diabetics that are exposed to SHS may have a higher likelihood for cardiovascular disease development through a diminished endothelial cell viability and an increased platelet activity, which are partially mediated by CD41 and not CD62P. This study provides support for an increased cardiovascular risk for diabetic patients that are exposed to SHS. This study also provides a new experimental technique to monitor platelet-endothelial cell interactions.     

Oxidation of cysteine and homocysteine by bovine albumin

The autooxidation of cysteine and homocysteine to their disulfide forms was determined by measuring the time course of thiol groups disappearance. We found the oxidative chemistry of cysteine and homocysteine to be quite different. In the absence of added Cu(II), cysteine autooxidized at a slower rate than homocysteine, though in its presence cysteine oxidation was much faster, homocysteine being found to be a poor responder to copper catalysis. Albumin speeded up the spontaneous oxidation of both aminothiols, the reaction being faster with cysteine than with homocysteine. The copper content of different albumins was found to be highly variable, ranging from 12.75 to 0.64 microg Cu(II)/g albumin. We propose that copper bound to albumin possesses redox cycling activity to perform cysteine oxidation since: (i) copper elimination by copper chelators markedly reduces oxidation; and (ii) a positive correlation exists between the albumin copper content and the oxidation reaction rate.     

Glycation of apolipoprotein E impairs its binding to heparin: identification of the major glycation site.

The increased glycation of plasma apolipoproteins represents a possible major factor for lipid disturbances and accelerated atherogenesis in diabetic patients. The glycation of apolipoprotein E (apoE), a key lipid-transport protein in plasma, was studied both in vivo and in vitro. ApoE was shown to be glycated in plasma very low density lipoproteins of both normal subjects and hyperglycemic, diabetic patients. However, diabetic patients with hyperglycemia showed a 2-3-fold increased level of apoE glycation. ApoE from diabetic plasma showed decreased binding to heparin compared to normal plasma apoE. The rate of Amadori product formation in apoE in vitro was similar to that for albumin and apolipoproteins A-I and A-II. The glycation of apoE in vitro significantly decreased its ability to bind to heparin, a critical process in the sequestration and uptake of apoE-containing lipoproteins by cells. Diethylenetriaminepentaacetic acid, a transition metal chelator, had no effect on the loss of apoE heparin-binding activity, suggesting that glycation rather than glycoxidation is responsible for this effect. In contrast, glycation had no effect on the interaction of apoE with amyloid beta-peptide. ApoE glycation was demonstrated to be isoform-specific. ApoE(2) showed a higher glycation rate and the following order was observed: apoE(2)>apoE(4)>apoE(3). The major glycated site of apoE was found to be Lys-75. These findings suggest that apoE is glycated in an isoform-specific manner and that the glycation, in turn, significantly decreases apoE heparin-binding activity. We propose that apoE glycation impairs lipoprotein-cell interactions, which are mediated via heparan sulfate proteoglycans and may result in the enhancement of lipid abnormalities in hyperglycemic, diabetic patients.     

Localization of the ezrin binding epitope for advanced glycation endproducts

Glycated proteins/advanced glycation endproducts contribute to the development of diabetic complications but the precise pathway from glycated proteins to complications is still being delineated. The ezrin, radixin and moesin protein family is a new class of advanced glycation endproduct-binding protein and we hypothesize that advanced glycation endproducts mediate some of their detrimental effects leading to diabetic complications by inhibiting ezrin's actions. Our previous study revealed that glycated proteins bind to the N-terminal domain of ezrin (aa 1–324) and this study further defines the ezrin binding epitope. Binding of glycated albumin to recombinant N-ezrin deletion constructs (aa 1–280, 1–170 and 1–144) and glutathione-S-transferase-N-ezrin fusion proteins, (aa 200–324 and 270–324) was analysed using ligand and far Western blotting, and surface plasmon resonance. Glycated albumin binding was markedly reduced on removal of amino acids 280–324, while binding was preserved in the fusion proteins. A series of peptides based on residues 280–324 was synthesized and those containing residues 277–299 of ezrin bound maximally. Peptide binding to glycated albumin was glycation-specific. An ezrin peptide (aa 277–299) dose-dependently reversed the inhibitory effect of glycated albumin on ezrin (1–324) phosphorylation in vitro, suggesting that binding of advanced glycation endproducts to ezrin changes the conformation of the latter sufficiently to alter binding interactions distant from the advanced glycation endproduct-binding site. This may have consequences for subcellular ezrin localization and signalling pathways. Altogether, these studies provide important structural knowledge for developing peptide antagonists that may be therapeutically useful in preventing advanced glycation endproduct:ezrin interactions in diabetes.     

The role of glycation and autoxidation on crystallin aggregation

SUMMARY

Incubation of α-crystallin with glucose and CuSO₄ resulted in crystallin changes similar to those observed in cataracts. Examination of the reaction mixtures by polyacrylamide slab gel electrophoresis showed progressive crystallin aggregation through non-disulfide covalent bonds and parallel increases in ultraviolet absorbance and non-tryptophan fluorescence. Both glucose and copper were required; iron was less effective. The reaction can be accelerated by increasing glucose concentration or by utilizing ribose which has a higher percentage of free aldehyde groups than glucose. These observations are consistent with a mechanism involving crystallin glycation. The reaction is mediated by hydrogen peroxide and transition metals since it is inhibited by catalase and by chelating agents. These results, in turn, are consistent with copper-catalyzed autoxidation of glucose and of glycated crystallin. This reaction generates superoxide free radical which dismutates to yield hydrogen peroxide. The latter, in turn, generates hydroxyl radicals in presence of transition metal ions (Fenton reaction). Hydroxyl radical attack leads to cross-linking which is enhanced in glycated proteins. Under hyperglycemic conditions, such as in diabetes mellitus, high levels of glucose occur in insulin-independent tissues such as the lens. Elevated cupremia and oxidative stress are also known to occur in diabetic patients. There-fore, our findings are consistent with crystallin glycation and superimposed oxyradical generation during diabetic cataractogenesis.     

Effects of glycated albumin on mesangial cells: evidence for a role in diabetic nephropathy

Nonenzymatically glycated proteins are preferentially transported across the glomerular filtration barrier, and the glomerular mesangium in diabetes is bathed with serum containing increased concentrations of glycated albumin. We investigated effects of glycated albumin on mesangial cells, which are involved in diabetic nephropathy. [³H]-thymidine incorporation was significantly inhibited when murine mesangial cells were grown in culture media containing human serum that had been nonenzymatically glycated by incubation for 4 days with 28 mM glucose. This inhibition was reversed when monoclonal antibodies that selectively react with Amadori products of glycated albumin were added to the culture media. Purified glycated albumin containing Amadori adducts of the glycation reaction induced significant inhibition of thymidine incorporation and stimulation of Type IV collagen secretion compared with cells cultured in the presence of purified nonglycated albumin. These changes were prevented when monoclonal antibodies specifically reactive with fructosyl-lysine epitopes in glycated albumin were added to the cultures. The antibodies had no effect on growth or collagen production in the presence of nonglycated albumin. The results provide the first evidence directly implicating Amadori adducts in glycated albumin in the pathogenesis of diabetic nephropathy, which is characterized by decreased cellularity in association with expansion of the mesangial matrix.     

Effects of oral ethinyl oestradiol and norethisterone on plasma copper and zinc complexes in post-menopausal women

Orally administered ethinyl oestradiol increased the plasma total copper concentration and reduced the albumin concentration in post-menopausal women. Approximately 80% of the increase in copper was due to a rise in caeruloplasmin-bound copper and 20% to an increase in the amount of copper bound per gram of albumin. The plasma total zinc concentration was reduced, due partly to the decrease in albumin concentration and partly to a reduction in the amount of zinc bound per gram of albumin. Norethisterone had no significant effect on plasma copper but it reduced plasma zinc and albumin, though to a lesser extent than ethinyl oestradiol. When administered sequentially with ethinyl oestradiol, norethisterone diminished the effects of the former on plasma copper, zinc and albumin.     

Inhibitory effect of copper on cystathionine beta-synthase activity: protective effect of an analog of the human albumin N-terminus

Copper was added to truncated, recombinant cystathionine beta-synthase (CBS), and the enzyme activity was assessed by measuring the production of cystathionine. 10 microM copper significantly decreased CBS activity by 50% while 25 microM copper decreased CBS activity by 70%. This inhibition was negated when an analog of the N-terminus of human albumin, Asp-Ala-His-Lys (DAHK), a strong transition metal binding peptide, was added. The use of copper chelators could significantly reduce in vivo homocysteine levels.     

Circulating Glycated Albumin and Glomerular Anionic Charges

Aiming to discern the mechanisms by which circulating glycated albumin alters the glomerular filtration properties that lead to glomerular dysfunction in diabetes, the authors studied the distribution and densities of anionic charges through the rat glomerular wall upon intravascular infusion of Amadori products, as well as in various conditions of increased glomerular permselectivity. Polylysine-gold was used as the probe to reveal the anionic charges. The study was carried on renal tissue sections of bovine serum albumin (BSA)- and glycated BSA–injected, normoglycemic animals. Results were generated through morphometrical evaluations of the gold labeling. Changes in glomerular anionic distribution were corroborated on renal tissue sections of short- and long-term diabetic rats and of normal newborn rats, situations known for abnormal glomerular filtration. Altered renal function in these conditions was clearly associated with changes in glomerular anionic charges. On the other hand, the infusion of glycated albumin in the circulation of normal rats, though altering glomerular filtration properties, did not modify the distribution and density of the polylysine-gold labeling through the glomerular basement membrane. Thus, anionic charges seem not to be the factor involved in the early changes of glomerular permeability induced by circulating glycated albumin.     

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.