Dipicolinic acid prevents the copper-dependent oxidation of low density lipoprotein

Effect of dipicolinic acid (pyridine 2,6-dicarboxylic acid) and pyridine compounds on the copper-dependent oxidation of human low density lipoprotein was analyzed in relation to the inhibition of copper reduction. Dipicolinic acid inhibited copper-dependent LDL oxidation completely, but the LDL oxidation was slightly inhibited by pyridine compounds with one carboxyl group at 2 or 6-position. Reduction of copper by LDL itself and ascorbate was inhibited completely by dipicolinic acid, but only partially by picolinic acid, quinolinic acid and isocinchomeronic acid with 2- or 6-carboxylic group. Pyridine compounds without 2- or 6-carboxyl group did not show any inhibitory effect on the LDL oxidation and the copper reduction. Protective effect of dipicolinic acid on the LDL oxidation was closely correlated with the copper-reducing activity. Dipicolinic acid shows an antioxidant action by the formation of a chelation complex with copper. This may have implications in understanding mechanisms of preventing LDL oxidation during the early phase of atherosclerosis.     

Inhibition by thiols of copper(II)-induced oxidation of oxyhemoglobin.

The ability of thiols, 2-imidazolethiones and uric acid to protect bovine oxyhemoglobin from copper(II)-induced oxidation to methemoglobin was investigated. The oxidation of oxyhemoglobin by Cu(II) proceeded in two phases: (1) an initial rapid reaction (less than 30 s) followed by (2) a slower reaction that carried it to completion. Thiols, including N-acetyl-L-cysteine, DL-dithiothreitol, reduced glutathione, DL-homocysteine, 2-mercaptoethanol and 2- and 3-mercaptopropionic acid, whose sulfhydryl groups were slowly oxidized by Cu(II) (with the exception of 2-mercaptopropionic acid), protected oxyhemoglobin in both phases of the reaction. Other thiols, including L-cysteine, cysteamine, and D-penicillamine, whose sulfhydryl groups were readily oxidized by Cu(II), protected hemoglobin initially, but within 2-4 min, the rate of methemoglobin formation was the same as Cu(II)-treated oxyhemoglobin. 2-Mercaptoimidazole and 1-methyl-2-mercaptoimidazole, which complex Cu(II) and inhibit Cu(II)-catalyzed oxidation of ascorbic acid, also protected hemoglobin in the initial phase, but not in the second phase. Uric acid, L-ergothioneine, and thiourea did not protect oxyhemoglobin in either the fast or slow phase. Cu(II) may have a coordination site involved in the oxidation of hemoglobin that is not blocked by the 2-imidazolethiones, uric acid, or the oxidized thiols. It is concluded that certain thiols that complex Cu(II) and are not rapidly oxidized will protect oxyhemoglobin from Cu(II)-induced oxidation, but the thiols are no longer effective once they are oxidized.     

Stabilization of ascorbate solution by chelating agents that block redox cycling of metal ions

Among the seven chelating agents tested, ethylenediamine di(o-hydroxyphenylacetic acid) and diethylenetriamine pentaacetic acid were found to almost completely inhibit ascorbate oxidation catalyzed by iron ions. The inhibition with the former chelator is due to the prevention of the reduction of Fe3+ by ascorbate, while the inhibition with the latter is caused by the strong inhibition of both this reductive reaction and the oxidation of Fe2+ by O2. These chelators almost completely inhibit ascorbate oxidation catalyzed by copper ions as well. These results indicate that the blocking of redox cycling of metal ions is important to prevent the oxidation of ascorbate.     

The interaction of ascorbate oxidase with L-dopa,L-tyrosine and 3,4-dihydroxycinnamic acid. Evidence for irreversible damage of the enzyme during catechol oxidase activity

The aerobic interaction between ascorbate oxidase and L-tyrosine, L-3,4-dihydroxyphenylalanine or 3,4-dihydroxycinnamic acid in 1:10 molar ratio was followed by optical absorption, CD and EPR spectroscopy in 0.1 M phosphate buffer at pH 5.0. While the spectra of the system ascorbate oxidase—L-tyrosine remain practically unaffected after several hours, indicating that no oxidation of the amino acid occurs in the conditions employed, rather drastic changes can be observed in the spectra of the ascorbate oxidase-catechol systems. In particular, while the optical absorption below 500 nm increases markedly due to the formation of the substrate oxidation products, an irreversible decrease in intensity of the absorption, CD and EPR spectral features associated with the blue copper(II) chromophores indicates that a partial loss of Type 1 copper by ascorbate oxidase has occurred during this secondary catechol oxidase activity. A copper species characterized by weak positive CD activity at 370 nm and EPR signal at intermediate field between those of the Type 2 and Type 1 coppers can be detected in the early stages of the reaction. The irreversible damage undergone by the protein during catechol oxidase activity may have biological significance and accounts for the low yield of purified enzyme obtained when the crude enzyme extract is left in prolonged contact with low molecular weight cell components, rich in σ-diphenolic compounds.     

Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.

Chain-breaking antioxidants such as butylated hydroxytoluene, alpha-tocopherol, and probucol have been shown to decrease markedly the oxidative modification of low density lipoprotein (LDL). Their mechanism of action appears to involve scavenging of LDL-lipid peroxyl radicals. The purpose of this study was to investigate the occurrence of radical reactions produced during oxidation of LDL and LDL-containing probucol initiated by lipoxygenase or copper. In addition, we have investigated the possibility of a synergistic interaction between ascorbate and probucol in inhibiting the oxidation of LDL. Incubation of LDL-containing probucol and lipoxygenase produced a composite electron spin resonance (ESR) spectrum due to the endogenous alpha-tocopheroxyl radical and probucol-derived phenoxyl radical. The spectral assignment was further verified by chemical oxidation of alpha-tocopherol and probucol. In the presence of ascorbic acid, these radicals in the LDL particle were reduced to their parent compounds with concomitant formation of the ascorbate radical. In both the peroxidation of linoleic acid and the copper-initiated peroxidation of LDL, the antioxidant activity of probucol was significantly increased by low (3-6 microM) concentrations of ascorbate. The probucol-dependent inhibition of LDL oxidation was enhanced in the presence of ascorbic acid. We conclude that the reaction between the phenoxyl radical of probucol and ascorbate results in a synergistic enhancement of the antioxidant capacity of these two compounds and speculate that such reactions could play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.     

Effect of purines on the oxidation of ascorbic acid induced by copper

Summary Uric acid and other purines including 1-methyl-, 7-methyl-, and 1,7-dimethyluric acid, adenine, guanine, xanthine, hypoxanthine, purine, and the structurally similar compound allopurinol protected ascorbic acid from oxidation catalyzed by copper. If the hydrogen at either the 3 or 9 nitrogen of the uric acid was replaced by a methyl group, the compound did not protect ascorbate. 3-Ribosyluric acid, xanthosine, adenosine, and guanosine also failed to protect ascorbate. It was concluded that in order for purines to complex with copper to protect ascorbate from copper-catalyzed oxidation, the nitrogens at both positions 3 and 9 of the purine must be unsubstituted.     

The analogous mechanisms of enzymatic inactivation induced by ascorbate and superoxide in the presence of copper

The mechanism of enzymatic inactivation of purified and membrane-bound acetylcholine esterase by ascorbate and copper was investigated. While the exposure of the enzyme to either ascorbate or copper did not cause enzymatic inactivation, the incubation of the enzyme with a combination of both ascorbate and copper resulted in a loss in acetylcholine esterase activity, which was time dependent. The enzymatic inactivation required either molecular oxygen or hydrogen peroxide under anaerobic conditions. Scavengers of hydroxyl radicals at concentrations of up to 100 mM did not provide protection to acetylcholine esterase. Only mannitol at very high concentrations (above 1 M) efficiently prevented the inactivation of the enzyme. The kinetics of the aerobic oxidation of reduced ascorbate in the presence of acetylcholine esterase and copper closely followed the rate of enzyme inactivation. Addition of the chelating agents EDTA and diethylenetriaminepentaacetic acid prevented both the oxidation of ascorbate and the inactivation of the enzyme. In the presence of low concentrations of histidine (0.5-2.0 mM), which forms high affinity complexes with copper, the rate of ascorbate oxidation was similar to that recorded in its absence. On the other hand, no enzyme inactivation was indicated in the presence of histidine. Low temperature EPR measurements have demonstrated the binding of copper to the enzyme, and have shown the reduction of the cupric enzyme to the corresponding cuprous complex. In view of these results, a general "site-specific" mechanism for biological damage can be offered, in which copper(II) ions are bound to enzymes or other biological macromolecules. Ascorbate plays a dual role: it reduces the cupric complex to the corresponding cuprous state and serves as a source for H2O2, which, in turn, reacts with the reduced copper complex, in a Fenton reaction. In this reaction, secondary hydroxyl radicals are site specifically formed, and react preferentially with the protein, at the site of their formation, causing its inactivation. This mechanism is analogous to that previously proposed (Samuni, A., Chevion, M., and Czapski, G. (1981) J. Biol. Chem. 256, 12632-12635) for the enhancement of the biological damage caused by superoxide in the presence of copper.     

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane,by ascorbate/dihydrolipoate in living HL-60 cells

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.     

Kinetic studies of copper-induced oxidation of urate, ascorbate and their mixtures

Urate and ascorbate are the major water-soluble low molecular weight antioxidants in serum. Much attention has been devoted to the effect of these antioxidants on lipoprotein peroxidation in vivo and on their effect on copper-induced peroxidation ex vivo. These studies revealed that urate inhibits ascorbate oxidation in vitro, whereas the effect of ascorbate on urate oxidation has not been systematically studied thus far. The present study addresses mechanistic aspects of the kinetics of copper-induced oxidation of both these antioxidants and their mutual effects in aqueous solutions. We found that: (i) ascorbate becomes oxidized much faster than urate. (ii) Urate inhibits the oxidation of ascorbate but, even in the presence of excess urate, ascorbate becomes oxidized much faster than urate. (iii) Ascorbate, as well as the products of its oxidation (and/or hydrolysis) inhibit the copper-induced oxidation of urate. All these results are consistent with the hypothesis that the rate of ascorbate oxidation is determined by the rate of reoxidation of reduced copper (Cu(I)) to Cu(II) by molecular oxygen, whereas the rate of urate oxidation is governed by the rate of oxidation of urate within a 2:1 urate/copper complex. We think that the mutual effects of urate and ascorbate on each other's oxidation are likely to enhance their inhibitory effect on lipid peroxidation in biologically relevant systems including membranes and lipoproteins.     

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.     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.     

Copper-induced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we have studied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes (small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were prepared by sonication and exposed to CuCl(2) in the absence or presence of naturally occurring reductants (ascorbic acid (AA) and/or alpha-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). The resultant oxidation process was monitored by recording the time-dependence of the absorbance at several wavelengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively high copper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentrations. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introduced into the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions, ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol. Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to the high oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biological significance of these results has yet to be evaluated.     

An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable pro-duction of AFR was detected, which was increased by a 10 M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive inter-actions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. © Rapid Science 1998     

Factors affecting in vivo electrochemistry: electrode-tissue interaction and the ascorbate amplification effect

While in vivo electrochemistry has been shown to be useful for discovering new neurophysiological phenomena, there is still considerable controversy about the identity of the compounds being measured and the concentration of those compounds in extracellular fluid in brain. We have found that carbon paste electrodes undergo changes in sensitivity and specificity for dopamine and other compounds after being implanted in brain. We have also examined the effect of ascorbate on the selective enhancement of catecholamine peaks to provide an explanation for the apparently very high concentrations of dopamine measured in the extracellular fluid space. After temporary brain implantation (20 min), carbon paste electrodes tested in vitro showed increased sensitivity and lower oxidation potentials for dopamine, norepinephrine and serotonin. These brain-treated electrodes also detected 3,4-dihydroxyphenylacetic acid (DOPAC) as a distinct peak at +0.16 V, although the electrode sensitivity for DOPAC was some 25 times lower than that for dopamine. Brain treatment did not alter electrode sensitivity or oxidation potential for 5-HIAA. The oxidation current for ascorbic acid when processed as the semiderivative showed no distinct peak in the potential range -0.2 to +0.4V for either untreated or brain-treated electrodes. However ascorbic acid amplified the electrochemical peaks of catechols in direct proportion to the ratio of the concentration of ascorbate to the concentration of the catechol. In the physiologic concentration range of 300 microM ascorbate, the electrochemical signal for 1 microM dopamine was amplified 4250%. While ascorbate amplification improves detectability of dopamine and norepinephrine, it also introduces ambiguity since changing catechol concentrations cannot be distinguished from changing ascorbate concentrations.     

Free radical mediated interaction of ascorbic acid and ascorbate/Cu(II) with viral and plasmid DNAs

Previous studies indicate that ascorbic acid, when combined with copper or iron cleaves several viral DNA. ln this study, we generated the ascorbate radical anion electrochemically in a simple chemical environment without the participation of a metal ion. This solution possesses viral DNA scission activity. Ohe absence of catalytic metal ions [Fe (III) and Cu(II)] in the incubation medium was evidenced by metal chelating agents such as desferrioxamine and EDTA. Ohe radical quenching at high EDTA concentration was attributed to ionic strength of EDTA rather than metal chelation. Ohe effects of antioxidants, radical scavangers, catalase, superoxide dismutase and some proteins on DNA cleavage have been tested. Cleavage may not arise directly from ascorbate free radical but the reaction of the radical form of ascorbate with oxygen may produce the actual reactive species. Aerobic oxidation of ascorbate itself strictly requires transition metal catalysts, however electrochemically produced ascorbyl radical avoided the kinetic barrier that prevented direct oxidation of ascorbic acid with oxygen and eliminated the need for the transition metal ion catalysts.     

Factors affecting the ascorbate- and phenolic-dependent generation of hydrogen peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H

2

O

2

), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H

2

O

2

production. H

2

O

2

generation from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% CO

2

was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% CO

2

. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H

2

O

2

generated by ascorbate and phenolic compounds, but there was still substantial H

2

O

2

generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H

2

O

2

generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H

2

O

2

generation is discussed.     

Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

Synergistic inhibition of low-density lipoprotein oxidation by rutin, gamma-terpinene, and ascorbic acid.

Low-density lipoprotein (LDL) oxidation may play a significant role in atherogenesis. Flavonoids are well-known for their excellent antioxidative capacity in various model systems, therefore we examined the behaviour of rutin, a quercetin-3-rutinosid, in the copper-mediated LDL oxidation. Rutin alone has been shown to protect LDL against oxidation. Furthermore we investigated the combination of rutin with a hydrophilic (ascorbate) and a lipophilic antioxidant (gamma-terpinene) in copper-mediated LDL oxidation. In both cases we found a synergistic effect on lag phase prolongation. To elucidate whether this effect mainly depends on the copper chelating ability of rutin we examined its reaction in more detail. Although inhibiting the oxidation of alpha-linolenic acid in the "rose bengal system" no direct influence of a copper-rutin-complex was determined. We conclude that a redox active copper-rutin-complex is still able to initiate the LDL oxidation but may prevent copper from a reaction at the binding sites of apoB-100. The synergistic effect in preventing LDL oxidation is due to this trapping of copper in a complex in the case of ascorbate. The synergistic action of rutin and gamma-terpinene can be explained by different distribution of rutin and gamma-terpinene in, and around the LDL-particle, respectively.     

Factors Affecting the Ascorbate- and Phenolic-dependent Generation of Hydrogen Peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H?0?), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H?0? production. H?0? gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0? was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0?. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H?0? generated by ascorbate and phenolic compounds, but there was still substantial H?0? generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H?0? generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H?0? generation is discussed.