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.     

Antioxidative and prooxidative effects of coumarin derivatives on free radical initiated and photosensitized peroxidation of human low-density lipoprotein

The antioxidative and/or prooxidative activity of 4-methylcoumanrin (MC), 7-hydroxy-4-methylcoumarin (HMC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), respectively, in the peroxidation of human low-density lipoprotein (LDL) has been studied. The peroxidation was initiated either thermally by water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), or photochemically by a triplet sensitizer benzophenone (BP) or its water-soluble analogue disodium 3,3'-disulfobenzophenonate (DSBP). The reaction kinetics were monitored by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) present in the native LDL. Kinetic analysis of the peroxidation process demonstrated that DHMC is a good antioxidant for both the AAPH-initiated and BP- and DSBP-photosensitized peroxidation; HMC is a prooxidant for the AAPH-initiated and DSBP-photosensitized peroxidation, but an antioxidant for the BP-sensitized peroxidation; MC is a prooxidant in all of these initiation conditions. The antioxidative action of the coumarin derivatives may include trapping the initiating radicals, trapping the propagating lipid peroxyl radicals, recycling alpha-tocopherol and/or deactivating the excited photosensitizer.     

Action of quinolinic and indolinonic aminoxyls as radical-scavenging antioxidants.

The kinetic studies on the actions of quinolinic and indolinonic aminoxyls in the oxidation of lipid peroxidation induced by free radicals were carried out to evaluate their antioxidant activity. These aminoxyls showed a similar reactivity toward peroxyl radical with alpha-tocopherol. The antioxidant efficacies of aminoxyls against oxidation of methyl linoleate in homogeneous solution were smaller than that of alpha-tocopherol. Hydroxylamine, a reduced form of aminoxyl, possessed a comparative antioxidant efficacy with alpha-tocopherol and was capable of suppressing the consumption of alpha-tocopherol. Aminoxyls showed more potent antioxidant activity than alpha-tocopherol against the oxidation of methyl linoleate micelles induced by peroxyl radical or by a combination of copper ion and hydrogen peroxide. These results suggest that quinolinic and indolinonic aminoxyls may act as potent antioxidants against lipid peroxidation, especially in the presence of a good reductant which reduces aminoxyl radicals to hydroxylamines.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Vitamin E protects proteins against free radical damage in lipid environments

The fragmentation of the membrane protein monoamine oxidase in submitochondrial particles was induced by defined free radicals during radiolysis and by a system dependent on hydrogen peroxide and a transition metal. By injection of alpha-tocopherol in vivo, the levels of this physiological antioxidant in the mitochondrial preparations could be elevated more than ten-fold. In both radical-generating systems the presence of high levels of alpha-tocopherol in the membrane substantially retarded the protein fragmentation, in parallel with lipid peroxidation. It is suggested that membrane-bound proteins are damaged during lipid peroxidation and that alpha-tocopherol protects cells against both types of damage.     

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.     

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.     

Recycling and antioxidant activity of tocopherol homologs of differing hydrocarbon chain lengths in liver microsomes

Tocopherols (vitamin E) function as inhibitors of lipid peroxidation in biomembranes by donating a hydrogen atom to the chain propagating lipid radicals, thus giving rise to chromanoxyl radicals of the antioxidant. We have shown that alpha-tocopherol homologs differing in the lengths of their hydrocarbon side chains (alpha-Cn) manifest strikingly different antioxidant potencies in membranes. The antioxidant activity of tocopherol homologs during (Fe2+ + ascorbate)- or (Fe2+ + NADPH)-induced lipid peroxidation in rat liver microsomes increased in the order alpha-tocopherol (alpha-C16) less than alpha-C11 less than alpha-C6 less than alpha-C1. Chromanoxyl radicals generated from alpha-tocopherol and its more polar homologs by an enzymatic oxidation system (lipoxygenase + linolenic acid) can be recycled in rat liver microsomes by NAD-PH-dependent electron transport or by ascorbate. The efficiency of recycling increased in the same order: alpha-tocopherol (alpha-C16) less than alpha-C11 less than alpha-C6 less than alpha-C1. Thus the high efficiency of regeneration of short-chain homologs of vitamin E may account for their high antioxidant potency.     

Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron overload

Ascorbate is a strong antioxidant; however, it can also act as a prooxidant in vitro by reducing transition metals. To investigate the in vivo relevance of this prooxidant activity, we performed a study using guinea pigs fed high or low ascorbate doses with or without prior loading with iron dextran. Iron-loaded animals gained less weight and exhibited increased plasma beta-N-acetyl-D-glucosaminidase activity, a marker of tissue lysosomal membrane damage, compared with control animals. The iron-loaded animals fed the low ascorbate dose had decreased plasma alpha-tocopherol levels and increased plasma levels of triglycerides and F(2)-isoprostanes, specific and sensitive markers of in vivo lipid peroxidation. In contrast, the two groups of animals fed the high ascorbate dose had significantly lower hepatic F(2)-isoprostane levels than the groups fed the low ascorbate dose, irrespective of iron load. These data indicate that 1) ascorbate acts as an antioxidant toward lipids in vivo, even in the presence of iron overload; 2) iron loading per se does not cause oxidative lipid damage but is associated with growth retardation and tissue damage, both of which are not affected by vitamin C; and 3) the combination of iron loading with a low ascorbate status causes additional pathophysiological changes, in particular, increased plasma triglycerides.     

Antioxidant effects of alpha- and gamma-carboxyethyl-6-hydroxychromans

Carboxyethyl-6-hydroxychromans (CEHC), the major metabolites of both tocopherols (Toc) and tocotrienols (Toc-3), have been found in human plasma. In the present study, the antioxidant properties of alpha- and gamma-CEHC were measured and compared with alpha- and gamma- tocopherols. Following results were obtained: (1)alpha- and gamma-CEHC have the same reactivities toward radicals and exert the same antioxidant activities against lipid peroxidation in organic solution as the corresponding parent tocopherols respectively; (2) the partition coefficient decreased in the order alpha-Toc (3.36) > gamma-Toc (3.14) > alpha-CEHC (2.26) > pentamethyl-6-chromanol (1.92) > gamma-CEHC (1.83) > 0 > Trolox (-0.97); (3) alpha- and gamma-CEHC scavenge aqueous radicals more efficiently but they inhibit the lipid peroxidation within the membranes less efficiently than the corresponding alpha- and gamma-Toc, respectively; (4) alpha-CEHC inhibits the oxidation synergistically with ascorbate; and (5) alpha- and gamma-CEHC reduce Cu(II) to give Cu(I) and corresponding quinones as major product, but the prooxidant effect of CEHC in the presence of cupric ion was small. These results imply that CEHC may act as an antioxidant in vivo especially for those who take tocopherol supplement.     

Effect of Ascorbate on Red Blood Cell Lipid Peroxidation

The present study investigates the effect of ascorbate on red cell lipid peroxidation. At a concentration between 0.2 mmol-20 mmol/l ascorbic acid reduces hydrogen peroxide-induced red blood cell lipid peroxidation resulting in a marked decrease in ethane and pentane production as well as in haemolysis. Ascorbic acid also shows an antioxidant effect on chelated iron-catalyzed hydrogen peroxide-induced peroxidation of erythrocyte membranes. At a concentration of 10 mmol/l ascorbic acid totally inhibits oxidative break-down of polyunsaturated fatty acids by radicals originating from hydrogen peroxide.

Our results indicate that ascorbate at the chosen concentration has an antioxidant effect on red blood cell lipid peroxidation.     

Antioxidant activity of L-adrenaline: a structure-activity insight

L-adrenaline belongs to a group of the compounds known as catecholamines, which play an important role in the regulation of physiological process in living organisms. The antioxidant activity and antioxidant mechanism of L-adrenaline was clarified using various in vitro antioxidant assays including 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), N,N-dimethyl-p-phenylenediamine (DMPD(+)), and superoxide anion radicals (O(2)(-)) scavenging activity, hydrogen peroxide (H(2)O(2)), total antioxidant activity, ferric ions (Fe(3+)) and cupric ions (Cu(2+)) reducing ability, ferrous ions (Fe(2+)) chelating activity. L-adrenaline inhibited 74.2% lipid peroxidation of a linoleic acid emulsion at 30 microg/mL concentration. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox displayed 83.3, 82.1, 68.1 and 81.3% inhibition on the peroxidation of linoleic acid emulsion at the same concentration, respectively. BHA, BHT, alpha-tocopherol and trolox were used as reference antioxidants and radical scavenger compounds. Moreover, this study will bring an innovation for further studies related to antioxidant properties of L-adrenaline. According to present study, L-adrenaline had effective in vitro antioxidant and radical scavenging activity.     

Antioxidant and antiradical activities of L-carnitine

L-carnitine plays an important regulatory role in the mitochondrial transport of long-chain free fatty acids. In this study, the antioxidant activity of L-carnitine was investigated as in vitro. The antioxidant properties of the L-carnitine were evaluated by using different antioxidant assays such as 1, 1-diphenyl-2-picryl-hydrazyl free radical (DPPH.) scavenging, total antioxidant activity, reducing power, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities. Total antioxidant activity was measured according to ferric thiocyanate method. alpha-tocopherol and trolox, a water-soluble analogue of tocopherol, were used as the reference antioxidant compounds. At the concentrations of 15, 30 and 45 microg/mL, l-carnitine showed 94.6%, 95.4% and 97.1% inhibition on lipid peroxidation of linoleic acid emulsion, respectively. On the other hand, 45 microg/mL of standard antioxidant such as alpha-tocopherol and trolox indicated an inhibition of 88.8% and 86.2% on peroxidation of linoleic acid emulsion, respectively. In addition, L-carnitine had an effective DPPH. scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, total reducing power and metal chelating on ferrous ions activities. Also, those various antioxidant activities were compared to alpha-tocopherol and trolox as references antioxidants.     

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated with Cu(2+) ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu(2+) to Cu(+) and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu(+) oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides.     

Cooperation of antioxidants in protection against photosensitized oxidation

The aim of this study was to determine whether alpha-tocopherol and zeaxanthin offer synergistic protection against photosensitized lipid peroxidation mediated by singlet oxygen and free radicals. The antioxidant action of zeaxanthin and alpha-tocopherol was studied in liposomes made of phosphatidylcholine and cholesterol. Progress of lipid peroxidation, induced by aerobic photoexcitation of rose bengal, was monitored by the detection of lipid hydroperoxides and by electron spin resonance oximetry. In addition, cholesterol was employed as a mechanistic reporter molecule, which forms characteristic products of the interaction with singlet oxygen or free radicals. Cholesterol hydroperoxides were quantitatively determined by HPLC/electrochemical detection. HPLC/ultraviolet-visible (UV-VIS) absorption detection was used to measure concentrations of zeaxanthin and alpha-tocopherol. Zeaxanthin, even at concentrations of 2.5 microM, effectively protected against singlet oxygen-mediated lipid peroxidation but was rapidly consumed due to interaction with free radicals. alpha-Tocopherol alone was not effective in protecting against lipid peroxidation, even at concentration of 0.1 mM. Combinations of zeaxanthin and alpha-tocopherol exerted a synergistic protection against lipid peroxidation. The synergistic effect may be explained in terms of prevention of carotenoid consumption by effective scavenging of free radicals by alpha-tocopherol therefore allowing zeaxanthing to quench the primary oxidant-singlet oxygen effectively.     

Interaction of ascorbate and alpha-tocopherol enhances antioxidant reserve of erythrocytes during anemia in visceral leishmaniasis

Visceral leishmaniasis (V.L.) is associated with enhanced lipid peroxidation along with impaired function of antioxidant defense system in erythrocytes. The effect of chronic treatment with ascorbate and alpha-tocopherol was studied on erythrocytes in hamsters infected with Leishmania donovani. Combination treatment with both antioxidants proved to be a potential suppressor of lipid hydroperoxide formation as well as hypotonic osmotic lysis during the leishmanial infection. Positive correlations between the depleted levels of erythrocyte ascorbate, GSH and alpha-tocopherol exhibit proportionate alterations in the nonenzymatic antioxidant levels at different stages of infection. Indirect measurement of transmembrane electron transfer as ferricyanide reduction suggests an active participation of endogenous contents of ascorbate and alpha-tocopherol in the protection against oxidative damage of membrane lipids. Cooperative behavior of both antioxidants in the ferricyanide reducing capacity was further evinced by resealing the ghosts in presence of exogenous ascorbate and alpha-tocopherol. Furthermore, intravesicular ascorbate serves in the defense of extravesicular ferricyanide induced oxidation of endogenous alpha-tocopherol. The results suggest an interacting role of ascorbate and alpha-tocopherol in maintaining the antioxidant reserve of erythrocytes during anemia in V.L.     

Trace element levels in the experimental peritonitis

Electron transfer from iron or copper ions to oxygen is an important example of cellular free radical initiation. Oxygen derived free radicals have been implicated as mediators of cellular injury in several model systems. To evaluate the importance of iron, copper and zinc levels on lipid peroxidation in peritonitis, we measured peritoneum malondialdehyde (MDA) as a marker of lipid peroxidation, zinc, copper, and iron levels during an animal model of intraperitoneal sepsis. Additionally the effects of the free radical scavenger alpha-tocopherol administration was studied. The peritoneum MDA, iron, copper and zinc levels were increased after induction of peritonitis with Escherichia Coli. The treatment with alpha-tocopherol was decreased the peritoneum MDA, iron and copper levels significantly, except the zinc level (p < 0.001, p < 0.001, p < 0.001, respectively). Additionally the alpha-tocopherol treatment for three days prior to injection of E.Coli more decreased MDA, copper and iron levels than that of the treatment with alpha-tocopherol at the time of injection of E. Coli (p < 0.001, p < 0.001, p<0.001, respectively). Our results indicated that copper, iron and zinc had important effects on peroxidation events in E. Coli induced peritonitis, and alpha-tocopherol treatment can improve the oxidant status.     

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.     

Antioxidant protective effect of flavonoids on linoleic acid peroxidation induced by copper(II)/ascorbic acid system

Antioxidants are compounds that can delay or inhibit lipid oxidation. The peroxidation of linoleic acid (LA) in the absence and presence of Cu(II) ion–ascorbate combinations was investigated in aerated and incubated emulsions at 37 °C and pH 7. LA peroxidation induced by copper(II)–ascorbic acid system followed first order kinetics with respect to hydroperoxides concentration. The extent of copper-initiated peroxide production in a LA system assayed by ferric thiocyanate method was used to determine possible antioxidant and prooxidant activities of the added flavonoids. The effects of three different flavonoids of similar structure, i.e. quercetin (QR), morin (MR) and catechin (CT), as potential antioxidant protectors were studied in the selected peroxidation system. The inhibitive order of flavonoids in the protection of LA peroxidation was: morin > catechin ≥ quercetin, i.e. agreeing with that of formal reduction potentials versus NHE at pH 7, i.e. 0.60, 0.57 and 0.33 V for MR, CT, and QR, respectively. Morin showed antioxidant effect at all concentrations whereas catechin and quercetin showed both antioxidant and prooxidant effects depending on their concentrations. The structural requirements for antioxidant activity in flavonoids interestingly coincide with those for Cu(II)-induced prooxidant activity, because as the reducing power of a flavonoid increases, Cu(II)–Cu(I) reduction is facilitated that may end up with the production of reactive species. The findings of this study were evaluated in the light of structure–activity relationships of flavonoids, and the results are believed to be useful to better understand the actual conditions where flavonoids may act as prooxidants in the preservation of heterogeneous food samples containing traces of transition metal ions.