Antioxidant properties of some different molecular weight chitosans

Chitosan, a cationic polysaccharide, is widely employed as dietary supplement and in pharmacological and biomedical applications. Although numerous studies have focused on its applications as pharmaceutical excipients or bioactive reagents, relationships between molecular weight (Mr) and biological properties remain unclear. The focus of this study was on the antioxidant properties of several Mr chitosans. We measured the ability of seven Mr chitosans (CT1; 2.8 kDa, CT2; 17.0 kDa, CT3; 33.5 kDa, CT4; 62.6 kDa, CT5; 87.7 kDa, CT6; 604 kDa, CT7; 931 kDa) to protect plasma protein from oxidation by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). A comparison of the antioxidant action of high Mr chitosans (CT6–CT7) with that of low Mr chitosans (CT1–CT5) showed that low Mr chitosans (CT1–CT5) were more effective in preventing the formation of carbonyl groups in plasma protein exposed to peroxyl radicals. AAPH substantially increases plasma protein carbonyl content via the oxidation of human serum albumin (HSA). We also measured the ability of these chitosans to protect HSA against oxidation by AAPH. Low Mr chitosans (CT1–CT5) were found to effectively prevent the formation of carbonyl groups in HSA, when exposed to peroxyl radicals. Low Mr chitosans were also good scavengers of N-centered radicals, but high Mr chitosans were much less effective. We also found a strong correlation between antioxidant activity and the Mr of chitosans in vitro. These activities were also determined by using the ‘TPAC’ test. These results suggest that low Mr chitosans (CT1–CT3) may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patients plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia.     

Reactivity toward oxygen radicals and antioxidant action of thiol compounds

Thiol compounds exert diverse functions in the defense network against oxidative stress in vivo. Above all, the role of glutathione in the enzymatic removal of hydrogen peroxide and lipid hydroperoxides has been well established. The scavenging of reactive free radicals is one of the many functions. In this study, the reactivities of several thiol compounds toward oxygen- and nitrogen-centered radicals were measured from their reaction with galvinoxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and also from their sparing effects on the decay of fluorescein, pyrogallol red, and BODIPY induced by peroxyl radicals. Furthermore, the antioxidant capacity against lipid peroxidation was assessed in the oxidation of methyl linoleate induced by free radicals in micelle systems. Cysteine, homocysteine, and glutathione exhibited considerable reactivity toward galvinoxyl, DPPH, and peroxyl radicals in this order but methionine did not. Bovine serum albumin (BSA) was less reactive toward these radicals than cysteine on molar base. Cysteine, homocysteine, and glutathione suppressed the oxidation of methyl linoleate in micelle systems, but methionine did not. The reactivity toward free radicals and antioxidant capacity of these thiol compounds were less than that of ascorbic acid, but higher than that of uric acid.     

Quinolinic Aminoxyl Protects Albumin Against Peroxyl Radical Mediated Damage

A study of peroxyl radical-mediated bovine serum albumin oxidation in the presence of the quinolinic aminoxyl 1,2-dihydro-2,2-diphenyl-4-ethoxy-quinoline-1-oxyl (QAO) was carried out in order to test its efficiency as a protein antioxidant. Albumin oxidation was induced by the tert-butylhydroperoxide/PbO₂ system. The extent of protein oxidation, measured by monitoring the formation of carbonyl groups, was considerably reduced in the presence of QAO. ESR measurements were carried out to confirm the consumption of the nitroxide during oxidation and its incorporation in the protein. The data obtained indicate that the quinolinic aminoxyl function can be used as an effective antioxidant in biological systems.     

In vitro evidence for an antioxidant role of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in the brain

We investigated the in vitro effect of 3-hydroxykynurenine (3HKyn), 3-hydroxyanthranilic acid (3HAA), kynurenine (Kyn) and anthranilic acid (AA) on various parameters of oxidative stress in rat cerebral cortex and in cultured C6 glioma cells. It was demonstrated that 3HKyn and 3HAA significantly reduced the thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence measurements in rat cerebral cortex, indicating that these metabolites prevent lipid peroxidation in the brain. In addition, GSH spontaneous oxidation was significantly prevented by 3HAA, but not by the other kynurenines in cerebral cortex. We also verified that 3HKyn and 3HAA significantly decreased the peroxyl radicals induced by the thermolysis of 2,2'-azo-bis-(2-amidinopropane)-derived peroxyl radicals, and to a higher degree than the classical peroxyl scavenger trolox. 2-Deoxy-d-ribose degradation was also significantly prevented by 3HKyn, implying that this metabolite was able to scavenge hydroxyl radicals. Furthermore, the total antioxidant reactivity of C6 glioma cells was significantly increased when these cells were exposed from 1 to 48h to 3HKyn, being the effect more prominent at shorter incubation times. TBA-RS values in C6 cells were significantly reduced by 3HKyn when exposed from 1 to 6h with this kynurenine. However, C6 cell morphology was not altered by 3HKyn. Finally, we tested whether 3HKyn could prevent the increased free radical production induced by glutaric acid (GA), the major metabolite accumulating in glutaric acidemia type I, by evaluating the isolated and combined effects of these compounds on TBA-RS levels and 2',7'-dihydrodichlorofluorescein (DCFH) oxidation in rat brain. GA provoked a significant increase of TBA-RS values and of DCFH oxidation, effects that were attenuated and fully prevented, respectively, by 3HKyn. The results strongly indicate that 3HKyn and 3HAA behave as antioxidants in cerebral cortex and C6 glioma cells from rats.     

Inhibition of peroxyl radical-mediated lipid oxidation by plasmalogen phospholipids and alpha-tocopherol

The recently discovered peroxyl radical scavenging properties of plasmalogen phospholipids led us to evaluate their potential interactions with alpha-tocopherol. The oxidative decay of plasmalogen phospholipids and of polyunsaturated fatty acids as induced by peroxyl radicals (generated from 2,2'-azobis-2-amidinopropane hydrochloride; AAPH) was studied in micelles using 1H-NMR and chemical analyses. In comparison with alpha-tocopherol, a 20- to 25-fold higher concentration of plasmalogen phospholipids was needed to induce a similar inhibition of peroxyl radical-mediated oxidation of polyunsaturated fatty acids. Plasmalogen phospholipids and alpha-tocopherol protected each other from oxidative degradation. In low-density lipoproteins (LDL) and micelles supplemented with plasmalogen phospholipids plus alpha-tocopherol, the peroxyl radical-promoted oxidation was additively diminished. The differences in the capacities to inhibit oxidation processes induced by peroxyl radicals between the plasmalogen phospholipids and alpha-tocopherol were less pronounced in the LDL particles than in the micelles. In conclusion, plasmalogen phospholipids and alpha-tocopherol apparently compete for the interaction with the peroxyl radicals. Oxidation processes induced by peroxyl radicals are inhibited in an additive manner in the presence of the two radical scavengers. The contribution of the plasmalogen phospholipids to the protection against peroxyl radical promoted oxidation in vivo is expected to be at least as important as that of alpha-tocopherol.     

Antioxidant activity of liver growth factor,a bilirubin covalently bound to albumin

We previously reported that treatment of spontaneously hypertensive rats (SHR) with liver growth factor (LGF), an albumin–bilirubin complex with a covalent bond, reduces blood pressure, improves nitric oxide (NO)-dependent vasodilatation, and exerts vascular antifibrotic actions. Because bilirubin, albumin, and albumin-bound bilirubins have antioxidant properties, we hypothesize that LGF might exert its cardiovascular actions through an antioxidant mechanism. We have tested in vitro the capacity of LGF to scavenge ABTS cation and peroxyl and hydroxyl radicals and to protect vascular NO from degradation by superoxide anion. We have also compared the antioxidant capacity of LGF with that of its molecular components albumin and bilirubin and the reference antioxidant trolox. LGF exhibited antioxidant capacity against all free radicals tested at lower concentrations than albumin, bilirubin, and trolox. LGF, bilirubin, and albumin were also able to protect endothelial NO from superoxide anion degradation in a fashion similar to that of superoxide dismutase or tiron, but at much lower concentrations. These data, together with our previous results in SHR, suggest that LGF might exert its cardiovascular regenerative actions, at least in part, through an antioxidant mechanism and that LGF could be a relevant circulating antioxidant in situations of oxidative stress.     

Nitroxides inhibit peroxyl radical-mediated DNA scission and enzyme inactivation

Nitroxides are cell-permeable stable radicals that protect biomolecules from oxidative damage in several ways. The mechanisms of protection studied to date include removal of superoxide radicals as SOD-mimics, oxidation of transition metal ions to preempt the Fenton reaction, and scavenging carbon-centered radicals. However, there is no agreement regarding the reaction of piperidine nitroxides with peroxyl radicals. The question of whether they can protect by scavenging peroxyl radicals is important because these radicals are formed in the presence of oxygen abundant in biological tissues. To further our understanding of the antioxidative behavior of piperidine nitroxides, we studied their effect on biochemical systems exposed to the water soluble radical initiator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). AAPH thermally decomposes to yield tert-amidinopropane radicals (t-AP(*)) that readily react with oxygen to form peroxyl radicals (t-APOO(*)). It has recently been reported that piperidine nitroxides protect plasmid DNA from t-AP(*) though not from t-APOO(*). The present study was directed at the question of whether these nitroxides can protect biological systems from damage inflicted by peroxyl radicals. The reaction of nitroxides with AAPH-derived radicals was followed by cyclic voltammetry and electron paramagnetic resonance spectroscopy, whereas the accumulation of peroxide was iodometrically assayed. Assaying DNA damage in vitro, we demonstrate that piperidine nitroxides protect from both t-AP(*) and t-APOO(*). Similarly, nitroxides inhibit AAPH-induced enzyme inactivation. The results indicate that piperidine nitroxides protect the target molecule by reacting with and detoxifying peroxyl radicals.     

Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes

To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants—a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids—phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol—in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.     

Effect on rat arterial blood pressure of chemically generated peroxyl radicals and protection by antioxidants

Convincing evidence suggests that blood redox changes play a role in the development of various cardiovascular disorders including hypertension. Nutritional antioxidants have been suggested to play a role in cardiovascular disease prevention. In this study, we investigated in vivo changes in rat arterial blood pressure induced by acute exposition to an increased load of peroxyl radicals and by the administration of selected antioxidants after chemically induced oxidative stress. Hydrosoluble and liposoluble peroxyl radicals, generated by 2,2'-azobis-(2-amidinopropane) dihydrochloride and 2,2'-azobis 2,4-di-methylvaleronitrile, induced a dose-dependent decrease in rat blood pressure. All antioxidants tested (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, vitamin C, glutathione and dithiothreitol) returned peroxyl radical-induced hypotension to normal. Of the various antioxidants tested, glutathione was the most effective in restoring blood pressure after peroxyl radical generation. Treatment of rats with a thiol-chelating agent (N-ethylmaleimide) and an oxidizing agent (5,5'-dithiobis-2-nitrobenzoic) inhibited peroxyl radical-mediated hypotension. Our results suggest that acute exposition to peroxyl radicals have a hypotensive effect on blood pressure and that thiols play an active role in the redox regulation of blood pressure. Other experiments are needed to clarify the role played by oxidative potentials on blood pressure and the mechanism of action of nutritional antioxidants.     

Mode of action of sesame lignans in protecting low-density lipoprotein against oxidative damage in vitro

We investigated the antioxidant properties of sesaminol, a major component of sesame oil, on the oxidative modification of human low-density lipoprotein (LDL) in vitro. Sesaminol inhibited the Cu2+-induced lipid peroxidation in LDL in a concentration-dependent manner with an IC50 36.0 +/- 10.0 nM. Sesaminol was a more effective scavenger than either alpha-tocopherol or probucol in reducing the peroxyl radicals derived from 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in aqueous solution. In addition, as determined by the secondary products of lipid peroxidation identified by using immunochemical methods, sesaminol completely inhibited the formation of 4-hydroxy-nonenal (4-HNE)- and malondialdehyde (MDA)-adducts in a concentration-dependent manner. Probucol and alpha-tocopherol at the same concentration exhibited a lesser inhibitory effect. Our findings suggest that sesaminol is a potentially effective antioxidant that can protect LDL against the oxidation.     

Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence

Multiple reactive oxygen/nitrogen species induce oxidative stress. Mammals have evolved with an elaborate defense network against oxidative stress, in which multiple antioxidant compounds and enzymes with different functions exert their respective roles. Radical scavenging is one of the essential roles of antioxidants and vitamin E is the most abundant and important lipophilic radical-scavenging antioxidant in vivo. The kinetic data and physiological molar ratio of vitamin E to substrates show that the peroxyl radicals are the only radicals that vitamin E can scavenge to break chain propagation efficiently and that vitamin E is unable to act as a potent scavenger of hydroxyl, alkoxyl, nitrogen dioxide, and thiyl radicals in vivo. The preventive effect of vitamin E against the oxidation mediated by nonradical oxidants such as hypochlorite, singlet oxygen, ozone, and enzymes may be limited in vivo. The synergistic interaction of vitamin E and vitamin C is effective for enhancing the antioxidant capacity of vitamin E. The in vitro and in vivo evidence of the function of vitamin E as a peroxyl radical-scavenging antioxidant and inhibitor of lipid peroxidation is presented.     

Protein and thiol oxidation in cells exposed to peroxyl radicals is inhibited by the macrophage synthesised pterin 7,8-dihydroneopterin

Monocyte cells are exposed to a range of reactive oxygen species (ROS) when they are recruited to a site of inflammation. In this study, we have examined the damage caused to the monocyte-like cell line U937 by peroxyl radicals and characterised the protective effect of the macrophage synthesised compound 7,8-dihydroneopterin.Exposure of U937 cells to peroxyl radicals, generated by the thermolytic breakdown of 2,2'-azobis(amidinopropane) dihydrochloride (AAPH), resulted in the loss of cell viability as measured by thiazolyl blue (MTT) reduction, and lactate dehydrogenase (LDH) leakage. The major form of cellular damage observed was cellular thiol loss and the formation of reactive protein hydroperoxides. Peroxyl radical oxidation of the cells only caused a small increase in cellular lipid oxidation measured. Supplementation of the media with increasing concentrations of 7,8-dihydroneopterin significantly reduced the cellular thiol loss and inhibited the formation of the protein hydroperoxides. High performance liquid chromatography (HPLC) analysis showed 7,8-dihydroneopterin was oxidised by both peroxyl radicals and preformed protein hydroperoxides to predominately 7,8-dihydroxanthopterin.The possibility that 7,8-dihydroneopterin is a cellular antioxidant protecting macrophage proteins during inflammation is discussed.     

Reduction of protein radicals by GSH and ascorbate: potential biological significance

The oxidation of proteins and other macromolecules by radical species under conditions of oxidative stress can be modulated by antioxidant compounds. Decreased levels of the antioxidants glutathione and ascorbate have been documented in oxidative stress-related diseases. A radical generated on the surface of a protein can: (1) be immediately and fully repaired by direct reaction with an antioxidant; (2) react with dioxygen to form the corresponding peroxyl radical; or (3) undergo intramolecular long range electron transfer to relocate the free electron to another amino acid residue. In pulse radiolysis studies, in vitro production of the initial radical on a protein is conveniently made at a tryptophan residue, and electron transfer often leads ultimately to residence of the unpaired electron on a tyrosine residue. We review here the kinetics data for reactions of the antioxidants glutathione, selenocysteine, and ascorbate with tryptophanyl and tyrosyl radicals as free amino acids in model compounds and proteins. Glutathione repairs a tryptophanyl radical in lysozyme with a rate constant of (1.05 ± 0.05) × 10⁵ M^–1 s^–1, while ascorbate repairs tryptophanyl and tyrosyl radicals ca. 3 orders of magnitude faster. The in vitro reaction of glutathione with these radicals is too slow to prevent formation of peroxyl radicals, which become reduced by glutathione to hydroperoxides; the resulting glutathione thiyl radical is capable of further radical generation by hydrogen abstraction. Although physiologically not significant, selenoglutathione reduces tyrosyl radicals as fast as ascorbate. The reaction of protein radicals formed on insulin, β-lactoglobulin, pepsin, chymotrypsin and bovine serum albumin with ascorbate is relatively rapid, competes with the reaction with dioxygen, and the relatively innocuous ascorbyl radical is formed. On the basis of these kinetics data, we suggest that reductive repair of protein radicals may contribute to the well-documented depletion of ascorbate in living organisms subjected to oxidative stress.     

The influence of medium components on Cu(2+)-dependent oxidation of low-density lipoproteins and its sensitivity to superoxide dismutase.

The extent of in vitro Cu(2+)-dependent oxidation of low-density lipoproteins (LDL) has been reported to vary widely depending upon reaction conditions. In this study, the effect of proteins and amino acids on Cu(2+)-induced LDL oxidation was examined. Treatment of LDL with 5 microM CuSO4 for 18 h in either phosphate-buffered saline (PBS) or Ham's F-10 medium resulted in extensive oxidation as determined by the content of thiobarbituric acid reactive substances (TBARS) and by increased lipoprotein electronegativity. In PBS, oxidation was entirely blocked by histidine and the tripeptide, gly-his-lys (GHK). Oxidation was also prevented by bovine serum albumin, but superoxide dismutase (SOD) provided only 20% protection. Both proteins bound similar amounts of Cu2+, but albumin appeared to be a more effective peroxyl radical trap as evidenced by its ability to prevent LDL oxidation induced by 2,2'-azo-bis(2-amidinopropane hydrochloride). In F-10 medium, SOD had marked inhibitory effects, in contrast to PBS. The addition of disulfides to PBS markedly enhanced the ability of SOD to inhibit oxidation. These results indicate that medium components which affect Cu2+ availability influence LDL oxidation and suggest that albumin is ideally suited as a plasma antioxidant to prevent oxidative modification of LDL. Furthermore, in certain instances, the inhibitory effects of SOD may be attributable to effects such as Cu2+ binding rather than dismutation of superoxide.     

Aromatic and aliphatic mono- and bis-nitroxides: a study on their radical scavenging abilities

Nitroxide radicals are an emerging class of interesting compounds with versatile antioxidant and radioprotective properties. All literature studies have so far concentrated on compounds bearing only one nitroxide function. Here, we now investigate and compare the radical scavenging behaviour and antioxidant activity of aromatic indolinonic and aliphatic piperidine bis-nitroxides, i.e compounds bearing two nitroxide functions. Their corresponding mono-derivatives were also studied for comparison. Radical scavenging activity was investigated using EPR and UV-Vis spectroscopy by following spectral changes in acetonitrile of the nitroxides in the presence of alkyl and peroxyl radicals generated, respectively, under anoxic or aerobic conditions from thermal decomposition of AMVN [2,2'-azobis(2,4-di-methylvaleronitrile)]. Antioxidant activity of the nitroxides was evaluated by monitoring conjugated dienes (CD) formation during methyl linoleate micelles peroxidation and by measuring carbonyl content in oxidized bovine serum albumin (BSA). The results show that: (a) each nitroxide moiety in bis-nitroxides scavenges radicals independent of each other; (b) aliphatic nitroxides do not scavenge peroxyl radicals, at least under the experimental conditions used here, whereas indolinonic aromatic ones do: their stoichiometric number is 1.14 and 2.17, respectively, for mono- and bis-derivatives; (c) bis-nitroxides are roughly twice more efficient at inhibiting lipid peroxidation compared to their corresponding mono-derivatives. Although this study provides only comparative information on the relative radical-scavenging abilities of mono- and bis-nitroxides, it helps in understanding further the interesting reactivity of these compounds especially with regards to peroxyl radicals where many controversies in the literature exist.     

Aromatic and aliphatic mono- and bis-nitroxides: A study on their radical scavenging abilities

Nitroxide radicals are an emerging class of interesting compounds with versatile antioxidant and radioprotective properties. All literature studies have so far concentrated on compounds bearing only one nitroxide function. Here, we now investigate and compare the radical scavenging behaviour and antioxidant activity of aromatic indolinonic and aliphatic piperidine bis-nitroxides, i.e compounds bearing two nitroxide functions. Their corresponding mono-derivatives were also studied for comparison. Radical scavenging activity was investigated using EPR and UV–Vis spectroscopy by following spectral changes in acetonitrile of the nitroxides in the presence of alkyl and peroxyl radicals generated, respectively, under anoxic or aerobic conditions from thermal decomposition of AMVN [2,2′-azobis(2,4-di-methylvaleronitrile)]. Antioxidant activity of the nitroxides was evaluated by monitoring conjugated dienes (CD) formation during methyl linoleate micelles peroxidation and by measuring carbonyl content in oxidized bovine serum albumin (BSA). The results show that: (a) each nitroxide moiety in bis-nitroxides scavenges radicals independent of each other; (b) aliphatic nitroxides do not scavenge peroxyl radicals, at least under the experimental conditions used here, whereas indolinonic aromatic ones do: their stoichiometric number is 1.14 and 2.17, respectively, for mono- and bis-derivatives; (c) bis-nitroxides are roughly twice more efficient at inhibiting lipid peroxidation compared to their corresponding mono-derivatives. Although this study provides only comparative information on the relative radical-scavenging abilities of mono- and bis-nitroxides, it helps in understanding further the interesting reactivity of these compounds especially with regards to peroxyl radicals where many controversies in the literature exist.     

Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies

In this study, free radical scavenging abilities of ferulic acid in relation to its structural characteristics were evaluated in solution, cultured neurons, and synaptosomal systems exposed to hydroxyl and peroxyl radicals. Cultured neuronal cells exposed to the peroxyl radical initiator AAPH die in a dose-response manner and show elevated levels of protein carbonyls. The presence of ferulic acid or similar phenolic compounds, however, greatly reduces free radical damage in neuronal cell systems without causing cell death by themselves. In addition, synaptosomal membrane systems exposed to oxidative stress by hydroxyl and peroxyl radical generators show elevated levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement. Ferulic acid greatly attenuates these changes, and its effects are far more potent than those obtained for vanillic, coumaric, and cinnamic acid treatments. Moreover, ferulic acid protects against free radical mediated changes in conformation of synaptosomal membrane proteins as monitored by EPR spin labeling techniques. The results presented in this study suggest the importance of naturally occurring antioxidants such as ferulic acid in therapeutic intervention methodology against neurodegenerative disorders such as Alzheimer's disease in which oxidative stress is implicated.     

Antioxidant activity of NADH and its analogue--an in vitro study

The antioxidant activities of NADH and of its analogue, 1,4-dihydro-2,6-dimethyl-3,5-dicarbethoxy-pyridine (PyH(2)), were evaluated in vitro. NADH was found to be oxidized by the peroxyl radical derived from 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH) decomposition, in a pH-dependent manner. Both NADH and PyH(2) inhibited the peroxidation of egg yolk lecithin (EYL) liposomes, although PyH(2) was more effective than NADH when 2,2'-azobis-4-methoxy-2,4-dimethyl-valeronitrile (methoxy-AMVN) was employed to induce EYL liposome peroxidation. The antioxidant activities of NADH and PyH(2) were also evaluated by measuring their influences on 1,3-diphenylisobenzofuran (DPBF) fluorescence decay in the presence of peroxyl radicals. NADH and PyH(2) were much more effective at inhibiting DPBF quenching in Triton X-100 micelles than in liposomes. These results indicate that NADH can inhibit lipid peroxidation despite being hydrophilic. Nevertheless, membrane penetration is an important factor and limits its antioxidant activity.     

Peroxidation of proteins and lipids in suspensions of liposomes, in blood serum, and in mouse myeloma cells

There is growing evidence that proteins are early targets of reactive oxygen species, and that the altered proteins can in turn damage other biomolecules. In this study, we measured the effects of proteins on the oxidation of liposome phospholipid membranes, and the formation of protein hydroperoxides in serum and in cultured cells exposed to radiation-generated hydroxyl free radicals. Lysozyme, which did not affect liposome stability, gave 50% protection when present at 0.3 mg/ml, and virtually completely prevented lipid oxidation at 10 mg/ml. When human blood serum was irradiated, lipids were oxidized only after the destruction of ascorbate. In contrast, peroxidation of proteins proceeded immediately. Protein hydroperoxides were also generated without a lag period in hybrid mouse myeloma cells, while at the same time no lipid peroxides formed. These results are consistent with the theory that, under physiological conditions, lipid membranes are likely to be effectively protected from randomly-generated hydroxyl radicals by proteins, and that protein peroxyl radicals and hydroperoxides may constitute an important hazard to biological systems under oxidative stress.     

Broad-spectrum antioxidant peptides derived from His residue-containing sequences present in human paraoxonase 1

Hydroxyl or peroxyl radicals and hypochlorous acid (HOCl) are known to cause the oxidation of lipoproteins. Here, we examined Cu²⁺-binding property of paraoxonase 1 (PON1), and antioxidant actions of peptides, resembling His residue-containing sequences in PON1, against oxidations by Cu²⁺, peroxyl radicals or HOCl. When Cu²⁺-binding property of PON1 was examined spectrophotometrically, the maximal Cu²⁺ binding was achieved at 1:1 molar ratio of PON1: Cu²⁺. Additionally, Cu²⁺-catalyzed oxidative inactivation of PON1 was prevented by Ca²⁺-depleted PON1 at 1:1 ratio, but not diethylpyrocarbonate (DEPC)-modified PON1, suggesting the participation of His residue in Cu²⁺-binding. When His-containing peptides were examined for antioxidant actions, those with either His residue at N-terminal position 2 or 3, or His-Pro sequence at C-terminal remarkably prevented Cu²⁺-mediated low density lipoprotein (LDL) oxidation and PON1 inactivation. Especially, FHKALY, FHKY or NHP efficiently prevented Cu²⁺-induced LDL oxidation (24 h), indicating a tight binding of Cu²⁺ by peptides. In support of this, the peptide/Cu²⁺ complexes exhibited a superoxide-scavenging activity. Separately, in oxidations by 2,2'-azobis-2-amidinopropane hydrochloride or HOCl, the presence of Tyrosine (Tyr) or Cysteine (Cys) residue markedly enhanced antioxidant action of His-containing peptides. These results indicate that His-containing peptides with Tys or Cys residues correspond to broad spectrum antioxidants in oxidation models employing Cu²⁺, 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) or HOCl.