Joseph Kanner

Nitric oxide as an antioxidant

Benzoate monohydroxy compounds, and in particular salicylate, were produced during interaction of ferrous complexes with hydrogen peroxide (Fenton reaction) in a N2 environment. These reactions were inhibited when Fe complexes were flushed, prior to the addition in the model system, by nitric oxide. Methionine oxidation to ethylene by Fenton reagents was also inhibited by nitric oxide. Myoglobin in several forms such as metmyoglobin, oxymyoglobin, and nitric oxide-myoglobin were interacted with an equimolar concentration of hydrogen peroxide. Spectra changes in the visible region and the changes in membrane (microsomes) lipid peroxidation by the accumulation of thiobarbituric acid-reactive substances (TBA-RS) were determined. The results showed that metmyoglobin and oxymyoglobin were activated by H2O2 to ferryl myoglobin, which initiates membrane lipid peroxidation; but not nitric oxide-myoglobin, which, during interaction with H2O2, did not form ferryl but metmyoglobin which only poorly affected lipid peroxidation. It is assumed that nitric oxide, liganded to ferrous complexes, acts to prevent the prooxidative reaction of these complexes with H2O2.

Nitric oxide an inhibitor of lipid oxidation by lipoxygenase cyclooxygenase and hemoglobin

The present study demonstrated that nitric oxide, which is an important mammalian metabolite, can inhibit oxidation by lipoxygenase, cyclooxygenase and hemoglobin. The inhibition is manifested as a lag-phase that is reversible. The inhibitory effect of nitric oxide on lipoxygenase and cyclooxygenase seems to derive from i) the capability of ·NO to reduce the ferric enzyme to the ferrous form, which is inactive; ii) competition for the iron site available for exogenous ligands; and iii) the radical scavenging ability of the nitroxide radical. Nitric oxide may act as a modulator of the arachidonic acid cascade and in the generation of oxygen-active species.

The stomach as a bioreactor: dietary lipid peroxidation in the gastric fluid and the effects of plant-derived antioxidants.

Atherosclerosis may result partly from processes that occur following food consumption and that involve oxidized lipids in chylomicrons. We investigated reactions that could occur in the acidic pH of the stomach and accelerate the generation of lipid hydroperoxides and co-oxidation of dietary constituents. The ability of dietary polyphenols to invert catalysis from pro-oxidation to antioxidation was examined. The acidic pH of gastric fluid amplified lipid peroxidation catalyzed by metmyoglobin or iron ions. Metmyoglobin catalyzed peroxidation of edible oil, resulting in 8-fold increase of hydroperoxide concentration. The incubation of heated muscle tissue in simulated gastric fluid for 2 h enhanced hydroperoxides accumulation by 6-fold to 1200 microM. In the presence of catechin or red wine polyphenols, metmyoglobin catalyzed the breakdown of hydroperoxides to zero, totally preventing lipid peroxidation and beta-carotene cooxidation. We suggest that human gastric fluid may be an excellent medium for enhancing the oxidation of lipids and other dietary constituents. The results indicate the potentially harmful effects of oxidized fats intake in the presence of endogenous catalysts found in foods, and the major benefit of including in the meal plant dietary antioxidants.

A novel function of red wine polyphenols in humans: prevention of absorption of cytotoxic lipid peroxidation products

Current evidence supports a contribution of polyphenols to the prevention of cardiovascular disease, but their mechanisms of action are not understood. We investigated the impact of red wine polyphenols on postprandial cytotoxic lipid peroxidation products (MDA) levels in humans. In a randomized, crossover study, the effect of red wine polyphenols on postprandial levels of plasma and urine MDA was investigated. Three meals of 250 g turkey cutlets supplemented by water (A); soaked in red wine after heating plus 200 ml of red wine (B); or soaked in red wine prior to heating plus 200 ml of red wine (C) were administered to 10 healthy volunteers. Subject baseline plasma levels of MDA were 50 ± 20 nM. After a meal of turkey meat cutlets, plasma MDA levels increased by 160 nM (P<0.0001);after (B) there was a 75% reduction in the absorption of MDA (P<0.0001). However, after (C), the elevation of plasma MDA was completely prevented (P<0.0001). Similar results were obtained for MDA accumulation in urine. Our study suggests that red wine polyphenols exert a beneficial effect by the novel new function, absorption inhibition of the lipotoxin MDA. These findings explain the potentially harmful effects of oxidized fats found in foods and the important benefit of dietary polyphenols in the meal.— Gorelik, S., Ligumsky, M., Kohen, R., Kanner, J. A novel function of red wine polyphenols in humans: prevention of absorption of cytotoxic lipid peroxidation products. FASEB J. 22, 41–46 (2008)

The Stomach as a “Bioreactor”: When Red Meat Meets Red Wine

To determine the stomach bioreactor capability for food oxidation or antioxidation, rats were fed red turkey meat cutlets (meal A) or red turkey meat cutlets and red wine concentrate (meal B). The hydroperoxides (LOOH) and malondialdehyde (MDA) levels of the stomach contents were evaluated during and after digestion; the postprandial plasma MDA level was also evaluated. In independently fed rats, the stomach LOOH concentration fell substantially 90 min following the meal, and the addition of red wine polyphenols enhanced LOOH reduction 3-fold. A similar trend was obtained for MDA. After pyloric ligation, the stomach contents of rats fed red meat homogenate showed >2-fold increases in LOOH and MDA accumulation. The postprandial plasma MDA level increased significantly by 50% following meal A and was maintained or even fell by 34% below basal level following meal B. The findings show that consumption of partially oxidized food could increase lipid peroxidation in the stomach and the absorption of cytotoxic lipid peroxidation products into the body. The addition of antioxidants such as red wine polyphenols to the meal may alter these outcomes. These findings explain the potentially harmful effects of oxidized fats in foods and the important benefit of consuming dietary polyphenols during the meal.

Lipid peroxidation and oxidation of several compounds by H2O2 activated metmyoglobin.

Activated metmyoglobin (MetMb) by H2O2 initiates oxidation of microsomal unsaturated fatty acids, β-carotene and methional but not formate. Lipid peroxidation by activated MetMb was not inhibited by catalase. The activated species which initiaes lipid peroxidation appears to be a porphyrin cation radical, P∸+FeIV=O, and not a hydroxyl radical.

Iron Release from Metmyoglobin, Methaemoglobin and Cytochrome C by A System Generating Hydrogen Peroxide

The reaction of H2O2 with resting metmyoglobin (MetMb), methaemoglobin (MetHb) and cytochrome-c (Cyt-c) was studied in the Soret and visible regions. The differences between the original and the final peak heights of the native haemproteins at 408 nm was found to be directly proportional to the loss of iron from the molecule. The release of iron from haemproteins was studied in a system generating H2O2 continuously at a low rate by an enzymic system, or by addition of large amounts of H2O2. Cytochrome-c, methaemoglobin and metmyoglobin during interaction with H2O2 at a concentration of 200 microM release 40%, 20% and 3%, respectively, of molecular iron after 10 min. The inhibition of haem degradation and iron release by enzymatically-generated H2O2 was determined using several hydroxyl radical scavengers, reducing agents and antioxienzymes, such as superoxide dismutase, catalase and caeruloplasmin.

Desferrioxamine as an Electron Donor. Inhibition of Membranal Lipid Peroxidation Initiated by H202 –Activated Metmyoglobin and Other Peroxidizing Systems

Desferrioxamine (DFO) involvement in several peroxidative systems was studied. These systems included: a) membranal lipid peroxidation initiated by H2O2-activated metmyoglobin (or methemoglobin); b) phenol-red oxidation by activated metmyoglobin or horseradish peroxidase (HRP): c) beta-carotene-linoleate couple oxidation stimulated by lipoxygenase or hemin. Desferrioxamine was found to inhibit all these systems but not ferrioxamine (FO). Phenol-red oxidation by H2O2-horseradish peroxidase was inhibited competitively with DFO. Kinetic studies using the spectra changes in the Soret region of metmyoglobin suggest a mechanism by which H2O2 reacts with the iron-heme to form an intermediate of oxy-ferryl myoglobin that subsequently reacts with DFO to return the activated compound to the resting state. These activities of DFO resemble the reaction of other electron donors.

Polyphenols activate Nrf2 in astrocytes via H2O2, semiquinones, and quinones.

Polyphenols, which occur both in edible plants and in foodstuff, have been reported to exert a wide range of health effects; however, the mechanism of action of these molecules is not fully understood. One important cellular pathway affected by polyphenols is the activation of the transcription factor Nrf2 via the electrophile response element, which mediates generation of phase 2 detoxifying enzymes. Our study found that Nrf2 nuclear translocation and the activity of NAD(P)H quinone oxidoreductase (NQO1) were increased significantly after treatment of astrocytes with tert-butylhydroquinone (tBHQ), resveratrol, or curcumin, at 20-50μM. Incubation of tBHQ, resveratrol, and curcumin in the growth medium in the absence of astrocytes caused the accumulation of H(2)O(2). Treatment of cells with either glutathione or metmyoglobin was found to decrease Nrf2 translocation and NQO1 activity induced by polyphenols by up to 40 and 60%, respectively. Addition of both glutathione and metmyoglobin to growth medium decreased Nrf2 translocation and NQO1 activity by up to 100 and 80%, respectively. In conclusion, because metmyoglobin, in the presence of polyphenols and glutathione, is known to interact with H(2)O(2), semiquinones, and quinones, the up-regulation of the antioxidant defense of the cells through activation of the Nrf2 transcription factor, paradoxically, occurs via the generation of H(2)O(2) and polyphenol-oxidized species generated from the exogenous microenvironment of the cells.

THE GENERATION OF FERRYL OR HYDROXYL RADICALS DURING INTERACTION OF HAEMPROTEINS WITH HYDROGEN PEROXIDE

The oxidation of 2-keto-4-thiomethyl butyric acid (KTBA) and methionine to ethylene has been used to evaluate generation of ferryl species or hydroxyl radicals by H2O2-activated haemproteins or free ferric ions. Hydrogen peroxide was generated by a glucose oxidase-glucose system at a rate of 1 microM/min. Free ferric in the presence of H2O2 oxidizes KTBA, and this was highly inhibited by hydroxyl radical scavengers, caeruloplasmin, superoxide dismutase (SOD) and EDTA. However, when metmyoglobin, methaemoglobin (MtHb) or horseradish peroxidase (HRP) were tested in the same model system, hydroxyl radical scavengers suppressed partially KTBA oxidation and caeruloplasmin, SOD and EDTA failed to inhibit the reaction. Cytochrome-c was found to be a weak promoter of KTBA oxidation in the presence of H2O2. Methionine was oxidized to ethylene by an active system which generates hydroxyl radicals, but not by H2O2-activated metmyoglobin. Ferric ions chelated to membranes or ADP in the presence of H2O2 generated enzymatically, initiated membranal lipid peroxidation only in the presence of ascorbic acid, and this was inhibited by EDTA. In contrast, metmyoglobin and methaemoglobin activated by H2O2 generated by the same system, initiated membranal lipid peroxidation and this was not inhibited by EDTA. It is concluded that ferryl and not HO. is the main oxidant in systems containing myoglobin and haemoglobin activated by low concentrations of H2O2.