Inhibition of lipid peroxidation by ubiquinol in submitochondrial particles in the absence of vitamin E

The relationship between the antioxidant effects of reduced coenzyme Q10 (ubiquinol, UQH2) and vitamin E (alpha-tocopherol) was investigated in beef heart submitochondrial particles in which lipid peroxidation was initiated by incubation with ascorbate + ADP-Fe3+. These effects were examined after extraction of coenzyme Q10 (UQ-10) and vitamin E from the particles and reincorporation of the same components alone or in combination. The results show that UQH2 efficiently inhibits lipid peroxidation even when vitamin E is absent. It is concluded that UQH2 can inhibit lipid peroxidation directly, without the mediation of vitamin E.     

Initiation of lipid peroxidation in submitochondrial particles: effect of respiratory inhibitors

Initiation of lipid peroxidation in the inner mitochondrial membrane was investigated using respiratory substrates and inhibitors and various iron chelates. An iron chelate was required for initiation of lipid peroxidation in the presence of either NADH or NADPH. The two nicotinamide nucleotides exhibited different activities in initiating lipid peroxidation with regard to concentration and to the effects of rotenone and rhein. Succinate and both nicotinamide nucleotides supported lipid peroxidation in the presence of thenoyl trifluoroacetone (TTFA), without a requirement for exogenously added iron. ADP stimulated lipid peroxidation in the case of NAD(P)H and TTFA, but inhibited it in the case of succinate and TTFA. Lipid peroxidation is thought to be enzymatically induced in both the NADH and the succinate dehydrogenase regions of the respiratory chain, and evidence is presented for a novel pathway of NADPH oxidation that may also be involved. Possible initiation mechanisms are discussed.     

Leaf aging and lipid peroxidation: The role of the antioxidants vitamin C and E

To study age-related peroxidation in annual beech ( Fagus silvatica Mill.) leaves and perennial fir ( Abies alba Mill.) needles, the concentration changes of the antioxidants vitamin C (ascorbic acid) and vitamin E (α-tocopherol) present in leaves and needles were determined, and the formation of thiobarbituric acid reactants was measured as an index of age-related lipid peroxidation. With age, the content of the lipid-soluble antioxidant vitamin E increased, and the increase was higher in beech leaves than in fir needles. A comparable age-dependent increase of the vitamin C content was found neither in leaves nor in needles. The concentration ratio of the vitamin C to the vitamin E present in leaves and needles, which determines the potency of the anti oxidative system consisting of both vitamins, declined with age. This decline was directly related to a higher peroxidation of lipids. The extent of age-related peroxidative damage of cells seems to be controlled by the potency of anti oxidative systems.     

NADH- and NADPH-dependent lipid peroxidation in bovine heart submitochondrial particles. Dependence on the rate of electron flow in the respiratory chain and an antioxidant role of ubiquinol.

Malondialdehyde formations by bovine heart submitochondrial particles supported by NADH or NADPH in the presence of ADP and FeCl3 was studied. The NADH-dependent reaction was maximal at very low rate of electron input from NADH to the respiratory chain and it decreased when the rate became high. The reaction was stimulated by rotenone and inhibited by antimycin A when the input was fast, whereas it was not affected by the inhibitors when the input was slow. The input rate of the electrons from NADPH was also so low that the reaction supported by NADPH was not affected by the inhibitors. Most of the endogenous ubiquinone in the particles treated with antimycin A was reduced by NADH even in the presence of ADP-Fe3+ chelate, but uniquinone was not reduced by NADPH when ADP-Fe3+ was present. Succinate strongly inhibited both NADH- and NADPH-dependent lipid peroxidation. The inhibition was abolished when uniquinone was removed from the particles, and it appeared again when uniquinone was reincorporated into the particles. Reduced uniquinone-2 also inhibited the peroxidation, but duroquinol, which reduces cytochrome b without reducing endogenous uniquinone, did not. Thus the malondialdehyde formation appeared to be inversely related to the extent of the reduction of endogenous uniquinone. These observations suggest that both NADH- and NADPH-dependent liquid-peroxidation reactions are closely related to the respiratory chain and that the peroxidation is controlled by the concentration of reduced ubiquinone.     

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.     

Rat lung microsomal lipid peroxidation: effects of vitamin E and reduced glutathione

Lung microsomal membranes that contain the redox active components associated with the mixed-function oxidase system can be peroxidized in vitro. To investigate the characteristics of rat lung microsomal lipid peroxidation, we performed experiments using a variety of peroxidation initiators and microsomes obtained from normal and vitamin E-deficient rats. We found that lung microsomes obtained from normal rats are peroxidized much less than liver microsomes obtained from the same animals. Only initiation systems using very high concentrations of ferrous iron produced any significant peroxidation of normal rat lung microsomes. Lung microsomes obtained from vitamin E-deficient rats were found to be much more susceptible to peroxidation. Glutathione (GSH) was effective in inhibiting peroxidation when lung microsomes from normal rats were peroxidized. GSH was not effective in decreasing peroxidation when microsomes from vitamin E-deficient rats were peroxidized in the same system. We conclude that both GSH and vitamin E protect lung microsomal membranes from peroxidation. Glutathione protection appears to be related to the presence of a sulfhydryl group.     

Cobaltous ion inhibition of lipid peroxidation in biological membranes.

The effect of cobalt on lipid peroxidation in biological membranes, phospholipid liposomes and fatty acid micelles was investigated. Cobaltous ion, at micromolar concentrations, inhibited iron-ascorbate induced lipid peroxidation in erythrocyte ghosts, microsomes and phosphatidylserine liposomes at pH 7.4. The pH seemed to be important for the anti-peroxidative effect of cobalt, because under slightly acidic conditions cobalt did not inhibit peroxidation. Cobalt was less effective in inhibiting peroxidation stimulated by organic hydroperoxides. Iron-ascorbate induced lipid peroxidation was also inhibited by EDTA. However, certain ratios of EDTA: cobalt in the reaction mixture stimulated peroxidation. Cobalt did not inhibit lipid peroxidation in linoleic acid micelles and phosphatidylethanolamine liposomes. The presence of phosphatidylserine, however, rendered these micelles and liposomes to cobalt inhibition. We conclude that the cobaltous ion is a potent inhibitor of lipid peroxidation in biological membranes and that the binding of cobalt to phosphatidylserine is necessary for the inhibitory effect of this metal ion.     

Succinate-dependent lipid peroxidation and its prevention by reduced ubiquinone in beef heart submitochondrial particles.

When succinate and ADP-Fe3+ chelate were added to beef heart submitochondrial particles pretreated with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase of the mitochondrial respiratory chain, the formation of malondialdehyde was observed. No formation was observed without the pretreatment. Oxaloacetate competitively inhibited the malondialdehyde formation with an apparent Ki of 3.4 microM. The malondialdehyde formation seemed to be initiated at the location between the p-hydroxymercuribenzoate-sensitive site and the 2-thenoyltrifluoroacetone-sensitive site of the succinate dehydrogenase because it was inhibited by the mercurial. Ubiquinone-10 was rapidly destroyed during the malondialdehyde-forming reaction when it was in the oxidized form, while the ubiquinone was not destroyed and the malondialdehyde formation was abolished when about 50% of the ubiquinone in the particles was in the reduced state. These observations suggest that the succinate-dependent peroxidation is strongly controlled by the redox state of ubiquinone.     

Dietary antioxidants as inhibitors of the heme-induced peroxidation of linoleic acid: mechanism of action and synergism

In this work, a quantitative kinetic model for investigating the heme-induced peroxidation of linoleic acid and its inhibition by two important dietary antioxidants, quercetin and alpha-tocopherol, is developed. The main conclusions of this work are: (1) The time dependence of the lipid hydroperoxide concentration is critically dependent on the rate constant for lipid hydroperoxide cleavage, initial fraction of lipid hydroperoxides among the pool of conjugated dienes, and rate of heme degradation. (2) The lipophilic antioxidant alpha-tocopherol acts as a chain-breaking antioxidant that quickly reduces 1-2 eq of lipid peroxyl radicals (inhibition of propagation), whereas the more hydrophilic antioxidant quercetin is only marginally chain-breaking but capable of reducing 4-5 eq of iron-oxo initiator (inhibition of initiation). (3) Based on comparisons between experimental peroxidation curves and simulated curves assuming additivity, it can be concluded that combinations of alpha-tocopherol and quercetin are generally synergistic. The kinetic analysis and HPLC titrations of the antioxidants both suggest that synergism mainly arises from a capacity of alpha-tocopherol to regenerate some quercetin oxidation products still endowed with a reducing activity.     

The specificity of lipoxygenase-catalyzed lipid peroxidation and the effects of radical-scavenging antioxidants

The oxidation of low density lipoprotein (LDL) by lipoxygenase has been implicated in the pathogenesis of atherosclerosis. It has been known that lipoxygenase-mediated lipid peroxidation proceeds in general via regio-, stereo- and enantio-specific mechanisms, but that it is sometimes accompanied by a share of random hydroperoxides as side reaction products. In this study we investigated the oxidation of various substrates (linoleic acid, methyl linoleate, phosphatidylcholine, isolated LDL, and human plasma) by the arachidonate 15-lipoxygenases from rabbit reticulocytes and soybeans aiming at elucidating the effects of substrate, lipoxygenase and reaction milieu on the contribution and mechanism of random oxidation and also the effect of antioxidant. The specific character of the rabbit 15-lipoxygenase reaction was confirmed under all conditions employed here. However, the specificity by soybean lipoxygenase was markedly dependent on the conditions. When phosphatidylcholine liposomes and LDL were oxygenated by soybean lipoxygenase, the product pattern was found to be exclusively regio-, stereo-, and enantio-random. When free linoleic acid was incorporated into PC liposomes and oxidized by soybean lipoxygenase, the free acid was specifically oxygenated, whereas esterified linoleate gave random oxidation products exclusively. Radical-scavenging antioxidants such as alpha-tocopherol, ascorbic acid and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol selectively inhibited the random oxidation but did not influence specific product formation. It is assumed that the random reaction products originate from free radical intermediates, which have escaped the active site of the enzyme and thus may be accessible to radical scavengers. These data indicate that the specificity of lipoxygenase-catalyzed lipid oxidation and the inhibitory effects of antioxidants depend on the physico-chemical state of the substrate and type of lipoxygenase and that they may change completely depending on the conditions.     

The ability of melatonin to counteract lipid peroxidation in biological membranes

This paper reviews recent data relevant to the antioxidant effects of melatonin with special emphasis on the changes produced in polyunsaturated fatty acids located in the phospholipids of biological membranes. The onset of lipid peroxidation within cellular membranes is associated with changes in their physicochemical properties and with the impairment of protein functions located in the membrane environment. All cellular membranes are especially vulnerable to oxidation due to their high concentration of polyunsaturated fatty acids. These processes combine to produce changes in the biophysical properties of membranes that can have profound effects on the activity of membrane-bound proteins. This review deals with aspects for lipid peroxidation of biological membranes in general, but with some emphasis on changes of polyunsaturated fatty acids, which arise most prominently in membranes and have been studied extensively in our laboratory. The article provides current information on the effect of melatonin on biological membranes, changes in fluidity, fatty acid composition and lipid-protein modifications during the lipid peroxidation process of photoreceptor membranes and modulation of gene expression by the hormone and its preventive effects on adriamycin-induced lipid peroxidation in rat liver. Simple model systems have often been employed to measure the activity of antioxidants. Although such studies are important and essential to understand the mechanisms and kinetics of antioxidant action, it should be noted that the results of simple in vitro model experiments cannot be directly extrapolated to in vivo systems. For example, the antioxidant capacity of melatonin, one of the important physiological lipophilic antioxidants, in solution of pure triglycerides enriched in omega-3 polyunsaturated fatty acids is considerably different from that in subcellular membranes.     

The effect of vitamin E on lipid peroxidation in the copper-deficient rat

The present study was designed to determine whether the supplementation of vitamin E in the copper-deficient diet would ameliorate the severity of copper deficiency in fructose-fed rats. Lipid peroxidation was measured in the livers and hearts of rats fed a copper-deficient diet (0.6 microg Cu/g) containing 62% fructose with adequate vitamin E (0.1 g/kg diet) or supplemented with vitamin E (1.0 g/kg diet). Hepatic lipid peroxidation was significantly reduced by vitamin E supplementation compared with the unsupplemented adequate rats. In contrast, myocardial lipid peroxidation was unaffected by the level of vitamin E. Regardless of vitamin E supplementation, all copper-deficient rats exhibited severe signs of copper deficiency, and some of the vitamin E-supplemented rats died of this deficiency. These findings suggest that although vitamin E provided protection against peroxidation in the liver, it did not protect the animals against the severity of copper deficiency induced by fructose consumption.     

Diffusional effects in the steady state kinetics of ubiquinol cytochrome c reductase in bovine heart submitochondrial particles

The steady-state kinetics of ubiquinol cytochrome c reductase was investigated in submitochondrial particles using ubiquinol-1 as electron donor in media of increasing viscosities obtained by water-polyethylene glycol mixtures. The minimum association rate constant, kmin = kcat/km, for cytochrome c was strongly viscosity dependent, whereas kmin for ubiquinol-1 was only weakly affected by viscosity. It is concluded that the interaction of cytochrome c with the membranous reductase is largely under diffusion control, whereas the oxidation of ubiquinol by the enzyme is not significantly controlled by diffusion in either the aqueous medium or the membrane. The results are compatible with the presence of a diffusion limited step in cytochrome c but not in ubiquinone in mitochondrial electron transfer.     

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes.

Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co²⁺, Mn²⁺, Hg²⁺, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional to lipid peroxidation.     

The relationship between cell membrane damage and lipid peroxidation under the condition of hypoxia‐reoxygenation: analysis of the mechanism using antioxidants and electron transport inhibitors

We consecutively observed lipid peroxidation and cell membrane damage under the condition of hypoxia‐reoxygenation (H/R) in cells and analyzed their mechanisms by using electron transport inhibitors and an antioxidant. In H/R experiments, lipid peroxidation and cell membrane damage were observed during the hypoxia phase. In the reoxygenation phase, lipid peroxidation stopped, while cell membrane damage did not. An antioxidant, n‐acetylcystein (NAC), and potassium cyanide (KCN) inhibited lipid peroxidation and cell membrane damage, while rotenone did not inhibit either of them. Although antimycin A did not inhibit lipid peroxidation, it inhibited cell membrane damage during the hypoxia phase but not during the reoxygenation phase. These results suggested that lipid peroxidation can affect cell membrane damage as a trigger during the hypoxia phase and the generation of oxidative stress can vary depending on the inhibition locations in the electron transport system. Copyright © 2009 John Wiley & Sons, Ltd.     

Radiation-induced lipid peroxidation, a fish diet and modulation of the effects by vitamin E

Data are presented in this paper on the effect of vitamin E on rats given a fish diet after whole-body gamma-irradiation. The content of lipid peroxidation products in rat plasma, brain and liver and also the content of vitamin E have been investigated. Irradiation increases lipid peroxidation in the studied tissues and decreases vitamin E content. This process is aggravated by the fish diet. Vitamin E given in addition to fish diet helps the organism to stabilize the antioxidant homeostasis at a qualitatively different level.     

Kinetic analysis of the free-radical-induced lipid peroxidation in human erythrocyte membranes: evaluation of potential antioxidants using cis-parinaric acid to monitor peroxidation.

cis-Parinaric acid (PnA), cis-trans-trans-cis-9, 11, 13, 15-octadecatetraenoic acid, is fluorescent (epsilon = 74,000 at 324 nm) when partitioned into a lipid environment and the fluorescence is destroyed upon reaction with free radicals. It has been used to monitor semiquantitatively free-radical-induced lipid peroxidation in human erythrocyte membranes. We have applied this assay to the quantitative evaluation of potential antioxidants. The kinetics of the reaction of PnA with free radicals were measured in erythrocyte ghosts. After initiation of free radical generation by cumene hydroperoxide and cupric ion, a steady-state rate of fluorescence decay is rapidly established. In the steady state the oxidation of PnA and, hence, the loss of fluorescence is a first-order process. In the presence of antioxidants, such as vitamin E, the rate constant of fluorescence loss decreases, thereby indicating that the antioxidant decreases the steady-state concentration of free radicals. By adding various concentrations of potential antioxidants, pseudo-first-order rate constants [k1] which measure the reactivity of antioxidants with free radicals were determined. Results show that, when incorporated into erythrocyte membranes, U-78, 517f, a vitamin E analog, is a potent free radical scavenger, being approximately 50% as effective as vitamin E and 10-15 times more potent than the aminosteroids evaluated (see Table 1).     

The effects of diazinon on lipid peroxidation and antioxidant enzymes in rat heart and ameliorating role of vitamin E and vitamin C

Diazinon is one of the most widely used organophosphate insecticides (OPIs) in agriculture and public health programs. Reactive oxygen species (ROS) caused by OPIs may be involved in the toxicity of various pesticides. The aim of this study was to investigate how diazinon affects lipid peroxidation (LPO) and the antioxidant defense system in vivo and the possible ameliorating role of vitamins E and C. For this purpose, experiments were done to study the effects of DI on LPO and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in adult rat heart. Experimental groups were: (1) control group, (2) diazinon treated (DI) group, (3) DI+vitamins E and C-treated (DI+Vit) group. The levels of malondialdehyde (MDA) and the activities of SOD and CAT increased significantly in the DI group compared with the control group. The activity of SOD and the levels of MDA decreased significantly in the DI+Vit group compared with the DI group. The differences between the DI+Vit and control groups according to the MDA levels and the activities of both SOD and CAT were statistically significant. These results suggest that treating rats with a single dose of diazinon increases LPO and some antioxidant enzyme activities in the rat myocardium and, in addition, that single-dose treatment with a combination of vitamins E and C after the administration of diazinon can reduce LPO caused by diazinon, though this treatment was not sufficiently effective to reduce the values to those in control group.