Substituted p-hydroquinones as inhibitors of lipid peroxidation

The technique based on monitoring oxygen consumption was applied to study 12 alkyl- and methoxy-substituted p-hydroquinones (QH(2)) as a chain-breaking antioxidant during the oxidation of styrene and methyl linoleate (ML) in bulk as well as ML oxidation in micellar solution of sodium dodecyl sulfate (SDS) at 37 degrees C. The antioxidant activities of QH(2) were characterized by two parameters: the rate constant k(1) for reaction of QH(2) with the peroxy radical LO(2)*: QH(2)+LO(2)*-->QH*+LOOH and the stoichiometric factor of inhibition, f, which shows how many kinetic chains may be terminated by one molecule of QH(2). In the case of styrene and ML oxidation in bulk, f values never exceed two; for the majority of QH(2), f was found to be significantly less than two due to the interaction of QH* with molecular oxygen. In the absence of superoxide dismutase (SOD), all the studied QH(2) displayed a very moderate if any antioxidant capability during ML oxidation in SDS micelles. When 20U/ml SOD was added, the majority of QH(2) showed a pronounced ability to inhibit ML oxidation, f parameter being ca. one. The features of QH(2) as an antioxidant in aqueous environment are suggested to associate with the reactivity of semiquinone (Q*(-)). Q*(-) reacts readily with molecular oxygen with formation of superoxide (O(2)*(-)); further reactions of O(2)*(-) result in fast depleting QH(2) and chain propagation. The addition of SOD results in purging a reaction mixture from O(2)*(-) and, as a corollary, in depressing undesirable reactions with the participation of O(2)*(-). With all the oxidation models, QH(2) were found to be very reactive to LO(2)*. The rate constants k(1) decreased progressively when going from the oxidation of styrene to ML oxidation in bulk and further to ML oxidation in SDS micelles.     

Oxygen-dependent antagonism of lipid peroxidation

Measurements of the rates for formation of conjugated dienes, malonylaldehyde, and lipid hydroperoxides show that increasing the concentration of O2 from 0.11 mM to 0.35 mM or 0.69 mM can slow the rate of linoleic acid peroxidation in a xanthine oxidase/hypoxanthine system. This effect is seen at pH 7.0 but not 7.4 and depends on the presence of monounsaturated fatty acids (oleic, cis, or trans vaccenic acid). Oxygen antagonism of ascorbic acid-iron-EDTA mediated lipid peroxidation is similarly dependent on fatty acid mixtures and occurs at pH 5.0 and 6.0 but not 7.0. The efficiency of initiation of peroxidation in the xanthine oxidase system is unaffected by monounsaturated fatty acids and O2 concentration. Increasing the O2 concentration increases the rate of superoxide radical production, but there is no change in salicylate hydroxylation (e.g., OH. production) or ferrous ion concentration. Oxygen-mediated slower rates of lipid peroxidation are associated with either increased H2O2 production or, based on an indirect assay, singlet O2 production. Increased O2 concentrations increase the rate of azobisisobutyronitrile-initiated lipid peroxidation as expected but addition of exogenous superoxide radicals slows the rate. Under similar conditions superoxide reacts with fatty acids to produce singlet O2. Overall, the data suggest that O2-mediated antagonism occurs because of termination reactions between hydroperoxyl (HO2.) and organic radicals, and singlet O2 or H2O2 are products of these reactions.     

The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H(2)O(2)), hydroxyl radical (HO*) or superoxide anion (O(2)*(-) ) on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO* slightly decreased F1F0-ATPase activity and HO* and O(2)*(-) partially inhibited the activity of adenine nucleotide translocase; H(2)O(2) failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for O(2)*(-) and alpha-ketoglutarate dehydrogenase for H(2)O(2) and HO*. Our results show that O(2)*(-), H(2)O(2) and HO* act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.     

Formation of M1G-dR from endogenous and exogenous ROS-inducing chemicals

The present study provides fundamental information regarding the production of M1G-dR by ROS. To investigate the production of M1G-dR from deoxyribose damage as caused by ROS, calf thymus DNA (CT-DNA) was incubated with NAD(P)H, CuCl2, and various concentrations of hydrogen peroxide (H2O2). The incubation of CT-DNA with H2O2 resulted in concentration-dependent increases in the number of M1G-dR adducts. In subsequent experiments, 1,4-tetrachlorobenzoquinone or catechol estrogens were evaluated for their effects on M1G-dR formation. In addition, the role of lipid peroxidation in the formation of M1G-dR was verified using an in vitro lipid peroxidation model which consisted of methyl esters of either fish oil or purified fatty acids found in cellular membranes. This experiment confirmed that M1G-dR is a major DNA adduct produced by lipid peroxidation. Furthermore, the number of double bonds in polyunsaturated fatty acids was found to be the key factor in the formation of M1G-dR. The findings obtained from this study provide important information regarding the molecular pathways for M1G-dR formation by ROS, which is an essential element in understanding and evaluating the genotoxicity of a variety of ROS-inducing chemicals.     

Formation of active forms of oxygen by rat peritoneal macrophages under the effect of cytotoxic dust]

The velocity of superoxide radicals (O2) production by rat peritoneal macrophages, phagocyting the dust particles (quartz and crocidolite-asbestos was measured by using the method of cytochrome c reduction. Generation of hydroxyl radicals (HO) by cells and intensity of lipid peroxidation in the membranes of phagocytes were also investigated. It was found, that under the action of quartz the cells form mainly O2, and under the action of crocidolite--O2 and HO(.). The differences observed were caused by catalytic properties of the surface of asbestos fiber, where the reaction of HO. formation from O2 takes place. The quartz particles increased the concentration of malondialdehyde in macrophages by 53% as compared with control; and lipid peroxidation intensity in the presence of crocidolite-asbestos fibers increased fourfold. The role of hydroxyl radicals in initiating of lipid peroxidation, cytotoxicity and mutagenicity of asbestos is discussed.     

Inhibition of complex I of the electron transport chain causes O2-. -mediated mitochondrial outgrowth

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O(2)(-)* production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O(2)(-)* production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY(581/591). Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O(2)(-)* to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O(2)(-)* production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O(2)(-)* production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O(2)(-)* to prevent alterations in mitochondrial morphology.     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

In vivo breath alkane as an index of lipid peroxidation

Interaction of active oxygen species with polyunsaturated fatty acids (PUFA) results in a series of reactions called lipid peroxidation. During the process of peroxidation of polyunsaturated fatty acids there is a scission of an alkane fragment extending from the methyl end of the fatty acid to the double bond. Thus, with a w-6 polyunsaturated fatty acid pentane is released, and with a w-3 polyunsaturated fatty acid ethane is released. These hydrocarbons are distributed in the body, partly metabolized, and excreted in the breath, making it possible to estimate the magnitude of in vivo lipid peroxidation by measuring pentane and ethane exhaled in breath. Advantages of this method are discussed as well as limitations and possible sources of error.     

Site-specific mechanisms of initiation by chelated iron and inhibition by alpha-tocopherol of lipid peroxide-dependent lipid peroxidation in charged micelles.

To obtain information on the role of iron-catalyzed lipid peroxidation in the presence of the small amount of lipid peroxide in deterioration of biological membranes, we examined factors affecting peroxidation of fatty acids in charged micelles. Peroxidation of linoleic acid (LA) was catalyzed by Fe2+ via reductive cleavage of linoleic acid hydroperoxide (LOOH) in negatively charged sodium dodecyl sulfate micelles, but not in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles. However, this Fe2(+)-induced, LOOH-dependent lipid peroxidation could be induced in TTAB micelles in the presence of a negatively charged iron chelator, nitrilotriacetic acid (NTA). The linoleic acid alkoxy radical (LO.) generated by the LOOH-dependent Fenton reaction was also trapped by N-t-butyl-alpha-phenylnitrone at the surface of TTAB micelles in the presence of NTA, but not in its absence. The degradation rates of two spin probes, N-oxyl-4,4'-dimethyloxazolidine derivatives of stearic acid (5-NS and 16-NS), were investigated to determine the site of production of radicals formed during LOOH-dependent lipid peroxidation. The rate of consumption of 16-NS during the LOOH-dependent Fenton-like reaction was higher in TTAB micelles containing LA than in those containing lauric acid (LauA), although the rates of formation of LO. in the two types of fatty acid micelles were similar. The rates of 5-NS consumption in LA and LauA micelles were almost the same and were as low as that of 16-NS consumption in LauA micelles. 16-NS was more inhibitory than 5-NS of LOOH-dependent lipid peroxidation, and this inhibition was associated with its higher consumption of 16-NS than of 5-NS. alpha-Tocopherol inhibited NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation in TTAB micelles, and was oxidized during this inhibition process. The rate and amount of alpha-tocopherol oxidized by the LOOH-dependent Fenton reaction were higher in LA micelles than in LauA micelles. alpha-Tocopherol inhibited the consumption of 16-NS during NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation more effectively than that of 5-NS. The distribution of the chromanol moiety of alpha-tocopherol was studied by the fluorescence quenching method. There was no difference between Stern-Volmer plots of the quenchings of alpha-tocopherol fluorescence by 5-NS and 16-NS. From these results, we discuss the mechanism of induction of LOOH-dependent peroxidation of LA and the mechanism of the antioxidant effects of alpha-tocopherol on it from the viewpoint of site-specific reaction.     

N-acyl dehydroalanines scavenge oxygen radicals and inhibit in vitro free radical mediated processes

N-substituted dehydroalanines react with and scavenge oxygen radicals. One of those compounds, the para-methoxyphenylacetyl dehydroalanine derivative, indexed as AD-5, inhibits the reduction of ferricytochrome c by superoxide anion (O2-.). It can also inhibit the oxidation of linolenic acid, another chemical process, which is mediated by hydroxyl radical (HO.). Furthermore, microsomal lipid peroxidation induced by iron salts was also inhibited by AD 5, but with a different degree of efficacy. In fact, lipid peroxidation initiated by a ferrous-oxygen complex (as in iron/NADPH-dependent peroxidation) was inhibited by AD 5 in a range of concentration of 2-4 mM. On the contrary, iron/NADPH-independent lipid peroxidation, where alkoxy radicals (RO.) have principally been involved, was inhibited in a range of concentration of 6-10 mM. The ESR studies by using the spin trapping agent DMPO, show that AD-5 reacts with HO. with a second order constant of 2.8 X 10(9)-4.5 X 10(9) M-1 s-1.     

Effect of pH on low-density lipoprotein oxidation by O2-./HO2. free radicals produced by gamma radiolysis.

This study aimed to analyze quantitatively the initiation and the consequences of low-density lipoprotein (LDL) peroxidation by O2-./HO2. free radicals produced by gamma radiolysis. The action of increasing radiation doses on aqueous LDL solutions has been monitored simultaneously by several parameters: a decrease in endogenous vitamin E, the formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes, the appearance of a differential fluorescence (excitation wavelength = 360 nm), and an increase of the relative electrophoretic mobility. Initial radiation yields (decrease in vitamin E, formation of TBARS) have been determined at pH 7 and pH 5.7 as a function of LDL concentration (from 0.75 to 9 g liter-1). From the comparison of these yields with those of O2-. radicals produced by water radiolysis, we have deduced reaction mechanisms for LDL peroxidation initiated by O2-./HO2. free radicals.     

Nonesterified fatty acids inhibit iron-dependent lipid peroxidation

The effect of various fatty acids on lipid peroxidation of liver microsomes induced by different methods in vitro was studied using oxygen uptake and malonaldehyde (MDA) production. It was observed that fatty acids with a single double bond are effective inhibitors of peroxidation. Stereo and positional isomers of oleic acid were equally effective as oleic acid. There was an absolute requirement for a free carboxyl group, since methyl esters of fatty acids and long-chain saturated and unsaturated hydrocarbons could not inhibit peroxidation. Saturated fatty acids with a chain length of 12-16 carbon atoms showed inhibition, whereas more than 18 carbon atoms reduced the inhibitory capacity. Fatty acids of lower chain length such as capric and caprylic acids did not show inhibition. Fatty acid inhibition was partially reversed by increasing the concentration of iron in the system. Peroxidation induced by methods which were independent of iron was not inhibited by fatty acids. It was observed that intestinal microsomes which were resistant to peroxidation due to the presence of nonesterified fatty acids in their membrane lipids were able to peroxidise by methods which do not require iron. These results suggest that certain fatty acids inhibit peroxidation by chelating available free iron. In addition, they may also be involved in competing with the esterified fatty acids in the membrane lipids which are the substrates for peroxidation.     

The role of radical reactions in organomercurials impact on lipid peroxidation

The organomercury compounds RHgX and R(2)Hg are broad-spectrum biocidal agents acting via diverse mechanisms in biological systems. Despite the enormous amount of studies carried out in last decades to elucidate the detailed mechanisms of organomercurials toxicity their biomolecular mode of action is still under debate. Among various toxicity mechanisms the action of RHgX and R(2)Hg at the membrane level due to the lipophilic properties of their molecules is discussed. Organomercurials are supposed to induce membrane associated oxidative stress in living organism through different mechanisms including the enhancement of the lipid peroxidation and intracellular generation of reactive oxygen species (ROS), H(2)O(2), O(2)(-), HO(). The perturbation of antioxidative defense system and the peroxidation of unsaturated fatty acids in membrane lipid bilayer are consequences of this impact. On the other hand, the involvement of organomercurials in radical and redox biochemical processes is manifested in carbon to metal bond cleavage that leads to the generation of reactive organic radicals R(). This pathway is discussed as one of the multiple mechanisms of organomercurials toxicity. The goal of this review is to present recent results in the studies oriented towards the role of organomercurials in the xenobiotic-mediated enhancement of radical production and hence in the promotion of lipids peroxidation. The application of natural and synthetic antioxidants as detoxification agents is presented.     

The effect of alpha-tocopherol on the lipid peroxidation of mitochondria and microsomes obtained from rat liver and testis.

The effect of intraperitoneal administration of alpha-tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the alpha-tocopherol treated group were not observed. The light emission was significantly higher in the control than in the alpha-tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the alpha-tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with alpha-tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbate-Fe2+ lipid peroxidation. The protector effect observed by alpha-tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

The upstream oxylipin profile of Arabidopsis thaliana: a tool to scan for oxidative stresses

Various physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.     

Perhydroxyl radical (HOO.) initiated lipid peroxidation. The role of fatty acid hydroperoxides

It is demonstrated that the perhydroxyl radical (HOO., the conjugate acid of superoxide (O2-], initiates fatty acid peroxidation (a model for biological lipid peroxidation) by two parallel pathways: fatty acid hydroperoxide (LOOH)-independent and LOOH-dependent. Previous workers (Gebicki, J. M., and Bielski, B. H. J. (1981) J. Am. Chem. Soc. 103, 7020-7025) demonstrated that HOO., generated by pulse radiolysis, initiates peroxidation in ethanol/water fatty acid dispersions by abstraction of the bis-allylic hydrogen atom from a polyunsaturated fatty acid. Addition of O2 to the fatty acid radicals forms peroxyl radicals (LOO.s), the chain-propagating species of lipid peroxidation. In this work it is demonstrated that HOO., generated either chemically (KO2) or enzymatically (xanthine oxidase), is a good initiator of fatty acid peroxidation in linoleic acid ethanol/water dispersions; O2- serves only as the source of HOO., and HOO. initiation can be observed at physiologically relevant pH values. In contrast to the previous results, the initiating effectiveness of HOO. is related directly to the initial concentrations of LOOHs in the lipids to be peroxidized. This defines a LOOH-dependent mechanism for fatty acid peroxidation initiation by HOO., which parallels the previously established LOOH-independent pathway. Since the LOOH-dependent pathway is much more facile than the LOOH-independent pathway, LOOH is the kinetically preferred site of HOO. attack in these systems. Experiments comparing HOO./LOOH-dependent fatty acid peroxidation with transition metal- and peroxyl radical-initiated peroxidation rule out the participation of the latter two species as initiators, which defines the HOO./LOOH initiation system as mechanistically unique. LOOH product studies are consistent with either a direct or indirect hydrogen atom transfer between LOOH and HOO. to yield LOO.s, which propagate peroxidation. The LOOH-dependent pathway of HOO.-initiated fatty acid peroxidation may be relevant to mechanisms of lipid peroxidation initiation in vivo.     

Rearing system and oleic acid supplementation effect on carcass and lipid characteristics of two muscles from an obese pig breed

Quality of pork depends on genotype, rearing and pre- and post-slaughter conditions. However, no information is available on rearing system changes and oleic acid supplementation on carcass characteristics and fatty acid (FA) profile of pork from the Alentejano (AL) pig, an obese breed. This study evaluates the effects of feeding low (LO) or high oleic acid diets (HO) to AL pigs reared in individual pens (IND) or outdoor (OUT) with access to pasture. Carcass composition was obtained and longissimus dorsi and semimembranosus samples were collected to analyse chemical composition and neutral and polar intramuscular lipids FA profile by gas chromatography. Statistical analysis was performed by a two-way ANOVA for rearing system and diet effects. OUT-reared pigs presented leaner carcasses than IND-reared ones. Both muscles presented lower intramuscular lipid content in OUT-reared pigs. Treatments affected the FA profile of muscles. Overall, OUT-reared pigs presented lower n-6/n-3 FA ratios, whereas pigs fed the HO diet exhibited lower saturated fatty acids (SFA), higher monounsaturated fatty acids (MUFA) levels and lower thrombogenic indexes on neutral intramuscular lipids than LO-fed pigs. On the polar fraction, OUT-reared pigs presented lower SAT and n-6/n-3 FA ratio, and higher polyunsaturated fatty acids (PUFA) levels on both muscles. Pigs fed the HO diet exhibited higher MUFA and lower PUFA levels on both muscles, and lower SAT levels on semimembranosus. This study shows rearing system and oleic acid supplementation have complementary effects and influence carcass composition and the nutritional quality of meat.     

Initiation of lipid peroxidation as a result of hemoglobin transformation into hemichrome induced by free fatty acids

The effects of free fatty acids on hemoglobin conversion and lipid peroxidation were studied in hemoglobin-containing liposomes (hemosomes) formed from an equimolar mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). It was shown that in hemosomes oxyhemoglobin is converted into hemichrome by the interaction of saturated fatty acids (arachidic, stearic, palmitic, myristic and lauric). This is accompanied by accumulation of primary and secondary products of lipid peroxidation. All fatty acids, except for lauric acid, have a stabilizing effect on lipid peroxidation in liposomes prepared from an equimolar mixture of PC and PE. The formation of lipid peroxidation products is inhibited by superoxide dismutase, D-alpha-tocopherol, D-mannitol and thiourea. The relationships between hemoglobin conversion and lipid peroxidation in hemosomes under effects of fatty acids were studied. The mechanisms of these reactions are discussed.     

Polyunsaturated fatty acids promote 8-hydroxy-2-deoxyguanosine formation through lipid peroxidation under the glutamate-induced GSH depletion in rat glioma cells

It has been reported that glutamate decreased the intracellular glutathione (GSH) concentration and thereby induced cell death in C6 rat glioma cells. Polyunsaturated fatty acids such as arachidonic acid, gamma-linolenic acid, and linoleic acid enhanced lipid peroxidation promoting 8-hydroxy-2'-deoxyguanosine (8-OH-dG) formation under the glutamate-induced GSH-depletion. The enhancement of lipid peroxidation by polyunsaturated fatty acids was species-dependent. Some antioxidants capable of scavenging oxygen and lipid radicals and some iron or copper scavengers inhibited both the lipid peroxidation and the 8-OH-dG formation, consequently protecting against cell death induced by glutamate-induced GSH depletion. These results suggest that GSH depletion caused by glutamate induces lipid peroxidation and consequently 8-OH-dG formation and that polyunsaturated fatty acids enhance lipid peroxidation associated with mediated 8-OH-dG formation through a chain reaction.     

Free fatty acids,lipid peroxidation,and lysosomal enzymes in experimental focal cerebral ischemia in primates: Loss of lysosomal latency by lipid peroxidation

Experimental focal cerebral ischemia was produced in monkeys (Macaca radiata) by occlusion of the right middle cerebral artery (MCA). The release of the lysosomal glycosidases, -d-hexosaminidase, -l-fucosidase and -d-mannosidase into the soluble fraction in the right basal ganglia of the experimental animals was measured at different periods from 30 min to 12 hr after occlusion and compared with the corresponding sham operated control animals. There was a significant increase in the released lysosomal enzymes in the MCA occluded animals at all periods and particularly at 4 hr after occlusion. The CSF from the experimental animals also showed elevated levels of hexosaminidase and fucosidase. The free fatty acids (FFA) measured in the basal ganglia at 30 min and 2 hr after occlusion showed a 100 fold increase in the experimental animals. The predominant fatty acid released was linoleic acid (18:2) followed by arachidonic acid (20:4). Lipid peroxidation in the basal ganglia measured by the thiobarbituric acid (TBA) reaction in the presence or absence of ascorbic acid also showed a significant increase in the experimental animals at all periods with a maximum at 30 min to 2 hr after occlusion. In order to assess whether lipid peroxidation causes damage to the lysosomes and release of the enzymes, a lysosome enriched P₂ fraction from the normal monkey basal ganglia was prepared and the effect of peroxidation studied. Maximum peroxidation in the P₂ fraction was observed in the presence of arachidonic acid, ascorbic acid and Fe²⁺. There was a good correlation between the extent of lipid peroxidation and the in vitro release of lysosomal hexosaminidase from the P₂ fraction. Anti-oxidants which strongly inhibited lipid peroxidation in the P₂ fraction prevented the release of hexosaminidase. The results suggested that in ischemia produced by MCA occlusion lipid peroxidation which damages the lysosomal membrane causes the release of lysosomal hydrolytic enzymes.Abbreviations used BHA butylated hydroxyanisole - BHT butylated hydroxytoluene - FFA free fatty acids - MCA middle cerebral artery - MDA malonaldehyde - PUFA polyunsaturated fatty acids - TBA thiobarbituric acid