Inhibition of lipid peroxidation by casein Evidence of molecular encapsulation of 1,4-pentadiene fatty acids

The capability of cyclodextrins to form molecular inclusion complexes with linoleate appeared in a lipoxygenase-linoleate model reaction as inhibition of oxygenation. The inhibited rates were established instantaneously upon addition of the complexant and maintained until linoleate was exhausted. Total cessation of the reaction was not obtained with cyclodextrins. All these features were reproduced also in casein-inhibited reaction mixtures. Both casein and cyclodextrins protected linoleate also against autoxidation although they did not change free radical generation by xanthine oxidase or Fe²⁺ reactions. Since neither of the inhibitors affected the enzyme directly, casein may also act by forming linoleate complexes which via a standing equilibrium reduce the oxidizable monomer fatty acids and cause substrate-limited reaction rates. Comparisons at acidic and alkaline pH, in the presence of increasing amounts of the complexants, detergent and hydroperoxides supported this view.     

Nonesterified fatty acids and lipid peroxidation

Oxygen free radicals damage cells through peroxidation of membrane lipids. Gastrointestinal mucosal membranes were found to be resistant to in vitro lipid peroxidation as judged by malonaldehyde and conjugated diene production and arachidonic acid depletion. The factor responsible for this in this membrane was isolated and chemically characterised as the nonesterified fatty acids (NEFA), specifically monounsaturated fatty acid, oleic acid. Authentic fatty acids when tested in vitro using liver microsomes showed similar inhibition. The possible mechanism by which NEFA inhibit peroxidation is through iron chelation and iron-fatty acid complex is incapable of inducing peroxidation. Free radicals generated independent of iron was found to induce peroxidaton of mucosal membranes. Gastrointestinal mucosal membranes were found to contain unusually large amount of NEFA. Circulating albumin is known to contain NEFA which was found to inhibit iron induced peroxidation whereas fatty acid free albumin did not have any effect. Addition of individual fatty acids to this albumin restored its inhibitory capacity among which monounsaturated fatty acids were more effective. These studies have shown that iron induced lipid peroxidation damage is prevented by the presence of nonesterified fatty acids.     

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.     

Essential fatty acids, lipid peroxidation and apoptosis

Essential fatty acids (EFAs) and their metabolites, especially gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid and decosahexaenoic acid are known to induce apoptotic death of tumour cells. But the exact mechanism by which these fatty acids are able to induce apoptosis is not clear. Recent studies suggest that these fatty acids are able to induce apoptosis in cells over expressing cytochrome P450 following depletion of cellular glutathione and inhibition of carnitine palmitoyl transferase I (CPTI) activity. On the other hand, BCL-2 prevented apoptosis induced by these long-chain fatty acids, where as n-3 fatty acids suppressed ras expression leading to suppression of development of overt neoplasia. Phosphorylation of BCL-2 inhibits its ability to interfere with apoptosis and enhances lipid peroxidation leading to the occurrence of apoptosis. Tumour cells treated with long-chain fatty acids show increase in lipid peroxidation process, depletion of antioxidants and phosphorylation of proteins. Based on these results, it is suggested that long-chain fatty acids induce apoptosis by enhancing lipid peroxidation, suppressing BCL-2 expression possibly by phosphorylation and augmentation of P450 activity. Thus, these long-chain fatty acids may, infact act at the level of gene/oncogene expression in producing their cytotoxic action on tumour cells.     

Inhibition of lipid peroxidation by prostaglandin oligomeric derivatives.

The inhibition of lipid peroxidation by oligomeric derivatives synthesized from prostaglandin E1 (PGE1) and PGB2 was studied using two rat models. In an in vitro model, the brain was exposed to decapitation-ischemia, the cortex was removed and homogenized, and the formation of thiobarbituric acid reactive substances (TBAR) was measured after exposing the homogenate to in vitro reoxygenation either in the presence or absence of oligomers. It was found that these oligomers could inhibit lipid peroxidation, and that their activities were higher than that of superoxide dismutase (SOD). In an in vivo administration model, either the oligomer or the vehicle was injected i.p. 30 min before decapitation. The brain was exposed to decapitation-ischemia, the cortex was homogenized and exposed to 'in vitro' reoxygenation, after which TBAR value was determined. Ester-type compounds had a greater activity than free-acid type compounds in inhibiting lipid peroxidation. A possible mechanism of the protective effect of these oligomers in ischemia/reperfusion injury may be to scavenge oxygen free radicals.     

Inhibition of nonenzymic lipid peroxidation by benzylisoquinoline alkaloids.

A group of benzylisoquinoline alkaloids, including five simple benzylisoquinolines, three phtalideisoquinolines, six aporphines, three protoberberines, and two benzophenanthridines, have been studied as inhibitors of lipid peroxidation stimulated by Fe2+/cysteine in rat liver microsomal fractions. Protopapaverine, apomorphine, laudanosoline, tetrahydroberberine, isoboldine, bulbocapnine, boldine, anonaine, glaucine, and stepholidine showed antiperoxidative effects, and structure-activity relationships were established. In simple benzylisoquinolines, the presence of phenolic hydroxyls or similar reactive groups is necessary for inhibition of peroxidation, while in aporphines and protoberberines nonhydroxylated compounds can exert antiperoxidative effects. The phtalideisoquinolines and benzophenanthridines tested were inactive.     

Inhibition of lipid peroxidation

Lipid peroxy radicals (ROO-) were detected by electron spin resonance (ESR) at low temperature after formation by addition of H₂O₂ into a suspension of mice lymphocites. If lymphocytes were treated with selenomethionine (Se-Met) prior to addition of H₂O₂, ROO-formation was inhibited in a fashion that was dependent on Se-Met concentration. Formation of ROO- in the spleen of mice was induced by⁶⁰Co irradiation. Animals that were supplemented with Na₂SeO₃ prior to irradiation exhibited a lower ROO-concentration than that of nontreated animals. Based on our experiments, we have concluded that Se has an oxygen-free radical scavenging effect. This should be a protective effect against lipid peroxy radical cellular attack.     

Albumin bound nonesterified fatty acids inhibit in vitro lipid peroxidation

Individual nonesterified fatty acids were bound to albumin in vitro and these fatty acid albumin complexes were used to study their effect on lipid peroxidation in liver microsomes. Peroxidation was induced by various methods and malondialdehyde (MDA) was measured as an index of peroxidation. Among the fatty acids tested, albumin-bound monounsaturated fatty acids showed more inhibition of peroxidation as compared to other fatty acids. Increasing the concentration of iron in the peroxidizing system, partially reversed the inhibition by fatty acids. Moreover, albumin-bound fatty acid did not inhibit iron independent peroxidation. This suggests that, like nonesterified fatty acids, albumin-bound fatty acids inhibit peroxidation by chelating the iron. Albumin fatty acid complex, similar to the fatty acid composition present in the circulating albumin, also showed inhibition of peroxidation. These data indicate that nonesterified fatty acids even when bound to albumin are capable of inhibiting peroxidation and circulating albumin, which contains various fatty acids bound to it, may impart some antioxidant effect in addition to other plasma antioxidants.     

Inhibition of mouse liver lipid peroxidation by high molecular weight phlorotannins from Sargassum kjellmanianum

The inhibitory effects of high molecularweight phlorotannins (HMP) from Sargassum kjellmanianum on mouse liverlipid peroxidation were investigated byspectrophotometric methods. The content ofmalondialdehyde (MDA) in liver samples wasmeasured by TBA (thiobarbituric acid)assay. It showed that HMP significantlyinhibited the generation of MDA invivo and in situations induced byCCl₄ and Fe²⁺-Vc (ascorbic acid),and significantly decreased membraneswelling of mouse liver mitochondria,compared with controls (p<0.01). HMP werefound to have strong anti-oxidativeactivity in inhibiting mouse liver lipidperoxidation.     

Inhibition of lipid peroxidation by alpha-tocopherolquinone and alpha-tocopherolhydroquinone

The antioxidant effect of alpha-tocopherolquinone and alpha-tocopherolhydroquinone was studied in liposomes and rat liver submitochondrial particles. Both alpha-tocopherolquinone and alpha-tocopherolhydroquinone inhibit lipid peroxidation induced by ascorbate/Fe2+ in liposomes and by cumene hydroperoxide in submitochondrial particles. Alpha-tocopherolhydroquinone is much more effective than alpha-tocopherolquinone in inhibiting lipid peroxidation. Submitochondrial particles, depleted of ubiquinones and reincorporated with alpha-tocopherolquinone, are protected from lipid peroxidation only in the presence of succinate. Alpha-tocopherolquinone cannot replace endogenous ubiquinones in the respiratory chain function, nevertheless it can be reduced by the mitochondrial respiratory chain substrates, presumably through the reduced ubiquinones.     

Inhibition of lipid peroxidation by S-nitrosoglutathione and copper

The antioxidant properties of S -nitrosoglutathione, a nitric oxide-derived product were studied in different experimental systems. By using the crocin bleaching test, S -nitrosoglutathione, in the presence of copper ions, shows an antioxidant capacity about six times higher than that of Trolox c and referable to the interception of peroxyl radicals by nitric oxide. Copper alone shows a modest inhibitory action, which is about seven times lower than that of Trolox c. S -nitrosoglutathione prevents lipid peroxidation induced by the well-known Fe 2+ /ascorbate system (IC 50 =450 μM) and the inhibitory effect is strongly reinforced by the presence of copper ions (IC 50 =6.5 μM). In addition, cumene hydroperoxide-induced lipid peroxidation is markedly decreased by S -nitrosoglutathione, provided that copper ions, maintained reduced by ascorbate, are present. Decomposition of S -nitrosoglutathione through metal catalysis and/or the presence of reducing agents and the consequent release of nitric oxide are of crucial importance for eliciting the antioxidant power. In this way, copper ions and/or reducing species with low antioxidant potency are able to promote the formation of an extremely strong antioxidant species such as nitric oxide.     

Inhibition of rat lipoprotein lipid peroxidation by the oral administration of D003, a mixture of very long-chain saturated fatty acids

Previous results have demonstrated that policosanol, a mixture of aliphatic primary alcohols isolated and purified from sugar cane wax, whose main component is octacosanol, inhibited lipid peroxidation in experimental models and human beings. D003 is a defined mixture of very long-chain saturated fatty acids, also isolated and purified from sugar cane wax, whose main component is octacosanoic acid followed by traicontanoic, dotriacontanoic, and tetracontanoic acids. Since very long-chain fatty acids are structurally related to their corresponding alcohols, we investigated the effect of oral treatment with D003 (0.5, 5, 50, and 100 mg/kg) over 4 weeks in reducing the susceptibility of rat lipoprotein to oxidative modification. The combined rat lipoprotein fraction VLDL + LDL was subjected to several oxidation systems, including those containing metal ions (CuSO4), those having the capacity to generate free radicals 2,2-azobis-2-amidinopropane hydrochloride (AAPH), and a more physiological system (resident macrophages). D003 (5, 50, and 100 mg/kg) significantly inhibited copper-mediated conjugated-diene generation in a concentration-dependent manner. D003 increased lag phase by 53.1, 115.3, and 119.3%, respectively, and decreased the rate of conjugate-diene generation by 16.6, 21.5, and 19.6%, respectively. D003 also inhibited azo-compound initiated and macrophage-mediated lipid peroxidation as judged by the significant decrease in thiobarbituric acid reactive substance (TBARS) generation. In all the systems the maximum effect was attained at 50 mg/kg. There was also a parallel attenuation in the reduction of lysine amino groups and a significant reduction of carbonyl content after oxidation of lipoprotein samples. Taken together, the present results indicate that oral administration of D003 protects lipoprotein fractions against lipid peroxidation in the lipid as well in the protein moiety.     

Inhibition of topoisomerases by fatty acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure activity relationships and mechanism of action were studied. Saturated fatty acids (C6:0 to C22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C16:1 to C22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Inhibition of protein synthesis by products of lipid peroxidation

Effects of lipid peroxidation products on in vivo and in vitro protein synthesis have been studied. Malondialdehyde (MDA), a product, and a routinely used index of lipid peroxidation, inhibits in vivo protein synthesis in the two mosses, Tortula ruralis and Cratoneuron filicinum, and in pea (Pisum sativum) leaf discs. When wheat germ supernatant or poly(A)-rich mRNA of T. ruralis was incubated with MDA its subsequent activity in a cell-free protein-synthesizing system was reduced. When MDA was added directly to the in vitro protein-synthesizing mixture containing moss polyribosomes, the inhibition of amino acid incorporation was small. However, when simultaneous lipid peroxidation was allowed to occur along with in vitro protein synthesis there was a strong inhibition of amino acid incorporation and MDA accumulated in the reaction mixture indicating that products of lipid peroxidation other than, and apparently more toxic than, MDA were involved. It was concluded that lipid peroxidation inhibits protein synthesis probably by releasing toxic products which may react with and inactivate some components of the protein-synthesizing complex.     

Inhibition of lipid peroxidation by the iron-binding protein lactoferrin

Lactoferrin containing physiological amounts of iron is an inhibitor of lipid peroxidation induced by iron(III) salts and ascorbic acid. It might therefore help to protect neutrophils, inflammatory foci and secretions from metal-ion-dependent oxidative damage.     

Inhibition of superoxide and ferritin-dependent lipid peroxidation by ceruloplasmin

Ceruloplasmin (CP) was found to inhibit xanthine oxidase and ferritin-dependent peroxidation of phospholipid liposomes, as evidenced by decreased malondialdehyde formation. Ceruloplasmin was also shown to inhibit superoxide-mediated mobilization of iron from ferritin, in a concentration-dependent manner, as measured spectrophotometrically using the iron(II) chelator bathophenanthroline sulfonate. Ceruloplasmin failed to function as a peroxyl radical-scavenging antioxidant as evidenced by its inability to inhibit free radical-initiated peroxidation of linoleic acid, suggesting that CP inhibited lipid peroxidation by affecting the availability of ferritin-derived iron. In addition, CP scavenged xanthine oxidase-derived superoxide as measured spectrophotometrically via its effect on cytochrome c reduction. However, the extent of the superoxide scavenging of CP did not quantitatively account for its effects on iron release, suggesting that CP inhibits superoxide-dependent mobilization of ferritin iron independently of its ability to scavenge superoxide. The effects of CP and apoferritin on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. In the absence of apoferritin, CP exhibited a concentration-dependent prooxidant effect. However, CP-dependent, iron-catalyzed lipid peroxidation was inhibited by the addition of apoferritin. Apoferritin did not function as a peroxyl radical-scavenging antioxidant but was shown to incorporate iron in the presence of CP. These data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation largely via its ability to reincorporate reductively mobilized iron back into ferritin.     

Evidence for mitochondrial DNA damage by lipid peroxidation

When mitochondria of rat liver were incubated in an in vitro system containing NADPH and ferrous chloride, marked lipid peroxidation occurred, as evidenced by the evolution of malonic dialdehyde. DNA isolated from these peroxidized mitochondrial preparations had completely different electrophoretic mobility than DNA isolated from mitochondria protected from peroxidation. Scavengers of superoxide anion, hydrogen peroxide and hydroxyl radicals offered no protection against either lipid peroxidation or DNA damage. However, alpha-tocopherol protected against both lipid peroxidation and damage to the mitochondrial genome. These results support the hypothesis that lipid peroxidation can mediate DNA damage.     

Inhibition of Listeria monocytogenes by fatty acids and monoglycerides.

Fatty acids and monoglycerides were evaluated in brain heart infusion broth and in milk for antimicrobial activity against the Scott A strain of Listeria monocytogenes. C12:0, C18:3, and glyceryl monolaurate (monolaurin) had the strongest activity in brain heart infusion broth and were bactericidal at 10 to 20 micrograms/ml, whereas potassium (K)-conjugated linoleic acids and C18:2 were bactericidal at 50 to 200 micrograms/ml. C14:0, C16:0, C18:0, C18:1, glyceryl monomyristate, and glyceryl monopalmitate were not inhibitory at 200 micrograms/ml. The bactericidal activity in brain heart infusion broth was higher at pH 5 than at pH 6. In whole milk and skim milk, K-conjugated linoleic acid was bacteriostatic and prolonged the lag phase especially at 4 degrees C. Monolaurin inactivated L. monocytogenes in skim milk at 4 degrees C, but was less inhibitory at 23 degrees C. Monolaurin did not inhibit L. monocytogenes in whole milk because of the higher fat content. Other fatty acids tested were not effective in whole or skim milk. Our results suggest that K-conjugated linoleic acids or monolaurin could be used as an inhibitory agent against L. monocytogenes in dairy foods.     

Inhibition of in vitro cholesterol synthesis by fatty acids.

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.