Lipid Peroxidation In Phosphatidylcholine Liposomes Research Articles

A marked stimulation of Fe 3+-dependent lipid peroxidation in phospholipid liposomes under acidic conditions

Lipid peroxidation in phosphatidylcholine liposomes induced by Fe 3+ alone, assessed by thiobarbituric acid-reactive substances (TBARS) production, was markedly enhanced as the solution pH was lowered from 7.4 to 5.5. On the other hand, at physiological pH, TBARS production by Fe 3+ was almost negligible. Results of the radical scavenger experiments with superoxide dismutase, catalase and hydroxyl radical ( OH) scavengers (sodium benzoate, mannitol and dimethylthiourea), deoxyribose degradation and ESR spectrometry suggest that the stimulation of Fe 3+-dependent lipid peroxidation under acidic conditions is involved in generation of superoxide anion (O 2 − ), hydrogen peroxide (H 2O 2) and OH during the reaction. The stimulation of Fe 3+-dependent TBARS production by increasing the [H +] completely disappeared by triphenylphosphine (TPP) treatment of the liposomes, but the reaction was reversible with either incorporation of cumen hydroperoxide (CumOOH) into the TPP-treated liposomes or the addition of CumOOH to the treated liposomes. Incubation of the CumOOH-incorporated TPP-treated liposomes with Fe 3+ at pH 5.5 also resulted in OH generation. Based on these results, a possible mechanism of stimulatory effect of Fe 3+ on lipid peroxidation under acidic conditions is discussed.

Carboxymethylated glucan inhibits lipid peroxidation in liposomes.

Protective capabilities were studied of carboxymethylated (1-->3)-beta-D-glucan from Saccharomyces cerevisiae cell wall against lipid peroxidation in phosphatidylcholine liposomes induced by OH radicals produced with Fenton's reagent (H2O2/Fe2+) and also by microwave radiation using absorption UV-VIS spectrophotometry. A significant decrease in the conjugated diene production, quantified as Klein oxidation index, was observed in the presence of a moderate amount of added glucan. Increase of the oxidation index was accompanied with enhanced carboxyfluorescein leakage as a result of liposome membrane destabilization. This process was markedly suppressed with glucan present in the liposome suspension. Therefore, glucan may be considered as a potent protector against microwave radiation-induced cell damage.

Glycosylation, esterification and polymerization of flavonoids and hydroxycinnamates: effects on antioxidant properties.

Dietary flavonoids and hydroxycinnamates are effective antioxidants that may affect health and are also important for food preservation. Of the flavonoids, quercetin is a common representative and is found in many plant foods, especially onions, apples, tea, and broccoli. Quercetin is glycosylated in most plants, and the position and the nature of substitution of the sugar are species specific. Catechins are a well-studied group of flavonoids found at high levels in tea. Hydroxycinnamates are also found at exceptionally high levels in many foods including coffee and cereal brans and include ferulic, sinapic, p-coumaric, and caffeic acids. These compounds are commonly ester-linked to sugars or organic acids. This chapter reviews the action of flavonoids and hydroxycinnamates in two antioxidant assays: direct scavenging of the ABTS radical in the aqueous phase1 and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidylcholine liposomes.2

Effect of Bile Acids on Lipid Peroxidation: The Role of Iron

The toxic effect of hydrophobic bile acids is claimed to be in part mediated by lipid peroxidation. Conversely, antioxidant properties of tauroursodeoxycholic acid (TUDC), a hydrophilic bile acid, have been suggested as a possible mechanism by which TUDC confers its beneficial effect in a variety of diseases. We have investigated the effect of taurodeoxycholic acid (TDC), a hydrophobic bile acid and TUDC on lipid peroxidation using a pure lipid system both in the presence and absence of iron ions. Neither TDC nor TUDC showed any effect on spontaneous lipid peroxidation of phosphatidylcholine liposomes or sodium arachidonate solution. This lack of effect excludes the possibility of direct prooxidant or antioxidant properties for TDC and TUDC. Addition of ferrous ions (0.1 mM) to the lipid system brought about a linear increase in lipid peroxidation with time. The presence of TDC caused an increase in the rate and extent of iron-stimulated lipid peroxidation. The propensity of bile acids to increase iron-induced lipid peroxidation was related to hydrophobicity of the individual bile acids, with the highest effect observed with taurolithocholic acid, whereas TUDC did not have any influence. The TDC-induced increase in the iron-stimulated lipid peroxidation was concentration dependent. Addition of TUDC (10 mM) completely abolished the effect of TDC (2 mM) on iron-induced lipid peroxidation. This finding suggests that TUDC does not function as an antioxidant per se but may prevent lipid peroxidation caused by TDC. In conclusion, only in the presence of iron ions, hydrophobic bile acids may enhance lipid peroxidation. TUDC has no antioxidant activity per se but may counter the TDC-induced increase in iron-stimulated lipid peroxidation.

Effect of aluminum ion on Fe(2+)-induced lipid peroxidation in phospholipid liposomes under acidic conditions.

The effects of Al3+ on Fe(2+)-induced lipid peroxidation in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) were examined under acidic conditions. The stimulatory effect of Al3+ on Fe(2+)-induced lipid peroxidation in the liposomes showed a biphasic response against pH variation, and the maximum stimulation was observed around pH 6.0. In addition, it was found that the stimulatory effect of Al3+ on the lipid peroxidation was dependent on the proportion of PS in the liposomes. On the other hand, the lipid peroxidation in PC liposomes was not stimulated by the addition of Al3+. From these findings, it is suggested that the Al3+ effect on Fe(2+)-induced lipid peroxidation under acidic conditions is largely dependent on the phospholipid composition. Trivalent cations such as Tb3+ and Ga3+ also stimulated Fe(2+)-induced lipid peroxidation in PC/PS liposomes under acidic conditions, but divalent cations (Zn2+ and Mn2+) showed no stimulatory effect. The extents of Fe2+ disappearance and Fe3+ formation during the reaction were enhanced by the addition of Al3+ or Ga2+, but Tb3+ had no effect on Fe2+ disappearance. The results with 1,6-diphenyl-1,3,5-hexatriene (DPH) showed that the fluorescence anisotropy of DPH-labeled PC/PS liposomes under acidic conditions was increased by the addition of Al3+. Furthermore, there is a relation between the extents of the fluorescence anisotropy of the complex and TBARS production. In contrast, the fluorescence anisotropy of DPH molecules embedded in PC liposomes was not changed by the addition of Al3+. Based on these results, a possible mechanism of the stimulatory effect of Al3+ on Fe(2+)-induced lipid peroxidation under acidic conditions is discussed.

Characterization of the Intermediate Products of Lipid Peroxidation in Phosphatidylcholine Liposomes by Fast-atom Bombardment Mass Spectrometry and Tandem Mass Spectrometry Techniques

Characterization of the Intermediate Products of Lipid Peroxidation in Phosphatidylcholine Liposomes by Fast-atom Bombardment Mass Spectrometry and Tandem Mass Spectrometry Techniques

Enhancement of peroxidase-induced lipid peroxidation by indole-3-acetic acid: effect of antioxidants.

Indole-3-acetic acid (IAA) enhanced the peroxidase-induced lipid peroxidation in phosphatidylcholine liposomes, as measured by loss of fluorescence of cis-parinaric acid. α-Tocopherol or β-carotene in the lipid phase or ascorbate or Trolox in the aqueous phase inhibited the loss of fluorescence induced by the peroxidase + IAA system, but glutathione had only a small inhibitory effect. The peroxyl radical formed by one-electron oxidation of IAA, followed by decarboxylation and reaction with oxygen, is suggested to act as the initiator of lipid peroxidation. The protection by ascorbate or Trolox is explained by the reactivity of these compounds with the IAA indolyl radical, as shown by pulse radiolysis experiments, whereas the weak effect of glutathione agrees with its low reactivity towards the IAA-derived peroxyl radical and its precursors.

Synergistic Interactions between Vitamin A and Vitamin E against Lipid Peroxidation in Phosphatidylcholine Liposomes

Interactions between α-tocopherol and all-transretinol in suppressing lipid peroxidation were studied in a unilamellar liposomal system of phosphatidylcholine from either egg or soybean, in which peroxidation was initiated by the water-soluble azo initiator 2,2′-azobis(2-amidino-propane)hydrochloride and peroxidation was measured as production of conjugated diene hydroperoxides. While all-transretinol alone was poorly effective, the combination of all-transretinol with α-tocopherol caused an inhibition period far beyond the sum of the inhibition periods observed with individual antioxidants, providing evidence of synergistic interactions. Furthermore, the inhibition rate calculated in the presence of both all-transretinol and α-tocopherol,Rinh(E+A), was lower thanRinh(E)observed with α-tocopherol alone, suggesting that the extension of the inhibition time cannot be ascribed only to the antioxidant activity of α-tocopherol. The extent of synergism was linear with a molar ratio all-transretinol/α-tocopherol ranging from 0.1 to 1.0, whereas a drop was observed at a ratio of 2.0. Synergistic antioxidant interactions between all-transretinol and α-tocopherol were also evident when peroxidation was evaluated as production of malondialdehyde. A time course study, in which peroxidation of liposomes and depletion of antioxidants were concomitantly monitored, while showing that most of α-tocopherol was consumed to bring about the inhibition period, indicated that autooxidative reactions substantially contributed to the rapid depletion of all-transretinol, when the antioxidants were allowed to act separately. On the other hand, when α-tocopherol and all-transretinol were combined, the consumption of both antioxidants was significantly delayed, indicating reciprocal protection. Regeneration mechanisms cannot be accounted for by our results. The observed synergism between all-transretinol and α-tocopherol does not appear as the result of specific structural interactions in the lipid bilayer. Combination of all-transretinol with butylated hydroxytoluene, which reduced markedly all-transretinol oxidation, resulted in a synergistic antioxidant activity greater than that observed with comparable amounts of α-tocopherol. In light of the known antioxidant mechanism of retinoids, the data suggest that by limiting autooxidation of all-transretinol, α-tocopherol strongly promotes its antioxidant effectiveness. The concerted radical scavenging action in turn results in a synergistic protection of the lipid system against peroxidative stress and, ultimately, slows down the α-tocopherol consumption.

The mechanism of iron (III) stimulation of lipid peroxidation.

A study conducted on Fe2+ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe3+ addition but the stimulation did not present a maximum at a Fe2+/Fe3+ ratio approaching 1:1. The species generated during Fe(3+)-catalyzed Fe2+ autoxidation was able to oxidize deoxyribose; the increased Fe2+ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe(3+)-catalyzed Fe2+ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triphenylphosphine. Neither Fe2+ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe3+ effect on Fe2+ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe2+/Fe3+ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe3+ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe3+ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.

Scavenging of free radicals by tenoxicam: a participating mechanism in the antirheumatic/antiinflammatory efficacy of the drug.

The radical scavenging activity of tenoxicam against hydroxyl (HO.), superoxide (O2.-), and peroxyl (LOO.) radicals, all of them involved in the inflammatory reactions, has been tested in different cell-free systems and by different techniques. Tenoxicam is a good scavenger of both HO. radicals (IC50 = 56.7 microM), as determined by Electron Spin Resonance (ESR) spectroscopy with the spin trapping (5,5-dimethyl-1-pyrroline N-oxide, DMPO) technique, and O2.- radicals generated by the phenazine methosulfate/reduced beta-nicotinamide adenine dinucleotide (PMS/NADH) system. The high reactivity of the drug towards HO. was confirmed by the rate constant of reaction with HO. (k approximately 10(10) M-1s-1), determined by competition kinetic studies with N,N-dimethyl-4-nitrosoaniline. In addition at a microM level (1-5 microM) it dose-dependently prevents the phycoerythrin peroxidation induced by the water-soluble azoinitiator 2,2-azobis (2-amidinopropane) dihydrochloride (ABAP), indicating a quenching effect on aqueous peroxyl radicals. The HO.-entrapping capacity was confirmed in models more close to the in vivo situation: tenoxicam inhibits the HO.-induced depolymerization of hyaluronic acid already at 15 microM and the HO.-driven lipid peroxidation in phosphatidylcholine liposomes (PCL) with an IC50 of 10 microM. In this membrane model it delays at 1-10 microM level the decomposition of phosphatidylcholine hydroperoxides to short-chain alkenals (markers: total carbonyl functions as 2,4-dinitrophenylhydrazones and conjugated dienes). The high susceptibility of the drug to HO. attack is also demonstrated by its extensive degradation (HPLC studies) when irradiated with HO. radicals. The antioxidant component of tenoxicam evidenced in this study sheds some light on the hitherto undefined mechanism of the antiinflammatory action of the drug.

Stimulation of Fe 2+-induced lipid peroxidation in phosphatidylcholine liposomes by aluminium ions at physiological pH

The effects of aluminium ions on Fe 2+-induced lipid peroxidation in egg yolk phosphatidylcholine liposomes were examined under various conditions. The degree of Fe 2+-induced lipid peroxidation of the liposomes was dependent on pH of the reaction mixture: pH 5.0 > pH 7.4. However, Fe 2+ did not induce lipid peroxidation in the liposomes at pH 9.0. The addition of AlCl 3, to the liposomal suspension resulted in a marked stimulation of Fe 2+-induced liposomal peroxidation at pH 7.4, depending on the concentration of AlCl 3. On the other hand, Fe 3+, Cu 2+, Pb 2+, Cd 2+ and Cr 6+ did not induce lipid peroxidation in the liposomes at pH 7.4 regardless of the presence and absence of AlCl 3. Fe 3+ enhanced Fe 2+-induced liposomal peroxidation at pH 7.4 but is unrelated to the stimulatory effect of AlCl 3. In the absence of AlCl 3, Fe 2+-induced liposomal peroxidation was observed after a lag phase of about 15 min. The lag phase of the reaction was shortened by the addition of AlCl 3 in a dose-dependent fashion. The shortening of the lag phase was also observed by the decrease of Fe 2+ concentration or by the co-presence of Fe 3+ in the reaction mixture. In addition, it was found that AlCl 3 stimulates Fe 2+ disappearance and Fe 3+ formation. The addition of AJC1 3 to the liposomal suspension at pH 7.4 resulted in a marked increase of the turbidity of the suspension. On the other hand, the turbidity of the liposomal suspension at pH 5.0 did not change by the addition of AlCl 3. Furthermore, addition of AlCl 3 at the same concentration to the buffer system without the liposomes did not cause an appreciable increase of the turbidity. From these results, it is suggested that Al 3+ promotes aggregation of the liposomes. In addition, it was found that there is a good correlation between the stimulatory effect of Al 3+ and the turbidity change of the suspension over the concentration range of AlCl 3 tested (0.1–1.0 mM). On the basis of these results, the stimulatory effect of Al 3+ on Fe 2+-induced lipid peroxidation of the liposomes is discussed in relation to the liposomal aggregation.

Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5μM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.

Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine

Nifedipine, verapamil and stobadine were tested and compared with butylated hydroxytoluene (BHT) as possible free radical scavengers inhibiting lipid peroxidation in phosphatidylcholine liposomes. Liposomes were peroxidized by incubation in air at 50 degrees C. Verapamil less than nifedipine less than BHT less than stobadine depressed the lipid peroxidation as detected spectroscopically for conjugate diene and thiobarbituric acid product formation. Verapamil and stobadine were tested as OH radical scavengers in a Fenton-type reaction against spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO), as detected by ESR spectroscopy. The tested drugs competed with DMPO in trapping OH radicals, with stobadine being more effective than verapamil. ESR spectra of nifedipine in the incubated liposomes revealed that nifedipine could be involved in free radical reactions in the liposomes leading to nifedipine-stable radical(s) which were immobilized in the membrane. The obtained results suggest that some of the beneficial effects of the studied drugs can be mediated in disease by their ability to scavenge free radicals and by their protective effect on lipid peroxidation.

Quinone formation from carcinogenic benzo[ a]pyrene mediated by lipid peroxidation in phosphatidylcholine liposomes

The behavior of benzo[ a]pyrene (B[ a]P) during peroxidation of phosphatidylcholine (PC) liposomes initiated by an azo compound was investigated to examine the mechanism of quinone formation from carcinogenic B[ a]P mediated by nonenzymatic lipid peroxidation occurring in vivo. B[ a]P had a retarding effect on the peroxidation of polyunsaturated fatty acid moiety of PC. The major oxidation products which accumulated in the peroxidized liposomes were B[ a]P 1,6-, 3,6-, and 6,12-quinone. Antioxidants acting as scavengers of chain-propagating lipid peroxy radicals effectively prevented not only lipid peroxidation but also B[ a]P oxidation in the liposomal suspension. PC hydroperoxides, the primary products of PC oxidation, did not react with B[ a]P in the absence of the azo compound, indicating that lipid peroxy radicals, not lipid hydroperoxides, are responsible for the formation of these quinones. The experiments using 18O 2 gas and 18O-labeled methyl linoleate hydroperoxides demonstrated that B[ a]P quinones are formed by incorporating molecular oxygen and their origin is partly due to the lipid peroxy radical. The mechanism proposed for the formation of B[ a]P quinones mediated by peroxidation of membrane lipids involves a direct attack of the lipid peroxy radical on B[ a]P and subsequent autocatalytic oxidation. Weak carcinogenic and noncarcinogenic pentacyclic aromatic hydrocarbons showed little reactivity to the lipid peroxy radical in the liposomes. Thus, the facility of the peroxidative attack on B[ a]P may be related to the powerful carcinogenic activity of this substance.

Enhanced lipid peroxidation of erythrocyte membranes and phosphatidylcholine liposomes induced by a xanthine oxidase system in the presence of catalase.

When rat erythrocyte membranes or phosphatidylcholine (PC) liposomes were incubated in a xanthine oxidase system, lipid peroxidation was stimulated by the addition of catalase. Addition of a large amount of xanthine oxidase caused no significant increase in the lipid peroxidation of PC liposomes unless catalase was present in the reaction system. Enhanced peroxidations of both membranes and liposomes observed in the presence of catalase were strongly inhibited by superoxide dismutase (SOD), suggesting that O-2 is an essential species in these reactions. Several iron-chelators strongly inhibited the lipid peroxidations, suggesting an involvement of iron in the peroxidation reaction. Unless catalase was present, the rate of liposome peroxidation was stimulated only slightly by addition of Fe3+, but in the presence of catalase, the addition of Fe3+ markedly accelerated the peroxidation reaction, which was inhibited by SOD or desferrioxamine. Furthermore, the addition of Fe3+ to the xanthine oxidase system in the presence of catalase caused a significant decrease in the rate of cytochrome c reduction, suggesting that Fe3+ may be reduced to Fe2+ by O-2. The peroxidations enhanced by catalase may be induced by supply of sufficient O-2 and Fe2+ for lipid peroxidation. Histidine and 1, 4-diazabicyclo-(2, 2, 2)-octane, quenchers of 1O2, inhibited the peroxidation of membranes and liposomes. Mannitol and benzoate, scavengers of OH·, showed no significant effect on the peroxidation of membranes or liposomes. These results indicate possible participation of 1O2 species in the O-2-dependent lipid peroxidation induced by the xanthine oxidase system in the presence of catalase.