Ferritin, a physiological iron donor for microsomal lipid peroxidation

In the process of lipid peroxidation of microsomes induced either by oxygen radicals generated by xanthine oxidase or by NADPH, ferritin is able to donate the necessary iron. The amount of ferritin necessary to catalyze the process of lipid peroxidation is in the physiological range. In contrast to the finding with phospholipid liposomes, catalase hardly stimulates the lipid peroxidation of microsomes.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Importance of iron in lipid peroxidation in the tyrosinase/4-hydroxyanisole system: possible mechanism of killing of malignant melanoma cells by 4-hydroxyanisole.

Phospholipid peroxidation of unsaturated phospholipid liposomes in the tyrosinase(mushroom)-4-hydroxyanisole system was studied in both the presence and absence of Fe3+, as a model of melanocyte damage by this agent. Ferric ion is required for the lipid peroxidation, and maximal lipid peroxidation was achieved with a molar ratio of [Fe3+]/[4-hydroxyanisole] of about 1. The lipid peroxidation was significantly inhibited by ceruloplasmin (a ferroxidase), indicating that Fe3+, which would be coordinated with metabolites, catechols, should be reduced to express its oxidant property. Judging from the results obtained with inhibitors or scavengers of active oxygen species, O2-, H2O2, and .OH would not mainly involve in the lipid peroxidation.     

Transferrin-dependent lipid peroxidation

The potential for iron bound to transferrin to be released and promote the peroxidation of phospholipid liposomes was investigated using ADP as a low molecular weight chelator and Superoxide generated by the xanthine/ xanthine oxidase system as the reducing agent. Lipid peroxidation in this system was dependent upon transferrin as the source of iron; increasing the transferrin concentration resulted in increased rates of lipid peroxidation. Increasing the xanthine oxidase activity also caused increased rates of peroxidation. Catalase stimulated rates of peroxidation at all xanthine oxidase activities tested. Conditions resulting in the most rapid release of iron from transferrin (low pH, high ADP) did not promote the greatest rates of lipid peroxidation, indicating that at neutral pH, rates of lipid peroxidation may be limited by the availability of iron. It is concluded that transferrin is not a likely source of iron for catalysis of deleterious biological oxidations such as lipid peroxidation in vivo.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe²⁺ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than α-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than α-tocopherol; (e) to be a weaker antiradical than α-tocopherol in the reduction of the stable radical DPPH^·. Resveratrol reduced Fe³⁺ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe³⁺, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like α-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

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.     

Steady magnetic fields effect on lipid peroxidation kinetics

The effect of steady magnetic fields (ranging from 0 to 280 mT) has been investigated on the kinetics of non-enzymatic lipid peroxidation occurring in a model system consisting of liposomes obtained from 1, 2-dioleoylphosphatidylcholine by oxygen consumption. The process was found to be accelerated by weak steady magnetic fields. A computer simulation method was employed to detect the reactions that govern the process kinetics, to elucidate magneto-sensitive stages (initiation and reduction of iron(III), as well as lipid peroxide radical recombination) and to determine their rate constants at various external magnetic fields. The kinetics of peroxidation of lipid cell membranes have been modeled mathematically at oxygen and ‘free’ iron concentrations close to those in the cells and also at increased free iron concentrations at different external magnetic field values.     

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.     

A new and suitable reconstructed system for NADPH-dependent microsomal lipid peroxidation

In order to evaluate the O-2 participation in NADPH-dependent microsomal lipid peroxidation, we used reconstructed system which contained detergent-solubilized NADPH-dependent cytochrome P-450 reductase, cytochrome P-450, phospholipid liposomes, NADPH and Fe3+-ADP. Lipid peroxidation, monitored by the formation of thiobarbituric acid-reactive substance, was increased with increasing concentration of detergent-solubilized NADPH cytochrome P-450 reductase, cytochrome P-450 or Fe3+-ADP. Cytochrome P-450-dependent lipid peroxidation was parallel to O-2 generation monitored by chemiluminescence probe with 2-methyl-6-(p-methoxyphenol)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one. Lipid peroxidation was significantly inhibited by superoxide dismutase, but not by catalase or sodium benzoate. The reconstructed system herein described is considered to be very close to NADPH-dependent microsomal lipid peroxidation system.     

Neutrality of amiodarone on the initiation and propagation of membrane lipid peroxidation.

Amiodarone is an iodinated benzofuran derivative largely used as an antiarrhythmic. Owing to the sensitivity of heart tissue to radicals, amiodarone was assayed for putative effects on lipid peroxidation studied in liposomes of soybean phosphatidylcholine and of bovine heart mitochondrial lipids used as model systems. Lipid peroxidations were initiated with Fe2+/ascorbic acid, and with peroxyl radicals generated from the azocompounds, AAPH and AMVN. These assays were carried out by following the quenching of the fluorescent probe cis-parinaric acid and by monitoring oxygen consumption. It has been ascertained that amiodarone does not protect or potentiate significantly the lipid peroxidation both lipidic systems. To fully ascertain the neutral behaviour of amiodarone in the lipid peroxidation process, the degradation of phospholipid acyl chains has been checked by GLC. These data confirm that amiodarone does not protect or potentiate lipid peroxidation to a significant extent. It is concluded that the limited effects of amiodarone might be related only indirectly with the lipid peroxidation. It is possible that the drug causes limited conformational and biophysical alterations in membrane phospholipid bilayers that can affect the process of peroxidation. Therefore, it is concluded that the therapeutic effects and benefits as a heart antiarrhythmic agent are independent of lipid peroxidation processes. Furthermore, the interaction of the drug with lipid bilayers does not induce significant conformational perturbations that could significantly favour or depress the peroxidation process.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

Effect of ferritin-containing fractions with different iron loading on lipid peroxidation.

Ferritin-containing fractions with different degrees of iron loading were prepared. All ferritin fractions stimulated the peroxidation of bovine brain phospholipid liposomes, as measured by the formation of thiobarbituric acid-reactive material. This stimulation was increased in the presence of ascorbate. Iron salts of equivalent concentration to those of the ferritin fractions were more stimulatory to lipid peroxidation at the higher iron concentrations. None of the fractions inhibited ascorbate-dependent peroxidation in the presence of added iron salts.     

Docosahexaenoic and arachidonic acid peroxidation: It's a within molecule cascade

Peroxidation is a well-known natural phenomenon associated with both health and disease. We compared the peroxidation kinetics of phosphatidylcholine (PC) molecules with different fatty acid compositions (i.e. 18:0, 18:1n-9, 18:2n-6, 20:4n-6 and 22:6n-3 at the sn-2 and 16:0 at sn-1 position) either as molecules free in solution or formed into liposomes. Fatty acid levels, oxygen consumption plus lipid hydroperoxide and malondialdehyde production were measured from the same incubations, at the same time during maximal elicitable peroxidation. PCs with highly peroxidizable fatty acids (i.e. 20:4n-6 and 22:6n-3) in the same incubation were found to be either fully peroxidized or intact. Rates of peroxidation of PCs with multiple bisallylic groups (i.e. 20:4n-6 and 22:6n-3) peroxidized at 2-3 times the rate per bisallylic bond than the same phospholipid with 18:2n-6. The results suggest that propagation of peroxidation (H-atom transfer) is firstly an intramolecular process that is several-fold faster than intermolecular peroxidation. PCs in solution peroxidized twice as fast as those in liposomes suggesting that only half of the phospholipids in liposomes were available to peroxidize i.e. the outer leaflet. Experiments on liposomes suggest that even after heavy peroxidation of the outer leaflet the inner leaflet is unaffected, indicating how cells may protect themselves from external peroxidation and maintain control over internal peroxidation. Intramolecular peroxidation may produce highly concentrated, localized sites of peroxidation product that together with internal control of peroxidation of the inner leaflet of membranes provide new insights into how cells control peroxidation at the membrane level.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Liposomes modulate docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells

The aim of this study was to investigate the effect of liposomes on docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells. Cytotoxicity of free docetaxel and docetaxel-containing liposomes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay in human hepatoma cell lines HepG2 and SMMC-7721. To the cell lines, blank liposomes prepared with soybean phosphatidylcholine (SPC), dimyristoylphosphocholine (DMPC), and dioleoylphosphocholine (DOPC) did not show any significant toxicity below a 0.02-mg/mL phospholipid concentration. On the other hand, free docetaxel showed IC₅₀ values of 9.13?×?10^?6?±?1.54?×?10^?5 and 1.58?×?10^?2?±?2.71?×?10^?2 mg/mL in HepG2 cells and SMMC-7721 cells, respectively, after of 24 hours of incubation. IC₅₀ values of docetaxel-encapsulating liposomes, measured in terms of total docetaxel concentration, were at least 1.5-fold higher than those of free docetaxel. SPC liposomes reduced cellular damage caused by free docetaxel, as evidenced by the attenuation of docetaxel-induced lactate dehydrogenase (LDH) leakage by over 11% after liposome encapsulation at each dosage. Docetaxel-induced oxidative membrane damage was monitored by the formation of the lipid peroxidation product, malondialdehyde (MDA), and the antioxidative property of SPC liposome was monitored by the suppression of superoxide dismutase (SOD). These data demonstrated that free docetaxel facilitated MDA formation and suppressed SOD, and that these membrane-damaging effects were reduced by SPC liposomes.     

Alloxan- and glutathione-dependent ferritin iron release and lipid peroxidation

The diabetogenic action of alloxan is believed to involve oxygen free radicals and iron. Incubation of glutathione (GSH) and alloxan with rat liver ferritin resulted in release of ferrous iron as assayed by spectrophotometric detection of ferrous-bathophenanthroline complex formation. Neither GSH nor alloxan alone mediated iron release from ferritin. Superoxide dismutase (SOD) and catalase did not affect initial rates of iron release whereas ceruloplasmin was an effective inhibitor of iron release. The reaction of GSH with alloxan resulted in the formation of the alloxan radical which was detected by ESR spectroscopy and by following the increase in absorbance at 310nm. In both instances, the addition of ferritin resulted in diminished alloxan radical detection. Incubation of GSH, alloxan, and ferritin with phospholipid liposomes also resulted in lipid peroxidation. Lipid peroxidation did not occur in the absence of ferritin. The rates of lipid peroxidation were not affected by the addition of SOD or catalase, but were inhibited by ceruloplasmin. These results suggest that the alloxan radical releases iron from ferritin and indicates that ferritin iron may be involved in alloxan-promoted lipid peroxidation.     

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions.     

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.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe³⁺-chelates, which initiated reactions that were dependent on membrane charge: Fe³⁺-EDTA and Fe³⁺-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe³⁺-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe³⁺-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.