Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe(2+) or Fe(3+)) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe(2+) is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.     

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

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

Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation.

Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10^?5m) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe²⁺-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction.     

LIPID PEROXIDE FORMATION IN RAT BRAIN

Abstract— Lipid peroxide formation as measured by the thiobarbituric acid reaction was demonstrated in subcellular fractions of rat brain. The ascorbic acid induced nonenzymic lipid peroxidation was distributed in all the subcellular fractions with a maximum in microsomes. The NADPH dependent enzymic lipid peroxidation occurred mainly in microsomes and to a smaller extent in synaptosomes; NADH could replace NADPH for the enzymic lipid peroxidation under the assay conditions employed. Fe²⁺ but not Fe³⁺ stimulated the NADPH or NADH dependent lipid peroxide formation. The optimum conditions with respect to pH, ascorbic acid or NADPH concentration, time of incubation and protein concentration were studied. Heating the microsomes at 100^oCdid not influence the ascorbate-induced lipid peroxidation but completely abolished the NADPH linked peroxidation. Several heavy metal ions, surface active agents and EDTA were inhibitory to lipid peroxidation. The effect of thiol agents indicated that -SH groups were involved in the enzymic lipid peroxidation. Studies on subcellular fractions of developing rat brain showed an increasing trend in lipid peroxidation with the advancing age of the animal. No significant difference in lipid peroxidation was observed between brains from normal rats and those from rats affected by experimental allergic encephalomyelitis.     

Role of cytochrome P-450 in ochratoxin a-stimulated lipid peroxidation

The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe³⁺, ethylenediaminetetra-acetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe³⁺. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.     

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber–Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe³⁺ and Cu²⁺, using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu²⁺/ascorbate elicited more oxygen consumption than Fe³⁺/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu²⁺/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe³⁺/ascorbate. Finally, Fe³⁺/ascorbate and Cu²⁺/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu²⁺ decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe³⁺/ascorbate and Cu²⁺/ascorbate but not by Cu²⁺ alone. N-Acetylcysteine completely prevented the damage elicited by Cu²⁺/ascorbate, Fe³⁺/ascorbate and Cu²⁺ alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe³⁺/ascorbate, partially reversed that of Cu²⁺/ascorbate but failed to reverse the damage promoted by Cu²⁺ alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.     

Studies on the interaction of bleomycin A2 with rat lung microsomes. III. Effect of exogenous iron on bleomycin-mediated DNA chain breakage

The interaction of bleomycin A₂ with rat lung microsomes results in bleomycin-mediated DNA chain breakage due to the mixed-function oxidase catalyzed activation of bleomycin. This study demonstrates that the addition of exogenous Fe³⁺ significantly enhances the bleomycin-mediated cleavage of DNA deoxyribose, that the enhancing effect of Fe³⁺ is maximum when a 1:1 ratio of bleomycin to Fe³⁺ is achieved and that either NADPH or NADH can serve as pyridine cofactors for this reaction. Since the activation of bleomycin can be facilitated by iron in the Fe²⁺ form, these observations support the hypothesis that the mixed-function oxidase system may serve to maintain either adventitious or exogenous iron in the Fe²⁺ form. In the absence of DNA, the interaction of bleomycin with rat lung microsomes results in the self-inactivation of bleomycin, a reaction which is also enhanced by the addition of exogenous Fe³⁺. Thus, the microsomal mixed-function oxidase system represents an efficient biological system for the ‘activation-inactivation’ of bleomycin.     

NADPH-dependent drug redox cycling and lipid peroxidation in microsomes from human term placenta

1. NADPH-dependent iron and drug redox cycling, as well as lipid peroxidation process were investigated in microsomes isolated from human term placenta. 2. Paraquat and menadione were found to undergo redox cycling, catalyzed by NADPH:cytochrome P-450 reductase in placental microsomes. 3. The drug redox cycling was able to initiate microsomal lipid peroxidation in the presence of micromolar concentrations of iron and ethylenediaminetetraacetate (EDTA). 4. Superoxide was essential for the microsomal lipid peroxidation in the presence of iron and EDTA. 5. Drastic peroxidative conditions involving superoxide and prolonged incubation in the presence of iron were found to destroy flavin nucleotides, inhibit NADPH:cytochrome P-450 reductase and inhibit propagation step of lipid peroxidation. 6. Reactive oxo-complex formed between iron and superoxide is proposed as an ultimate species for the initiation of lipid peroxidation in microsomes from human term placenta as well as for the destruction of flavin nucleotides and inhibition of NADPH:cytochrome P-450 reductase as well as for impairment of promotion of lipid peroxidation under drastic peroxidative conditions.     

Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation

Our previous results indicated that cytochrome P450 destruction by benzene metabolites was caused mainly by benzoquinone (Soucek et al., Biochem. Pharmacol. 47 (1994) 2233-2242). The aim of this study was to investigate the interconversions between hydroquinone, semiquinone, and benzoquinone with regard to both spontaneous and enzymatic processes in order to test the above hypothesis. We have also studied the participation of hydroquinone and benzoquinone in OH radicals formation and lipid peroxidation as well as the role of ascorbate and transition metals. In buffered aqueous solution, hydroquinone was slowly oxidized to benzoquinone via a semiquinone radical. This conversion was slowed down by the addition of NADPH and completely stopped by microsomes in the presence of NADPH. Benzoquinone was reduced to semiquinone radical at a significantly higher rate and this conversion was stimulated by NADPH and more effectively by microsomes plus NADPH while semiquinone radical was quenched there. In microsomes with NADPH. both hydroquinone and benzoquinone stimulated the formation of OH radicals but inhibited peroxidation of lipids. Ascorbate at 0.5-5 mM concentration also produced significant generation of OH radicals in microsomes. Neither hydroquinone nor benzoquinone did change this ascorbate effect. On the contrary, 0.1-1.0 mM ascorbate stimulated peroxidation of lipids in microsomes whereas presence of hydroquinone or benzoquinone completely inhibited this deleterious effect of ascorbate. Iron-Fe2+ apparently played an important role in lipid peroxidation as shown by EDTA inhibition, but it did not influence OH radical production. In contrast, Fe3+ did not influence lipid peroxidation, but stimulated OH radical production. Thus, our results indicate that iron influenced the above processes depending on its oxidation state, but it did not influence hydroquinone/benzoquinone redox processes including the formation of semiquinone. It can be concluded that interconversions between hydroquinone and benzoquinone are influenced by NADPH and more effectively by the complete microsomal system. Ascorbate, well-known antioxidant produces OH radicals and peroxidation of lipids. On the other hand, both hydroquinone and benzoquinone appear to be very efficient inhibitors of lipid peroxidation.     

Clofibric acid or diethylmaleate supplemented diet decrease blood pressure in DOCA-salt treated male Sprague-Dawley rats - relation with liver antioxidant status

The effects of ascorbate and a-tocopherol as antioxidants and as co-operative factors against NADPH-dependent lipid peroxidation in human placental mitochondria have been studied. The addition of ascorbate at low concentration (up to 50 M) to the NADPH-generating system resulted in increasing lipid peroxidation and Fe³⁺ to Fe²⁺ reduction. High concentration of ascorbate (150 M), which produced maximal rate of ascorbate-dependent lipid peroxidation, was found to inhibit almost completely NADPH-dependent lipid peroxidation by maintaining too much iron in its reduced form. Either stimulatory or inhibitory effect of ascorbate on NADPH-dependent lipid peroxidation depends on the appropriate Fe³⁺/Fe²⁺ ratio. -Tocopherol caused a decrease of NADPH-dependent lipid peroxidation, inhibiting completely this process at 150 M concentration. The inhibitory effect of -tocopherol increased rapidly with the increasing ascorbate concentration, almost complete inhibition of NADPH-dependent lipid peroxidation being obtained at 25 M -tocopherol and 50 M ascorbate. This strong inhibitory combined effect of -tocopherol and ascorbate was independent of the Fe³⁺/Fe²⁺ ratio, as a-tocopherol is not able to reduce Fe³⁺ to Fe²⁺ under the conditions employed. These findings suggest that antioxidant effects of ascorbate in placental mitochondria are mediated by recycling of a-tocopherol rather than by strong reduction of Fe³⁺ to Fe²⁺. On the basis of the results obtained, we assume that adequate concentrations of a-tocopherol and ascorbate in placental tissue may prevent the release of lipid peroxide from placental mitochondria and therefore could be protective against the development of preeclampsia.     

Cytosolic factors which affect microsomal lipid peroxidation in lung and liver

Studies were carried out to determine the effects of lung and liver cytosol on pulmonary and hepatic mierosomal lipid peroxidation, to determine the cytosolic concentrations of various substances which affect lipid peroxidation, and to determine which of these substances is responsible for the effects of the cytosol on lipid peroxidation. Lung cytosol inhibits both enzymatic (NADPH-induced) and nonenzymatic (Fe²⁺-induced) lung microsomal lipid peroxidation. In contrast, liver cytosol stimulates lipid peroxidation in hepatic microsomes during incubation alone, enhances Fe²⁺-stimulated lipid peroxidation, and has no effect on the NADPH-induced response. Substances which are known to be involved in inhibition of lipid peroxidation, including glutathione, glutathione reductase, glutathione peroxidase, and superoxide dismutase, are found in greater concentrations in liver cytosol than in lung cytosol. However, ascorbate is found in approximately equal concentrations in pulmonary and hepatic cytosol. Most of the effects of the cytosol on lipid peroxidation seem to be due to ascorbate and glutathione. For example, ascorbate, in concentrations found in lung cytosol, inhibits lung microsomal lipid peroxidation to about the same extent as the cytosol. The effects of liver cytosol on hepatic microsomal lipid peroxidation can be duplicated by concentrations of ascorbate and glutathione normally found in the cytosol; i.e., ascorbate stimulates and glutathione inhibits lipid peroxidation with the net effect being similar to that of liver cytosol. The results indicate that ascorbate has opposite effects on pulmonary and hepatic microsomal lipid peroxidation and suggest that ascorbate plays a major role in protecting pulmonary tissue against the harmful effects of lipid peroxidation.     

Regional lipid peroxidation in rat brain in vitro: Possible role of endogenous iron

Lipoperoxidative capacity of various brain areas of aging rats was examined in vitro using the thiobarbituric acid test. Significant regional differences in the generation of lipid peroxides were found in freshly prepared homogenates from different areas of brain incubated under air. Incubation under oxygen resulted in marked stimulation of lipid peroxidation, with highest increases in hypothalamus (144%). Addition of exogenous Fe²⁺ and ascorbic acid resulted in stimulation of lipid peroxidation ranging from 10-fold in cortex to 20-fold in hypothalamus homogenates during incubation in air. A linear relationship was found between endogenous iron content in brain regions and their ability to produce lipid peroxides in vitro under oxygen for all areas except striatum. Several iron chelating agents effectively inhibited lipid peroxidation under hyperbaric oxygen whereas oxygenfree radical scavengers, as well as catalase and superoxide dismutase were not effective. It is concluded that regional differences in lipoperoxidative capacity of brain areas in vitro are in part governed by local endogenous iron content and may indicate regional susceptibility to oxidative damage.     

Studies on lipid peroxidation in normal and tumour tissues. The Yoshida rat liver tumour.

Reduced rates of lipid peroxidation have been observed in Yoshida hepatoma cells and microsomes when compared with appropriate control tissue (normal rat liver) under the same pro-oxidant conditions. The pro-oxidant conditions used were incubation with NADPH+ADP+iron or ascorbate+iron or exposure to gamma-irradiation. As previously shown with the Novikoff hepatoma, the relative concentrations of alpha-tocopherol and polyunsaturated fatty acids are important in conferring resistance to lipid peroxidation in the Yoshida hepatoma. Furthermore, NADPH-cytochrome c reductase and the NADPH-cytochrome P-450 electron transport chain, which are involved in the initiation and propagation of certain types of lipid peroxidation, are found at very much reduced levels in the Yoshida hepatoma. The relative importance of these aberrations are discussed.     

Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation

The mechanism of the formation of 4-hydroxynonenal through the NADPH-linked microsomal lipid peroxidation was investigated. The results were as follows: 4-hydroxynonenal arises exclusively from arachidonic acid contained in the polar phospholipids, neither arachidonic acid of the neutral lipids nor linoleic acid of the polar or neutral lipids are substrates for 4-hydroxynonenal generation. This finding results from the estimation of the specific radioactivity of 4-hydroxynonenal produced by microsomes prelabelled in vivo with [U-14C]arachidonic acid. Phospholipid-bound 15-hydroperoxyarachidonic acid would have the structural requirements needed for 4-hydroxynonenal (CH3-(CH2)4-CH(OH)-CH=CH-CHO). Microsomes supplemented with 15-hydroperoxyarachidonic acid and NADPH, ADP/iron converted only minimal amounts (0.6 mol%) of 15-hydroperoxyarachidonic acid into 4-hydroxynonenal; similarly, 15-hydroperoxyarachidonic acid incubated at pH 7.4 in the presence of ascorbate/iron yielded only small amounts of 4-hydroxynonenal with a rate orders of magnitude below that observed with microsomes. Phospholipid-bound 15-hydroperoxyarachidonic acid is therefore not a likely intermediate in the reaction pathway leading to 4-hydroxynonenal. The rate of 4-hydroxynonenal formation is highest during the very initial phase of its formation and the onset does not show a lag phase, suggesting a transient intermediate predominantly formed during the early phase of microsomal lipid peroxidation. After 60 min of incubation, 204 nmol polyunsaturated fatty acids (20 nmol 18:2, 143 nmol 20:4, 41 nmol 22:6) were lost per mg microsomal protein and the incubation mixture contained 206 nmol lipid peroxides, 71.6 nmol malonic dialdehyde and 4.6 nmol 4-hydroxynonenal per mg protein. Under artificial conditions (pH 1.0, ascorbate/iron, 20 h of incubation) not comparable to the microsomal peroxidation system, 15-hydroperoxyarachidonic acid can be decomposed in good yields (15 mol%) into 4-hydroxynonenal. Autoxidation of arachidonic acid in the presence of ascorbate/iron gave after 25 h of incubation 2.8 mol% (pH 7.4) and 1.5 mol% (pH 1.0) 4-hydroxynonenal. The most remarkable difference between the non-enzymic system and the enzymic microsomal system is that the latter forms 4-hydroxynonenal at a much higher rate.     

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.     

Effect of ascorbate in the reduction of transferrin-associated iron in endocytic vesicles

Externally added ascorbate or NADH effectively reduced ferricyanide and promoted the exit of Fe³⁺ originated from acid-destabilized transferrin contained inside endocytic vesicles. The effect of ascorbate was mediated by an ascorbate uptake system, and the effect of NADH was mediated by the membrane-associated oxidoreductase. At physiological concentrations of both ascorbate and NADH, the ascorbate transport and the NADH-oxidoreductase system were additive as measured by the rate of reduction of ferricyanide and by the mobilization of transferrin-associated iron. The results indicate that Fe³⁺ reduction may occur by a nonenzymatic reaction with ascorbate transported into the vesicle lumen. The ascorbate-mediated reduction of iron derived from transferrin occurring in the endosome could play a major role in cellular iron uptake.     

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.     

Lactoperoxidase-catalyzed lipid peroxidation of microsomal and artificial membranes

Lactoperoxidase, in the presence of H₂O₂, I^?, and rat liver microsomes, will peroxidize membrane lipids, as evidence by malondialdehyde formation. Fe³⁺ assists in the formation of malondialdehyde. Fe³⁺ can be added at the end of the reaction period as well as at the beginning with equal effectiveness, suggesting that it only acts to assist in the conversion of lipid peroxides, previously formed by lactoperoxidase, to malondialdehyde. The addition of EDTA to the microsomal reaction mixture results in a 40% decrease in malondialdehyde formation. The antioxidant butylated hydroxytoluene will completely block the formation of malondialdehyde. Malondialdehyde formation is not dependent upon the production of superoxide, singlet oxygen, or hydroxyl radicals. Peroxidation of membrane lipids by this system is equally effective in both intact microsomes and in liposomes, indicating that iodination of microsomal protein is not required for lipid peroxidation to occur.     

Antioxidant mechanism of Mn(II) in phospholipid peroxidation.

Antioxidant action of Mn2+ on radical-mediated lipid peroxidation without added iron in microsomal lipid liposomes and on iron-supported lipid peroxidation in phospholipid liposomes or in microsomes was investigated. High concentrations of Mn2+ above 50 microM inhibited 2,2'-azobis (2-amidinopropane) (ABAP)-supported lipid peroxidation without added iron at the early stage, while upon prolonged incubation, malondialdehyde production was rather enhanced as compared with the control in the absence of Mn2+. However, in a lipid-soluble radical initiator, 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN)-supported lipid peroxidation of methyl linoleate in methanol Mn2+ apparently did not scavenge lipid radicals and lipid peroxyl radicals, contrary to a previous report. At concentrations lower than 5 microM, Mn2+ competitively inhibited Fe(2+)-pyrophosphate-supported lipid peroxidation in liposomes consisting of phosphatidylcholine with arachidonic acid at the beta-position and phosphatidylserine dipalmitoyl, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in the presence of iron complex in microsomes. Iron reduction responsible for lipid peroxidation in microsomes was not influenced by Mn2+.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.