Lipid peroxide formation in microsomes. The role of non-haem iron

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 (Fe²⁺ or Fe³⁺) 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 Fe²⁺ 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.     

Oxidation of 7-dehydrocholesterol by a mouse liver microsomal system dependent on reduced pyridine nucleotides

Aerobic incubation of 7-dehydrocholesterol with mouse liver microsomes in the presence of a detergent, an iron salt, and NADH or NADPH resulted in the conversion of the sterol to more polar products. In the presence of Fe(3+) or low levels of Fe(2+) the reaction was dependent upon reduced pyridine nucleotide and a microsomal enzyme system. At high levels of Fe(2+) or in the presence of Fe(2+) or Fe(3+) and ascorbic acid, nonenzymatic oxidation of 7-dehydrocholesterol occurred in the absence of NADH or NADPH. Chromatograms of products resulting from the enzyme-dependent and enzyme-independent reactions were similar. The enzymatic reaction was inhibited by certain chelating agents, by antioxidants, and by menadione, phenazine methosulfate, and ferricyanide. Low concentrations of EDTA stimulated the reaction and high concentrations inhibited it. In the complete system sterol oxidation was correlated with the peroxidation of microsomal lipids, but peroxidation of microsomal lipids proceeded more rapidly when either the sterol, the detergent, or both were omitted. Ergosterol was resistant to oxidation under conditions that caused extensive loss of 7-dehydrocholesterol. Microsomes from tissues other than liver were relatively inactive.     

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+.     

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.     

Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms

Treatment of hepatic microsomes with Fe(3+)/ascorbate activates UDP-glucuronyltransferase (UGT), a phenomenon totally prevented and reversed by reducing agents. At microM concentrations, iron and copper ions catalyze the formation of ROS through Fenton and/or Haber-Weiss reactions. Unlike iron ions, indiscriminate binding of copper ions to thiol groups of proteins different from the specialized copper-binding proteins may occur. Thus, we hypothesize that incubation of hepatic microsomes with the Cu(2+)/ascorbate system will lead to both UGT oxidative activation and Cu(2+)-binding induced inhibition, simultaneously. We studied the effects of Cu(2+) alone and in the presence of ascorbate on rat liver microsomal UGT activity. Our results show that the effects of both copper alone and in the presence of ascorbate were copper ion concentration- and incubation time-dependent. At very low Cu(2+) (25nM), this ion did not modify UGT activity. In the presence of ascorbate, however, UGT activity was increased. At higher copper concentrations (10 and 50microM), this ion led to UGT activity inhibition. In the presence of ascorbate, 10microM Cu(2+) activated UGT at short incubation periods but inhibited this enzyme at longer incubation times; 50microM Cu(2+) only inhibited UGT activity. Thiol reducing agent 2,4-dithiothreitol prevented and reversed UGT activation while EDTA prevented both, UGT activation and inhibition. Our results are consistent with a model in which Cu(2+)-induced oxidation of UGT leads to the activation of the enzyme, while Cu(2+)-binding leads to its inhibition. We discuss physiological and pathological implications of these findings.     

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.     

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.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

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.     

Melatonin and N-acetyl serotonin inhibit selectively enzymatic and non-enzymatic lipid peroxidation of rat liver microsomes

Melatonin (N-acetyl-5-methoxytryptamine) and its immediate precursor N-acetyl serotonin in the metabolism of tryptophan are free radical scavengers that have been found to protect against non-enzymatic lipid peroxidation in many experimental models. By contrast, little is known about the antioxidant ability of these indoleamines against NADPH enzymatic lipid peroxidation. The light emission produced by rat-liver microsomes, expressed as total cpm during 180 min of incubation at 37 degrees C, was two-fold greater in the presence of ascorbate (0.4mM) when compared with NADPH (0.2 mM). Maximal peaks of light emission produced by microsomes lipid peroxidized with ascorbic-Fe(2+) or NADPH and expressed as cpm were 354,208 (at 60 min) and 135,800 (at 15 min), respectively. During non-enzymatic lipid peroxidation a decrease of total chemiluminescence (inhibition of lipid peroxidation) was observed when increasing concentrations of melatonin were added to liver microsomes. The protective effect was concentration-dependent. The inhibition observed in light emission was coincident with the protection of the most PUFAs. Preincubation of microsomes with N-acetyl serotonin reduced these changes very dramatically. Thus, in the presence of both antioxidants (0.36, 0.75, 1.5 mM), light emission percent inhibition during non-enzymatic (ascorbate-Fe(2+)) lipid peroxidation of rat liver microsomes was for melatonin: 6.12, 16.20, 34.88 and for N-acetyl serotonin: 85.10, 88.48, 84.4 respectively. The incubation of rat liver microsomes in the presence of NADPH (0.36, 0.75, 1.5 mM) produce a sudden increase of chemiluminescence that gradually increased and reached a maximal value at about 15 min; however, N-acetyl serotonin reduced these changes very efficiently.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.     

Mechanisms of lipid peroxide formation in animal tissues

1. Homogenates of rat liver, spleen, heart and kidney form lipid peroxides when incubated in vitro and actively catalyse peroxide formation in emulsions of linoleic acid or linolenic acid. 2. In liver, catalytic activity is distributed throughout the nuclear, mitochondrial and microsomal fractions and is present in the 100000g supernatant. Activity is weak in the nuclear fraction. 3. Dilute (0·5%, w/v) homogenates catalyse peroxidation over the range pH5·0–8·0 but concentrated (5%, w/v) homogenates inhibit peroxidation and destroy peroxide if the solution is more alkaline than pH7·0. 4. Ascorbic acid increases the rate of peroxidation of unsaturated fatty acids catalysed by whole homogenates of liver, heart, kidney and spleen at pH6·0 but not at pH7·4. 5. Catalysis of peroxidation of unsaturated fatty acids by the mitochondrial and microsomal fractions of liver is inhibited by ascorbic acid at pH7·4 but the activity of the supernatant fraction is enhanced. 6. Inorganic iron or ferritin are active catalysts in the presence of ascorbic acid. 7. Lipid peroxide formation in linoleic acid or linolenic acid emulsions catalysed by tissue homogenates is partially inhibited by EDTA but stimulated by o-phenanthroline. 8. Cysteine or glutathione (1mm) inhibits peroxide formation catalysed by whole homogenates, mitochondria or haemoprotein. Inhibition increases with increase of pH.     

Free radical mediated interaction of ascorbic acid and ascorbate/Cu(II) with viral and plasmid DNAs

Previous studies indicate that ascorbic acid, when combined with copper or iron cleaves several viral DNA. ln this study, we generated the ascorbate radical anion electrochemically in a simple chemical environment without the participation of a metal ion. This solution possesses viral DNA scission activity. Ohe absence of catalytic metal ions [Fe (III) and Cu(II)] in the incubation medium was evidenced by metal chelating agents such as desferrioxamine and EDTA. Ohe radical quenching at high EDTA concentration was attributed to ionic strength of EDTA rather than metal chelation. Ohe effects of antioxidants, radical scavangers, catalase, superoxide dismutase and some proteins on DNA cleavage have been tested. Cleavage may not arise directly from ascorbate free radical but the reaction of the radical form of ascorbate with oxygen may produce the actual reactive species. Aerobic oxidation of ascorbate itself strictly requires transition metal catalysts, however electrochemically produced ascorbyl radical avoided the kinetic barrier that prevented direct oxidation of ascorbic acid with oxygen and eliminated the need for the transition metal ion catalysts.     

Studies on the hyperplasia (''regeneration'') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.     

The regeneration with ascorbate of Helix pomatia methaemocyanin mediated by hydrogen peroxide

The regeneration of the copper bands of H. pomatia haemocyanin proceeds much more slowly with an excess of ascorbate than with a slight excess of hydrogen peroxide. The regenerating agent with ascorbate is hydrogen perioxide, formed in its autoxidation at the air. This was concluded after regeneration experiments with ascorbate under strictly anaerobic conditions and at the air in the presence of catalase. The autoxidation of ascorbate was catalysed by Fe and Cu ions. In the presence of EDTA there is still metal catalysis, especially in slightly alkaline medium, due to the Fe(III)-EDTA complex. Addition of diethylenetriamine pentaacetate completely abolished the metal catalysis.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b(561) enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.     

Trace transition metal-catalyzed reactions in the microsomal metabolism of alkyl hydrazines to carbon-centered free radicals.

Radical production from alkyl hydrazines (i.e. phenelzine and benzylhydrazine) in rat liver microsomes has been proposed to occur via cytochrome P-450-catalyzed one-electron oxidation followed by beta-scission of an alkyl radical. In microsomes treated with phenelzine (2-phenylethylhydrazine), NADPH, and the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN), the 4-POBN/2-phenylethyl radical adduct was detected by electron paramagnetic resonance spectroscopy. The addition of catalase and superoxide dismutase resulted in a 28.5 and 24% decrease in radical production, respectively. The concentration of the 4-POBN/2-phenylethyl radical adduct decreased significantly in the presence of metal chelators, i.e. EDTA, diethylenetriaminepentaacetic acid (DTPA), or deferoxamine mesylate. When phenelzine was incubated with deferoxamine mesylate-washed microsomes and NADPH in Chelex-treated incubation buffer, no significant radical adduct formation was detected. Addition of iron-chelator complexes (either Fe(3+)-DTPA or Fe(3+)-EDTA) greatly stimulated production of the 4-POBN/2-phenylethyl radical adduct in this system. These results show that the 2-phenylethyl radical produced from phenelzine in a microsomal system arises via a trace transition metal-catalyzed reaction. This reaction may occur through oxidation of phenelzine by the hydroxyl radical, which has also been spin-trapped with 4-POBN in this system.     

Lipid peroxidation initiated by superoxide-dependent hydroxyl radicals using complexed iron and hydrogen peroxide

Iron salts stimulate lipid peroxidation by decomposing lipid peroxides to produce alkoxyl and peroxyl radicals which initiate further oxidation. In aqueous solution ferrous salts produce OH. radicals, a reactive species able to abstract hydrogen atoms from unsaturated fatty acids, and so can initiate lipid peroxidation. When iron salts are added to lipids, containing variable amounts of lipid peroxide, the former reaction is favoured and OH. radicals contribute little to the observed rate of peroxidation. When iron is complexed with EDTA, however, lipid peroxide decomposition is prevented, but the complex reacts with hydrogen peroxide to form OH. radicals which are seen to initiate lipid peroxidation. Superoxide radicals appear to play an important part in reducing the iron complex.     

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.