Oxidation of ferrous iron during peroxidation of lipid substrates

Oxidation of Fe2+ in solution was dependent upon medium composition and the presence of lipid. The complete oxidation of Fe2+ in 0.9% saline was markedly accelerated in the presence of phosphate or EDTA and the ferrous oxidation product formed was readily recoverable as Fe2+ by ascorbate reduction. In contrast, in the presence of either brain synaptosomal membranes, phospholipid liposomes, fatty acid micelles or H2O2, less than 50% of the Fe2+ oxidized during an incubation could be recovered as Fe2+ via reduction with ascorbate. In the presence of unsaturated lipid, oxidation of Fe2+ was associated with peroxidation of lipid, as assessed by the uptake of O2 and formation of thiobarbituric acid-reactive products during incubations. Although relatively little Fe2+ oxidation or lipid peroxidation occurred in saline with synaptosomes or linoleic acid micelles during an incubation with Fe2+ alone, significant Fe2+ oxidation and lipid peroxidation occurred in incubations containing a 1:1 ratio of Fe2+ and Fe3+. Extensive Fe2+ oxidation and lipid peroxidation also occurred with Fe2+ alone in saline incubations with either linolenic or arachidonic acid acid micelles or liposomes prepared from dilinoleoylphosphatidylcholine. While a 1:1 ratio of Fe2+ and Fe3+ enhanced thiobarbituric acid-reactive product formation in incubations containing linolenic or arachidonic micelles, it reduced the rate of O2 consumption as compared with Fe2+ alone. The results demonstrate that oxidation of Fe2+ in incubations containing lipid substrates is linked to and accelerated by peroxidation of those substrates. Furthermore, the results suggest that oxidation of Fe2+ in the presence of lipid or H2O2 creates forms of iron which differ from those formed during simple Fe2+ autoxidation.     

Effect of pH and oxalate on hydroquinone-derived hydroxyl radical formation during brown rot wood degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe2+ and H2O2 in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe3+ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe3+ at a rate constant of 4.5 x 10(3) M(-1)s(-1) at pH 4.0. Fe2+ is also very stable at a low pH. H2O2 generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe3+. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe3+. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 micro M), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

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.     

The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation

Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions.     

Microsomal reduction of low-molecular-weight Fe3+ chelates and ferritin: enhancement by adriamycin, paraquat, menadione, and anthraquinone 2-sulfonate and inhibition by oxygen

Reduction of iron is important in promoting xenobiotic-enhanced, microsomal lipid peroxidation, yet there is little evidence that Fe3+ chelates that promote lipid peroxidation can be reduced by the microsomal system. We have shown that rat liver microsomes catalyse NADPH-dependent reduction of Fe3+ without chelator, as well as Fe3+(ADP), Fe3+(ATP), Fe3+(citrate), Fe3+(EDTA), and ferrioxamine in N2. The NADPH oxidation that accompanied Fe3+ reduction was inhibited by CO for all chelates, except Fe3+ (EDTA). This implies that, except for Fe3+ (EDTA), cytochrome P450 was involved in reduction of the complexes. Adriamycin, paraquat, and anthraquinone 2-sulfonate (AQS) enhanced reduction of all the Fe3+ chelates, whereas menadione enhanced reduction only of Fe3+(ADP) and Fe3+(citrate). All the compounds enhanced oxidation of NADPH in the presence or absence of iron. This was not inhibited by CO, and the results are compatible with Fe3+ reduction occurring via the xenobiotic radicals produced by cytochrome P450 reductase. Microsomal reduction of the xenobiotics, except menadione, enabled the reduction and release of iron from ferritin. Fe3+ chelate reduction, both with and without xenobiotic, was inhibited by O2, although it still proceeded in air at 10-20% of the rate in N2. Iron-dependent lipid peroxidation was promoted by ADP and ATP, inhibited 50% by citrate, and completely inhibited by EDTA and desferrioxamine. Of the xenobiotics, only Adriamycin enhanced microsomal lipid peroxidation. These results indicate that the effects of chelators and xenobiotics on Fe3+ reduction do not correlate with lipid peroxidation and, although reduction is necessary, there must be other factors involved.     

Antioxidant properties of S-adenosyl-L-methionine in Fe(2+)-initiated oxidations

S-Adenosylmethionine (SAM) is protective against a variety of toxic agents that promote oxidative stress. One mechanism for this protective effect of SAM is increased synthesis of glutathione. We evaluated whether SAM is protective via possible antioxidant-like activities. Aerobic Hepes-buffered solutions of Fe2+ spontaneously oxidize and consume O2 with concomitant production of reactive oxygen species and oxidation of substrates to radical products, e.g., ethanol to hydroxyethyl radical. SAM inhibited this oxidation of ethanol and inhibited aerobic Fe2+ oxidation and consumption of O2. SAM did not regenerate Fe2+ from Fe3+ and was not consumed after incubation with Fe2+. SAM less effectively inhibited aerobic Fe2+ oxidation in the presence of competing chelating agents such as EDTA, citrate, and ADP. The effects of SAM were mimicked by S-adenosylhomocysteine, but not by methionine or methylthioadenosine. SAM did not inhibit Fe2+ oxidation by H2O2 and was a relatively poor inhibitor of the Fenton reaction. Lipid peroxidation initiated by Fe2+ in liposomes was associated with Fe2+ oxidation; these two processes were inhibited by SAM. However, SAM did not show significant peroxyl radical scavenging activity. SAM also inhibited the nonenzymatic lipid peroxidation initiated by Fe2+ + ascorbate in rat liver microsomes. These results suggest that SAM inhibits alcohol and lipid oxidation mainly by Fe2+ chelation and inhibition of Fe2+ autoxidation. This could represent an important mechanism by which SAM exerts cellular protective actions and reduces oxidative stress in biological systems.     

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.     

Microsomal lipid peroxidation: mechanisms of initiation. The role of iron and iron chelators

The role of iron and iron chelators in the initiation of microsomal lipid peroxidation has been investigated. It is shown that an Fe3+ chelate in order to be able to initiate enzymically induced lipid peroxidation in rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with O2; and (c) formation of a relatively stable perferryl radical. NADH can support lipid peroxidation in the presence of ADP-Fe3+ or oxalate-Fe3+ at rates comparable to those obtained with NADPH but requires 10 to 15 times higher concentrations of the Fe3+ chelates for maximal activity. The results are discussed in relation to earlier proposed mechanisms of microsomal lipid peroxidation.     

Cytochrome c-catalyzed membrane lipid peroxidation by hydrogen peroxide.

Cytochrome c(3+)-catalyzed peroxidation of phosphatidylcholine liposomes by hydrogen peroxide (H2O2) was indicated by the production of thiobarbituric acid reactive substances, oxygen consumption, and emission of spontaneous chemiluminescence. The iron chelator diethylenetriaminepentaacetic acid (DTPA) only partially inhibited peroxidation when H2O2 concentrations were 200 microM or greater. In contrast, iron compounds such as ferric chloride, potassium ferricyanide, and hemin induced H2O2-dependent lipid peroxidation which was totally inhibitable by DTPA. Cyanide and urate, which react at or near the cytochrome-heme, completely prevented lipid peroxidation, while hydroxyl radical scavengers and superoxide dismutase had very little or no inhibitory effect. Changes in liposome surface charge did not influence cytochrome c3+ plus H2O2-dependent peroxidation, but a net negative charge was critical in favoring cytochrome c(3+)-dependent, H2O2-independent lipid auto-oxidative processes. These results show that reaction of cytochrome c with H2O2 promotes membrane oxidation by more than one chemical mechanism, including formation of high oxidation states of iron at the cytochrome-heme and also by heme iron release at higher H2O2 concentrations. Cytochrome c3+ could react with mitochondrial H2O2 to yield "site-specific" mitochondrial membrane lipid peroxidation during tissue oxidant stress.     

Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.

Endothelial cells, macrophages, neutrophils, and neuronal cells generate superoxide (O2-) and nitric oxide (.NO) which can combine to form peroxynitrite anion (ONOO-). Peroxynitrite, known to oxidize sulfhydryls and to yield products indicative of hydroxyl radical (.OH) reaction with deoxyribose and dimethyl sulfoxide, is shown herein to induce membrane lipid peroxidation. Peroxynitrite addition to soybean phosphatidylcholine liposomes resulted in malondialdehyde and conjugated diene formation, as well as oxygen consumption. Lipid peroxidation was greater at acidic and neutral pH, with no significant lipid peroxidation occurring above pH 9.5. Addition of ferrous (Fe+2) or ferric (Fe+3) iron did not enhance lipid peroxide formation over that attributable to peroxynitrite alone. Diethylenetetraminepentacetic acid (DTPA) or iron removal from solutions by ion-exchange chromatography decreased conjugated diene formation by 25-50%. Iron did not play an essential role in initiating lipid peroxidation, since DTPA and iron depletion of reaction systems were only partially inhibitory. In contrast, desferrioxamine had an even greater concentration-dependent inhibitory effect, completely abolishing lipid peroxidation at 200 microM. The strong inhibitory effect of desferrioxamine on lipid peroxidation was due to direct reaction with peroxynitrous acid in addition to iron chelation. We conclude that the conjugate acid of peroxynitrite, peroxynitrous acid (ONOOH), and/or its decomposition products, i.e., .OH and nitrogen dioxide (.NO2), initiate lipid peroxidation without the requirement of iron. These observations demonstrate a potential mechanism contributing to O2-(-)and .NO-mediated cytotoxicity.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

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+.     

Reactions of Adriamycin with Microsomal Iron and Lipids

Iron plays a central role in oxidative injury, reportedly because it catalyzes superoxide- and hydrogen peroxide-dependent reactions yielding a powerful oxidant such as the hydroxyl radical. Iron is also thought to mediate the cardiotoxic and antitumour effects of adriamycin and related compounds. NADPH-supplemented microsomes reduce adriamycin to a semiquinone radical, which in turn re-oxidizes in the presence of oxygen to form superoxide and hence hydrogen peroxide. During this redox cycling membrane-bound nonheme iron undergoes superoxide dismutase- and catalase-insensitive reductive release. Membrane iron mobilization triggers lipid peroxidation, which is markedly enhanced by simultaneous addition of superoxide dismutase and catalase. The results indicate that : i) lipid peroxidation is mediated by the release of iron, yet the two reactions are governed by different mechanisms; and ii) oxygen radicals are not involved in or may actually inhibit adriamycin-induced lipid peroxidation. Microsomal iron delocalization and lipid peroxidation might represent oxyradical-independent mechanisms of adriamycin toxicity.     

Lipid peroxidation dependent aldrin epoxidation in liver microsomes, hepatocytes and granulation tissue cells

Lipid peroxidation activity was determined in liver microsomes, hepatocytes and cultured granuloma cells by measuring ethane and pentane production with an improved capillary gas chromatographic method. Lipid peroxidation initiated by ferrous ions and NADPH produced significantly more hydrocarbons at 4% O2 than under atmospheric (21% O2), hyperoxic or hypoxic conditions. In liver microsomes ferrous ions and ascorbic acid stimulated the non-enzymatic lipid peroxidation and concomitantly the epoxidation of aldrin. The results demonstrate that epoxidation of aldrin can be triggered by the iron initiated lipid peroxidation.     

Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue

Free radicals and reactive oxygen species (ROS) participate in physiological and pathological processes in the thyroid gland. Bivalent iron cation (ferrous, Fe(2+)), which initiates the Fenton reaction (Fe(2+) + H2O2 --> Fe(3+) + *OH + OH(-)) is frequently used to experimentally induce oxidative damage, including that caused by lipid peroxidation. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of carcinogenesis. In turn, melatonin is a well-known antioxidant and free radical scavenger. The aim of the study was to estimate the effect of melatonin on basal and iron-induced lipid peroxidation in homogenates of the porcine thyroid gland. In order to determine the effect of melatonin on the auto-oxidation of lipids, thyroid homogenates were incubated in the presence of that indoleamine in concentrations of 0.0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, or 5.0 mM. To study melatonin effects on iron-induced lipid peroxidation, the homogenates were incubated in the presence of FeSO(4) (40 microM) plus H2O2 (0.5 mM), and, additionally, in the presence of melatonin in the same concentrations as above. The degree of lipid peroxidation was expressed as the concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. Melatonin, in a concentration-dependent manner, decreased lipid peroxidation induced by Fenton reaction, without affecting the basal MDA + 4-HDA levels. In conclusion, melatonin protects against iron + H2O2-induced peroxidation of lipids in the porcine thyroid. Thus, the indoleamine would be expected to prevent pathological processes related to oxidative damage in the thyroid, cancer initiation included.     

Effects of deferrioxamine on iron-catalyzed lipid peroxidation.

The kinetics of iron binding by deferrioxamine B mesylate and the ramifications of this process upon iron-catalyzed lipid peroxidation were assessed. The relative rates of Fe(III) binding by deferrioxamine varied for the chelators tested as follows: ADP greater than AMP greater than citrate greater than histidine greater than EDTA. The addition of a fivefold molar excess of deferrioxamine to that of Fe(III) did not result in complete binding (within 10 min) for any of the Fe(III) chelates tested except ADP:Fe(III). The rates of Fe(III) binding by deferrioxamine were greater at lower pH and when the competing chelator concentration was high in relationship to iron. The relatively slow binding of Fe(III) by deferrioxamine also affected lipid peroxidation, an iron-dependent process. The addition of deferrioxamine to an ascorbate- and ADP:Fe(III)-dependent lipid peroxidation system resulted in a time-dependent inhibition or stimulation of malondialdehyde formation (i.e., lipid peroxidation), depending on the ratio of deferrioxamine to iron. Converse to Fe(III), the rates of Fe(II) binding by deferrioxamine from the chelators tested above were rapid and complete (within 1 min), and resulted in the oxidation of Fe(II) to Fe(III). Lipid peroxidation dependent on Fe(II) autoxidation was stimulated by the addition of deferrioxamine. Malondialdehyde formation in this system was inhibited by the addition of catalase, and a similar extent of lipid peroxidation was achieved by substituting hydrogen peroxide for deferrioxamine. Collectively, these results suggest that the kinetics of Fe(III) binding by deferrioxamine is a slow, variable process, whereas Fe(II) binding is considerably faster. The binding of either valence of iron by deferrioxamine may result in variable effects on iron-catalyzed processes, such as lipid peroxidation, either via slow binding of Fe(III) or the rapid binding of Fe(II) with concomitant Fe(II) oxidation.     

The consequences of lipid peroxidation in isolated hepatocytes.

Lipid peroxidation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to isolated rat hepatocytes and the resultant biochemical and morphological alterations investigated. As previously observed with microsomes, malonaldehyde formation was associated with the inactivation of glucose-6-phosphatase. Inhibition of microsomal oxidative drug metabolism was correlated with the release and subsequent inactivation of NADPH-cytochrome c reductase, whereas cytochrome P-450 destruction occurred only in the presence of high concentrations of the organic hydroperoxide which were associated with extensive malonaldehyde formation. Under these conditions there were also marked ultrastructural alterations in the hepatocytes which were not apparent after incubation in the presence of iron (less than or equal to 187 muM Fe3+). The latter treatment was, however, associated with moderate biochemical effects such as glucose-6-phosphatase inactivation and increased membrane permeability. The cellular defence system against lipid peroxidation is discussed and it is concluded that the isolated liver cell system provides a valuable tool for the study of lipid peroxidation and its pathological implications.     

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

Oxygen-radical-mediated lipid peroxidation and inhibition of ADP-induced platelet aggregation

The effects of lipid peroxidation on ADP-induced aggregation of washed rat platelets were examined using a oxygen-radical-generating system consisting of H2O2 and ferrous ion. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS). Incubation of the platelets with various concentrations of H2O2 (2-10 mM) in the presence of 10 microM Fe2+ resulted in a decrease of the aggregating capacity and an increase of TBARS value, depending on the concentrations of H2O2. Addition of catalase (0.1 mg/ml) to the incubation medium containing 10 microM Fe2+ and 10 mM H2O2 effectively protected the aggregating capacity, but superoxide dismutase (0.1 mg/ml) did not protect H2O2/Fe(2+)-induced inhibition of the platelet aggregation. The results of kinetic studies on the platelet aggregation with varying ADP and Ca2+ concentrations suggested that treatment of the platelets with H2O2/Fe2+ causes decreases in the binding affinities of ADP and Ca2+ for the platelets. On the basis of these results, change in the aggregating capacity of the platelets by treatment with H2O2/Fe2+ is discussed in relation to lipid peroxidation.