Antioxidant capacity of desferrioxamine and ferrioxamine in the chemically-initiated lipid peroxidation of rat erythrocyte ghost membranes

2,2'-Azo-bis-(2-amidinopropane) induces the thermal lipid peroxidation of red blood cells membranes by a mechanism that is not iron dependent. The peroxidation rate, as assessed by oxygen uptake or visible chemiluminescence measurements, can be diminished by micromolar concentrations of desferrioxamine (DF), with a median inhibitory concentration (the concentration of DF that reduces the lipid peroxidation rate to 50% of that observed without scavengers addition) of 10 microM. In these conditions, the DF/Fe3+ (1:2) complex is nearly five times less efficient than DF. The present data show that DF is able to trap the initiator radicals and/or the free radicals involved in the lipid peroxidative chain at micromolar concentrations, range in which the agent cannot be used as a general test for iron involvement.     

Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation

The generation of oxygen radicals and the process of lipid peroxidation have become a focus of attention for investigators in the fields of central nervous system (CNS) trauma and stroke (e.g., ischemia). Considering our level of understanding of free radical and lipid peroxidation chemistry, absolute proof for their involvement in the pathophysiology of traumatic and ischemic damage to the CNS has been meager. While direct, unequivocal evidence for the participation of free radicals and lipid peroxidation as primary contributors to the death of neuronal tissue waits to be established, numerous recent studies have provided considerable support for the occurrence of free radical and lipid peroxidation reactions in the injured or ischemic CNS. In addition, the pharmacological use of antioxidants and free radical scavengers in the treatment of experimental CNS trauma and ischemia has provided convincing, although indirect evidence, for the involvement of oxygen radicals and lipid peroxidation in these conditions. The intent of this and its companion paper is to review: 1) the biochemical processes which may give rise to free radical reactions in the CNS, 2) the environment of the ischemic cell as it may affect the generation of oxygen radicals and the catalysis of lipid peroxidation reactions, 3) the evidence for the involvement of free radical mechanisms in CNS trauma and ischemia, and 4) the pathophysiological consequences of these phenomena.     

A comparison of the kinetics of low-density lipoprotein oxidation induced by copper or by gamma-rays: influence of radiation dose-rate and copper concentration

The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis. The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or by oxygen free radicals generated by 60Co gamma-rays. The effects of copper concentration and irradiation dose-rate on LDL peroxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene, hydroperoxides, and thiobarbituric acid reactive substance formation as well as alpha-tocopherol disappearance. In the case of gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiation dose-rate. The radiation chemical rates (nu) increased with increasing dose-rate. Copper-induced LDL peroxidation followed two kinetic patterns: a slow kinetic for copper concentrations between 5-20 microM, and a fast kinetic for a copper concentration of 40 microM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copper binding site on apo-B. When compared with gamma-rays, copper ions act as drastic and powerful oxidants only at higher concentrations (> or = 40 microM).     

Protective effects of lazaroids against oxygen-free radicals induced lysosomal damage

Lipid peroxidation of membranes by oxygen free radicals has been implicated in various disease states. Different antioxidants and iron chelators have been used to reduce lipid peroxidation. Lazaroids have been used for the acute treatment of central nervous system disorders such as trauma and ischemia wherein lipid peroxidative processes take place.In this study we evaluated the effect of lazaroids (U-785 18F and U-74389F) on the release of acid phosphatase activity and formation of malondialdehyde (MDA) in rat liver lyosomes subjected to exogenously generated oxygen free radicals. There was a significant increase in the acid phosphatase release and MDA formation in the presence of oxygen free radicals. This was prevented by both the lazaroids. In a separate study the effect of lazaroid U-74389F was seen on the zymosan-stimulated polymorphonuclear (PMN) leukocyte-derived chemiluminescence. The PMN leukocyte chemiluminescent activity was attenuated by the lazaroid in a dose-dependent manner. These studies suggest that lazaroids may inhibit lipid peroxidation and stabilize the membrane.     

Influence of histidine on lipid peroxidation in sarcoplasmic reticulum.

The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidine's effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo.     

Carotenoids are degraded by free radicals but do not affect lipid peroxidation in unilamellar liposomes under different oxygen tensions

It has been questioned whether carotenoids can act as antioxidants in biological membranes. Biological membranes can be modeled for studies of lipid peroxidation using unilamellar liposomes. Both carotenoid depletion and lipid peroxidation were increased with increasing oxygen tension in unilamellar liposomes. Carotenoids in such liposomes were found to be very sensitive to degradation by free radicals generated from iron and 2,2'-azobis(2-amidinopropane) dihydrochloride, but they were not protective against lipid peroxidation. Lycopene and beta-carotene were more sensitive to free radical attack than lutein, zeaxanthin, and beta-cryptoxanthin.     

Acidic pH amplifies iron-mediated lipid peroxidation in cells

The goal of our study was to investigate the mechanism by which changes in extracellular pH influence lipid peroxidation processes. Ferrous iron can react with hydroperoxides, via a Fenton-type reaction, to initiate free radical chain processes. Iron is more soluble at lower pH values, therefore we hypothesized that decreasing the environmental pH would lead to increased iron-mediated lipid peroxidation. We used Photofrin, a photosensitizer that produces singlet oxygen, to introduce lipid hydroperoxides into leukemia cells (HL-60, K-562, and L1210). Singlet oxygen reacts with the PUFA of cells producing lipid hydroperoxides. Using EPR spin trapping with POBN, free radical formation from HL-60 cells was only detected when Photofrin, light, and ferrous iron were present. Free radical formation increased with increasing iron concentration; in the absence of extracellular iron, radical formation was below the limit of detection and lipid hydroperoxides accumulated in the membrane. In the presence of iron, lipid-derived radical formation in cells is pH dependent; the lower the extracellular pH (7.5-5.5), the higher the free radical flux; the lower the pH, the greater the membrane permeability induced in K-562 cells, as determined by trypan blue dye exclusion. These data demonstrate that lipid peroxidation processes, mediated by iron, are enhanced with decreasing extracellular pH. Thus, acidic pH not only releases iron from "safe" sites, but this iron will also be more damaging.     

Cooperation of antioxidants in protection against photosensitized oxidation

The aim of this study was to determine whether alpha-tocopherol and zeaxanthin offer synergistic protection against photosensitized lipid peroxidation mediated by singlet oxygen and free radicals. The antioxidant action of zeaxanthin and alpha-tocopherol was studied in liposomes made of phosphatidylcholine and cholesterol. Progress of lipid peroxidation, induced by aerobic photoexcitation of rose bengal, was monitored by the detection of lipid hydroperoxides and by electron spin resonance oximetry. In addition, cholesterol was employed as a mechanistic reporter molecule, which forms characteristic products of the interaction with singlet oxygen or free radicals. Cholesterol hydroperoxides were quantitatively determined by HPLC/electrochemical detection. HPLC/ultraviolet-visible (UV-VIS) absorption detection was used to measure concentrations of zeaxanthin and alpha-tocopherol. Zeaxanthin, even at concentrations of 2.5 microM, effectively protected against singlet oxygen-mediated lipid peroxidation but was rapidly consumed due to interaction with free radicals. alpha-Tocopherol alone was not effective in protecting against lipid peroxidation, even at concentration of 0.1 mM. Combinations of zeaxanthin and alpha-tocopherol exerted a synergistic protection against lipid peroxidation. The synergistic effect may be explained in terms of prevention of carotenoid consumption by effective scavenging of free radicals by alpha-tocopherol therefore allowing zeaxanthing to quench the primary oxidant-singlet oxygen effectively.     

Cellular signalling and free-radical modulating activities of the novel peptidomimetic L-glutamyl-histamine.

A novel histamine-containing peptidomimetic, L-glutamyl-histamine (L-Glu-Hist), has been synthesized and characterized as a possible cytokine mimic which might lead to cellular responses of improved specificity. The energy-minimized 3-D conformations of L-Glu-Hist derived from its chemical structure stabilize Fe2+-chelating complexes. L-Glu-Hist concentration-dependently accelerates a decrease in ferrous iron in ferrous sulfate solution and shows ferroxidase-like activity at concentrations less than 3 mM in the phenanthroline assay, whereas in the concentration range 3-20 mM it restricts the availability of Fe2+ to phenanthroline by chelation of iron ions. At low concentrations (less than or about 1 mM), L-Glu-Hist stimulates peroxidation of phosphatidylcholine in liposomes catalyzed by a superoxide anion radical (O2)-generating system (Fe2+ + ascorbate) and, at high concentrations (*10 mM), it suppresses lipid peroxidation (LPO) in liposomes. The stimulation of LPO by L-Glu-Hist is related to its ability at low concentrations (*0.05 mM) to release O2 free radicals as determined by the superoxide dismutase-inhibitable reduction of cytochrome c. The release of O2 by L-Glu-Hist might result from its ferroxidase-like activity, while its inhibition of LPO is due to chelation of Fe2+, prevention of the formation of free radicals, and degradation of lipid hydroperoxides at 5-20 mM L-Glu-Hist concentrations. L-Glu-Hist releases O2 at concentrations which stimulate [3H]thymidine incorporation into DNA and proliferation of mouse spleen lymphocytes and also of mononuclear cells from human blood. The induction of lymphocyte proliferation by L-Glu-Hist is dose-dependent in the 0.01-0.05 mM concentration range, although the maximal stimulation of LPO in the O2-dependent system is observed at higher L-Glu-Hist concentrations (*1 mM). Thus, low concentrations of oxygen free radicals released by L-Glu-Hist may provide a very fast, specific, and sensitive trigger for lymphocyte proliferation and immunoregulation.     

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.     

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.     

NADPH- and adriamycin-dependent microsomal release of iron and lipid peroxidation

In a previous study (Minotti, G., 1989, Arch. Biochem. Biophys. 268, 398-403) NADPH-supplemented microsomes were found to reduce adriamycin (ADR) to semiquinone free radical (ADR-.), which in turn autoxidized at the expense of oxygen to regenerate ADR and form O2-. Redox cycling of ADR was paralleled by reductive release of membrane-bound nonheme iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. In the present study, iron release was found to increase with concentration of ADR in a superoxide dismutase- and catalase-insensitive manner. This suggested that membrane-bound iron was reduced by ADR-. with negligible contribution by O2-. or interference by its dismutation product H2O2. Following release from microsomes, Fe2+ was reconverted to Fe3+ via two distinct mechanisms: (i) catalase-inhibitable oxidation by H2O2 and (ii) catalase-insensitive autoxidation at the expense of oxygen, which occurred upon chelation by ADR and increased with the ADR:Fe2+ molar ratio. Malondialdehyde formation, indicative of membrane lipid peroxidation, was observed when approximately 50% of Fe2+ was converted to Fe3+. This occurred in presence of catalase and low concentrations of ADR, which prevented Fe2+ oxidation and favored only partial Fe2+ autoxidation, respectively. Lipid peroxidation was inhibited by superoxide dismutase via increased formation of H2O2 from O2-. and excessive Fe2+ oxidation. Lipid peroxidation was also inhibited by high concentrations of ADR, which favored maximum Fe2+ release but also caused excessive Fe2+ autoxidation via formation of very high ADR:Fe2+ molar ratios. These results highlighted multiple and diverging effects of ADR, O2-., and H2O2 on iron release, iron (auto-)oxidation and lipid peroxidation. Stimulation of malondialdehyde formation by catalase suggested that lipid peroxidation was not promoted by reaction of Fe2+ with H2O2 and formation of hydroxyl radical. The requirement for both Fe2+ and Fe3+ was indicative of initiation by some type of Fe2+/Fe3+ complex.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes

Increased iron stores are associated with free radical generation and carcinogenesis. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of tumor initiation. Melatonin and structurally related indoles are effective in protecting against oxidative stress. The aim of the study was to compare the relative efficacies of melatonin, N-acetylserotonin (NAS), indole-3-propionic acid (IPA), and 5-hydroxy-indole-3-acetic acid (5HIAA) in altering basal and iron-induced lipid peroxidation in homogenates of hamster testes. To determine the effect of the indoles on the autoxidation of lipids, homogenates were incubated in the presence of each agent in concentrations of 0.0, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 2.5, or 5.0 mM. To study their effects on induced lipid peroxidation, homogenates were incubated with FeSO(4) (30 microM + H(2)O(2) (0.1 mM) + each of the indoles in the same concentrations as above. The degree of lipid peroxidation was expressed as concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. The indoles decreased both basal and iron-related lipid peroxidation in a concentration-dependent manner. Melatonin reduced basal MDA + 4-HDA levels when used at the concentrations of 0.25 mM or higher, and prevented iron-induced lipid peroxidation at concentrations of 1.0, 2.0, 2.5, or 5.0 mM. The lowest effective concentrations of NAS required to lower basal and iron-related lipid peroxidation were 0.05 mM and 0.25 mM, respectively. IPA, only when used in the highest concentrations of 2.5 mM or 5 mM inhibited basal lipid peroxidation levels and it was ineffective on the levels of MDA + 4-HDA due to iron damage. 5HIAA reduced basal lipid peroxidation when used at concentrations of 0.25 mM or higher, and it prevented iron-induced lipid peroxidation only at the highest applied concentration (5 mM). In conclusion, melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testes. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation.     

Trace element levels in the experimental peritonitis

Electron transfer from iron or copper ions to oxygen is an important example of cellular free radical initiation. Oxygen derived free radicals have been implicated as mediators of cellular injury in several model systems. To evaluate the importance of iron, copper and zinc levels on lipid peroxidation in peritonitis, we measured peritoneum malondialdehyde (MDA) as a marker of lipid peroxidation, zinc, copper, and iron levels during an animal model of intraperitoneal sepsis. Additionally the effects of the free radical scavenger alpha-tocopherol administration was studied. The peritoneum MDA, iron, copper and zinc levels were increased after induction of peritonitis with Escherichia Coli. The treatment with alpha-tocopherol was decreased the peritoneum MDA, iron and copper levels significantly, except the zinc level (p < 0.001, p < 0.001, p < 0.001, respectively). Additionally the alpha-tocopherol treatment for three days prior to injection of E.Coli more decreased MDA, copper and iron levels than that of the treatment with alpha-tocopherol at the time of injection of E. Coli (p < 0.001, p < 0.001, p<0.001, respectively). Our results indicated that copper, iron and zinc had important effects on peroxidation events in E. Coli induced peritonitis, and alpha-tocopherol treatment can improve the oxidant status.     

Effects of Ceruloplasmin on Superoxide-Dependent Iron Release from Ferritin and Lipid Peroxidation

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin. suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum. and inhibited at higher concentrations. However. the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.     

Neuroprotective activity of p-terphenyl leucomentins from the mushroom Paxillus panuoides

The neuroprotective mechanism of p-terphenyl leucomentins from the mushroom Paxillus panuoides was studied. Leucomentins showed potent inhibition of lipid peroxidation and H2O2 neurotoxicity, but free from any role as reactive oxygen species (ROS) scavengers. Iron-mediated oxidative damage has been implicated in these processes, as a provider of ROS via iron. Leucomentins can chelate iron when DNA is present with iron and H2O2, and so inhibiting DNA single strand breakage. These results suggest that the neuroprotective action of leucomentins is dependent on their ability to chelate iron.     

Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes

Exposure of red blood cells to oxygen radicals can induce hemoglobin damage and stimulate protein degradation, lipid peroxidation, and hemolysis. To determine if these events are linked, rabbit erythrocytes were incubated at 37 degrees C with various oxygen radical-generating systems and antioxidants. Protein degradation, measured by the production of free alanine, increased more than 11-fold in response to xanthine (X) + xanthine oxidase (XO). A similar increase in proteolysis occurred when the cells were incubated with acetaldehyde plus XO, with ascorbic acid plus iron (Asc + Fe), or with hydrogen peroxide (H2O2) alone. Upon addition of XO, increased proteolysis was evident within 5 min and was linear for up to 5 h. In contrast, lipid peroxidation, as shown by the production of malonyldialdehyde, conjugated dienes, or lipid hydroperoxides was observed only after 2 h of incubation with X + XO, acetaldehyde + XO, or H2O2. Ascorbate plus Fe2+ induced both protein degradation and lipid peroxidation; however, the addition of various antioxidants (urate, xanthine, glucose, or butylated hydroxytoluene) decreased lipid peroxidation without affecting proteolysis. Thus, these processes seem to occur by distinct mechanisms. Furthermore, at low concentrations of XO, protein degradation was clearly increased in the absence of detectable lipid peroxidation products. Hemolysis occurred only in a small number of cells (9%) and followed the appearance of lipid peroxidation products. Thus, an important response of red cells to oxygen radicals is rapid degradation of damaged cell proteins. Increased proteolysis seems to occur independently of membrane damage and to be a more sensitive indicator of cell exposure to oxygen radicals than is 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.