The effects of propofol or sevoflurane on free radical production after tourniquet induced ischaemia-reperfusion injury during knee arthroplasty.

BACKGROUND: We studied the effects of anesthesia with propofol or sevoflurane on the production of free oxygen radicals during total knee arthroplasty performed with the use of an ischemic tourniquet by measuring the levels of malondialdehyde (MDA). METHODS: We studied two groups of patients (20 patients in each group) who underwent total knee arthroplasty. To maintain anesthesia we delivered 66% nitrous oxide plus sevoflurane or propofol. Blood samples for the determination of the MDA levels were drawn before the application of the ischemic tourniquet and 5 and 30 minutes after its release. RESULTS: There were no differences between groups in regard to age, weight and duration of the tourniquet application. MDA levels decreased significantly in the propofol group 30 minutes after the release of the tourniquet (1.7 micromol litre(-1) vs 1.57 micromol litre(-1), Friedman's ANOVA, P = 0.007). In contrast, there was a small rise of the MDA levels in the sevoflurane group (1.82 micromol litre(-1) vs 1.96 micromol litre(-1), Friedman's ANOVA, P = 0.007). CONCLUSION: Propofol may have anti-oxidant properties in orthopaedic surgery requiring tourniquet application, but sevoflurane needs further study.

Oxidative stress in hepatic ischemia-reperfusion injury: the role of antioxidants and iron chelating compounds.

Ischemia-reperfusion (IR) injury is a multifactorial process triggered when the liver or other organs are transiently subjected to reduced blood supply followed by reperfusion. It has been shown that "reactive oxygen species" (ROS) are generated during ischemia and reperfusion and may represent pivotal mediators of the ensuing pathological complications. In some cases, however, moderate production of ROS may exert protective effects, a phenomenon presumably related to "ischemic preconditioning". This review will focus mainly on: a) describing the sources and the biochemical mechanisms of ROS generation during ischemia and reperfusion, b) discussing current developments in understanding the biochemical pathways by which ROS may induce toxic or protective effects, c) critically evaluating the results of previous attempts to counteract the toxic effects of ROS by using a variety of antioxidant and transition metal-chelating agents, and d) if feasible, proposing potential new pharmaceutical agents aimed at ameliorating ROS-inducing deleterious effects during reperfusion. It is concluded that ROS are generated from different sources, at different periods during IR, and may act by a variety of not well understood biochemical mechanisms which ultimately lead to cell damage and tissue failure.

Intracellular labile iron determines H2O2-induced apoptotic signaling via sustained activation of ASK1/JNK-p38 axis.

Hydrogen peroxide (H2O2) acts as a second messenger in signal transduction participating in several redox regulated pathways, including cytokine and growth factor stimulated signals. However, the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this work, using Jurkat T lymphoma cells and primary human umbilical vein endothelial cells, it was observed that changes in intracellular "labile iron" were able to modulate signal transduction in H2O2-induced apoptosis. Chelation of intracellular labile iron by desferrioxamine rendered cells resistant to H2O2-induced apoptosis. In order to identify the exact points of iron action, we investigated selected steps in H2O2-mediated apoptotic pathway, focusing on mitogen activated protein kinases (MAPKs) JNK, p38 and ERK. It was observed that spatiotemporal changes in intracellular labile iron, induced by H2O2, influenced the oxidation pattern of the upstream MAP3K ASK1 and promoted the sustained activation of JNK-p38 axis in a defined time-dependent context. Moreover, we indicate that H2O2 induced spatiotemporal changes in intracellular labile iron, at least in part, by triggering the destabilization of lysosomal compartments, promoting a concomitant early response in proteins of iron homeostasis. These results raise the possibility that iron-mediated oxidation of distinct proteins may be implicated in redox signaling processes. Since labile iron can be pharmacologically modified in vivo, it may represent a promising target for therapeutic interventions in related pathological conditions.

Uric acid and oxidative stress.

Uric acid is the final product of purine metabolism in humans. The final two reactions of its production catalyzing the conversion of hypoxanthine to xanthine and the latter to uric acid are catalysed by the enzyme xanthine oxidoreductase, which may attain two inter-convertible forms, namely xanthine dehydrogenase or xanthine oxidase. The latter uses molecular oxygen as electron acceptor and generates superoxide anion and other reactive oxygen products. The role of uric acid in conditions associated with oxidative stress is not entirely clear. Evidence mainly based on epidemiological studies suggests that increased serum levels of uric acid are a risk factor for cardiovascular disease where oxidative stress plays an important pathophysiological role. Also, allopurinol, a xanthine oxidoreductase inhibitor that lowers serum levels of uric acid exerts protective effects in situations associated with oxidative stress (e.g. ischaemia-reperfusion injury, cardiovascular disease). However, there is increasing experimental and clinical evidence showing that uric acid has an important role in vivo as an antioxidant. This review presents the current evidence regarding the antioxidant role of uric acid and suggests that it has an important role as an oxidative stress marker and a potential therapeutic role as an antioxidant. Further well designed clinical studies are needed to clarify the potential use of uric acid (or uric acid precursors) in diseases associated with oxidative stress.

Alterations in plasma oxidative stress markers after laparoscopic operations of the upper and lower abdomen.

The patient's position during laparoscopic surgery can have a clinically relevant effect on lower limb and splanchnic circulation; this factor has not yet been investigated with respect to oxidative stress markers. In order to assess this effect, a prospective clinical trial was designed wherein 2 groups of patients were studied. In group A, 15 patients underwent upper abdominal nonhepatobiliary operations (13 modified Nissen fundoplications and 2 Taylor vagotomies) in the head-up position. In group B, 15 patients underwent lower abdominal operations (10 laparoscopic colectomies and 5 inguinal hernia repairs) in the head-down position. The pneumoperitoneum was maintained at 14 mm Hg in all cases. Plasma concentrations of thiobarbituric-acid reactive substances (TBARS), a marker of lipid peroxidation, plasma total antioxidant status (TAS), and serum uric acid concentrations were measured preoperatively, 5 minutes after deflation of the pneumoperitoneum, and 24 hours postoperatively. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum activities were measured preoperatively and 24 hours postoperatively. In group A, there was a significant increase in TBARS levels (p<0.005) immediately after deflation of the pneumoperitoneum and a significant decrease in TAS and uric acid levels (p<0.005) in the first postoperative day. There was also a significant postoperative elevation in both ALT and AST activities (p<0.001). In group B, no significant increase was found in postoperative TBARS or transaminase levels. TAS and uric acid levels decreased significantly in the first postoperative day (p<0.05) and (p<0.005, respectively). In conclusion, these results show that a combination of pneumoperitoneum and the head-up position causes significant increase in lipid peroxidation, decrease in plasma TAS, and increase in transaminases. The mechanism responsible for these events could be the low-flow ischemia-reperfusion syndrome induced by the pneumoperitoneum and aggravated by the head-up position.

On the molecular mechanism of metmyoglobin-catalyzed reduction of hydrogen peroxide by ascorbate.

Hydrogen peroxide induces rapid oxidation of metmyoglobin with an apparent second order rate constant, k1 = 3.4 x 10(4) M-1 min-1. The product of this interaction is ferrylmyoglobin with an unstable free radical on the globin moiety. This activated form of myoglobin is able: (a) to initiate the peroxidation of erythrocyte membranes and (b) to form intra- and intermolecular covalent crosslinkings. The presence of ascorbic acid in amounts stoichiometric to H2O2 efficiently prevents all the above processes. Moreover, in the presence of ascorbic acid a cyclic process is taking place leading to H2O2 reduction, ascorbic acid oxidation, and unmodified metmyoglobin formation (reaction 1).

Glutathione-dependent reduction of peroxides during ferryl- and met-myoglobin interconversion: a potential protective mechanism in muscle.

Met-myoglobin is oxidized both by H2O2 and other hydroperoxides to a species with a higher iron valency state and the spectral characteristics of ferryl-myoglobin. Glutathione (GSH) reduces the latter species back to met-myoglobin with parallel oxidation to its disulfide (GSSG) but cannot reduce met-myoglobin to ferrous myoglobin. Under aerobic conditions, the GSH-mediated reduction of ferry-myoglobin is associated with O2 consumption and amounts of GSSG are formed far in excess over that of the peroxide added. Under anaerobic conditions, this ratio is close to unity. These results are interpreted in terms of a one-electron redox process involving the reduction of ferryl-myoglobin to met-myoglobin and the one-electron oxidation of GSH to its thiyl radical. Further reactions of thiyl radicals are influenced by the presence of oxygen which will be the determining factor in the ratio H2O2 added/GSSG formed. It is suggested that, when oxygen is limiting, myoglobin may serve as a protector of muscle cells against peroxides and other oxidants.

Glucose oxidase-produced H2O2 induces Ca2+-dependent DNA damage in human peripheral blood lymphocytes.

DNA of lymphocytes from human peripheral blood was analyzed by using the single cell gel electrophoresis technique (comet assay). The cells were used either as received from the donors or after treatment with various concentrations of the H2O2-generating enzyme glucose oxidase, in order to achieve a continuous flow of H2O2. The formation of single strand breaks (SSB) was dose-related but the time course of the induction of SSB by relatively low concentrations of glucose oxidase was of a biphasic mode with a fast increase 2 to 5 min after the addition of glucose oxidase followed by a gradual decrease toward the original base level during the next 35 to 60 min. This response of the cells appears to be based on the activation of already existing defense system(s) because it was shown that H2O2 is continuously released during the reaction time and the inhibition of protein synthesis does not affect the observed pattern. Supplementation of the growth medium with various antioxidants resulted in substantial protection only when the agents were taken up by the cells. The presence of the intracellular calcium chelator BAPTA protected the cells from H2O2-induced DNA damage in a dose-dependent manner. Only at the higher rate of H2O2-generation considerable DNA damage was observed in the presence of BAPTA. These results suggest that H2O2, at low concentrations induces DNA damage through intracellular Ca2+ -mediated processes, which lead to DNA strand breaks possibly by endonuclease activation.

SIN-1-induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay).

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1-15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca(2+)-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca(2+) changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H2O2-induced changes in Ca(2+) homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.

Effect of ambient oxygen concentration on lipofuscin accumulation in cultured rat heart myocytes--a novel in vitro model of lipofuscinogenesis.

The objective of this study was to elucidate the factors involved in the accumulation of lipofuscin in post-mitotic cells. The hypothesis that oxidative stress accelerates the rate of lipofuscin accumulation was tested by examining the effects of 5%, 20%, and 40% ambient oxygen concentration on lipofuscin content in cultured rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry at 7 and 12 days of in vitro age. Lipofuscin-emitted yellow autofluorescence increased in direct relationship to ambient oxygen concentration with age. Transmission electron microscopic examination of the cells after 3, 8, and 12 days in culture indicated a progressive time and oxygen dependent increase in the frequency and size of lipofuscin organelles. The results are interpreted to suggest that oxidative stress is one of the causal factors in the accumulation of lipofuscin.

Trimetazidine protects low-density lipoproteins from oxidation and cultured cells exposed to H(2)O(2) from DNA damage.

Trimetazidine is a well-established anti-ischemic drug, which has been used for long time in the treatment of pathological conditions related with the generation of reactive oxygen species. However, although extensively studied, its molecular mode of action remains largely unknown. In the present study, the ability of trimetazidine to protect low-density lipoproteins (LDL) from oxidation and cultured cells from H(2)O(2)-induced DNA damage was investigated. Trimetazidine, tested at concentrations 0.02 to 2.20 mM, was shown to offer significant protection to LDL exposed to three different oxidizing systems, namely copper, Fe/ascorbate, and met-myoglobin/H(2)O(2). The oxidizability of LDL was estimated by measuring, (i) the lag period, (ii) the maximal rate of conjugated diene formation, (iii) the total amount of conjugated dienes formed, (iv) the electrophoretic migration of LDL protein in agarose gels (REM), and (v) the inactivation of the enzyme PAF-acetylhydrolase present in LDL. In addition, the presence of trimetazidine decreased considerably the DNA damage in H(2)O(2)-exposed Jurkat cells in culture. H(2)O(2) was continuously generated by the action of glucose oxidase at a rate of 11.8 +/- 1.5 microM per min (60 ng enzyme per 100 microl), and DNA damage was assessed by the single cell gel electrophoresis assay (also called comet assay). The protection offered by trimetazidine in this system (about 30% at best) was transient, indicating modification of this agent during its action. These results indicate that trimetazidine can modulate the action of oxidizing agents in different systems. Although its mode of action is not clarified, the possibility that it acts as a lipid barrier permeable transition metal chelator is considered.

Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage.

The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or "comet" assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1-5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copper-chelating agents, did not protect against H(2)O(2)-induced cellular DNA damage. In fact, membrane permeable copper-chelating agents induced DNA damage in the absence of H(2)O(2). These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H(2)O(2)-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), an intracellular Ca(2+)-chelator, also protected against H(2)O(2)-induced DNA damage, it is likely that intracellular Ca(2+) changes are involved in this process as well. The exact role of Ca(2+) and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Toxic effects of daunorubicin on isolated and cultured heart cells from neonatal rats.

Various aspects of the cardiotoxicity of the anthracycline derivative and antineoplastic drug daunorubicin were investigated using isolated and cultured cells from neonatal rat hearts as a model system. Treatment of the cells with concentrations of daunorubicin of the same order of magnitude as those used in chemotherapy was accompanied by marked toxic effects, e.g. a decreased or abolished contraction, and release of lactate dehydrogenase, pyruvate and oxidized glutathione to the medium. A decreased frequency of contraction appeared to be the most sensitive probe of daunorubicin toxicity, followed by release of pyruvate and oxidized glutathione/lactate dehydrogenase. Daunorubicin and/or its metabolites also bound to cellular protein and DNA. Exposure to daunorubicin was shown to be accompanied by a rapid induction of primarily DT-diaphorase and a slower induction of glutathione transferase. The latter observations are interpreted to indicate a protective role of quinone- and peroxide-metabolizing enzymes, respectively, and support the hypothesis that daunorubicin toxicity involves generation of free radical derivatives, which initiate lipid peroxidation. This conclusion is further substantiated by the demonstration that addition of daunorubicin leads to an increased oxygen consumption.

Reduction of ferryl- and metmyoglobin to ferrous myoglobin by menadione-glutathione conjugate. Spectrophotometric studies under aerobic and anaerobic conditions.

Both metmyoglobin (MbIII) and ferrylmyoglobin (MbIV) are reduced by the menadiol-glutathione conjugate (GS-Q2-) to oxymyoglobin (MbIIO2) or deoxymyoglobin (MbII), depending whether the assay is carried out under aerobic or anaerobic conditions, respectively. Under aerobic conditions, the reduction of MbIII to MbIIO2 by GS-Q2- is associated with O2 consumption. The latter process is accounted for by (a) the autoxidation of the conjugate yielding H2O2 and (b) the rapid binding of O2 to MbII to yield MbIIO2. The ratio [O2]consumed/[MbIIO2]formed is approximately 1.5 at the time when MbIIO2 formation is maximal (at about 0.8 min). This ratio, higher than the unit, indicates that there is more than one O2-consuming reaction in this experimental model. The ratio of initial rates of O2 consumption and MbIIO2 formation is close to the unit [(-dO2/dt)/(+ dMbIIO2/dt) = 1.1]. The formation of H2O2 originating during the autoxidation of the GS-Q2- is substantially lower in the presence of MbIII, probably due to the heterolytic cleavage of the O--O bond of the peroxide by the hemoprotein. Although the latter reaction should yield MbIV, this species is not observed in the absorption spectrum, probably due to its rapid reduction by GS-Q2-. MbIV is reduced to MbIIO2 by the GS-Q2-. Whether this reaction takes place in one-electron transfer steps, that is, the sequence: MbIV----MbIII----MbIIO2 is difficult to evaluate by absorption spectral analysis, due to the rapid rate of the [MbIV----MbIIO2] transition. Under anaerobic conditions, the reduction of either MbIII or MbIV by GS-Q2- yields MbII as a stable molecular product. Anaerobic conditions prevent any further interaction of MbII with intermediates of O2 reduction derived from GS-Q2- autoxidation.

Laparoscopic surgery-induced changes in oxidative stress markers in human plasma.

BACKGROUND: The induction of the pneumoperitoneum increases intraabdominal pressure (IAP), causing splanchnic ischemia, whereas its deflation normalizes IAP and splanchnic blood flow. This procedure appears to represent an ischemia-reperfusion model in humans. METHODS: Thirty laparoscopic cholecystectomies (LC) were performed in 30 patients with a mean age of 54.6 +/- 15.6 years. A group of 20 patients mean age, 57.3 +/- 9.65 who underwent open cholecystectomy (OC) was also studied. Vein plasma levels of thiobarbituric acid-reactive substances (TBARS), a marker of free radical production; plasma total antioxidant status (TAS); and uric acid (UA) levels were measured preoperatively, 5 min after deflation of the pneumoperitoneum or at the end of operation, and 24 h postoperatively. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBL) levels were measured preoperatively and 24 h after the operation. RESULTS: In the LC group, significant elevations in the concentration of TBARS were observed in the early postoperative measurements in comparison with the preoperative measurements. TAS and UA levels were decreased significantly 24 h postoperatively compared to preoperative levels. The postoperative levels of AST, ALT, and TBL increased significantly in comparison with the preoperative levels. In the OC group, no alterations in the concentration of TBARS were observed in the postoperative period. The other parameters had changes similar to those recorded for the LC group. CONCLUSIONS: Free radical-induced lipid peroxidation associated with a decrease in plasma antioxidant capacity and UA levels as well as altered hepatic function is observed after deflation of the pneumoperitoneum. These results suggest that free radicals are generated at the end of a laparoscopic procedure, possibly as a result of an ischemia-reperfusion phenomenon induced by the inflation and deflation of the pneumoperitoneum.

Improved methods for automatic monitoring of contracting heart cells in culture.

The present communication describes improved methods for isolating and plating beating heart cells from neonatal rats using collagenase and collagen-coated petri dishes. The amplitude and frequency of contraction are continuously and simultaneously measured under well defined conditions and during prolonged periods of time with a highly sensitive and thermostated instrument. Additions, e.g. drugs and toxic agents, are made through an accessory pump system that involves extensive dilution of the added compound with medium; aliquots of medium can be withdrawn for estimation of metabolites. The system described is reliable and relatively inexpensive and allows a more extensive use of isolated heart cells, especially in studies of heart functions where small changes in amplitude and frequency of beating during prolonged periods of time are important.

Enzyme induction by daunorubicin in neonatal heart cells in culture.

The effect of the antineoplastic agent daunorubicin on beating heart cells from neonatal rats was investigated with respect to cell damage and induction of enzymes possibly involved in drug metabolism. Of the enzymes assayed DT-diaphorase and glutathione-S-transferase showed a two-to-four fold increase in activity: higher concentrations of daunorubicin inactivated glutathione-S-transferase. Daunorubicin toxicity increased in the presence of dicoumarol, a specific inhibitor of DT-diaphorase. These results indicate that both DT-diaphorase and glutathione-S-transferase may be involved in the metabolism of daunorubicin.

Effect of daunorubicin on subcellular pools of glutathione in cultured heart cells from neonatal rats.

Alterations in cellular GSH and its compartmentation were investigated as a possible mechanism of toxicity of the anthracycline derivative daunorubicin in neonatal heart cells. Cultured beating heart cells from neonatal rats were exposed to daunorubicin at therapeutically relevant concentrations and the resulting changes in cellular GSH as well as cytosolic and mitochondrial pools of GSH were determined. Toxicity was estimated as an increased permeability of the plasma membrane to cytosolic enzymes, e.g., lactate dehydrogenase. Control heart cells were found to contain 12.2 +/- 1.8 nmoles GSH/10(6) cells. Daunorubicin caused a rapid initial decrease followed by a transient increase in cellular GSH. The extent of the latter increase was dependent on the concentration of daunorubicin. High concentrations of daunorubicin gave only a slight increase followed by a pronounced decrease in cellular GSH. By applying a digitonin-based method the effect of daunorubicin on the cytosolic and mitochondrial pools of GSH were separated. The concentration of cytosolic and mitochondrial reduced GSH was estimated to be 8.9 +/- 1.5 nmoles/10(6) cells and 3.3 +/- 0.6 nmoles/10(6) cells, respectively. The results indicate that daunorubicin caused a decrease of cytosolic GSH and, after a short lag period, a release of alctate dehydrogenase. No decrease of mitochondrial GSH occurred under these conditions indicating that daunorubicin influences selectively cytosolic GSH. No lipid peroxidation products were detected in DRB-treated cells under conditions when lactate dehydrogenase was released. Likewise, addition of the iron-chelator desferrioxamin did not influence the release of lactate dehydrogenase, whereas dithiothreitol offered partial protection.(ABSTRACT TRUNCATED AT 250 WORDS)

Redox cycling of myoglobin and ascorbate: a potential protective mechanism against oxidative reperfusion injury in muscle.

Metmyoglobin catalyzes the decomposition of H2O2 as well as other hydroperoxides by using ascorbic acid as a substrate. The ratio of H2O2 reduced to ascorbate oxidized is close to one, whereas the rate of oxidation is directly proportional to both H2O2 and metmyoglobin concentrations. Ascorbate also prevents the protein modifications and the O2 evolution that accompany the reaction of metmyoglobin with hydroperoxides. In the absence of ascorbate, myoglobin and H2O2 promote the peroxidation of unsaturated fatty acids and, thus, may cause damage to cellular constituents. However, lipid peroxidation is inhibited in the presence of ascorbate and, for this reason, it is suggested that this heme protein functions in the opposite manner. The redox cycling of myoglobin by ascorbate may act as an important electron "sink" and defense mechanism against peroxides during oxidative challenge to muscle.