Measurement and characterization of postischemic free radical generation in the isolated perfused heart

Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the postischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g parallel = 2.033 and g perpendicular = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and aN = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.     

Alkyl free radicals from the beta-scission of fatty acid alkoxyl radicals as detected by spin trapping in a lipoxygenase system

2-Methyl-2-nitrosopropane (tNB)-radical adducts from incubation mixtures of fatty acids and soybean lipoxygenase in borate buffer (pH 9.0) were measured by electron paramagnetic resonance (EPR). In addition to the previously reported six-line signal of secondary carbon-centered radicals (RCHR'), a weak signal submerged in the baseline was detected after the peroxidation phase was finished. We propose that this radical is a decomposition product formed via beta-scission of fatty acid alkoxyl radicals. EPR spectra of tNB-radical adducts formed in mixtures of either linoleic acid, arachidonic acid, or 15-hydroperoxyeicosatetraenoic acid with lipoxygenase exhibited hyperfine structure characteristic of tNB/.CH2CH2-R with hyperfine coupling constants: aN = 17.1 G; aH beta = 11.2 G (2H); and aH gamma = 0.6 G (2H). In the case of linolenic acid, this radical tNB/.CH=CH-R' with hyperfine coupling constants: aN = 17.1 G; aH beta = 10.9 G (2H); aH gamma = 1.1 G; and aH delta = 0.5 G. In accord with the decomposition scheme of hydroperoxides derived from unsaturated fatty acids, the radical adducts tNB/.CH2CH2-R and tNB/.CH2-CH=CH-R' were assigned as the pentyl and 2-pentenyl radicals, respectively.     

EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.     

Real-time continuous-flow spin trapping of hydroxyl free radical in the ischemic and post-ischemic myocardium

Real-time monitoring of spin-trapped oxygen-derived free radicals released by the isolated ischemic and reperfused rat heart has been achieved by ESR analysis of the coronary effluents using continuous flow detection and high-speed acquisition techniques. Two nitrone spin traps 5,5-dimethyl pyrroline 1-oxide (Me2PnO) and 3,3,5,5-tetramethyl pyrroline 1-oxide (MePnO) have been separately perfused at a concentration of 40 mM during a sequence of 50 min of low-flow ischemia (1 ml/min) followed by 30 min of global ischemia and subsequent reperfusion at the control flow rate (14 ml/min). ESR spectra were sequentially obtained in 5-min or 30-s blocks during low-flow ischemia and reperfusion, respectively. 1. The results show the formation of OH. free radicals in the ischemic and reperfused heart, as demonstrated by the observation of Me2PnO-OH (aN = aH = 14.9 G; g = 2.0053) and Me4PnO-OH (aN = 15.2 G, aH = 16.8 G; g = 2.0055) spin adducts. There is no evidence of significant biological carbon-centered or peroxyl free radicals spin-adduct formation in the coronary effluents or in lipid extracts analyzed after reflow. 2. The OH. generation began 15-20 min after the onset of ischemia and was moderate, peaking at 30-40 min. During reperfusion, an intense formation of OH. spin adducts was observed, with a maximum at 30-60 s and a further gradual decrease over the following 2 min. 3. Cumulative integrated values of the amount of spin adducts released during the ischemic period show a Me2PnO-OH level fourfold greater than that of Me4PnO-OH. It was 2.5 times greater during reflow, reflecting slower kinetics with the more stable Me4PnO. 4. The original ESR detection technique developed in this study allows accurate real-time quantitative monitoring of the oxygen-derived free radicals generated during myocardial injury. It might provide a quick and reliable new means for assessing the efficacy of free-radical inhibitors.     

The enzymatic reduction of actinomycin D to a free radical species

Actinomycin D is an antitumor antibiotic in current clinical use. The ability of this and other antitumor antibiotics to undergo a reductive metabolism to produce free radical species has raised considerable interest in the literature in the past few years. The ability of actinomycin D to undergo a reductive metabolism was investigated using a ferredoxin reductase/NADPH system. This enzyme system has been used by a number of authors as a model for an enzymatic drug reducing system. In this study radical production was measured using direct ESR spectroscopy, the spin trapping technique, and oxygen consumption. It was shown that under anaerobic conditions the ferredoxin reductase/NADPH system could reduce actinomycin D to produce a semiquinone-imine free radical (aN = 2.8 (2N); aH = 2.8 (3H)). This radical production was found to be both drug and NADPH dependent. The effect of DNA on the drug's metabolism was also investigated. This was thought to be important because the proposed therapeutic action of the drug is centered on the DNA. Addition of calf thymus DNA to the reaction system abolished the signal produced by the actinomycin D, suggesting that intercalated actinomycin D is not a suitable substrate for ferredoxin reductase. Under aerobic conditions the ferredoxin reductase/NADPH/actinomycin D system generated the superoxide anion radical by reducing molecular oxygen. Evidence for this was obtained by spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO-superoxide radical adduct was produced (aN = 14.4 G; aH beta = 11.4 G; aH gamma = 1.3 G). Production of this adduct was drug and NADPH dependent, and was inhibited by superoxide dismutase. Superoxide production was also monitored by oxygen consumption studies.     

Photodegradation of hyaluronic acid: EPR and size exclusion chromatography study.

Photochemically induced radical reactions involving the lateral sequences and the end macromolecular chain groups of hyaluronic acid in aqueous solutions at 293K were studied by EPR spin trapping technique with DMPO (5,5-dimethylpyrroline-1-oxide). In the first 1-10 minutes of irradiation EPR indicates spin adducts of two carbon centered radicals with the splitting constants of aN = 1.60 mT, aH = 2.51 mT and aN = 1.56 mT, aH = 2.28 mT. After longer irradiation time (over 10 minutes) dominate two further DMPO adducts of radicals centered on hetero-atoms with splitting constants of aN = 1.44 mT, aH = 1.60 mT and of aN = 1.49 mT, aH = 1.49 mT. Simultaneously, molecular weight followed by SEC decreases, suggesting that UV irradiation leads to the breaking of interglycosidic bonds of hyaluronic acid main macromolecular chain.     

The NoH value in EPR spin trapping: a new parameter for the identification of 5,5-dimethyl-1-pyrroline-N-oxide spin adducts.

The ratio of the nitrogen to hydrogen hyperfine splittings (aN/aH) of spin adducts derived from the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) has been found to be a useful parameter for adduct identification. For example, this parameter makes it possible to distinguish between the superoxide (aN/aH = 1.22-1.26) and peroxyl (aN/aH = 1.33-1.40) radical adducts of DMPO in aqueous solution. Since the aN to aH ratio corrects for minor differences in EPR spectrometer calibration, it is a more reproducible parameter than the aN and aH values themselves.     

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.     

An ESR study of the nitroxide radical of pentastarch-conjugated deferoxamine

At higher concentrations, deferoxamine (DFO) reacts with hydroxyl radicals to produce a stable nitroxide free radical. Formation and decay of this nitroxide radical was investigated and compared with a novel modified pentastarch conjugate of DFO (MPS-DFO). Photolytic generation of hydroxyl radicals from H2O2 in the presence of free DFO produced a nitroxide radical with coupling constants of aN = 8.0 G and aH = 6.5 G. Under the same experimental conditions, equimolar concentrations of MPS-DFO produced an ESR signal of reduced intensity while iron-saturated MPS-DFO produced no signal. Incubation of free DFO with pentastarch (i.e., without conjugation) greatly decreased the intensity of the nitroxide radical signal. Using a spin-trapping technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), the pentastarch vehicle was shown to inhibit the DMPO-OH adduct formation. The decay of the DFO nitroxide radical decayed with a second-order rate constant while that of MPS-DFO decayed with a first-order rate constant. Thus, a novel derivative of DFO may provide some additional benefit in limiting DFO nitroxide radical formation and might explain the reported reduced in vivo toxicity of MPS-DFO relative to free DFO.     

Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1

Glycation, the nonenzymatic reaction between protein amino groups and reducing sugars, induces protein damage that has been linked to several pathological conditions, especially diabetes, and general aging. Here we describe the direct identification of a protein-bound free radical formed during early glycation of histone H1 in vitro. Earlier EPR analysis of thermal browning reactions between free amino acids and reducing sugars has implicated the sugar fragmentation product glycolaldehyde in the generation of a 1,4-disubstituted pyrazinium free radical cation. In order to evaluate the potential formation of this radical in vivo, the early glycation of BSA, lysozyme, and histone H1 by several sugars (D-glucose, D-ribose, ADP-ribose, glycolaldehyde) under conditions of physiological pH and temperature was examined by EPR. The pyrazinium free radical cation was identified on histone H1 glycated by glycolaldehyde (g = 2.00539, aN = 8.01 [2N], aH = 5.26 [4H], aH = 2.72 [4H]), or ADP-ribose. Reaction of glycoaldehyde with poly-L-lysine produced an identical signal, whereas reaction with BSA or lysozyme produced only a minor unresolved singlet signal. In the absence of oxygen the signal was stable over several days. Our results raise the possibility that pyrazinium radicals may form during glycation of histone H1 in vivo.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Oxygen-derived free radicals and 'stunned myocardium'

A brief, transient period of coronary artery occlusion (less than 20 minutes in duration) followed by reperfusion does not result in irreversible myocyte injury or death, yet the regional contractile function and high energy phosphate content of the previously ischemic tissue remains depressed or 'stunned' for hours to days following reperfusion. It has been suggested that this prolonged postischemic dysfunction of viable, previously ischemic myocardium may be a consequence of oxygen-derived free radicals generated during occlusion or at the time of reperfusion. Recent evidence demonstrates that free radical scavenging agents such as superoxide dismutase (SOD) + catalase, N-2-mercaptopropionylglycine, and allopurinol, administered prior to coronary artery occlusion, significantly enhance recovery of regional contractile function of the stunned, previously ischemic tissue. This improved contractile function was not, however, accompanied by improvements in high energy phosphate metabolism: infusion of SOD + catalase did not preserve ATP stores in the previously ischemic tissue. These data support the hypothesis that oxygen-derived free radicals contribute, at least in part, to the phenomenon of the stunned myocardium. The source or mechanisms of free radical production in the setting of brief, transient ischemia, however, remains to be elucidated.     

Use of Spin Traps in Intact Animals Undergoing Myocardial Ischemia/Reperfusion: A New Approach to Assessing the Role of Oxygen Radicals in Myocardial “Stunning”

Numerous studies have indirectly, suggested that oxygen-derived free radicals play an important path-ogenetic role in the prolonged depression of contractile function observed in myocardium reperfused after reversible ischemia (myocardial “stunning”). In order to provide direct evidence for the oxy-radical hypothesis of stunning, we administered the spin trap, α-phenyl N-tert-butyl nitrone (PBN), to open-chest dogs undergoing a 15-min coronary artery occlusion followed by reperfusion. Plasma of local coronary venous blood was analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR signals characteristic of radical adducts of PBN appeared during ischemia and increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of adducts abated but did not cease, persisting up to 3 h after reflow. The production of PBN adducts after reperfusion was inversely related to collateral flow during ischemia. PBN itself enhanced recovery of contractile function. indicating that the radicals trapped may play a pathogenetic role in myocardial stunning. Superoxide dismutase plus catalase attenuated PBN adduct production and, at the same time, improved recovery of contractile function. Antioxidant therapy given 1 min before reperfusion suppressed PBN adduct production and improved contractile recovery; however, the same therapy given 1 min after reperfusion did not suppress early radical production and did not attenuate contractile dysfunction. After i.v. administration, the elimination half-life of PBN was estimated to be approximately 4–5 h. The results demonstrate that 1) free radicals are produced in the stunned myocardium in intact animals; 2) inhibition of free radical production results in improved contractile recovery; and 3) the free radicals important in causing dysfunction are produced in the first few minutes of reperfusion. Taken together, these studies provide cogent evidence supporting the oxy-radical hypothesis of stunning in open-chest dogs. It is now critical to determine whether these results can be reproduced in conscious animal preparations.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

Detection of free radicals during the cellular metabolism of adriamycin

Experiments were conducted to determine which free radicals are generated during the metabolism of adriamycin (ADM) by canine tracheal epithelial (CTE) cells, guinea pig enterocytes, and rat hepatocytes. The technique employed in this study was spin trapping; the spin trap utilized was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). The spin adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed during the metabolism of ADM by CTE cells. However, the addition of dimethyl sulfoxide to the in vitro system suggested that superoxide is initially spin trapped by the nitrone, and that the adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) is rapidly bioreduced to afford DMPO-OH. The addition of superoxide dismutase to the system indicated that superoxide generation was primarily intracellular. The adriamycin semiquinone free radical (ADM-SQ) was produced during the metabolism by enterocytes and hepatocytes. The rate of the production of ADM-SQ was enhanced under anaerobic conditions, suggesting that molecular oxygen was responsible for the degradation of this carbon-centered free radical. However, spin trapping of oxygen radicals was not observed; this observation suggests that these reactive intermediates are not produced at concentrations sufficient for detection by spin-trapping experiments.     

Oxygen consumption and electron spin resonance studies of free radical production by alveolar cells exposed to anoxia: inhibiting effects of the antibiotic ceftazidime

By EPR spectroscopy, we investigated free radical production by cultured human alveolar cells subjected to anoxia/re-oxygenation (A/R), and tested the effects of ceftazidime, an antibiotic previously demonstrated to possess antioxidant properties. Two A/R models were performed on type II pneumocytes (A549 cell line), either on cells attached to culture dishes (monolayer A/R model; 3.5 h of anoxia, 30 min of re-oxygenation) or after cell detachment (suspension A/R model; 1 h of anoxia, 10 min of re-oxygenation). Ceftazidime and selective inhibitors (SOD, Tiron, L-NMMA) were added before anoxia. Free radical production was assessed by the EPR spin trapping technique. Oxygen consumption was monitored, in parallel with EPR studies, in the suspension A/R model. The production of free radical species was demonstrated by the generation of PBN-radical adducts: (a(N) = 15.2 G) in the monolayer A/R model and a six-line EPR spectrum (a(N) = 15.7 G and a(H) = 2.7 G) in the suspension A/R model. A kinetic study performed by oximetry, in parallel with EPR spectroscopy, demonstrated marked alterations of the cell respiratory function and that the free radical production started during anoxia and increased during re-oxygenation. In the suspension A/R model, the amplitude of EPR spectra were decreased upon the addition of 200 U/ml SOD (37% inhibition), 0.1 mM Tiron (67% inhibition) and 1 mM L-NMMA (43% inhibition). Addition of 1 mM ceftazidime decreased the amplitude of EPR spectra (37% inhibition) in both A/R models. Complementary in vitro EPR studies demonstrated that CAZ scavenged the hydroxyl radical (produced by the Fenton reaction). The protective effect of ceftazidime in the cell model could thus be linked to its ability to scavenge superoxide anions, nitrogen-derived species and hydroxyl radicals.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Reperfusion injury

Several lines of evidence point to a major role of oxygen free radicals in the pathogenesis of cell death or dysfunction in a variety of disease processes. Recent studies from this as well as other laboratories have demonstrated that oxygen free radicals play a major role in the pathogenesis of post-ischemic reperfusion injury in the heart. We have recently developed methods for direct measurement of radical species and/or specific byproducts of radical injury. Timely administration of oxygen radical scavengers reduced the quantity of free radicals generated following reperfusion and in addition improved recovery of post-ischemic ventricular function and metabolism. In a regionally ischemic model the free radical scavenger recombinant human superoxide dismutase also administered at the time of reflow was shown to limit infarct size. In this article we review the biophysical and molecular mechanisms of oxygen free radical generation that are viewed as contributing to post-ischemic reperfusion injury. We also discuss the mechanisms by which the body defends against free radical attack and the interrelationships of free radical injury to other mechanisms of tissue injury.     

Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study

The real-time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous-flow ESR using high-speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous-flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH = 0.188 mT and g = 2.0054). 1. The results showed that ascorbyl free radical release occurred mainly during sequences of low-flow ischemia (90 min) coupled or not with 30 min of zero-flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl-free-radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen-derived-free-radical-induced cellular damage. 2. The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia. 3. Perfusion of superoxide dismutase during low-flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free-radical-induced myocardial injury. 4. The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.     

Mechanisms of generation of oxygen radicals and reductive mobilization of ferritin iron by lipoamide dehydrogenase.

The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.