Recent evidence suggests that postischemic myocardial dysfunction ("stunning") may be mediated by oxygen free radicals, but the exact time window during which the critical radical-mediated damage develops remains unknown. Furthermore, the evidence for the oxyradical hypothesis is indirect and, therefore, inconclusive. Thus, the potent and cell-permeable antioxidant N-(2-mercaptopropionyl)-glycine (MPG) was administered as an intra-coronary infusion (8 mg/kg/hr) to three groups of open-chest dogs undergoing a 15-minute coronary occlusion followed by 4 hours of reperfusion. In group I (n = 8), the infusion of MPG was started 15 minutes before occlusion and ended 2 hours after reperfusion; in group II (n = 9), MPG was started 1 minute before reperfusion and ended 2 hours thereafter; in group III (n = 10), MPG was started 1 minute after reperfusion and ended 2 hours and 15 minutes thereafter. Control dogs (group IV) (n = 10) received vehicle. Recovery of contractile function (assessed as systolic wall thickening) was equivalent in groups I and II, and in both groups it was substantially greater than in controls (p less than 0.005 at 4 hours). In contrast, in group III recovery of function was indistinguishable from controls. To determine whether the protection afforded by MPG was due to inhibition of free radical reactions, myocardial production of free radicals was directly assessed by intracoronary infusion of the spin trap alpha-phenyl N-tert-butyl nitrone (PBN). In control dogs (group VII, n = 6), radical adducts of PBN were released in the coronary venous blood after reperfusion, with a burst occurring in the first 5 minutes. MPG given as in group II (group V, n = 5) markedly suppressed myocardial production of PBN adducts (delta = -98% over 3 hours, p less than 0.01 vs. controls); this effect was evident immediately after reperfusion. MPG given as in group III (group VI, n = 5) also suppressed PBN adduct production (delta = -83% over 3 hours, p less than 0.025 vs. controls), but this effect was delayed. Hence, the radicals important in myocardial stunning appear to be those generated immediately after reperfusion. In vitro studies demonstrated that MPG is an exceptionally powerful scavenger of .OH (rate constant = 8.1 x 10(9) M-1 sec-1 by pulse radiolysis) but has no significant effect on .O2- (rate constant less than 10(3) M-1 sec-1), H2O2 (rate constant = 1.6 M-1 sec-1), or non-.OH-initiated lipid peroxidation, suggesting that removal of .OH is the major mechanism of the beneficial effects of MPG.(ABSTRACT TRUNCATED AT 400 WORDS)
Recent studies suggest that oxygen free radicals may mediate postischemic myocardial dysfunction ("stunning"), but all the evidence for this hypothesis is indirect. Thus, we used electron paramagnetic resonance (EPR) spectroscopy and the spin trap, a-phenyl N-tert-butyl nitrone (PBN), to directly investigate whether free radicals are produced after a 15-min coronary artery occlusion and subsequent reperfusion in 30 open-chest dogs. After intracoronary infusion of PBN, EPR signals characteristic of oxygen-and carbon-centered radical adducts were detected in the venous blood draining from the ischemic/reperfused vascular bed. The myocardial release of PBN adducts began during coronary occlusion but increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of radicals abated but did not cease, persisting up to 3 h after reflow. The EPR spectra (as6 = 2.67-2.79 G, aN = 14.75-15.00 G) were consistent with the trapping by PBN of secondary oxygen-and carbon-centered radicals, such as alkoxy and alkyl radicals, which could be formed by reactions of primary oxygen radicals with membrane lipids. There was a linear, direct relationship between the magnitude of PBN adduct production and the degree of ischemic flow reduction. Recovery of contractile function (measured as systolic wall thickening) after reperfusion was greater (P < 0.05) in dogs given PBN than in controls.This study demonstrates that reversible regional myocardial ischemia in the intact animal is associated with prolonged free radical generation, and that the intensity of such generation is related to the severity of ischemia. The results provide direct evidence to support the hypothesis that reactive oxygen metabolites contribute to the persistent contractile dysfunction (myocardial stunning) observed after brief ischemia in vivo.
Electron paramagnetic resonance (EPR) spectroscopy was used to investigate whether (i) the free radicals produced in the "stunned" myocardium (myocardium with postischemic contractile dysfunction) are derived from 02, (ii) inhibition of radical reactions improves function, and (iii) i.v. spin traps are effective. Open-chest dogs undergoing a 15-min coronary occlusion received an i.v. infusion of the spin trap, a-phenyl N-tert-butylnitrone (PBN) (50 mg/kg). In group I (n = 6), EPR signals characteristic of radical adducts of PBN appeared in the coronary venous blood during ischemia and increased dramatically after reperfusion. In group II (n = 6), which received PBN and i.v. superoxide dismutase (SOD; 16,000 units/kg) plus catalase (12,000 units/kg), myocardial production of PBN adducts was undetectable during ischemia (A = -100%, P < 0.01 vs. group I) and markedly inhibited after reperfusion (A = -86%, P < 0.001). This effect was seen at all levels of ischemic zone flow but was relatively greater in the low-flow range. In group III (n = 8), the same dosages of SOD and catalase without PBN markedly enhanced contractile recovery (measured as systolic wall thickening) after reperfusion [P < 0.01 at 3 hr vs. controls (group IV, n = 7)]. Systemic plasma activity of SOD and catalase averaged 127 ± 24 and 123 ± 82 units/ml, respectively, 2 min after reperfusion. PBN produced no apparent adverse effects and actually improved postischemic contractile recovery in group I (P < 0.05 at 3 hr vs. controls). This study shows that (i) SOD and catalase are highly effective in blocking free radical reactions in vivo, (ii) the radicals generated in the "stunned" myocardium are derived from univalent reduction of 02, and (iu) inhibition of radical reactions improves functional recovery. The results provide direct, in vivo evidence to support the hypothesis that reactive oxygen metabolites play a causal role in the myocardial "stunning" seen after brief ischemia.Periods of myocardial ischemia that are too brief to cause necrosis are nevertheless followed by prolonged depression of contractility (1-3) or "stunning" (4), which is associated with numerous functional abnormalities (1)(2)(3)(4). Recent studies have shown that myocardial stunning can be attenuated by antioxidants (5-11), suggesting that the accumulation of reactive oxygen metabolites, such as superoxide radical (02-), hydrogen peroxide (H202), and hydroxyl radical (HO-), may play an important role in the pathogenesis of postischemic dysfunction. However, demonstration of the free radical hypothesis of myocardial stunning remains inconclusive because the evidence is indirect. In particular, it has not been determined whether oxygen-derived radicals are actually generated after a brief coronary occlusion and, if so, whether their generation is actually necessary for dysfunction to occur. An unambiguous link in vivo between oxyradical formation and myocardial stunning could be established if one could directly quantitate free radical generation with and without antio...
Rats fed a high-fat ethanol-containing diet for 2 weeks were found to generate free radicals in liver and heart in vivo. The radicals are believed to be carbon-centered radicals, were detected by administering spin-trapping agents to the rats, and were characterized by electron paramagnetic resonance spectroscopy. The radicals in the liver were demonstrated to be localized in the endoplasmic reticulum. Rats fed ethanol in a low-fat diet showed significantly less free radical generation. Control animals given isocaloric diets without ethanol showed no evidence of free radicals in liver and heart. When liver microsomes prepared from rats fed the high-fat ethanol diet were incubated in a system containing ethanol, NADPH, and a spin-trapping agent, the generation of 1-hydroxyethyl radicals was observed. The latter was verified by using "3C-substituted ethanol. Microsomes from animals fed the high-fat ethanol-containing diet had higher levels of cytochrome P-450 than microsomes from rats fed the low-fat ethanol-containing diet. The results suggest that the consumption of ethanol results in the production of free radicals in rat liver and heart in vivo that appear to initiate lipid peroxidation.Chronic excessive use of alcohol by humans results in liver disease (1, 2) characterized by fatty infiltration, which can lead to fibrotic degeneration and necrosis (1,3,4). However, the mechanisms leading to the development of alcoholic liver disease remain unclear. Lipid peroxidation was linked to ethanol consumption by Di Luzio and coworkers (5, 6), who reported that antioxidants prevented the development of fatty liver after a large acute dose of ethanol. Investigations by others have subsequently indicated that lipid peroxidation appears to occur in the livers of animals soon after the administration of an acute dose of alcohol. Much of the evidence for these claims has been based on the thiobarbituric acid assay for malondialdehyde in livers of animals treated with ethanol (7, 8), but increases in conjugated dienes and chemiluminescence as well as decreases in hepatic glutathione have also been reported (8). In addition, Muller and Sies (9) have reported an enhanced production of ethane and n-pentane, which are believed to be products of the peroxidative degradation of membrane lipids, during the metabolism of ethanol by perfused livers. These various findings have been interpreted as evidence that lipid peroxidation has occurred in the liver as a result of ethanol metabolism. Lipid peroxidation has also been proposed as a mechanism of ethanol-induced toxicity in the heart (10), gastric mucosa (11), and testes (12). However, the role of peroxidative events in the development of human alcoholrelated disease is highly controversial.Mechanisms that may initiate lipid peroxidation after ethanol exposure are also uncertain. Several laboratories have presented evidence that chronic ethanol feeding stimulates the production of hydroxyl radicals by liver microsomes (13,14). If this type of radical production occurs in the...
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