C. Coudray

ASSESSMENT OF RADICAL ACTIVITY DURING THE ACUTE PHASE OF MYOCARDIAL INFARCTION FOLLOWING FIBRINOLYSIS : UTILITY OF ASSAYING PLASMA MALONDIALDEHYDE

Numerous experimental and clinical studies have reported a role of radical forms of oxygen in the etiology of the manifestations of reperfusion of the ischemic myocardium. However, clinical results remain controversial. The aim of this study was to ascertain the existence of reperfusion-related radical stress after thrombolysis with a marker that is easy to use and reliable. Thirty patients hospitalized for acute myocardial infarction were involved in the study. Of these, 18 had been subjected to intravenous thrombolysis (Group I) and 12 had not (Group II). They were compared to two control groups who had no history of myocardial infarction. Of these, 16 were patients with coronary heart disease hospitalized for stable angina (Group III) and 17 were patients free of any known cardiovascular disease (Group IV). Radical activity was assessed in plasma samples taken from a peripheral vein over a 10-day period of hospitalization by measuring (1) malondialdehydes (MDA) concentrations using fluorometry techniques or HPLC, (2) the antioxidant activity of glutathione peroxidase (GPx) and (3) the concentration of various antiradical compounds (beta-carotene, vitamins A and E, uric acid). All patients in Group I had a patent artery on coronary angiography and showed a significant increase in plasma MDA when compared to those who had not been subjected to thrombolysis (3.15 +/- 0.62 and 2.70 +/- 0.40 mole/l of plasma, respectively). Furthermore, GPx plasma activity was also significantly increased following thrombolysis. By contrast, there was no significant alteration in the antiradical compounds measured. These data suggest that MDA measurements (an early measurement 1-2 days and a late measurement 5-7 days after reperfusion) by fluorometry is a good marker of radical stress during reperfusion in man. The assessment of this marker in patients might represent a simple and reliable test of reperfusion efficacy following thrombolysis, and it might enable one to test the effect of various antioxidant therapies associated with thrombolytic treatment.

Effects of adriamycin on chronic cardiotoxicity in selenium-deficient rats

SummaryAdriamycin (doxorubicin) is an antineoplastic drug used to treat various cancers; however, its chronic use is unfortunately accompanied by cardiotoxicity. This toxicity can be reduced by antioxidant agents such as selenium, and it is particularly interesting that cancer patients are usually deficient in this trace element, which suggests that its supplementation could contribute to beneficial treatment.We have examined the effect of adriamycin on chronic cardiotoxicity in 6-week selenium deficiency in rats. Selenium-deficient rats showed a considerable reduction of selenium levels and of selenium-containing glutathione peroxidase. Cardiac lipid peroxides increased slightly in the deficient rats, whereas plasma and heart lipid peroxides increased markedly in adriamycin-treated rats. This increase was greatly accentuated in selenium deficiency. These results suggest that free radical mechanism may be contributing to adriamycin toxicity, and above all show the importance of balancing the selenium levels in adriamycin-treated subjects to limit its harmful myocardial action. A decrease in adriamycin cardiotoxicity with no concomitant decrease in its antineoplastic activity would be of considerable value by improving the therapeutic benefit of the drug.

Effect of zinc deficiency on lipid peroxidation status and infarct size in rat hearts

The objective of this study was to investigate the effect of dietary zinc on endogenous production of lipid peroxides, and on myocardial infarct size in rats. Male rats were fed a zinc-deficient diet containing 4 ppm zinc, or a standard diet containing 60 ppm zinc. After 3 weeks of diet, half of the animals underwent occlusion of the left coronary artery. The remaining animals underwent sham operation without occlusion. Forty-eight hours later, the hearts were sampled and lipid peroxide levels and infarct size were evaluated. Coronary occlusion was associated with an increase in cardiac lipid peroxide levels which were more pronounced in the zinc deficient group. However, infarct size appeared to be independent from zinc deficiency, despite the free radical-mediated lipid peroxide augmentation reported here. The pharmacological limitation of infarct size in rats with permanent coronary occlusion is discussed.

Evidence of cytosolic iron release during post‐ischaemic reperfusion of isolated rat hearts Influence on spin‐trapping experiments with DMPO

Previous studies of oxygen‐derived free radical generation based on spin‐trapping methods have shown a signal formed of six bands (sextet) using electron spin resonance spectrometry (ESR) or coronary effluents collected during post‐ischaemic reperfusion of isolated hearts perfused with 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO). The origin of this signal has recently become controversial. In the present study we show that, in the rat, this sextet and cytosolic iron release occur simultaneously, and that this signal can be inhibited by the iron chelator desferrioxamine. It also appears that the iron release is not protein bound, and could therefore have a marked catalytic activity. This may be responsible for the production or an artefactural signal observed as the sextet.

Ischemia and reperfusion injury in isolated rat heart: Effect of reperfusion duration on xanthine oxidase, lipid peroxidation, and enzyme antioxidant systems in myocardium

SummaryThe aim of this work was to assess the catalytic activity of xanthine oxidase, the level of lipid peroxides and enzymic antioxidant systems in isolated rat heart muscle subjected to a globally partial ischemia followed by varying durations of reperfusion.After 40 min of globally partial ischemia (residual perfusion flow rate: 0.1 ml/min), four different durations of reperfusion were investigated (0, 20, 40, and 60 min). After each experimental ischemia/reperfusion sequence, the heart was frozen in liquid nitrogen. Lipid peroxides were assayed in the cardiac homogenate and the catalytic of xanthine oxidase and enzymic antioxidant systems (glutathione peroxidase, superoxide dismutase and catalase) were determined in the centrifuged supernatant. In the different experimental protocols studied in this work, there was no significant increase in the activity of cardiac xanthine oxidase or in the level of lipid peroxides when compared to the non reperfused or to the continuously perfused hearts. Indeed, enzymic antioxidant systems were also not significantly modified in the different periods of reperfusion when compared to control hearts (continuously perfused hearts).These results suggest that xanthine oxidase is apparently not a major source of free radicals in the course of an ischemia-reperfusion sequence in heart muscle, in particular, if we consider the early phases of reperfusion. The process of lipid peroxidation, assessed by assaying thiobarbituric acid reactants, is not a predominant phenomenon of reperfusion-induced injury, at least in the experimental model used here. However, enzymic antioxidant systems investigated in this study do not seem modified. This could mean that the small quantity of oxygen free radicals produced does not overwhelm the enzymic antioxidant systems of myocardium which is in agreement with peroxidatized lipid results.

Effect of selenium supplementation on biological constants and antioxidant status in rats

Oxygen-derived free radicals are currently suspected to be widely involved in the aetiology of several clinical disorders. In animals as well as in man, antioxidant trials are often undertaken to prevent oxidative stress. Among antioxidant molecules selenium has been largely studied. This study shows that plasma Se level is not a good index of Se status in the organism, at least at high levels of selenium. Red blood cell Se seems to be a more reliable index of Se status and could replace plasma Se level in the supplementation trials both in animals and humans. Se supplementation did not result in a significant decrease in oxidative stress markers as evaluated by blood and tissue malondialdehyde contents in healthy animals. Furthermore, heart function was altered and plasma Alanine aminotransferase activity was significantly increased in the selenium-supplemented group, which could reflect a slight subtoxic effect of selenium supplementation at the level used here. In view of the results presented, the maximum selenium content in animal diet in selenium supplementation experiments should not be higher than 2 mg/kg.

Selenium supplementation decreases the pro-oxidant and cardiotoxicity effects of adriamycin in the rat.

Adriamycin (doxorubicin; ADR) is an antineoplastic drug used to treat various cancers; however, its chronic us is unfortunately accompanied by cardiotoxicity. Previous results suggested that a free radical mechanism may contribute to ADR toxicity. Because it is often reported that cancer patients are deficient in selenium (Se), we hypothesised that ADR toxicity might be reduced by antioxidant agents such as vitamin E and Se. ADR-induced cardiotoxicity was examined in rats maintained on a Se-supplemented diet. The animals were kept on either a standard (0.22 mg/kg) or a Se-supplemented (2.5 mg/kg) diet starting 4 weeks prior to the first ADR treatment. ADR, or its excipient, was administered intraperitoneally in six equal injections over a period of 3 weeks giving a cumulative dose of 15 mg/kg body weight. Blood was collected in the thoracic cavity and the heart was subjected to a sequence of perfusion/partial ischemia/reperfusion ex vivo. Se status, GPx activities, vitamins E and C and MDA contents were determined on RBC and cardiac homogenates. ADR-treated rats showed a significant decrease in RBC Se (-29%) and in RBC Se-GPx (-34%) compared to the placebo group, and a significant increase in RBC MDA content (+2000%). This latter increase was attenuated by the Se supplementation (+1600%). In parallel, RBC vitamin E decreased markedly in the ADR-treated group (-50%) and was totally restored by the Se supplementation. Cardiac biochemical analyses confirmed the blood results. The present data confirm that a free radical mechanism does contribute to ADR toxicity, and show the importance of balancing the Se levels in ADR-treated subjects to limit its harmful myocardial action. Adecrease in ADR toxicity with no concomitant decrease in its antineoplastic activity would be of considerable value by improving the therapeutic benefit of the drug.

Xanthine oxidase activity and lipid peroxide content following different types of ischemia in the isolated rat heart

It is currently believed that reactive oxygen species are produced in the heart post-ischemia-reperfusion, causing pathophysiological disorders. Studies reported in the literature dealing with this subject have generated contradictory findings. The aim of this study was to assess the catalytic activity of the superoxide anion-producing enzyme xanthine oxidase, and the level of lipid peroxides in isolated rat heart muscle undergoing ischemia of varying duration and severity followed by reperfusion.Three levels of ischemia were investigated: total, and partial at either 0.10 or 0.35 ml/min (residual flow rate). Three different periods of ischemia were examined in each case. After each period of ischemia, followed by 10 min of reperfusion, the heart was frozen in liquid nitrogen. Xanthine oxidase activity and lipid peroxide levels were assayed in the cardiac homogenate and in the centrifuged supernatant, respectively. In the different experimental protocols studied here, both cardiac xanthine oxidase and lipid peroxide levels remained statistically unchanged compared to the continuously perfused control hearts. Moreover, in a recent study (Boucher et al., FEBS Lett. 203, 261–264, 1992), we were unable to detect reactive oxygen species in perfusate upon reperfusion of ischemic rat hearts.These results suggest that changes in xanthine oxidase activity during myocardial ischemia-reperfusion, and lipid peroxidation, as assessed by measuring thiobarbituric acid reactants and lipid hydroperoxides, are not predominant phenomena in ischemia-reperfusion-induced injury, at least in the experimental model used in this study. The significance of these results is discussed in the light of the popular point of view suggested first by McCord in 1985 concerning the conversion of xanthine dehydrogenase to xanthine oxidase in heart in the course of ischemia.

Stimulation of Peroxidative Reactions by a High Dose of Allopurinol in the Rat Myocardium

Dear Sir, Xanthine oxidase-superoxide production has been implicated in the initiation of the peroxidative process in the myocardium under certain pathophysiological conditions [1]. In line with this hypothesis are reports which show that allopurinol [4-hydroxypyrazolo-(3,4d) pyrimidine], a xanthine oxidase inhibitor, can prevent or significantly reduce myocardial reperfusion injury [2]. In most of these studies, however, the doses of allopurinol required are very high, and in all cases, they are far above those used in humans for the treatment of gout. Different reports have shown that overdosing with allopurinol can exert certain toxic effects in the rat [3,4]. In a previous study [4] we have shown that chronic administration of allopurinol (20 mg/kg twice a day for 5 days) fails to exert any cardioprotective effect in rats submitted to permanent coronary artery ligation. Moreover, our results suggested the occurrence of a toxic effect of the treatment, as shown by an increase in tissue edema in the nonischemic portion of the myocardium in treated animals. In the present study we examined the effects of chronic administration of allopurinol (according to the protocol previously described [4]) on (1) myocardial malondialdehyde (MDA) content, taken as an index of lipid peroxidation, (2) superoxide dismutase (SOD) and catalase activities, which are the main enzymatic systems involved in the clearance of oxygen free radicals, and (3) xanthine oxidase activity, which is supposed to be inhibited by the treatment. The experiment was carried out on male adult Wistar rats (220-250 g). Allopurinol (Sigma Chemical Company), suspended in an aqueous solution of gum arabic (20 g/l), was administered orally at 20 mg/kg twice a day for 5 days. Control animals received a similar treatment using vehicle only (gum arabic at 2%). Volumes of administration were adjusted to 1 ml for 100 g body weight. On the last day of treatment, animals (n = 9/group) were anesthetized with sodium pentobarbital (40 mg/kg i.p., 1 ml/kg) and heparin was administered via a femoral vein (100 IU/100 g). The hearts were cut out and perfused via the aorta for 4 minutes to remove any blood from the vascular beds. The perfusate was a Krebs-Henseleit buffer (Ca 2÷ = 2.4 mM; K ÷ = 5.6 mM; glucose 11 mM) equilibrated with a gas mixture of 95% 02 and 5% C02 at 37°C, pH 7.4. Following this perfusion, the hearts were quickly frozen in liquid nitrogen. MDA myocardial content, as well as SOD, catalase, and xanthine oxidase activities, were measured using conventional assays (using the method of Ohkawa et al. [5] for MDA measurement, of Marklund [6] for SOD activity, of Beers and Sizer [7] for catalase activity, and of Sugiura et al. [8] for xanthine oxidase activity). Results were expressed as mean _+ standard error of the mean (SEM) and compared using Students t test following analysis of variance by Fishers test. Treatment with allopurinol is likely to be associated with stimulation of a peroxidative process, shown by the elevation of myocardial MDA content and by the increase of SOD and catalase activities. Moreover, the activity of xanthine oxidase that has the potential to generate superoxide anions appears paradoxically to be increased in the myocardium of treated animals. Allopurinol is both a substrate and an inhibitor of xanthine oxidase [9]; during the reaction it is transformed into alloxanthine (oxypurinol). This metabolite is itself an inhibitor of the enzyme and is slowly cleared by the kidney. Suzuki and Sudo [3] have reported an elevation of both MDA content and xanthine oxidase activity in the kidney of rats 3 days after the onset of treatment with allopurinol (100 mg/kg b.w./ day, per os). These authors suggested that allopurinol could lead, in the early phase of the treatment, to an elevation of superoxide anion production because of the oxidation of allopurinol to oxypurinol. In a second phase, oxypuriliol produced by the reaction could reach a sufficient level to inhibit the enzyme. However, there is still no experimental evidence showing that either longer durations of treatment with allopurinol or oxypurinol administration can lead to an inhibition of xanthine oxidase-dependent superoxide production. Thus, the mechanism of the toxic effect of allopurinol remains unclear. In the light of our results it appears that, in our experimental conditions, allopurinol toxicity might involve an overproduction of oxy-