Basic Research in
Cardiology
Basic Res Cardio187:478--488 (1992)
Ischemia and reperfusion injury in isolated rat heart: Effect of reperfusion duration on xanthine oxidase, lipid peroxidation, and enzyme antioxidant systems in myocardium C. Coudray*, S. Pucheu**, F. Boucher**, J. de Leiris**, a n d A. Favier* * L a b o r a t o i r e de Biochimie C, C e n t r e H o s p i t a l i e r R6gional de G r e n o b l e , F r a n c e ** L a b o r a t o i r e de Physiologie cellulaire cardiaque, U R A C. N. R.S. 632, Universit6 J o s e p h Fourier, Grenoble, France
Summaly: The 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 activity 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 perfnsed 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. Key words: Ischemia -reperfusion, xanthine oxidase, _oxygen frce radicals, lipid peroxidation, _enzymic antioxidant systems
Abbreviations index: DMPO: dimethyl pyrroline-N-oxide, EPR: electron paramagnetic resonance, GPx: glutathione peroxidase, LDH: lactate dehydrogenase, LP: lipid peroxides, OFR: oxygen free radicals, SOD: superoxide dismutase, TBAR: thiobarbituric acid reactants, XD: xanthine dehydrogenase, XO: xanthine oxidase 745
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Introduction
It is currently considered that oxygen free radicals (OFR) are produced during cardiac ischemia, but, in particular, upon repeffusion (19, 22, 33). However, their mechanisms of production and involvement in the development of cellular lesions related to these pathophysiological situations remain to be demonstrated. McCord et al. (11) suggested that xanthine dehydrogenase (XD) underwent an irreversible proteolytic conversion to xanthine oxidase (XO) during intestinal ischemia. Subsequently, some workers placed the XO system at the forefront of potential O F R producers in the isolated ischemic and reperfused rat heart (10, 6, 42). In contrast to these results, other works on the transformation of XD to XO in isolated rat heart models generated contradictory findings (16, 58). Similarly, work involving the direct m e a s u r e m e n t of O F R by electron paramagnetic resonance (EPR) in the coronary effluent collected in the initial phases of reperfusion (1, 2, 74) led to contradictory results and are today open to criticism (48, 49). O n the other hand, work based on the measurement of myocardial lipid peroxide breakdown products following an ischemia-reperfusion sequence ex vivo have shown only very slight variations in the myocardial content of thiobarbituric acid reactants ( T B A R ) (51, 67) or even no changes whatsoever (9, 44, 45). Finally, extensive literature data have reported the beneficial effects of enzymic antioxidant treatments in the protection of O F R induced myocardial disorders. Unfortunately, other works have led to contradictory results according to the experimental model. However, the role and the response of endogene enzymic antioxidant systems are little dealt with and available results are still poor (25, 53, 63). In light of the contradictory reports on this subject, it was decided to investigate ex vivo, the effects of different durations of reperfusion which follow a globally partial ischemia of 40 rain with a residual perfusion flow-rate of 0.1 ml/min, on three parameters: myocardial XO activity (a potential generator of superoxide anions) on myocardial T B A R levels (commonly used as an index of lipid peroxidation), and on enzymic antioxidant system status. Lactate dehydrogenase (LDH) activity in the coronary effluent was evaluated and used as an index of cellular lesions during a partial 40-rain ischemia followed by up to 60 rain of reperfusion. Material and methods Animals
Adult male Wistar rats (Iffa Credo, 69210 l'Arbresle, France), weighing 300-350 g were used and were fed a standard laboratory diet (UAR, 91360 Villemoisson-sur-Orge, France). Experimental groups considered of six rats each (random selection). Perfusion protocol
Animals deprived of food for 18 h were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), heparinized (100 IU/rat, i.v.), and sacrificed. Hearts were removed and mounted on a perfusion apparatus via the aorta. Aortic perfusion was then started at a constant flow-rate of 11 ml/min for 10 rain. The perfusion liquid used was derived from that described by Krebs and Henseleit (35), containing Ca2+ = 2.4 raM, K + = 5.6 mM and glucose = 11 mM, equilibrated with a gas mixture of 95 % 02 and 5 % CO 2 at 37 ~ pH 7.4. After a stabilization period (10 min), the hearts were subjected to a globally partial ischemia of 40 rain (residual perfusion flow rate 0.l ml/min) followed by the studied reperfusion periods. In order to verify that the perfusion duration itself had no effect on the parameters studied, three control groups were perfused in standard normoxic conditions (11 ml/min) for 30, 50, and 110 min. The release of myocardial LDH during ischemia-reperfusion in a group of rats was compared to that noted in hearts in normoxie perfusion (control group). At the end of the ischemia/reperfusion protocols, the hearts of the experimental groups were frozen in liquid nitrogen according to the method of Wollenberger (72) until assay.
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Biochemical analyses One gram of heart tissue was homogenized in 10 ml of 60 mM Tris-HCl, pH 7.4, containing 1 mM diethyltriaminopentaacetic acid, using a motorized Teflon Potter tissue homogenizer. TBAR was assayed in the complete tissue homogenate by the method of Ohkawa (50): briefly, the cardiac homogenate was mixed with sodium dodeeyl sulfate to disperse aggregates and the sample was then treated with thiobarbituric acid in acid medium and heated at 95 ~ for 60 rain. The colored complex was extracted by butanol and absorbance at 532 nm was determined against TBAR standard range. The activity of XO was measured in the supernatant using a colorimetric method (65). The method assays hydrogen peroxide formed by the action of XO on hypoxanthine added to the sample in buffer. Cardiac glutathione peroxidase (GPx) activity was assayed spectrophotometrically according to the method described by Gunzler (27). It is based on the reduction of oxidized glutathion coupled to the oxidation of NADPH2. The disappearance of NADPH2 is followed at 340 rim. Cardiac superoxide dismutase (SOD) was assessed with the technique of Marklund (43). It involves the inhibition of pyrogallol autoxidation by SOD at pH 8.2. One unit of SOD corresponds to the SOD quantity which is capable of inhibition, by 50 %, of the spontaneous autoxidation of pyrogallol in 1 ml of reagent mixture under the conditions defined in our SOD assay. Cardiac eatalase was also estimated according to the technique described by Beers (3). H202 disappearance was followed during I rain in the presence of cardiac supernatant. H202 destroyed by cardiac catalase is then calculated using the molar extinction coefficient of 4300 M - i c m 1 for H202. LDH activity in the coronary effluent was assayed at different times (Fig. 3) according to the method of Wroblewski and La Due (73) by following the disappearance of NADH2 at 334 nm (Boehringer kit). The method of Lowry (39) was employed to determine the protein content of tissue homogenates, using bovine serum albumin as standard.
Statistics Standard procedures were used to calculate means and standard deviations. The PCSM statistics program was used to automatically compare the mean variances in the different groups with the F test. In the case of groups with heterogeneous mean variances, the program applied the "Aspin Welch" correction test before analysis. An analysis of variance (ANOVA) was used, followed by the NewmanKeuls test. Differences between groups were considered to be significant when the value of p was less than 0.05.
Results T h e first salient o b s e r v a t i o n is that perfusion itself up to 110 rain did not cause any significant modification in the p a r a m e t e r s studied in this work (Table 1).
Effect o f different durations o f reperfusion on X O activity T h e r e was no significant effect of globally, partial, n o r m o t h e r m i c ischemia o n the catalytic activity of X O as c o m p a r e d to continuously p e r f u s e d hearts. O n the o t h e r h a n d , 20, 40 or 60 rain of r e p e r f u s i o n do n o t lead to significant changes in the catalytic activity of x a n t h i n e oxidase as c o m p a r e d to n o n r e p e r f u s e d or to c o n t i n u o u s l y p e r f u s e d hearts (Fig. 1). Table 1. Effect of duration of perfusion on the level of thiobarbituric acid reactants, on the myocardial activity of xanthine oxidase, and on the enzymic antioxidant system. Results are expressed as mean + standard deviation (M + SD) (n = 5 to 6 hearts per group). Perfusion
XO*
TBAR $
SOD*
Catalase*
GPx*
30ran 50 mn 110 mn
506_+142 466 • 40 532 _+ 184
462_+79 430 • 58 420 • 96
33.6_+4.44 30.7 • 2.66 34.8 + 2.54
15.7_+2.60 14.8 • 1.39 18.8 _+3.06
1.57+0.19 1.68 • 0.16 1.6l _+0.29
* mIU/g protein. $ nmol/g protein. * IU/mg protein.
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Coudray et al., Free radicals and ischemia/repertbsion ex vivo >, > o-
800
0)
600
mlU/g
"o >r o
protein
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r 4O0
rJ:: C
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iiiiiii!i!!i!!!ii
20O
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0 Perfusion
0
2O
4 0
60
min
Figure 1. Effect of a globally, partial ischemia followed by different durations of reperfusion on the myocardial activity of xanthine oxidase. Results are expressed as mean • standard deviation (M _+ SD) (n = 5 to 6 hearts per group). After a stabilization )eriod (10 min), the isolated hearts were subjected to a globally, partial, isothermal ischemia (40 min), followed by different duration of reperfusion.
E f f e c t o f d i f f e r e n t durations o f reperfusion on T B A R c o n t e n t A f t e r 40 min of globally, partial (0.1 ml/min) ischemia, the cardiac T B A R level was u n c h a n g e d c o m p a r e d to the continuously p e r f u s e d group, w h e r e a s cardiac T B A R levels u n d e r w e n t a non-significant increase at 20 a n d 40 rain of r e p e f f u s i o n (Fig. 2) c o m p a r e d to n o n - r e p e r f u s e d or continuously p e r f u s e d hearts.
c: 800 t D.
~ 600t ~E
400] o
o-
~:
Perfusion 0
20
40
60 min
Figure 2. Effect of a globally, partial ischemia followed by different durations of reperfusion on the level of thiobarbituric acid reactants. Results are expressed as mean _+ standard deviation (M _+ SD) (n = 5 to 6 hearts per group). After a stabilization period (10 rain), the isolated hearts were subjected to a globally, partial ischemia (40 rain), followed by different durations of reperfusion.
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Table 2. Effect of globally partial ischemia followed by different durations of reperfusion on the enzymic antioxidant status. Results are expressed as mean _+standard deviation (M _+SD) (n = 5 to 6 hearts per group). After a stabilization period (10 rain), the isolated hearts were subjected to a globally partial, isothermal ischemia (40 rain), followed by different duration of reperfusion.
SOD* Catalase* GPx*
Perfusion
Reperf 0 mn
Reperf 20 mn
Reperf 40 mn
Reperf 60 mn
30.7 + 2.66 14.8 _+ 1.39 1.68_+0.16
32.6 _+3.50 18.9 _+2.57 1.54+0.09
32.6 _+2.84 18.2 _+2.99 1.79_+0.24
35.4 _+2.76 19.8 + 3.11 1.53_+0.25
33.2 _+3.33 17.8 _+1.56 1.32_+0.26
* IU/mg protein.
Effect o f different durations of reperfusion on enzymic antioxidant systems GPx and S O D activities remain unchanged after a globally partial ischemia of 40 rain compared to continuously perfused hearts, whereas catalase activity was non significantly augmented after this ischemia period. On reperfusion, the increase in catalase activity persists in the course of the different periods of reperfusion. Glutathione peroxidase and S O D activities remain, however, unchanged in the different studied periods of reperfusion compared to the non-reperfused hearts (Table 2).
Release of L D H The release of L D H during partial ischemia at 0.1 ml/min was 10 times greater than during normoxic perfusion prior to the ischemia. This release was accentuated during the initial
3"
LDH
Activity*
i
2
1
/ .-, ---T- ~,, .,-, 7 n, I 120min 20 40 60 B0 100 t lschemic and reperfused hearts [] Continuously perfused hearts * IU/ml in coronary effluent per g of heart tissue 0
p,.
Figure 3. Relcase of LDH during a globally partial ischemia (40 min) followed by up to 60 min of reperfusion in isolated rat hearts. Results are expressed as mean standard deviation (M -+ SD) (n = 5 to 6 hearts per group). After a stabilization period (10 min), the isolated hearts were subjected to 40 min of partial ischemia (0.1 ml/min), followed by a 60-min reperfusion. At the indicated times, perfusates were assayed for LDH activity.
Coudray et al., Free radicals and ischemia/reperfusion ex vivo
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phases of reperfusion, with a maximum after 4 min. Coronary L D H levels then decreased rapidly, reaching a plateau at approximately 4 - 5 times of their basal level at reperfusion (Fig. 3).
Discussion
Most published work attributes a role to OFR in the genesis of pathophysiological disorders occurring in ischemia and reperfusion (12, 19, 22, 33, 53, 74). The release of LDH into the coronary effluent is a reflection of membrane alterations which cause leakage of the cytoplasmic enzyme. The detection of radical species which could exert these deleterious effects and the identification of the biochemical consequences of their actions in the heart are far from satisfying. Questions concerning the sites of OFR production, the quantities produced, and the mechanisms of action still remain without an unambiguous answer. Levels of XO apparently vary among tissues and among animal species (18, 24, 29, 47, 52). Direct approaches to the detection of OFR in coronary effluents following experimental myocardial ischemia (7, 23, 74) have recently elicited controversy (48, 49). In the present study, the catalytic activity of XO, T B A R level, and enzymic antioxidant status were assayed as a function of the duration of reperfusion in globally partially isothermic ischemic rat heart. We were unable to observe any significant increase in XO activity in our working hypotheses. Indeed, regardless of the ischemia-reperfusion protocol applied in our study, the activity of the superoxide anion-producing form of XO never significantly increased. These results tend to confirm the findings of Das (16) and Reimer (58), who showed that XO does not participate in myocardial lesions following ischemiareperfusion. In another context, assays of XO and XD in rat hearts exposed to > 98 % 02 for 48 h (20) did not differ significantly from controls. Finally, measurements of the catalytic activity of XO revealed no significant difference in the activities of XO or XD after ischemias of different durations (10 to 40 min) (69). On the other hand, this author reported an approximately 300 % increase in XO activity and an approximately 300 % decrease in that of XD after reperfusion. Moreover, these phenomena appeared to be independent of the duration of the applied ischemia. A recent report by Thompson-Gorman and Zweier (66) did not show any significant difference in the catalytic activity of XO after a 30-min ischemia. They, however, concluded that OFR production by XO was high during reperfusion, based on the effect of inhibitors (allopurinol, oxypurinol) of the enzyme, as well as on the detection of hydroxyl radicals by EPR, in the form of a DMPO-OH spin adduct. Validation of the biochemical concept of OFR production based on the inhibitory effect on XO and on the detection of DMPO-OH signals in coronary perfusion outlet liquids, however, calls for certain reservations: Numerous results implicating XO as the prime source of OFR in ischemia have been obtained indirectly, by studying the effects of the inhibition of the enzyme, and in this perspective the results were discordant (5, 8, 16, 58). The precise mechanism of action of allopurinol is imperfectly understood and inhibition of XO is only one possibility (34, 37, 46). However, it is surprising that no D M P O - O O H signal would be reported in the coronary effluent on reperfusion, in spite of the relative high half-life of the superoxide anions, if considerable production of superoxide anions really occurred. Moreover, the presence of the D M P O - O H spin adduct in the coronary effluent is not a confirmation of the extravascular formation of the OH radical. It follows that radicals detected in the coronary effluent probably arise from vascular endothelial cells (75). In a recent study, Nohl et al. (49) have proposed that the D M P O - O O H adduct might result from the release of cytosolic iron and
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ascorbic acid into the coronary effluent. Indeed, Arroyo et al. (1) have seen ascorbyl radicals exclusively under aerobic conditions, thus, in the presence of oxygen (5). Finally, the DMPO-OH signal can form as a result of heating, acid treatment, ukra-violet irradiation, the use of non-purified DMPO or the presence of traces of iron in the effluent (30, 40, 41). As a result of these arguments, it is justified to question the importance of XO in the production of free radicals in ischemia-reperfusion ex vivo. Even though most work tends to defend the hypothesis of a conversion of XD during ischemia, this remains to be confirmed. It is more probable that XO is not a major source of superoxide anion production during post-ischemia reperfusion. In any case, XO did not participate in post-ischemic lesions in our experimental studies. In addition, LP were assayed in order to assess the intensity of the peroxidative process due to OFR eventually produced during ischemia or reperfusion. There was no significant modification of lipid peroxidation intermediates (TBAR) among the different groups of hearts subjected to the varying durations of reperfusion. These results confirm our prior work (13, 14, 15), as well as previous work of others (9, 44, 45, 68), who reported no changes in lipid peroxidation parameters. In studies by Otani (51) and T6r6k (67), assays of lipid peroxidation intermediates showed only slight increases of TBAR, while other studies (26, 64) reported an approximately 300 % increase in TBAR after reoxygenating the hypoxic rat heart. Several interpretations may be formulated for our results, which are in apparent disagreement with some published data: 1) Lipid peroxidation could be limited or nonexistent in our ex vivo model, since potential OFR-generating systems could be absent or inactive (e.g., XO) in these preparations. Also, the production of OFR and subsequently that of LP could be much greater in vivo than ex vivo because of the presence of other production mechanisms, i.e. leukocytes (21, 70), platelets (36) or the release of catecholamines (31, 71). 2) Even though considerable experimental findings in vivo have shown the increase in myocardial TBAR in the underperfused zone of regionally ischemic hearts (56, 57, 60, 67), this occurred only when the ischemia lasted for more than 45 min (61). These results support the hypothesis according to which lipid peroxidation in pathophysiological situations of ischemia and reperfusion requires relatively long periods of ischemia and is mediated primarily by biological mechanisms which are absent or inactive ex vivo. 3) Some studies have reported that cardiac peroxidation is controlled differently than in other tissues (4). In general, and even in the presence of a sufficient quantity of OFR, myocardial radical defense systems are more effective. Thus, iron overload in the rat heart does not cause an important increase in TBAR (55), in contrast to the liver. 4) Numerous studies using EPR technique have shown that the overproduction of radicals in isolated rat hearts occurs during the early phase of post-ischemic reperfusion. It could be suggested that the phases of initiation and propagation of the lipid peroxidation reaction, as well as the degradation of lipid peroxides, are steps that can delay TBAR production in tissues. Finally, GPx, catalase and SOD have key roles in the enzymic defense system against OFR, they reduce superoxide anions, H202, and other lipid organic peroxides. Glutathione peroxidase can be inactivated and fragmented by OFR (54, 62). In this way, a sudden increase in OFR production can destroy the main defense system of the cell. If the attack is not too strong, a quick removal of the modified enzyme will occur and the native enzyme will be synthetized. But if the destruction of the enzyme by OFR is substantial, the antioxidant enzymes will be destroyed, leading to a low defense potential and a possible destruction of the cell components by peroxidative reactions. Guarnieri et al. (25) have
Coudray et al., Free radicals and ischemia/reperfusion ex vivo
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reported that glutathione peroxidase activity was decreased in the cytosol of rat hearts under hypoxic conditions. Peterson et al. showed a significant decrease in catalase activity in canine myocardium during experimental ischemia and reperfusion (60 rain of ischemia followed by 30 min of reperfusion). Finally, Simmons et al. have recently pointed out that inactivation of myocardium catalase or glutathione peroxidase led to increase in peroxidation in rat heart homogenates and mitochondrial suspensions. In our study, antioxidant enzymic system do not seem to be affected in the different durations of reperfusion as compared to the non-reperfused or to the continuously perfused hearts. This could mean that cells were not overwhelmed by OFR production, which is confirmed by a stable level of cardiac T B A R in the different durations of reperfusion. It is, thus, highly unlikely that sufficient quantities of OFR are generated ex vivo, and the hypothesis that XO is the prime cause of ischemia/reperfusion injury by massive generation of superoxide anions is not consistent with our data. In any case, our results do not mean that OFR do not play any role in processes leading to post-ischemic physiobiological disorders. Nor do they cast doubt on the results of other investigators, but rather suggest that other sources of radicals should be considered, as well as other biochemical results of OFR production (attack on proteins, for example). OFR could participate in the development of cellular lesions attributed to the reperfusion syndrome, particularly by affecting endothelial functions without damaging the cells (38). Finally, it is to be noted that OFR may be related to the loss of endothelium-dependent coronary arterial relaxation. However, an inn-eased lipid peroxidation is not the only process that could cause cellular lesions involving OFR. Indeed, direct radical attack on membrane proteins or enzymes has also been reported in numerous studies (12, 17, 28, 32). In summary, the results obtained under our experimental conditions suggest that XO does not participate in post-ischemic lesions and that the increase in lipid peroxides is not the major event that can lead to cell death. How might one reconcile these results with the enourmous number of experiments showing protection against ischemia/reperfusion injury by treatment with antioxidants, antioxidant enzymes, or allopurinol? It seems most likely that a small amount of reactive oxygen could be generated in a reduced acidic environment, such as occurs in ischemic tissue and on reperfusion. However, this production of free radicals or TBAR, even in minimal quantities, may be a signal for triggering biochemical processes localized primarily in membranes (59) and which can cause total disorganization of the membrane and possibly cell death. In view of the importance of the problem of ischemia/ reperfusion injury both theoretically and practically, we believe that these findings although negative, should be on record. Acknowledgements
This work was supported by a French Ministry of Research and Technology grant n" 88-C-0852 (Paris, France). The authors thank Dr J. M. C. Guneridge for helpful discussions. We gratefully acknowledge the generous technical assistance of Monique Rual and Jacqueline Meo.
Re[erences
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29. De Jong JW, Van Der Meer P, Nieukoop AS, Huizer T, Stroeve RJ, Bos E (1990) Xanthine oxidoreductase activity in perfused hearts of various species, including humans. Circulation Res 67:770-773 30. Janzen EG, Jandrisits LT, Shetty RV, Haire DL, Hilborn JW (1989) Synthesis and purification of 5,5' dimethyl-l-pyrroline-N-oxide for biological applications. Chem Biol Interac 70:167-172 31. Jewett SL, Eddy LJ, Hochstein P (1989) Is the autoxidation of catecholamines involved in ischemiareperfusion injury. Free Rad Biol Med 6:185 188 32. Kako KJ (1987) Free radical effects on membranes protein in myocardial ischemia/reperfusion injury. J Mol Cell Cardiol 19:209-2ll 33. Kloner RA, Przyklenk K, Whittaker P (1989) Deleterious effects of oxygen radicals in ischemia/ reperfusion Resolved and unresolved issues. Circulation, 80:1115-1127 34. Ko KM, Godin DV (1990) Inhibition of transition metal ion-catalysed ascorbate oxidation and lipid peroxidation by allopurinol. Biochem Pharmacol 40:803-809 35. Krebs HA, Henseleik K (1932) Untersuchungen fiber die HarnstofPoildung in Tierk~Srpern. Hoppe Seylers Z Physiol Chem 210:33-66 36. Kubes P, Ibbotson G, Russell J, Wallace JL, Granger DN (1990) Role of platelet-activating factor in ischemia-reperfusion-induced leucocyte adherence. Am J Physiol 22:G300-G305 37. Lasley RD, Ely SW, Berne RM, Mentzer RM (1988) Allopurinot enhanced adenine nucleotide repletion after myocardial ischemia in the isolated rat heart. J Clin Inves 81:16-20 38. Lawson DL, Metha JL, Nichols WW (1990) Coronary reperfusion in dogs inhibits endotheliumdependent relaxation; Role of superoxide radicals. Free Rad Biol Med 8:373-380 39. Lowry OH, Rosebrough NJ, Faar AL, Ranrall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem i93:265-75 40. Makino K, Imaishi H, Morinishi H, Hagiwara T, Takeuchi T, Murakami A, Nishi M (1989) An artifact in the ESR spectrum obtained by spin trapping with DMPO. Free Rad Res Commun 6:19-28 41. Makino K, Hagiwara T, Imaishi H, Nishi M, Fujii S, Ohya H. Murakami A (1990) DMPO spin trapping in the presence of Fe ion. Free Rad Res Commun 9:223-240 42. Manning AS (1988) Reperfnsion-induced arrhythmias: Do free radicals play a critical role? Free Rad Biol Med 4:305-316 43. Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469-474 44. Maupoil V, Rochette L (1.988) Evaluation of free radical and lipid peroxide formation during global ischemia and reperfusion in isolated perfused rat heart. Cardiovasc Drugs Therap 2:615-621 45. Mitchell JR, Smith CV, Hughes H, Lenz ML, Jaeschke H, Shappell SB, Michael LH, Entman ML (1987) No evidence for reactive oxygene damage in ischemia-reflow injury. Trans Assoc Am Physic 100:54-61 46. Moorhouse PC, Grootveld M, Halliwel B (1987) Allopurinol and oxypurinol are hydroxyl radical scavengers. FEBS Let 213:223 47. Muxfeldt M, Schaper W (1987) The activity of xanthine oxidase in heart and liver of rats, guinea pigs, rabbit and human beings. Circulation 76:198 48. Nakazawa H, Ichimori K, Shinozaki Y, Okino H, Hori S (1988) Is superoxide demonstration by electron-spin resonance spectroscopy really superoxide? Am J Physiol 255:H213-H215 49. Nohl H, Staze K, Napetsching S, Ishikawa T (1991) Is oxidative stress primarily involved in reperfusion injury of the ischemic heart. Free Rad Biol Med 11:581-588 50. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxide in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351-358 51. Otani H, Engelman RM, Rousou JA, Breyer RH, Lemeshow S, Das DK (1986) Cardiac performance during reperfusion improved by pretreatment with oxygen-free-radical scavengers. J Thorac Cardiovasc Surg 91:290-295 52. Parks DA, Granger DN (1986) Xanthine oxidase: Biochemistry, distribution and physiology. Acta Physiol Scand 548:87-99 53. Peterson DA, Asigner RW, Elsperger KJ, Homans DC, Eaton JW (1985) Reactive oxygen species may cause myocardial reperfusion injury. Biochem Biophys Res Commun 127:87-93 54. Pigeolet E, Remacle J (1991) Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals. Free Rad Biol Med 11:191-195
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55. Pucheu S, Coudray C, Favier A, de Leiris J (1991) Effects of iron-overload in experimental myocardial ischemia and reperfusion. J Mol Cell Cardiol 23:122 (Abstract) 56. Rao PS, Mueller HS (1983a) Lipid peroxidation in acute myocardium ischemia. Adv Exp Med Biol 161:347-363 57. Rao PS, Cohen MV, Mueller HS (1983b) Production of free radicals and lipid peroxidation in early experimental ischemia. J Mol Cell Cardiol 15:713-716 58. Reimer KA, Jennings RB (1985) Failure of xanthine oxidase inhibitor altopurinol to limit infarct size after ischemia and reperfusion in dogs. Circulation, 71:1069-1075 59. Richter C (1987) Biophysical consequences of lipid peroxidation in membranes. Chem Phys Lipids 44:175-189 60. Romaschin AD, Rebeyka I, Wilson GJ, Mickle D A G (1987) Conjugated dienes in ischemia and reperfused lnyocardium. An in vivo chemical signature of oxygen free radical mediated injury. J Mol Cell Cardiol 19:289-302 61. R6th E, T6r6k B, Zsoldos T, Matkovics B (1985) Lipid peroxidation and scavenger mechanism in experimentally induced infarcts. Basic Res Cardiol 80:530-536 62. Salo DC, Pacifici RE, Sin SW, Giulivi C, Davies KJA (1990) Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. Biol Chem 265:11919-11927 63. Simmons TW, Jamall IS (1989) Relative importance of intracellular glutathione peroxidase and catalase in vivo for prevention of peroxidation to the heart. Cardiovasc Res 23:774-779 64. Subramanian R, Plehn S, Noonan J, Schmidt M, Shug AL (1987) Free radical-mediated damage during myocardial ischemia and reperfusion and protection by carnitine esters. Z Kardiol 76:41-45 65. Sugiura M, Kato K, Adachi T, Ito Y, Hirano K (1981) A new method for the assay of xanthine oxidase activity. Chem Pharma Bull 29:430-432 66. Thompson-Gorman SL, Zweier JL (1990) Evaluation of the role of xanthine oxidase in myocardial reperfusion injury. J Biol Chem 265:6656-6663 67. T6r~Sk B, R6th E, B~r V, Pollak Z (1986) Effects of antioxidant therapy in experimentally induced heart infarcts. Basic Res Cardiol 81:167-179 68. Van Der Heide RS, Sobotka PA, Ganote CE (1987) Effects of the free radical scavenger DMTU and mannitol on the oxygen paradox in perfused rat hearts. J Mol Cell Cardiol 19:615-626 69. Van Jaarsveld H, Groenewald AJ, Potgieter GM, Barnad SP, Vermaak WJH, Barnard HC (1988) Effect of normothermic ischemic cardiac arrest and of reperfusion on the free oxygen radical scavenger enzymes and xanthine oxidase (A generator of superoxide anions). Enzyme 39:8-16 70. Werns SV, Lucchesi BR (1988) Leukocytes, oxygen radicals, and myocardial injury due to ischemia and reperfusion. Free Rad Biol Med 4:31-37 71. Wilde AA, Peters RJ, Janse MJ (1988) Catecholamine release and potassium accumulation in the isolated globally ischemic rabbit heart. J Mol CelI Cardiol 20:887-896 72. Wollenberger A, Ristou O, Schoffa G (1960) Eine einfache Technik der extremschnellen Abkt~hlung gr6gerer Gewebestticke. Pflfig Arch "Eur J Physiol". 270:399-412 73. Wroblewski F, La Due JS (1955) Lactic dehydrogenase activity in blood. Proc Soc Exp Biol Med 90:210-213 74. Zweier JL, Flaherty JT, Weisfeldt ML (1987) Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 84:1404-1408 75. Zweier JL, Kauppusamy P, Lutty G (1988) Measurement of endothelial cell free radical generation: Evidence for a central mechanism of free radical injury in post-ischemic tissue. Proc Natl Acad Sci USA 85:4046-4050 Received May 14, 1992 accepted June 12, 1992 Authors' address: Charles Coudray, (Pharm.D, Ph.D, HDR), Laboratoire de Biochimie C, C.H.R.U. de Grenoble, B.P. 217 X, 38043 Grenoble Cedex, France
Cardiology
Basic Res Cardio187:478--488 (1992)
Ischemia and reperfusion injury in isolated rat heart: Effect of reperfusion duration on xanthine oxidase, lipid peroxidation, and enzyme antioxidant systems in myocardium C. Coudray*, S. Pucheu**, F. Boucher**, J. de Leiris**, a n d A. Favier* * L a b o r a t o i r e de Biochimie C, C e n t r e H o s p i t a l i e r R6gional de G r e n o b l e , F r a n c e ** L a b o r a t o i r e de Physiologie cellulaire cardiaque, U R A C. N. R.S. 632, Universit6 J o s e p h Fourier, Grenoble, France
Summaly: The 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 activity 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 perfnsed 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. Key words: Ischemia -reperfusion, xanthine oxidase, _oxygen frce radicals, lipid peroxidation, _enzymic antioxidant systems
Abbreviations index: DMPO: dimethyl pyrroline-N-oxide, EPR: electron paramagnetic resonance, GPx: glutathione peroxidase, LDH: lactate dehydrogenase, LP: lipid peroxides, OFR: oxygen free radicals, SOD: superoxide dismutase, TBAR: thiobarbituric acid reactants, XD: xanthine dehydrogenase, XO: xanthine oxidase 745
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Introduction
It is currently considered that oxygen free radicals (OFR) are produced during cardiac ischemia, but, in particular, upon repeffusion (19, 22, 33). However, their mechanisms of production and involvement in the development of cellular lesions related to these pathophysiological situations remain to be demonstrated. McCord et al. (11) suggested that xanthine dehydrogenase (XD) underwent an irreversible proteolytic conversion to xanthine oxidase (XO) during intestinal ischemia. Subsequently, some workers placed the XO system at the forefront of potential O F R producers in the isolated ischemic and reperfused rat heart (10, 6, 42). In contrast to these results, other works on the transformation of XD to XO in isolated rat heart models generated contradictory findings (16, 58). Similarly, work involving the direct m e a s u r e m e n t of O F R by electron paramagnetic resonance (EPR) in the coronary effluent collected in the initial phases of reperfusion (1, 2, 74) led to contradictory results and are today open to criticism (48, 49). O n the other hand, work based on the measurement of myocardial lipid peroxide breakdown products following an ischemia-reperfusion sequence ex vivo have shown only very slight variations in the myocardial content of thiobarbituric acid reactants ( T B A R ) (51, 67) or even no changes whatsoever (9, 44, 45). Finally, extensive literature data have reported the beneficial effects of enzymic antioxidant treatments in the protection of O F R induced myocardial disorders. Unfortunately, other works have led to contradictory results according to the experimental model. However, the role and the response of endogene enzymic antioxidant systems are little dealt with and available results are still poor (25, 53, 63). In light of the contradictory reports on this subject, it was decided to investigate ex vivo, the effects of different durations of reperfusion which follow a globally partial ischemia of 40 rain with a residual perfusion flow-rate of 0.1 ml/min, on three parameters: myocardial XO activity (a potential generator of superoxide anions) on myocardial T B A R levels (commonly used as an index of lipid peroxidation), and on enzymic antioxidant system status. Lactate dehydrogenase (LDH) activity in the coronary effluent was evaluated and used as an index of cellular lesions during a partial 40-rain ischemia followed by up to 60 rain of reperfusion. Material and methods Animals
Adult male Wistar rats (Iffa Credo, 69210 l'Arbresle, France), weighing 300-350 g were used and were fed a standard laboratory diet (UAR, 91360 Villemoisson-sur-Orge, France). Experimental groups considered of six rats each (random selection). Perfusion protocol
Animals deprived of food for 18 h were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), heparinized (100 IU/rat, i.v.), and sacrificed. Hearts were removed and mounted on a perfusion apparatus via the aorta. Aortic perfusion was then started at a constant flow-rate of 11 ml/min for 10 rain. The perfusion liquid used was derived from that described by Krebs and Henseleit (35), containing Ca2+ = 2.4 raM, K + = 5.6 mM and glucose = 11 mM, equilibrated with a gas mixture of 95 % 02 and 5 % CO 2 at 37 ~ pH 7.4. After a stabilization period (10 min), the hearts were subjected to a globally partial ischemia of 40 rain (residual perfusion flow rate 0.l ml/min) followed by the studied reperfusion periods. In order to verify that the perfusion duration itself had no effect on the parameters studied, three control groups were perfused in standard normoxic conditions (11 ml/min) for 30, 50, and 110 min. The release of myocardial LDH during ischemia-reperfusion in a group of rats was compared to that noted in hearts in normoxie perfusion (control group). At the end of the ischemia/reperfusion protocols, the hearts of the experimental groups were frozen in liquid nitrogen according to the method of Wollenberger (72) until assay.
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Biochemical analyses One gram of heart tissue was homogenized in 10 ml of 60 mM Tris-HCl, pH 7.4, containing 1 mM diethyltriaminopentaacetic acid, using a motorized Teflon Potter tissue homogenizer. TBAR was assayed in the complete tissue homogenate by the method of Ohkawa (50): briefly, the cardiac homogenate was mixed with sodium dodeeyl sulfate to disperse aggregates and the sample was then treated with thiobarbituric acid in acid medium and heated at 95 ~ for 60 rain. The colored complex was extracted by butanol and absorbance at 532 nm was determined against TBAR standard range. The activity of XO was measured in the supernatant using a colorimetric method (65). The method assays hydrogen peroxide formed by the action of XO on hypoxanthine added to the sample in buffer. Cardiac glutathione peroxidase (GPx) activity was assayed spectrophotometrically according to the method described by Gunzler (27). It is based on the reduction of oxidized glutathion coupled to the oxidation of NADPH2. The disappearance of NADPH2 is followed at 340 rim. Cardiac superoxide dismutase (SOD) was assessed with the technique of Marklund (43). It involves the inhibition of pyrogallol autoxidation by SOD at pH 8.2. One unit of SOD corresponds to the SOD quantity which is capable of inhibition, by 50 %, of the spontaneous autoxidation of pyrogallol in 1 ml of reagent mixture under the conditions defined in our SOD assay. Cardiac eatalase was also estimated according to the technique described by Beers (3). H202 disappearance was followed during I rain in the presence of cardiac supernatant. H202 destroyed by cardiac catalase is then calculated using the molar extinction coefficient of 4300 M - i c m 1 for H202. LDH activity in the coronary effluent was assayed at different times (Fig. 3) according to the method of Wroblewski and La Due (73) by following the disappearance of NADH2 at 334 nm (Boehringer kit). The method of Lowry (39) was employed to determine the protein content of tissue homogenates, using bovine serum albumin as standard.
Statistics Standard procedures were used to calculate means and standard deviations. The PCSM statistics program was used to automatically compare the mean variances in the different groups with the F test. In the case of groups with heterogeneous mean variances, the program applied the "Aspin Welch" correction test before analysis. An analysis of variance (ANOVA) was used, followed by the NewmanKeuls test. Differences between groups were considered to be significant when the value of p was less than 0.05.
Results T h e first salient o b s e r v a t i o n is that perfusion itself up to 110 rain did not cause any significant modification in the p a r a m e t e r s studied in this work (Table 1).
Effect o f different durations o f reperfusion on X O activity T h e r e was no significant effect of globally, partial, n o r m o t h e r m i c ischemia o n the catalytic activity of X O as c o m p a r e d to continuously p e r f u s e d hearts. O n the o t h e r h a n d , 20, 40 or 60 rain of r e p e r f u s i o n do n o t lead to significant changes in the catalytic activity of x a n t h i n e oxidase as c o m p a r e d to n o n r e p e r f u s e d or to c o n t i n u o u s l y p e r f u s e d hearts (Fig. 1). Table 1. Effect of duration of perfusion on the level of thiobarbituric acid reactants, on the myocardial activity of xanthine oxidase, and on the enzymic antioxidant system. Results are expressed as mean + standard deviation (M + SD) (n = 5 to 6 hearts per group). Perfusion
XO*
TBAR $
SOD*
Catalase*
GPx*
30ran 50 mn 110 mn
506_+142 466 • 40 532 _+ 184
462_+79 430 • 58 420 • 96
33.6_+4.44 30.7 • 2.66 34.8 + 2.54
15.7_+2.60 14.8 • 1.39 18.8 _+3.06
1.57+0.19 1.68 • 0.16 1.6l _+0.29
* mIU/g protein. $ nmol/g protein. * IU/mg protein.
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Coudray et al., Free radicals and ischemia/repertbsion ex vivo >, > o-
800
0)
600
mlU/g
"o >r o
protein
_T_
r 4O0
rJ:: C
E 2 0 >,
iiiiiii!i!!i!!!ii
20O
/// /// /// /// /// /.// //-/ // // 7 / ///
5"/ /// 1//
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0 Perfusion
0
2O
4 0
60
min
Figure 1. Effect of a globally, partial ischemia followed by different durations of reperfusion on the myocardial activity of xanthine oxidase. Results are expressed as mean • standard deviation (M _+ SD) (n = 5 to 6 hearts per group). After a stabilization )eriod (10 min), the isolated hearts were subjected to a globally, partial, isothermal ischemia (40 min), followed by different duration of reperfusion.
E f f e c t o f d i f f e r e n t durations o f reperfusion on T B A R c o n t e n t A f t e r 40 min of globally, partial (0.1 ml/min) ischemia, the cardiac T B A R level was u n c h a n g e d c o m p a r e d to the continuously p e r f u s e d group, w h e r e a s cardiac T B A R levels u n d e r w e n t a non-significant increase at 20 a n d 40 rain of r e p e f f u s i o n (Fig. 2) c o m p a r e d to n o n - r e p e r f u s e d or continuously p e r f u s e d hearts.
c: 800 t D.
~ 600t ~E
400] o
o-
~:
Perfusion 0
20
40
60 min
Figure 2. Effect of a globally, partial ischemia followed by different durations of reperfusion on the level of thiobarbituric acid reactants. Results are expressed as mean _+ standard deviation (M _+ SD) (n = 5 to 6 hearts per group). After a stabilization period (10 rain), the isolated hearts were subjected to a globally, partial ischemia (40 rain), followed by different durations of reperfusion.
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Table 2. Effect of globally partial ischemia followed by different durations of reperfusion on the enzymic antioxidant status. Results are expressed as mean _+standard deviation (M _+SD) (n = 5 to 6 hearts per group). After a stabilization period (10 rain), the isolated hearts were subjected to a globally partial, isothermal ischemia (40 rain), followed by different duration of reperfusion.
SOD* Catalase* GPx*
Perfusion
Reperf 0 mn
Reperf 20 mn
Reperf 40 mn
Reperf 60 mn
30.7 + 2.66 14.8 _+ 1.39 1.68_+0.16
32.6 _+3.50 18.9 _+2.57 1.54+0.09
32.6 _+2.84 18.2 _+2.99 1.79_+0.24
35.4 _+2.76 19.8 + 3.11 1.53_+0.25
33.2 _+3.33 17.8 _+1.56 1.32_+0.26
* IU/mg protein.
Effect o f different durations of reperfusion on enzymic antioxidant systems GPx and S O D activities remain unchanged after a globally partial ischemia of 40 rain compared to continuously perfused hearts, whereas catalase activity was non significantly augmented after this ischemia period. On reperfusion, the increase in catalase activity persists in the course of the different periods of reperfusion. Glutathione peroxidase and S O D activities remain, however, unchanged in the different studied periods of reperfusion compared to the non-reperfused hearts (Table 2).
Release of L D H The release of L D H during partial ischemia at 0.1 ml/min was 10 times greater than during normoxic perfusion prior to the ischemia. This release was accentuated during the initial
3"
LDH
Activity*
i
2
1
/ .-, ---T- ~,, .,-, 7 n, I 120min 20 40 60 B0 100 t lschemic and reperfused hearts [] Continuously perfused hearts * IU/ml in coronary effluent per g of heart tissue 0
p,.
Figure 3. Relcase of LDH during a globally partial ischemia (40 min) followed by up to 60 min of reperfusion in isolated rat hearts. Results are expressed as mean standard deviation (M -+ SD) (n = 5 to 6 hearts per group). After a stabilization period (10 min), the isolated hearts were subjected to 40 min of partial ischemia (0.1 ml/min), followed by a 60-min reperfusion. At the indicated times, perfusates were assayed for LDH activity.
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phases of reperfusion, with a maximum after 4 min. Coronary L D H levels then decreased rapidly, reaching a plateau at approximately 4 - 5 times of their basal level at reperfusion (Fig. 3).
Discussion
Most published work attributes a role to OFR in the genesis of pathophysiological disorders occurring in ischemia and reperfusion (12, 19, 22, 33, 53, 74). The release of LDH into the coronary effluent is a reflection of membrane alterations which cause leakage of the cytoplasmic enzyme. The detection of radical species which could exert these deleterious effects and the identification of the biochemical consequences of their actions in the heart are far from satisfying. Questions concerning the sites of OFR production, the quantities produced, and the mechanisms of action still remain without an unambiguous answer. Levels of XO apparently vary among tissues and among animal species (18, 24, 29, 47, 52). Direct approaches to the detection of OFR in coronary effluents following experimental myocardial ischemia (7, 23, 74) have recently elicited controversy (48, 49). In the present study, the catalytic activity of XO, T B A R level, and enzymic antioxidant status were assayed as a function of the duration of reperfusion in globally partially isothermic ischemic rat heart. We were unable to observe any significant increase in XO activity in our working hypotheses. Indeed, regardless of the ischemia-reperfusion protocol applied in our study, the activity of the superoxide anion-producing form of XO never significantly increased. These results tend to confirm the findings of Das (16) and Reimer (58), who showed that XO does not participate in myocardial lesions following ischemiareperfusion. In another context, assays of XO and XD in rat hearts exposed to > 98 % 02 for 48 h (20) did not differ significantly from controls. Finally, measurements of the catalytic activity of XO revealed no significant difference in the activities of XO or XD after ischemias of different durations (10 to 40 min) (69). On the other hand, this author reported an approximately 300 % increase in XO activity and an approximately 300 % decrease in that of XD after reperfusion. Moreover, these phenomena appeared to be independent of the duration of the applied ischemia. A recent report by Thompson-Gorman and Zweier (66) did not show any significant difference in the catalytic activity of XO after a 30-min ischemia. They, however, concluded that OFR production by XO was high during reperfusion, based on the effect of inhibitors (allopurinol, oxypurinol) of the enzyme, as well as on the detection of hydroxyl radicals by EPR, in the form of a DMPO-OH spin adduct. Validation of the biochemical concept of OFR production based on the inhibitory effect on XO and on the detection of DMPO-OH signals in coronary perfusion outlet liquids, however, calls for certain reservations: Numerous results implicating XO as the prime source of OFR in ischemia have been obtained indirectly, by studying the effects of the inhibition of the enzyme, and in this perspective the results were discordant (5, 8, 16, 58). The precise mechanism of action of allopurinol is imperfectly understood and inhibition of XO is only one possibility (34, 37, 46). However, it is surprising that no D M P O - O O H signal would be reported in the coronary effluent on reperfusion, in spite of the relative high half-life of the superoxide anions, if considerable production of superoxide anions really occurred. Moreover, the presence of the D M P O - O H spin adduct in the coronary effluent is not a confirmation of the extravascular formation of the OH radical. It follows that radicals detected in the coronary effluent probably arise from vascular endothelial cells (75). In a recent study, Nohl et al. (49) have proposed that the D M P O - O O H adduct might result from the release of cytosolic iron and
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ascorbic acid into the coronary effluent. Indeed, Arroyo et al. (1) have seen ascorbyl radicals exclusively under aerobic conditions, thus, in the presence of oxygen (5). Finally, the DMPO-OH signal can form as a result of heating, acid treatment, ukra-violet irradiation, the use of non-purified DMPO or the presence of traces of iron in the effluent (30, 40, 41). As a result of these arguments, it is justified to question the importance of XO in the production of free radicals in ischemia-reperfusion ex vivo. Even though most work tends to defend the hypothesis of a conversion of XD during ischemia, this remains to be confirmed. It is more probable that XO is not a major source of superoxide anion production during post-ischemia reperfusion. In any case, XO did not participate in post-ischemic lesions in our experimental studies. In addition, LP were assayed in order to assess the intensity of the peroxidative process due to OFR eventually produced during ischemia or reperfusion. There was no significant modification of lipid peroxidation intermediates (TBAR) among the different groups of hearts subjected to the varying durations of reperfusion. These results confirm our prior work (13, 14, 15), as well as previous work of others (9, 44, 45, 68), who reported no changes in lipid peroxidation parameters. In studies by Otani (51) and T6r6k (67), assays of lipid peroxidation intermediates showed only slight increases of TBAR, while other studies (26, 64) reported an approximately 300 % increase in TBAR after reoxygenating the hypoxic rat heart. Several interpretations may be formulated for our results, which are in apparent disagreement with some published data: 1) Lipid peroxidation could be limited or nonexistent in our ex vivo model, since potential OFR-generating systems could be absent or inactive (e.g., XO) in these preparations. Also, the production of OFR and subsequently that of LP could be much greater in vivo than ex vivo because of the presence of other production mechanisms, i.e. leukocytes (21, 70), platelets (36) or the release of catecholamines (31, 71). 2) Even though considerable experimental findings in vivo have shown the increase in myocardial TBAR in the underperfused zone of regionally ischemic hearts (56, 57, 60, 67), this occurred only when the ischemia lasted for more than 45 min (61). These results support the hypothesis according to which lipid peroxidation in pathophysiological situations of ischemia and reperfusion requires relatively long periods of ischemia and is mediated primarily by biological mechanisms which are absent or inactive ex vivo. 3) Some studies have reported that cardiac peroxidation is controlled differently than in other tissues (4). In general, and even in the presence of a sufficient quantity of OFR, myocardial radical defense systems are more effective. Thus, iron overload in the rat heart does not cause an important increase in TBAR (55), in contrast to the liver. 4) Numerous studies using EPR technique have shown that the overproduction of radicals in isolated rat hearts occurs during the early phase of post-ischemic reperfusion. It could be suggested that the phases of initiation and propagation of the lipid peroxidation reaction, as well as the degradation of lipid peroxides, are steps that can delay TBAR production in tissues. Finally, GPx, catalase and SOD have key roles in the enzymic defense system against OFR, they reduce superoxide anions, H202, and other lipid organic peroxides. Glutathione peroxidase can be inactivated and fragmented by OFR (54, 62). In this way, a sudden increase in OFR production can destroy the main defense system of the cell. If the attack is not too strong, a quick removal of the modified enzyme will occur and the native enzyme will be synthetized. But if the destruction of the enzyme by OFR is substantial, the antioxidant enzymes will be destroyed, leading to a low defense potential and a possible destruction of the cell components by peroxidative reactions. Guarnieri et al. (25) have
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reported that glutathione peroxidase activity was decreased in the cytosol of rat hearts under hypoxic conditions. Peterson et al. showed a significant decrease in catalase activity in canine myocardium during experimental ischemia and reperfusion (60 rain of ischemia followed by 30 min of reperfusion). Finally, Simmons et al. have recently pointed out that inactivation of myocardium catalase or glutathione peroxidase led to increase in peroxidation in rat heart homogenates and mitochondrial suspensions. In our study, antioxidant enzymic system do not seem to be affected in the different durations of reperfusion as compared to the non-reperfused or to the continuously perfused hearts. This could mean that cells were not overwhelmed by OFR production, which is confirmed by a stable level of cardiac T B A R in the different durations of reperfusion. It is, thus, highly unlikely that sufficient quantities of OFR are generated ex vivo, and the hypothesis that XO is the prime cause of ischemia/reperfusion injury by massive generation of superoxide anions is not consistent with our data. In any case, our results do not mean that OFR do not play any role in processes leading to post-ischemic physiobiological disorders. Nor do they cast doubt on the results of other investigators, but rather suggest that other sources of radicals should be considered, as well as other biochemical results of OFR production (attack on proteins, for example). OFR could participate in the development of cellular lesions attributed to the reperfusion syndrome, particularly by affecting endothelial functions without damaging the cells (38). Finally, it is to be noted that OFR may be related to the loss of endothelium-dependent coronary arterial relaxation. However, an inn-eased lipid peroxidation is not the only process that could cause cellular lesions involving OFR. Indeed, direct radical attack on membrane proteins or enzymes has also been reported in numerous studies (12, 17, 28, 32). In summary, the results obtained under our experimental conditions suggest that XO does not participate in post-ischemic lesions and that the increase in lipid peroxides is not the major event that can lead to cell death. How might one reconcile these results with the enourmous number of experiments showing protection against ischemia/reperfusion injury by treatment with antioxidants, antioxidant enzymes, or allopurinol? It seems most likely that a small amount of reactive oxygen could be generated in a reduced acidic environment, such as occurs in ischemic tissue and on reperfusion. However, this production of free radicals or TBAR, even in minimal quantities, may be a signal for triggering biochemical processes localized primarily in membranes (59) and which can cause total disorganization of the membrane and possibly cell death. In view of the importance of the problem of ischemia/ reperfusion injury both theoretically and practically, we believe that these findings although negative, should be on record. Acknowledgements
This work was supported by a French Ministry of Research and Technology grant n" 88-C-0852 (Paris, France). The authors thank Dr J. M. C. Guneridge for helpful discussions. We gratefully acknowledge the generous technical assistance of Monique Rual and Jacqueline Meo.
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