T angioplasty has focused cardiac research on interventions

Myocardial Salvage With Trolox and Ascorbic Acid for an Acute Evolving Infarction Donald A. G. Mickle, MD, Ren-Ke Li, MD, Richard D. Weisel, MD, Peter L. Birnbaum, MD, Tai-Wing Wu, PhD, George Jackowski, PhD, M. Mindy Madonik, BSc, Graham W. Burton, PhD, and Keith U. Ingold, PhD Department of Clinical Biochemistry and Division of Cardiovascular Surgery, Toronto General Hospital, University of Toronto, and Division of Chemistry, National Research Council of Canada, Ottawa, Ontario, Canada Both Trolox (a water-soluble analogue of a-tocopherol) and ascorbic acid were more effective than superoxide dismutase or catalase in protecting myocyte cell cultures from free radical attack (induced by hypoxanthine and xanthine oxidase). In a canine model of two hours of left anterior descending coronary artery occlusion followed by four hours of reperfusion, Trolox and ascorbic acid reduced the area of infarction within the area at risk. The Trolox group received 500 ml of deoxygenated saline solution containing 2.0 g of Trolox, 3.0 g of ascorbic acid, and 18 mg of EDTA (ethylenediaminetetraacetic acid) infused into the ascending aorta 30 seconds before and four minutes after reperfusion. Saline controls received 500 ml of deoxygenated saline solution containing 18 mg of EDTA. The angioplasty group had unmodified reperfusion by simple release of the occlusion. The area at risk and the area infarcted were estimated with Evans blue and triphenyl tetrazolium hydrochloride stains, respectively. The ratio of the area infarcted to the area at risk was significantly lower with Trolox (angioplasty, 30.4% 2 5.1%; saline, 20.8% 2 2.9%; and Trolox, 8.7% 2 4.0%; p < 0.01). In summary, the antioxidants Trolox and ascorbic acid effectively reduced myocardial necrosis after ischemia. ( 1989;47:553-7) he advent of thrombolytic therapy and emergency T angioplasty has focused cardiac research on interventions intended to limit infarct size in patients seen within four hours of an acute evolving infarction. The interventions that have been demonstrated to reduce infarct size include emergency coronary artery bypass grafting [ 1-31, circulatory assistance during emergency angioplasty [3-51, and either reducing myocardial metabolic demands [6] or infusing superoxide dismutase (SOD) and catalase during reperfusion [7]. Experimental studies have indicated that oxygenmediated free radical injury [7-91 contributes to the myocardial damage associated with ischemia and reperfusion. Therefore, we compared the effectiveness of selected antioxidants in protecting canine ventricular myocytes from pharmacologically generated free radicals, and we evaluated the most effective scavengers in a canine model of regional myocardial ischemia and reperfusion intended to simulate a patient seen with an acute myocardial infarction. We found that Trolox, a water-soluble analogue of vitamin E, and ascorbic acid were effective in preventing myocyte necrosis in cell culture studies and in a canine model of two hours of left anterior descending coronary artery (LAD) occlusion followed by four hours of reperfusion. Presented at the Research Forum of the Twenty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Sep 26-28, 1988. Address reprint requests to Dr Mickle, Toronto General Hospital, Department of Clinical Biochemistry, ES3-404, 200 Elizabeth St, Toronto, Ont M5G 2C4 Canada. Material and Methods Cell Cultures Adult canine ventricular myocardium was dissociated with trypsin and collagenase. The supernatant was centrifuged to collect myocytes. After the supernatant was discarded, the cells were resuspended in culture medium containing 10% fetal bovine serum and Dulbecco s modified Eagle s medium (Gibco Laboratories, Life Technologies Inc, Grand Island, NY). The fibroblasts and myocytes were separated manually, and the identity of the myocytes was confirmed by the morphological appearance (Fig 1, left) and by fluorescent staining with antibodies to actin [lo] (Fig 2, left) and to ventricular myosin light chain 1 [ll] (Fig 2, right). Myocyte cultures of the same generation and age were employed for these studies. The number of myocytes tested in each culture dish was 2.25 * 0.06 x los cells, which was similar for each experiment. The time to myocyte necrosis from a pharmacological free radical injury was used to compare the effectiveness of the antioxidants. The morphological criteria for myocyte necrosis were sarcolemma1 rupture and cell shrinkage (Fig 1, right). Free Radical Studies The free radical studies were performed by removing the cell culture medium and adding 6.0 ml of phosphatebuffered saline solution (ph 7.4). Incubation (at 37 C) with phosphate-buffered saline solution alone did not produce any morphological changes for 45 minutes (n = 10). The addition of hypoxanthine (1 mmolil) and xanthine oxidase (300 IUIL) rapidly produced myocyte necro- 0 1989 by The Society of Thoracic Surgeons 0003-4975/89/$3.50

554 MICKLE ET AL 1989;47:55>7 Fig I. The morphological appearance of cultured canine myocytes in phosphate-buffered saline solution (left) and two minutes after the addition of hypoxanthine and xanthine oxidase to generate free radicals (right). The free radicals produced sarcolemmal rupture and cell shrinkage. (Magnification, ~200 before 53% reduction.) sis (n = 10) (see Fig 1, right). The addition of hypoxanthine (n = 10) or xanthine oxidase (n = 10) alone did not induce any morphological changes at 15 minutes. The substances evaluated for preventing free radical attack were Trolox, a water-soluble analogue of vitamin E (Aldrich Chemical Co, Milwaukee, WI); ascorbic acid (Hoffmann-La Roche, Brampton, Ont, Canada); SOD (Sigma Chemical Co, St. Louis, MO); and catalase (Boehringer-Mannheim, Montreal, Que, Canada). All antioxidants tested were administered immediately before the addition of the free radical generation system. The concentrations of each antioxidant were established from previous reports and from preliminary studies. We employed approximately twice the concentrations of SOD and catalase reported by Jolly and colleagues [7] to reduce myocardial infarct size by 50% in canine studies of regional ischemia (SOD = 14,500 IU/kg and catalase = 55,000 IU/kg). Preliminary studies were performed to establish the optimal concentrations of Trolox and ascorbic acid to be tested. In myocyte culture studies, we found that Trolox concentrations of less than 1.34 mmol/l reduced the time to myocyte necrosis. Therefore, we used this concentration for both Trolox and ascorbic acid in our myocyte culture studies. The effect of preincubating the myocytes with Trolox and ascorbic acid in phosphate buffered saline solution, before the addition of the free radical generation system, was studied to determine the optimal protection times for these antioxidants. Canine Studies Mongrel dogs weighing 15 to 25 kg were anesthetized with sodium pentobarbital (30 mg/kg administered intravenously), intubated, and ventilated with a Harvard respirator. The animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). A median sternotoiny was performed. Catheters were placed in the right femoral artery to monitor arterial pressure, and a micromanometer-tipped catheter was inserted into the left ventricle from the apex to monitor ventricular pressure. The LAD was isolated just distal to the first diagonal branch, and a 3 to 5 mm segment was dissected. The heart was carefully inspected, and any large collateral vessel:$ from the right or circumflex coronary artery were revlxsibly ligated near the ventricular apex to provide a reproducible region of ischemia. A Doppler flow probe was placed around the LAD to confirm the absence clf blood flow with ischemia and the restoration of blood Ilow with reperfusion in the LAD. The animals were randomly allocated to the three experimental groups: angioplasty group, saline group, and Trolox group. In the angioplasty group (n = 9, the LAD was occluded for two hours. Then the occlusion was released, and the ischemic myocardium was reperfused for four hours to simulate emergency a ngioplasty in a patient seen within two hours of an acute coronary occlusion. In the saline group (n = 7), 500 ml of deoxygenated saline solution containing 18 mg of EDTA (ethylenediaminetetraacetic acid) at a ph of 7.4 was rapidly infused into the ascending aorta, beginning 30 seconds before reperfusion, at a rate of approximately 100 ml/min. The Trolox group (n = 6) also received 500 ml, of deoxygenated saline solution containing 18 mg of EDTA, 2.0 g of Trolox (16 mmol/l), and 3.0 g of ascorbic xid (34 mmol/l) at a ph of 7.4. Arterial pressures were maintained near control values during reperfusion by fluid infusions. The animals were killed with an anesthetic overdose, and the hearts were excised after four hoc rs of reperfusion. Determination of Are2 at Risk and Area of Infarction The area at risk and the area of infarction were estimated by a dual-staining technique [7]. Evans blue (500 ml, 0.25%) was infused into the aortic root at a pressure of 80 mm Hg with the LAD occluded to assess the area at risk. At the same time, 500 ml of 1.5% triphenyl tetrazolium Fig 2. Cultured canine vettricular myocytes zuith fluorescent staining by antibodies to actin (left, and to ventricular myosin light chairi 1 (right). (Magnification, x 200 before 53% reduction.)

1989;47553-7 MICKLE ET AL 555 rn c *" 1 L E. 9 1 T * *p <0.01 I I I Free Troiox Ascorbic Trolox S& Catalase S&. Radical Acid and and System Ascobic Catalaae Acid Fig 3. The time from the addition of hypoxanthine and xanthine oxidase (to generate free radicals) until myocyte necrosis (no preincubation with antioxidants). Free radical generation rapidly produced tnyocyte necrosis. Trolox, ascorbic acid, and the combination of the two prolonged the time to niyocyte necrosis, but superoxide distirutase (SOD), catalase, and the cornbination of the two did not protect the myocytes from free radical injury. hydrochloride was infused into the LAD distal to the site of occlusion at 37 C and 80 mm Hg to delineate the area of infarction. The left ventricle was cut into slices 1 cm thick, and the areas at risk (the regions that did not stain with Evans blue) and the areas of infarction within the area at risk (the regions that did not stain with triphenyl tetrazolium) were measured with a planimeter. The area of infarction was expressed as a percentage of the area at risk. Statistical Analysis Statistical analysis was performed with the Statistical Analysis System programs (SAS Institute, Cary, NC). Differences between times to myocyte necrosis and the areas of infarction were evaluated by an analysis of variance, and the differences were specified by Duncan's multiple range test when the F ratio of the analysis of variance was significant ( p < 0.05). Canine hemodynamic measurements were assessed by a two-way analysis of variance (simultaneously evaluating time and treatment group), and differences were specified by a one-way analysis of variance and Duncan's test. bination of Trolox (0.67 mmovl) and ascorbic acid (0.67 mmol/l) significantly (p < 0.01) prolonged the time to myocyte necrosis (see Fig 3). However, the addition of SOD (24,200 IU/L), catalase (92,000 IU/L), or the combination of these at the same concentrations did not prolong myocyte survival after free radical generation (see Fig 3). Therefore Trolox and ascorbic acid were more successful than SOD and catalase in retarding free radical damage to the myocytes. In preincubation studies, the antioxidant was preincubated before the addition of hypoxanthine and xanthine oxidase. The antioxidant effect of Trolox peaked with three minutes of preincubation and then decreased to control levels with seven minutes of preincubation, whereas that of ascorbic acid decreased linearly with increasing duration of preincubation and also approached control levels with seven minutes of preincubation. These results indicated that the times of optimal protection for Trolox and ascorbic acid differed, and suggested that some Trolox may need to be incorporated into the lipid membranes of the myocyte to be most effective. Canine Study Hernodynamic measurements were not different between groups before reperfusion. Occlusion of the LAD decreased left ventricular end-systolic pressure from 110? 33 mm Hg before occlusion to 96 32 mm Hg after two hours of occlusion (p < 0.01) at similar left ventricular end-diastolic pressures (7 * 2 mm Hg before occlusion and 8 * 6 mm Hg after two hours of occlusion). Reperfusion decreased left ventricular end-systolic pressure in the angioplasty and saline groups but not in the Trolox group (at four hours of reperfusion: angioplasty group, 74? 29 mm Hg; saline group, 85 * 20 mm Hg; Trolox group, 110 * 25 mm Hg; p < 0.01). Reperfusion increased left ventricular end-diastolic pressure in the angioplasty group but not in the Trolox group (at four hours of reperfusion: angioplasty group, 11 * 2 mm Hg; saline group, 9 * 4 mm Hg; Trolox group, 5? 3 mm Hg; p < 0.01). The infusion of dopamine hydrochloride at 4 Pglkgi min restored left ventricular end-systolic pressure to I ~~ 7 *p <.01 Different than Trolox Results Myocyte Cell Cultures In control experiments with myocytes in phosphatebuffered saline solution but without hypoxanthine or xanthine oxidase, myocytes had no morphological changes until 45 minutes. The addition of either hypoxanthine or xanthine oxidase did not induce any morphological changes by 15 minutes. The addition of hypoxanthine and xanthine oxidase induced myocyte necrosis within three minutes (Fig 3; see Fig 2, left). The addition of Trolox (1.34 mmol/l), ascorbic acid (1.34 mmol/l), and the com- Angioplaaty Saline Trolox Fig 4. The area of infarction (AI) expressed as a percentage of the area at risk far) was significantly reduced with Trolox. An infusion of deoxygenated saline solution reduced the percentage of infarction compared with the angioplasty group, but the difference was not significant.

556 MICKLE ET AL 1989;4755>7 preischemic values in all groups, but the left ventricular end-diastolic pressure measurements remained different (angioplasty group, 14 * 6 mm Hg; saline group, 11 +- 2 mm Hg; Trolox group, 5 * 3 mm Hg; p < 0.05). The area at risk as a percentage of the area of the left ventricle was not significantly different between the three groups (angioplasty group, 22.3% * 3.1%; saline group, 20.8% -t 4.1%; Trolox group, 20.1% * 2.0%). Figure 4 illustrates the area of infarction expressed as a percentage of the area at risk. Trolox significantly reduced the area of infarction within the area at risk. In addition, the saline group had a lower percentage of infarction compared with the angioplasty group, but the difference was not significant ( p = 0.051). Comment Experimental studies have implicated oxygen-mediated free radical injury as an important mechanism of damage in the setting of ischemia and reperfusion (7-91. The myocardial defenses against free radical injury include two types of antioxidants: preventive antioxidants, which convert potential sources of free radicals such as H,O, into inert products, and chain-breaking antioxidants, which trap free radicals and prevent peroxidative damage. In biological systems, glutathione peroxidase and catalase are preventive antioxidants, whereas SOD, ascorbic acid, uric acid, bilirubin, reduced glutathione, and protein sulfhydryl groups are water-soluble chainbreaking antioxidants [12, 131. Vitamin E is the major and possibly the only lipid-soluble chain-breaking antioxidant [14, 151. Doroshow and colleagues [ 161 demonstrated that heart muscle contains only 27% of the SOD activity and less than 1.3% of the catalase activity found in the liver. Both heart and liver are equally active with respect to seleniumdependent glutathione peroxidase activity. Therefore, the heart muscle is not as well equipped to detoxify free radicals as the liver and may be more sensitive to reperfusion injury [16]. Guarnieri and colleagues [17] found that hypoxia decreases myocardial glutathione peroxidase and SOD activities, which persist for at least 60 minutes after reoxygenation. Das and associates [IS] found that the activity of SOD, catalase, and glutathione peroxidase is decreased after ischemia. Therefore, myocardial free radical injury may be aggravated during reperfusion because the cellular defenses are diminished by ischemia and hypoxia. Gauduel and Duvelleroy [19] demonstrated that a- tocopherol is a more effective antioxidant than SOD, catalase, reduced glutathione, or mannitol. They found that a-tocopherol is at least three times more effective than SOD and five times more effective than catalase in preventing malonaldehyde formation during reperfusion after global ischemia. Because a-tocopherol is lipid sohble, it cannot be given intravenously to patients with an acute myocardial infarction. The ideal antioxidant to prevent myocardial free radical injury should be both water soluble and lipid soluble to facilitate diffusion into both the lipid and aqueous compartments and prevent membrane lipid peroxidation. Trolox (6-hydroxy-2,5,7,8-teramethyl-chroman-2-carboxylic acid, an antioxidant originally designed for food preservation [20]) has a structural resemblance to a-tocopherol but is both water soluble and lipid soluble. In vitro studies [21] have demonstrated that Trolox is an excellent antioxidant for inhibiting peroxidation in phospholipid bilayers. Although a-tocopherol traps peroxyl radicals more rapidly than Trolox in hydrocarbon solvents [22,23], Trolox is much more soluble in water and is therefore able to rapidly equilibrate between the aqueous and lipid phases 1241. The much greater mobility of Trolox and its ready solubility in water me;n that it can be quickly recruited and used at sites of lipid peroxidation when local defenses including, in particular, the membrane-bound antioxidant a-tocophercil have been overwhelmed by free radical attack. Ascorbic acid has been demonstrated to be an effective water-soluble antioxidant [12, 211. In addition, ascorbic acid can regenerate a-tocopherol and Trolox after their partial oxidation by free radicals to the tocopheroxyl or Trolox radicals, respectively (21, 251. In this way, the membrane free radical is transported, in effect, from the lipid phase, which it could damage, out into the more or less inert aqueous phase [21, 251. Both Trolox and ascorbic acid were more protective in the myocyte culture studies against a pharmacological free radical injury than S,3D, catalase, or the combination of the two, despite the I act that we employed twice the concentrations recommended by Jolly and colleagues [7]. Because our preincubation studies appeared to reflect different action sites, we elected to use a combination of Trolox and ascorbic acid in our canine studies to optimize scavenger protection. In our canine studies, Trolox and ascorbic acid were prepared in deoxygenatecl saline solution, and EDTA was added to chelate any iron present in the solution. Both Trolox and ascorbic acid are readily oxidized, and anaerobic conditions must be present to maintain their antioxidant activity. In addition, administration of these agents into the ascending aorta may prevent oxidation prior to their delivery to the myocardium. We used the dual-staini.7g technique described by Jolly and coworkers [7] to asses.$ the extent of infarction within the region at risk. Previous studies from our laboratory [26] have shown that even one hour of reperfusion is sufficient to wash out the myocardial dehydrogenase enzymes required to convert tetrazolium to its red color. To confirm the adequacy of our staining technique, we performed electron microscopy on the areas that did and those that did not stain red with tetrazolium. The red areas had minimal disruption of cellular architecture, whereas areas without staining had marked deterioration of cellular architecture. Trolox and ascorbic acid effectively reduced infarction in this canine model intended to simulate successful thrombolysis and emergency angioplasty in a patient seen within two hours of an acute LAD occlusion. An infusion of deoxygenated saline solution during reperfusion also reduced the area of infarction within the area at risk, although the difference was not significant. Previous studies in our laboratory [2?] demonstrated that limiting

1989:47:553-7 MICKLE ET AL 557 the oxygen content of the initial reperfusate reduced the amount of necrosis of an isolated gracilis muscle that had been ischemic for 12 hours. The deoxygenated saline solution also contained EDTA, which may have reduced oxygen free radical formation by chelating iron. The combination of Trolox, ascorbic acid, and deoxygenated saline solution produced a 66% decrease in the myocardial infarction within the area at risk. This salvage is greater than has been reported with enzyme antioxidants. Our results suggest that an infusion of nonenzyme antioxidants during urgent revascularization may improve myocardial salvage with reperfusion. In conclusion, the antioxidants, Trolox and ascorbic acid, prepared in a deoxygenated medium effectively prevented myocardial necrosis both in ventricular myocyte cell cultures and in a canine model of two hours of LAD occlusion followed by four hours of reperfusion. 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