Pretreatment With Antioxidants and Allopurinol Diminishes Cardiac Onset Events in Coronary Artery Bypass Grafting

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1 Pretreatment With Antioxidants and Allopurinol Diminishes Cardiac Onset Events in Coronary Artery Bypass Grafting Tero Sisto, MD, Hannu Paajanen, MD, Timo Mets/i-Ketel~i, PhD, Aimo Harmoinen, PhD, Isto Nordback, MD, and Matti Tarkka, MD Departments of Surgery and Clinical Chemistry, Tampere University Hospital, and Department of Biomedical Sciences, University of Tampere, Tampere, Finland Oxygen-derived free radicals constitute one part of the etiologic factors for cardiac onset harmful events. Allopurinol is able to reduce the generation of free radicals. Vitamins E and C scavenge radicals after their formation. Eighty-one patients with coronary artery disease were randomized into four study groups: Group 1 (n = 20) patients had stable disease and received oral vitamin E for 4 weeks, and vitamin C and allopurinol 2 days before and 1 day after coronary artery bypass grafting. Group 2 (n = 25) consisted of their controls. Group 3 patients (n = 17) had more unstable disease and received the same medications as group 1, except that vitamin E was given only 2 days before the operation. Group 4 (n = 19) was their controls. Groups 1 and 3 had fewer ischemic electrocardiographic events and required less dopamine perioperatively than corresponding control groups 2 and 4. Group 3 had fewer perioperative infarctions and less creatine kinase-mb release than the respective controls (group 4). Plasma levels of vitamins E and C, urate, and total free radical trapping ability were considered to support the theory about the role of free radicals in reperfusion injury. Especially the unstable patients, but also patients with stable coronary artery disease requiring coronary artery bypass grafting benefit from perioperative allopurinol and vitamin E and C treatment. (Ann Thorac Surg 1995;59: ) O xygen-derived free radicals are important components in the multifactorial etiology of myocardial dysfunction after ischemia. A significant increase in the generation of free radicals measured directly [1] or as lipid peroxidation products [2] has been shown in the human myocardium after successful thrombolysis or angioplasty. These clinical studies support the experimental studies [3, 4] showing that free radicals are generated during reperfusion after ischemia. Controversy prevails as to whether similar free radical generation takes place during reperfusion in coronary artery bypass grafting (CABG) [5, 6]. The significance of reperfusion injury, ie, the harmful effect of free radicals on the myocardium, has been questioned, but recent experimental studies provide strong evidence that free radicals may mediate myocardial injury [4, 7, 8]. Attempts have been made to prevent this injury by a variety of agents [9]. In many animal studies [10-12], but also in human clinical studies [13-15[, allopurinol, by reducing the formation of free radicals, reduces harmful myocardial consequences following reperfusion after global ischemia. Free radical levels measured by indirect methods correlate inversely with the vitamin E level. Dietary vitamin E prevents a free radical rise during reperfusion in a coronary artery bypass operation [6]. Vitamin E synergistically with vitamin Accepted for publication Feb 28, Address reprint requests to Dr Sisto, Samoilijankatu 27, Tampere, Finland. C ameliorates the injury caused by free radicals by reducing the amount of free radicals after their generation [6, 16]. Thus, theoretically the strongest protection against free radical injury may be achieved by both reducing the generation (allopurinol) and increasing the scavenging after the generation of free radicals (vitamin E and C). The purpose of this clinical randomized study is to ascertain whether pretreatment with allopurinol, combined with vitamins E and C, has an effect on myocardial onset events after reperfusion in CABG. Material and Methods The study design was accepted by the Ethical Committee of Tampere University Hospital, Finland, and informed consent was obtained from all patients. Study participants consisted of 81 patients undergoing a standard coronary artery bypass operation. Two separate randomizations were performed. (1) Those in whom the coronary heart disease allowed more than 1 month delay before the operation were randomly divided into group 1 (n - 20) and the control group 2 (n = 25). Patients in group 1 received vitamin E (600 rng daily) 28 days before CABG, and in addition both vitamin C (2 g daily) and allopurinol (600 mg daily) for 2 days before and I day after CABG. (2) Those who had more unstable clinical condition were randomized into group 3 (n = 17) and the control group 4 (n = 19). Group 3 followed the same scheme as group 1, but taking vitamin E only 2 days 1995 by The Society of Thoracic Surgeons /95/$ (95)00197-S

2 1520 SISTO ET AL Ann Thorac Surg ANTIOXIDANT PRETREATMENT IN CABG 1995;59: Table 1. Patient Characteristics a Group 1 Group 2 Group 3 Group 4 Variable (n = 20) (n = 25) (n- 17) (n = 19) Age (y)b Women 6 (30) 7 (28) 7 (41) 5 (26) NYHA class II 3 (15) 6 (24) 3 (18) 4 (21) III 15 (75) 15 (60) 11 (65) 8 (42) 1V 2 (10) 4 (16) 3 (17) 7 (37) Diabetes 1 (5) 2 (8) 2 (12) 3 (16) Previous AMI 11 (55) 9 (36) 8 (47) 13 (68) Hypercholesterolemia 6 (30) 9 (36) 7 (41) 8 (42) (>6.5 retool/l) EF < a Values are shown as number of patients, with percentage in parentheses. b Mean -+ standard deviation. AMI - acute myocardial infarction; EF = ejection fraction; NYHA - New York Heart Association. before the operation. Control groups 2 and 4 did not receive any study medication. The randomization was performed by birth year: patients with uneven birth year belonged to the study group and patients with even birth year to the control group. If a patient could not eat on the first postoperative morning, the vitamins were given intravenously and allopurinol was given through a nasogastric tube. The following patient exclusion criteria were used: women of premenopausal age, myocardial infarction or the use of study medicines during 30 days before CABG, renal (creatinine level >150 ~g/l) or hepatic (aspartate aminotransferase level >40 IU/L) disease, and any known allergy to study medicines. Preoperative patient characteristics are shown in Table 1. The patients were perfused in moderate hypothermia (28 C) with nonpulsative flow from a bubble oxygenator (Shiley 100 A; Shiley Inc, Irvine, CA). The circuit was primed with 2,000 ml of Ringer's acetate. Blood cardioplegia (6 to 8 C) was delivered through the BCD-Plus device (Shiley), which mixed blood with asanguineous solution in a ratio of 4:1. Potassium concentration of the induction plegia was 21 mmol/l, and reinfusion given every 15 minutes contained 9 mmol/l of potassium. Warm blood cardioplegia was given at the end of the cross-clamp period. There was no statistical difference in operative data between the study groups (Table 2). All patients received in addition to vein grafts one internal mammary artery graft. Twelve-lead electrocardiogram was recorded before and just after completion of CABG, and daily for 5 postoperative days. Dissociation disturbances (yes or no) were detected, as well as ischemic electrocardiographic alterations (T-wave inversion, ST-segment alteration more than 1 mm and Q wave) by an independent cardiologist without knowledge of the study group of the patient. The criteria for myocardial infarction were a new Q wave in the electrocardiogram after operation, and an increase in the level of creatine kinase-mb (CK-MB) over the value of 125 IU/L. Blood samples for CK-MB mea- surement were taken before operation, 5 and 30 minutes after the removal of the aortic clamp, 8 hours after operation and daily thereafter for 2 postoperative days. Enzyme levels were determined by direct measurement of the CK-B subunit after immunoinhibition of the CK-M. During the operation and the stay in the intensive care unit the use of adrenaline, noradrenaline, dopamine, and nitroglycerin was recorded. Blood samples for measurement of plasma total free radical trapping ability (TRAP) as well as for measurement of tocopherol (vitamin E), urate, and ascorbic acid (vitamin C) levels were taken before operation and on the first postoperative morning. The plasma concentrations of vitamins E and C and uric acid were measured by high-performance liquid chromatography with an electrochemical detector. The TRAP determination is described in detail elsewhere [17]. Total free radical trapping ability is expressed as micromoles of peroxyl radicals trapped by I L of the sample. In addition to the direct measurement of TRAP, a calculated TRAP also was derived from the concentrations of individual peroxyl radical-trapping antioxidants in plasma with experimentally determined stoichiometric factors. The following stoichiometric factors were found earlier and now used: TRAP calculated = 2.0 (vitamin E) (uric acid) (vitamin C) (protein sulfhydryl [SH] groups). Results are expressed as means. Statistical analyses were made using Student's t test for parametric data and Mann-Whitney or )(2 tests for nonparametric data. For comparing data within the same group before and after operation a paired-samples t test was used. One-way analysis of variance with Bonferroni test was used when multiple comparisons were made between all four groups. Probabilities less than 5% (p < 0.05) were considered significant. Calculations were made with an SPSS statistical program (SPSS Inc, Chicago, IL) for personal computers. Results No operative or hospital mortality occurred. No patient required mechanical assistance for maintenance of hemodynamics after operation. No side effects of the study medicines were noted. Table 2. Operative Data Variable Group 1 Group 2 Group 3 Group 4 Distal anastomoses Cross-clamp time (min) CBP time (min) Plegia a Antegrade 12 (60) 19 (76) 11 (65) 13 (68) Retrograde 8 (40) 6 (24) 6 (35) 6 (32) a Number of patients, with percentage in parentheses. CBP = cardiopulmonary bypass.

3 Ann Thorac Surg SISTO ET AL ;59: ANTIOXIDANT PRETREATMENT IN CABG Table 3. Cardiac Events" p Value, p Value, Variable Group 1 Group 2 1 vs 2 Group 3 Group 4 3 vs 4 Postoperative infarction 1 (5) 2 (8) NS 1 (6) 6 (32) 0.06 Ischemic ECG events 4 (20) 13 (52) (18) 11 (58) 0.01 Dissociation disturbances 5 (25) 6 (24) NS 2 (12) 8 (42) 0.05 a Values are shown as number of patients with percentage from total number of patient group in parentheses. ECG = electrocardiographic; NS = not significant. Cardiac event rates are presented in Table 3. There were significantly more ischemic electrocardiographic events in the control groups 2 and 4 than in the respective study groups 1 and 3. There was a clear difference between groups 3 and 4 in the occurrence of arrhythmias. No difference was detected in the perioperative use of noradrenaline and nitroglycerin. Only I patient received adrenaline in group 3. Use of dopamine was more frequent and lasted longer in control group 4 compared with the respective study group 3. Between groups 1 and 2 there was no difference. Control groups had a tendency to require more dopamine for the support of hemodynamics than treatment groups (p = 0.057). Values of CK-MB measured preoperatively and 5 and 30 minutes after aortic clamp release did not differ from each other. Postoperative CK-MB release was higher in group 4 than in group 3 (68 versus 36 U; p = 0.01). Group 2 CK-MB values did not differ from those in group 1 (46 versus 41 IU; p = 0.49). Vitamin levels, urate levels, and TRAP before and after operation are shown in Figure 1. Preoperative values of vitamin C and urate were about the same in each group. In group 1 the preoperative vitamin E level was highest; the other three groups did not differ from each other. Calculated TRAP values were similar to measured TRAP values. Vitamin C level diminished in the control groups but not in the treatment groups. Urate levels decreased in the treatment groups but not in the control groups. Comment Combination therapy with allopurinol, vitamin C, and vitamin E appeared to inhibit ischemic electrocardiographic alterations. Allopurinol together with vitamin C also showed a trend toward protection from perioperatire infarction. Perioperative myocardial infarction was less frequent in the treatment groups. However, the protective effect of the present medication on the myocardium was not as marked as observed in a previous clinical study [14]. One study has shown that episodes of arrhythmia are fewer among patients treated with allopurinol [13]. We could not confirm this. Also, CK-MB release in our study was lower in groups treated with antioxidants and allopurinol than in the respective control groups. Opposite results also have been reported! 7,1 p o.ool 4g 1 p=0,03 p=0.03 p=0.05 g, A Ilbefore [~affer group 1 group 2 group 3 group 4 group 1 group 2 group 3 group 4 ~before [~aher B group 1 group 2 group 3 group 4 group 1 group 2 group 3 group 4 Fig 1. Vitamin E (A), vitamin C (B), urate (C), and total free radical trapping ability (D) concentrations (~tmol/l) in plasma before and after operation. The statistical difference between concentrations before and after operation in each group is shown. (NS = not significant.) D ~before ~]after

4 1522 SISTO ET AL Ann Thorac Surg ANTIOXIDANT PRETREATMENT IN CABG 1995;59: regarding CK-MB release [13] and perioperative infarction [15l. The ischemia-reperfusion-induced injury during and after open heart operation has been suggested to be mediated by free radical-induced peroxidation of structural lipids in the biomembranes of cells and organelles. The precise pathogenesis of reperfusion injury is not clear. However, these cell membrane alterations probably are responsible for the observed clinical consequences, such as arrhythmias, functional disturbances, and even stunning of the myocardium [4]. There are two means of reducing the cell injury caused by free radicals: either preventing their production or increasing the amount of agents that can scavenge these radicals after their generation. Allopurinol is a competitive inhibitor of xanthine oxidase, the enzyme that converts hypoxanthine to uric acid. Allopurinol, or its active metabolite oxypurinol, is also a weak radical scavenger. Several animal studies have proved that pretreatment with allopurinol in the cardioplegic solution has a beneficial, preventive effect on ischemic myocardial injuries [10, 12, 18]. Also, pretreatment with oral allopurinol has a beneficial clinical effect, for example in terms of arrhythmias, perioperative infarctions, the use of inotropes [14], and hospital mortality [15]. However, the mechanism involved is uncertain, because xanthine oxidase has been detected by current methods at very low amounts in the human heart [19]. Malkiel and associates [20] have pointed out that allopurinol decreases the rate of copper-mediated ascorbate oxidation. Thus, allopurinol may potentiate the possible beneficial effect of vitamin C. Urate, a salt of uric acid, is an indicator of the presence of uric acid. The preoperative levels of urate are similar in each group, but the effect of allopurinol after operation is seen in the treatment groups as decreased urate. Neither in earlier human nor in animal studies has the effect of allopurinol been confirmed by urate or uric acid measurements [13-15[. However, urate is a good peroxyl radical scavenger. Thus, reducing urate production by inhibition of xanthine oxidase may also be harmful, at least theoretically. This dualistic role of allopurinol may explain some of the contradictions between the allopurinol trials. Lipid soluble vitamin E is a free radical scavenger. It also acts synergistically with vitamin C, which produces vitamin E from its radicals [16]. Heart cell membranes contain vitamin E, which protects from oxidative injury in rats [21]. Vitamin E also has been found to prevent the formation of free radicals in humans [6]. This has been considered indirect indication that vitamin E also could diminish myocardial injury. Vitamin C is a primary factor in the defense against free radicals due to its free radical trapping ability and its indirect function via its interaction with vitamin E. It therefore is the most effective water-soluble antioxidant in the extracellular fluid and the cytoplasm [22]. We have found, as expected, that the plasma vitamin E level is highest in group 1, which received vitamin E supplementation I month before operation. We could not show any significant difference between the study groups in the change in plasma vitamin E levels before or after operation. In the study by Cavarocchi and associates [6] the vitamin E level before cardiopulmonary bypass was lower in the group receiving vitamin E substitution 12 hours before operation. It has been shown that at least 300 mg of vitamin E has to be taken 14 days before operation to double the myocardial vitamin E levels. Even 800 mg daily of oral vitamin E does not increase patients' vitamin E levels if taken only 2 days before operation [23]. Possibly due to this short interval between taking this lipid-soluble vitamin and operation, serum in the study by Cavarocchi and associates was not saturated with vitamin E. In the present study the amount of vitamin E decreased significantly in each group after operation similarly to the TRAP levels, possibly due to vitamin E consumption in the antioxidative process, as also has been suggested in earlier studies [61. However, another possible reason for this decrease in vitamin E level could be hemodilution during operation. In this study we observed that the highest vitamin E levels decreased the most after operation, suggesting that the greater the supply of vitamin E the greater the consumption of it. Total free radical trapping ability describes total interaction and effectiveness of antioxidant defense systems. Total free radical trapping ability activity involves four major secondary antioxidants: vitamins E and C, urate, and SH groups, of which vitamin E may be the most important factor. Thus, it is not surprising that the free radical trapping capacity is highest in group 1. In this study plasma vitamin C levels decreased in the control groups more than in the study groups. Klein and associates [24] have shown that in pigs receiving vitamins E and C a week before ischemia the size of myocardial infarction is reduced more than with the same treatment during ischemia, although both treatments are significantly effective. In the latter case the result may be the effect of vitamin C alone. The greater consumption of vitamin C in control groups than in treatment groups in our study probably indicates the need for it in the antioxidative process and the lack of supplementation. The biochemical findings described above seem in accord with the free radical theory. It should be borne in mind, however, that the biochemical measurements reflect plasma levels and not tissue (myocardium) levels. However, although the exact mechanism remains to be verified, these results demonstrate that antioxidants and allopurinol are able to significantly reduce perioperafive myocardial ischemia. References 1. Grech ED, Dodd NJF, Bellamy CM, Perry RA, Morrison WL, Ramsdale DR. Free-radical generation during angioplasty reperfusion for acute myocardial infarction. Lancet 1993;341: Roberts MJD, Young IS, Trouton TG, et al. Transient release of lipid peroxides after coronary artery balloon angioplasty. Lancet 1990;336: Garlick PB, Davies MJ, Hearse DJ, Slater TF. Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res 1987;61: Bolli R, Jeroudi MO, Patel BS, et al. Marked reduction of free

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