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1 Reduction of Reperfusion Injury With Mannitol Cardioplegia Ricardo Ferreira, MD, Mario Burgos, MD, Susana Llesuy, PhD, Luis Molteni, MD, Jose Milei, MD, Beatriz Gonzalez Flecha, PhD, and Albert0 Boveris, PhD Department of Cardiovascular Surgery (ECAVI), Sanatorio Colegiales, Buenos Aires; Institute of Histology and Embryology, School of Medicine, UNC, CONICET, Mendoza; Department of Biological Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires; and Department of Cardiology, Hospital Fernandez, Buenos Aires, Argentina Forty consecutive patients undergoing myocardial revascularization were divided into two equal groups: group 1 received standard cardioplegic solution, and group 2 received a solution containing mannitol, 59.8 mmol/l. In 6 patients in each group, myocardial biopsies were done before ischemia and at the time of reperfusion. Samples were assessed by chemiluminescence to determine oxidative stress and by electron microscopic studies. A significant reduction in atrial arrhythmias was observed in the mannitol group. Chemiluminescence in group 1 showed a photoemission of 37.6 f 3.5 cpm/mg of protein x for the preischemia samples and 74.8 f 16 cpdmg of protein x for the reperfusion samples (p < 0.001). In group 2, the values for chemiluminescence were 37.7 f 3.4 cpdmg of protein x lo- and 40 f 6.1 cpdmg of protein x respectively (p = not significant). Electron microscopic studies showed, for group 1, increased grades of damaged mitochondria in the reperfusion biopsy specimens compared with the preischemia biopsy specimens (p < 0.01). In group 2, differences for damaged mitochondria were not significant. These results support the hypothesis that mannitol reperfusate significantly reduces myocardial damage in patients undergoing open heart procedures. They also suggest that this protective effect may be in part secondary to the antioxidant property of mannitol, although other mechanisms may have accounted for or contributed to the improved outcome after ischemia. (Ann Thoruc Surg 1989;48:77-84) lthough topical cooling and cold potassium car- A dioplegia have markedly reduced mortality and morbidity in open heart procedures, there still exists a variable degree of myocardial injury as a consequence of the ischemia/reperfusion mechanism. Cell damage has been assessed by both ultrastructural and histochemical studies, and is clinically expressed by the presence of disturbances in the conduction system and supraventricular and ventricular arrhythmias. Moreover, in patients with a low ejection fraction preoperatively, the postoperative period can also be associated with impaired ventricular function. Evidence accumulated over the past several years indicates that oxygen-derived free radicals are in part responsible for the production of reperfusion injury. We [l] have recently observed the presence of myocardial oxidative stress caused by these metabolites in patients who underwent coronary artery bypass grafting. It has also been shown in animal models [2-51 that a variety of antioxidants or scavengers are able to reduce cell damage associated with the ischemiaheperfusion mechanism as well as the incidence of reperfusion-induced arrhythmias. Moreover, proarrhythmic effects were obtained when the heart was exposed to an oxygen-derived free radicalgenerating system [5]. In the present study, we tested the hypothesis that Accepted for publication Feb 9, 1989 Address reprint requests to Dr Ferreira, OHiggins 1780, Florida, 1602, Buenos Aires, Argentina. administration of mannitol in a cardioplegic solution reduces the incidence of postoperative arrhythmias and myocardial damage. Oxygen free radical activity was indirectly assessed by chemiluminescence and myocardial damage, by electron microscopic studies. Material and Methods This prospective trial involved 40 patients who underwent coronary artery bypass grafting between September 1, 1987, and February 1, To obtain a homogeneous group, patients with the following characteristics were considered for the study: an ejection fraction greater than 45%; absence of recent (less than 4 weeks) myocardial infarction; and complete or satisfactory revascularization at the time of operation. The patients were divided into two equal groups based on the cardioplegic solution used. Age, ejection fraction, aortic-cross clamp time, and number of grafts performed were similar in the two patient groups (Table 1). Written informed consent was obtained from those patients who were selected for myocardial biopsy. Cardiopulmonary bypass was instituted, and the perfusate was cooled to 28 C. Topical hypothermia with crushed ice saline solution was added. The two study groups were as follows: 20 patients (group 1) received 500 ml of standard cardioplegic solution, and 20 patients (group 2) received 500 ml of a cardioplegic solution containing mannitol (59.8 mmovl) (Laboratorios FADA, Buenos Aires, Argentina). The composition of the infu by The Society of Thoracic Surgeons /89/$3.50

2 78 FERREIRA ET AL Ann Thorac Surg 1989:48:77-84 Table 1. Characteristics of the Two Groupsa Group 1, Group 2, Control Mannitol Variable (n = 20) (n = 20) Age (yr) C Ejection fraction (W) 53 L Aortic cross-clamp time (min) No. of grafts LAD grafts a Data are shown as the mean k the standard error of the mean where applicable. LAD = left anterior descending coronary artery. sions is listed in Table 2. The osmolarity of both perfusates was measured with a vapor pressure osmometer (Wescor 5500, Wescor, Logan, UT). In both groups the solution was administered into the aortic root at a temperature of 4 C and a pressure ranging between 80 and 100 mm Hg. A needle thermistor (Yellow Springs Instrument Company, Yellow Springs, OH) was inserted into the septum for continuous monitoring of myocardial temperature, which was maintained at approximately 18 C. After each distal anastomosis was constructed, 50 ml of the respective cardioplegic solution was given into the vein graft. In all circumstances, at least one of the internal mammary arteries was used, and this anastomosis was done as the last step before the aortic clamp was removed. Six patients in each group were randomly chosen to undergo myocardial biopsies, which were done according to the following protocol. Before the aorta was crossclamped, full-thickness needle biopsy specimens (Travenol Tru-cut biopsy needle) were obtained from the apex of the left ventricle. Specimens for chemiluminescence determination were immersed in normal saline solution and immediately frozen, and specimens for electron microscopy were placed in cold 3% glutaraldehyde in 0.1 moyl of cacodylate buffer (ph 7.4). Both sets of specimens were designated as preischemia samples. After completion of the distal anastomoses, the aortic crossclamp was removed and defibrillation was performed at 30" to 33 C using a 40-W-s direct-current countershock. After ten minutes of reperfusion and with the patient rewarmed, new biopsy specimens were obtained and processed as described. These specimens were designated as reperfusion samples. The patient was weaned from cardiopulmonary bypass when the systemic temperature reached 37 C. Study of Rhythm Disturbances At the time of operation, the number of electric countershocks required to defibrillate the heart after the aortic cross-clamp was removed was registered. Rhythm disturbances that occurred within the first 24 hours after operation and required treatment were considered. They included the following: atrial fibrillation; supraventricular tachycardia, defined as six or more consecutive premature supraventricular complexes with a frequency greater than 100 beats per minute; ventricular tachycardia, defined as six or more consecutive premature ventricular complexes with a frequency greater than 100 beats per minute; frequent premature ventricular complexes, defined as 30 or more premature ventricular complexes per hour; and second-degree or third-degree atrioventricular block. Personnel of the intensive care unit were specially advised to continuously observe and register the type and frequency of rhythm disturbances appearing on a telemetric monitor. Hydroperoxide-Initiated Chemiluminescence Frozen biopsy specimens (approximate weight, 20 mg) were placed in saline solution at 0" to 2 C and rushed to the laboratory. The samples were homogenized in a small Potter Elvejhem Teflon homogenizer in 120 mmoyl of KCl and 30 mmol/l of phosphate buffer (ph 7.5) at 0" to 2 C. The homogenate was warmed to 30"C, combined with 3 mmol/l of tert-butyl hydroperoxide, and assayed for chemiluminescence in a Packard Tri-Carb model 3320 scintillation counter (Packard Instrument Co, Santa Ana, CA) in the out-of-coincidence mode [6, 71. The proteins of the homogenate were determined by the method of Lowry and associates [8], and chemiluminescence was expressed as counts per minute per milligram of homogenate protein. Determinations were conducted in a blind manner by two different observers. Electron Microscopy Tissues for transmission electron microscopy were fixed in cold 3% glutaraldehyde in 0.1 movl of cacodylate buffer (ph 7.4), postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon. Three blocks were selected from different depths of the biopsy specimen, and from each block, a section 1 pm thick was cut, stained with 1 % toluidine-borax, and examined by light microscopy to select appropriate areas for thin sectioning. Five ultrathin sections were obtained from each of the three blocks. Ultrathin sections mounted in copper grids were stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 1A electron microscope. Electron micrographs were systematically taken at Table 2. Composition of the Two Cardioplegic Solutions Variable Standard Mannitol Dextrose (mmol/l) NaCl (mmol/l) KCl (mmoul) CaCl (mmol/l) MgCl (mmoul) NaHCO, Mannitol (mmol/l) Glycine (mmovl) ph at 5 C Osmolarity (mosm/l)

3 Ann Thorac Surg 1989;48:77-84 FERREIRA ET AL 79 I lo TIME (min) Fig 1. Time course of hydroperoxide-initiated chemiluminescence shows the effect of mannitol reperfusate in the reperfusion sainples from group 2. (A = group 1 preischemic samples; = group 1 reperfusion samples; A = group 2 preischemia samples; 0 = group 2 reperfusion samples.) X2,500 and x 10,000 magnification for the comparative evaluation of the preischemia and reperfusion samples in both groups, A single score was given for each area, and the observations were conducted in a blind manner by two different observers. According to the criteria of Kloner and co-workers [9], the severity of mitochondrial swelling was graded 0 through 4 as follows: 0 = normal mitochondria; 1 = early swelling as manifested by separation of cristae and clearing of matrix density; 2 = more marked swelling than in grade 1; 3 = massive swelling with architectural disruption; and 4 = the findings in grade 3 plus rupture of inner and outer mitochondrial membranes. When a point on the grid fell on a mitochondrion, this mitochondrion was assigned a numerical value of 0 through 4 depending on its morphological degree of damage. A mean mitochondrial grade for each cell was obtained by calculating the weighted average (0 through 4) of mitochondrial swelling. Approximately 200 mitochondria per sample were graded in this manner. Stat is tical Analysis The paired t test was used to determine the significance of differences, and data were expressed as the mean t the standard error of the mean. Data from chemiluminescence were analyzed by variance factorial design. To compare the incidence of rhythm disturbances in the two groups, the,$ test was used. Results Clinical Outcome All patients recovered satisfactorily from the operation. There were no perioperative myocardial infarctions as measured by the appearance of new Q waves on the electrocardiogram or by increased values for serum creatine kinase. Four patients in group 1 and 3 in group 2 required inotropic support four to 18 hours after operation. No patient required a major dose of dopamine hydrochloride (more than 5 pg/kg/min). The hospital mortality was 2.5%; 1 patient in group 1 had uncontrolled gastrointestinal bleeding and died on the 13th postoperative day. Rhythm Disturbances The number of countershocks needed to reverse ventricular fibrillation at the time of reperfusion was 1.7? 0.4 in group 1 and 1.0? 0.27 in group 2 (p < 0.02). Rhythm disturbances developed in 7 patients (35%) in group 1: atrial fibrillation in 3, frequent premature ventricular Fig 2. Electron micrograph of preischemia biopsy specitnen demonstrates preserved architecture of the sarcornere. The mitochondria have intact membranes and tightly packed cristae. (x25,ooo before 38% reduction.) lriset shozos that mitochondria and sarcomere both have a normal appearance. (~20,000 before 38% reduction.)

4 80 FERREIRA ET AL Ann Thorac Surg 1989;48:77-&1 Fig 3. Electron micrograph of reperfusion biopsy specimen from a patient in group 1 shows areas of focal rnyofibrillar disorganization with myocytolysis. Mitochondria demonstrate slight to moderate edema, and in some areas, there is clarijication of the matrix with massive swelling (arrow). (X25,OOO before 38% reduction.) Znset shows mitochondria with moderate swelling. (~20,000 before 38% reduction.) complexes in 2, supraventricular tachycardia in 1, and transient third-degree atrioventricular block in 1. In group 2, 1 patient (5%) had transient third-degree atrioventricular block. This difference was significant (p < 0.05). Hydroperoxide-Initiated Chemiluminescence The mean value for hydroperoxide-initiated chemiluminescence in the preischemia biopsy specimens from the 6 group 1 patients was 37.6 f 3.5 cpdmg of protein X and in the reperfusion biopsy specimens, 74.8 f 16 cpdmg of protein x In group 2, the photoemission count for the preischemia biopsy specimens was cpdmg of protein x lop3 and for the reperfusion specimens, 40 f 6.1 cpdmg of protein x lop3. The photoemission count was significantly reduced in the reperfusion biopsy specimens from group 2 compared with those from group 1 ( p < 0.001). No significant differences were found between the preischemia samples from both groups or between the preischemia and postischemia samples from group 2. Figure 1 shows the markedly increased hydroperoxide-initiated chemiluminescence in the reperfusion samples from group 1 compared with those from group 2. Electron Microscopy QUALITATIVE ANALYSIS. The preischemia biopsy specimens from both groups showed well-preserved intercalated discs and similar mitochondria1 arrangement, density, and general morphology. The cytoplasmic myofilament, glycogen, and sarcolemma were well preserved (Fig 2). Morphological changes in the reperfusion biopsy specimens from group 1 showed areas of focal myo- Fig 4. Electron micrograph of reperfusion biopsy specimen from a patient in group I: details of an area showing (A) severe damage of mitochondria with massive swelling and disruption of cristae and (B) mitochondria with marked swelling and rupture of membranes. (~20,000 before 38% reduction.)

5 Ann Thorac Surg 1989;48:77-84 FERREIRA ET AL 81 REPERFUSION INJURY WITH MANNITOL CARDIOI'LEGIA Fig 5. Electron micrograph of reperfusion biopsy specimen from a patient in group 2. The cytoplasmic myofilaments and sarcolemma are well preserved. The mitochondria1 arrangement is similar to the preischemia biopsy specimen. (~2,500 before 38% reduction.) Inset shows mitochondria with normal arrangement of the matrix and minimal edema. (X20,OOO before 38% reduction.) fibrillar disorganization with myocytolysis and sarcoplasmic vacuolation. Mitochondria had minimal to moderate swelling in some myocardial areas (Fig 3) and massive swelling and disrupted cristae in others (Fig 4). Reperfusion biopsy specimens from group 2 showed mitochondria with well-preserved architecture, tightly packed cristae, and gray matrix density in most of the electron micrographs. In some scattered areas, moderate swelling with clearing of matrix density and separation of cristae was observed, but no abnormalities of myofibrillar organization were detected (Fig 5). QUANTITATIVE ANALYSIS. In group 1, the percentage of severely damaged mitochondria, grades 3 and 4, was 6% * 2% and 5% * 2%, respectively, for the preischemia samples. For the reperfusion samples, grades 3 and 4 were 16% * 5% and 15% 2 2%, respectively. These results were significantly different for grade 4 (p < 0.05) (Table 3). Values for normal (grade 0) mitochondria in the same group were significantly different when the preischemia and reperfusion samples were compared (p < 0.05). In group 2, similar values were observed in both the preischemia and reperfusion samples (see Table 3). Comment Our study shows that the addition of mannitol to a cardioplegic solution significantly decreased the number of countershocks required to return the heart to sinus rhythm. Also observed was a reduction in rhythm disturbances during the first 24 postoperative hours. In this respect, the complete disappearance of atrial fibrillation was remarkable in the mannitol group (group 2). Furthermore, our study suggests that mannitol or the potentiation effect of mannitol by glucose may reduce the occurrence of oxidative stress as shown by a marked decrease in the hydroperoxide-initiated chemiluminescence in the biopsy specimens of group 2. Similarly, a relatively pre- Table 3. Grading of Mitochondria1 Damage" Mitochondrial Grade (%) Group Sampleb No. of Mitochondria Checked 1 (n = 6) A 60 f 9 16? 5 13 f 3 6f2 5?2 209 f 17 B f c 5 15 f f 8 p Value <0.05 NS NS NS <0.01 NS 2 (n = 6) A 69 f c 3 9f4 5c f 16 B 60 f 4 16 t 2 11 f f 17 p Value NS NS NS NS NS NS a Data are shown as the mean k the standard error of the mean. minutes of reperfusion. NS = not significant. A represents the samples obtained before ischemia and 8, those obtained after 10

6 82 FERREIRA ET AL Ann Thorac Surg 1989:48:77-84 served ultrastructure, as shown by the morphometric analysis of electron micrographs, was characteristic of the mannitol biopsy specimens. To our knowledge, this is the first study of these properties of mannitol in humans. Hydroperoxide-initiated chemiluminescence constitutes a reliable method within the various techniques currently used for determining oxidative stress [lo, 111. This assay appears to have a higher degree of sensitivity than malondialdehyde formation. Hydroperoxide-initiated chemiluminescence has been used to detect decreased levels of endogenous antioxidants in brain and liver homogenates from ethanol-treated rats [12] and from tumor-bearing mice [13] and in heart homogenates from Adriamycin (doxorubicin hydrochloride)-treated rabbits and mice [14, 151. In this decade, considerable interest has arisen over the role of oxygen-derived free radicals in reperfusioninduced damage of the heart, and within this field, some investigators [2-51 focused their research on the implication of free radicals in the genesis of reperfusion-induced arrhythmias. These authors have shown the arrhythmogenic properties of an oxygen-derived free radicalgenerating system. They have also demonstrated that the addition of antioxidant agents such as superoxide dismutase, catalase, mannitol, glutathione, or desferrioxamine in the perfusion medium exerts an antiarrhythmic effect in the isolated animal heart subjected to regional ischemia and reperfusion. Oxygen free radicals, particularly the OH', attack unsaturated fatty acid chains and other cell structures such as proteins and deoxyribonucleic acid. Because unsaturated fatty acids are in abundance in cell membranes, they become critical targets of free radical activity, and a propagation reaction (lipoperoxidation) is initiated [ 161. The mechanism by which oxygen free radicals are generated on reperfusion remains unclear. The role of xanthine oxidase as a source of these metabolites has been observed in animals, but, as reported in 1986 [17], the human heart has little if any xanthine oxidase activity; therefore the hypothesis that xanthine oxidase catalyzes the generation of the superoxide anion (0,') may not be applied in the clinical setting. In the alternative hypothesis, on reperfusion, oxygen encounters a highly reduced mitochondrial respiratory chain, which triggers a high production of 0,' at the inner mitochondrial membrane "1. After dismutation of 0,' at the mitochondrial matrix, the resulting H,O, diffuses out of the cytosol, reacts with myoglobin, and produces HO' ~91. In a previous work, we [l] showed that in patients undergoing myocardial revascularization, a burst of oxygen free radicals is generated on reperfusion, and this was demonstrated by a significant rise in hydroperoxideinitiated chemiluminescence. This was associated with cell damage assessed both by ultrastructural studies and by the determination of histochemical succinic dehydrogenase activity. Encouraged by these results, we tested in the present study the hypothesis that the addition of mannitol to the cardioplegic solution would offer better protection to the heart. Mannitol has been shown to improve myocardial function after ischemia. One possible mechanism of action is related to its osmotic activity, decreasing cellular edema by creating a hyperosmotic extracellular milieu. Magovern and co-workers [20] tried to elucidate the mechanisms by which reperfusion with mannitol is beneficial. They reperfused rabbit hearts with two equally hyperosmotic solutions, one containing mannitol and the other containing glucose. The mannitol group demonstrated a better tolerance to ischemia on the basis of ventricular function determinations, measurement of coronary flow, and myocardial edema formation. These results provided suggestive evidence that mannitol may achieve a myocardial protective effect by some other property than its hyperosmotic action. These authors hypothesized that mannitol could act as a scavenger of free radicals. It has been reported [21] that mannitol is an oxygen free radical scavenger of the OH by forming a mannitol radical (MH2 + OH' -+ MH' + H,O) that undergoes disproportionation or dimerizes. The standard cardioplegic solution in our experience possesses higher osmolarity than the mannitol solution. Therefore the better preservation of ultrastructure observed in the mannitol samples should be explained by mechanisms other than hyperosmolarity. Another aspect that must be taken into consideration is the determination of the optimal concentration of mannitol in a cardioplegic solution. Bernier and associates [4] found effective reduction of ventricular fibrillation with 50 mmol/l of mannitol, and recently the same group [22] observed the potentiation of the antifibrillatory effects of mannitol (50 mmol/l) by low concentrations (11 mmol/l) of glucose. The mechanism for this effect remains to be elucidated. We introduced chemiluminescence as an indirect method to assess free radical activity, and a significant reduction in photoemission was observed in the biopsy specimens from the mannitol group. Our results suggest that the protective action of mannitol or the interaction of mannitol and glucose is in part secondary to a scavenging property. However, we concede that several aspects remain unclear. Is mannitol limited to the vascular space under these conditions, whether simply by membrane permeability factors or by reduced mannitol diffusion because of hypothermia? Or is it conceivable that with ischemia, there are sufficient increases of sarcolemmal permeability to allow the relatively small mannitol molecule to gain access to the cytosol? If mannitol could diffuse in amounts sufficient to reach an effective scavenging concentration within the cells, then it could be acting through the ascribed mechanism. The role of activated leukocytes as an oxidant source is also well known. In spite of the washout effect of the perfusate, an undetermined number of leukocytes reach the capillary system of the myocardium through the noncoronary flow, and mannitol may act over the free radicals generated by these cells. Further investigations are necessary to clarify the mechanisms by which antioxidant agents including mannitol exert their protective action when administered in a cardioplegic solution.

7 Ann Thorac Surg 1989;48:77-84 FERREIRA ET AL 83 References 1. Ferreira R, Llesuy S, Milei J, et al. Assessment of myocardial oxidative stress in patients after myocardial revascularization. Am Heart J 1988;115: Stewart JR, Blackell MS, Crute SL, et al. Inhibition of surgically induced ischemia/reperfusion injury by oxygen free radical scavengers. J Thorac Cardiovasc Surg 1983;86: Menasche P, Grousset C, Gauduel Y, Piwnica A. A comparative study of free radical scavengers in cardioplegic solution. Improved protection with peroxidase. J Thorac Cardiovasc Surg 1986;92:26& Bernier M, Hearse DJ, Manning AS. Reperfusion-induced arrhythmias and oxygen-derived free radicals. Studies with "anti-free radical" interventions and a free-radical generating system in the isolated perfused rat heart. Circ Res 1986; 58:331; Hearse DJ, Tosaki A. Free radicals and reperfusion-induced arrhythmias: protection by spin trap agent PBN in the rat heart. Circ Res 1987;60: Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;62: Boveris A, Fraga CG, Varsavski AI, Kooch OR. Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats. Arch Biochem Biophys 1983; , 8. Lowry OH, Rosenbrough AL, Farr AL, Randall RI. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193: Kloner RA, Fishbein MC, Braunwald E, Maroko PR. Effect of propranolol on mitochondrial morphology during acute myocardial ischemia. Am J Cardiol 1978;41: Boveris A, Cadenas E, Chance B. Ultraweak chemiluminescence: a sensitive assay for oxidative radical reactions. Fed Proc 1981;40:2=. 11. Boveris A, Cadenas E, Reiter R, Filipowsky M, Nakase Y, Chance 8. Organ chemiluminescence: noninvasive assay for oxidative radical reactions. Proc Natl Acad Sci USA 1980; 77: Cadenas E, Varsavsky A, Boveris A, Chance B. Oxygen of organic hydroperoxide-induced chemiluminescence of brain and liver homogenates. Biochem J 1981;198: Boveris A, Llesuy SF, Fraga CG. Increased liver chemiluminescence in tumor-bearing mice. J Free Radic Biol Med 1985;1: Llesuy SF, Milei J, Molina H, Boveris A, Milei S. Comparison of lipid peroxidation and myocardial damage induced by Adriamycin and 4'-epiadriamycin in mice. Tumori 1985; 71: Milei J, Boveris A, Llesuy S, et al. Amelioration of Adriamycin-induced cardiotoxicity in rabbits by prenylamine and vitamins A and E. Am Heart J 1986;111: Freeman BA, Crapo JD. Biology of disease. Free radicals and tissue injury. Lab Invest 1982;47: Downey J, Chambers D, Miura T, Yellon D, Jones D. Allopurinol fails to limit infarct size in a xanthine oxidase-deficient species. Circulation 1986;74(Suppl 2): Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide: general properties and effect of hyperbaric oxygen. Biochem J 1973;134: McCord JM. Free radicals and myocardial ischemia: overview and outlook. J Free Radic Biol Med 1988;4: Magovern GF, Bolling SF, Casale AS, Bulkley BH, Gardner TJ. The mechanism of mannitol in reducing ischemic injury: hyperosmolarity or hydroxyl scavenger? Circulation 1984; 7O(Suppl 1): Ouriel K, Ginsburg ME, Patti CS, Pearce FJ, Hicks GL. Preservation of myocardial function with mannitol reperfusate. Circulation 1985;72(Suppl 2): Bernier M, Hearse DJ. Reperfusion-induced arrhythmias: mechanisms of protection by glucose and mannitol. Am J Physiol 1988;254:H INVITED COMMENTARY This article by Ferreira and colleagues is provocative and potentially important. However, there are several caveats in its interpretation. First, patients were randomized to one of two treatment groups without informed consent. The rationale was that mannitol is commonly used in cardioplegic solutions anyway, and informed consent was, therefore, unnecessary. Moreover, it was not required by the local institutional review board. While not disagreeing with this contention, an important principle of human research is that when treatment is assigned, strictly on the basis of study participation, consent must be obtained. Responsibility for enforcement is both that of the authors and that of The Annals. Second, the occurrence of increased chemiluminescence in biopsy specimens from patients treated with mannitol is not specific for the occurrence of free radical moieties. For example, changes in the redox state of tissue iron can influence chemiluminescence. Moreover, using the current study design, no evidence is provided that the simple presence of mannitol in the myocardial tissues did not influence chemiluminescence. It is of interest that the extent of chemiluminescence did not correlate with the duration of aortic cross-clamp time; most hypotheses of free radical injury suggest that the extent of this phenomenon should relate directly to the extent of ischemic injury. Third, the potential dangers of subjecting a small sampling of the patient population to biopsy introduces statistical hazards not present when the biopsy technique is applied to greater numbers of the studied population. Fourth, side-by-side comparison of the two cardioplegic solutions reveals differences not limited to the inclusion of mannitol. Differences exist in four other components of the solution that cannot be excluded as contributing to the results. The sodium chloride concentration in the mannitol-containing cardioplegic solution was about 60% of that