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Ann Thorac Surg 2000;69:785-791
© 2000 The Society of Thoracic Surgeons


Original Articles

Off-pump bypass graft operation significantly reduces oxidative stress and inflammation

Bashir M. Matata, PhDa, Andrzej W. Sosnowski, MDa, Manuel Galiñanes, FRCSa

a Division of Cardiac Surgery, Department of Surgery, University of Leicester, Glenfield Hospital, Leicester, United Kingdom

Address reprint requests to Dr Galiñanes, Division of Cardiac Surgery, Department of Surgery, University of Leicester, Glenfield Hospital, Leicester LE3 9QP, United Kingdom
e-mail: mg50{at}le.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 References
 
Background. This study investigated whether off-pump coronary bypass graft operations on the beating heart under normothermic conditions reduces the systemic oxidative stress and inflammatory reaction seen in patients operated under cardiopulmonary bypass (CPB).

Methods. A cardiac stabilizer (Octopus Tissue Stabilizer; Medtronic Inc, Minneapolis, MN) was used to perform the coronary anastomoses on the normothermic beating heart with or without CPB. Serial blood samples were taken at various intervals. Plasma was analyzed for several oxidative stress and inflammatory markers.

Results. Significant increases from prior anesthesia values of lipid hydroperoxides (190% at 4 hours), protein carbonyls (250% at 0.5 hours) and nitrotyrosine (510% at 0.5 hours) were seen in the CPB group, but they were abolished or significantly reduced in the off-pump group. Complement C3a and elastase levels were rapidly increased upon the institution of CPB, and this was followed by increases in IL-8, TNF-{alpha}, and sE-selectin. In contrast, the rise of these factors was blunted in patients operated without CPB.

Conclusions. Off-pump coronary bypass graft operation on a beating heart significantly reduces oxidative stress and suppresses the inflammatory reaction associated with the use of CPB.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 References
 
Patients undergoing cardiac operations under cardiopulmonary bypass (CPB) have a systemic inflammatory reaction that is believed to result in increased postoperative morbidity and prolonged hospital stay [1]. Important features of this inflammatory reaction include the activation of complement [2] and leukocytes [1], the release of proinflammatory cytokines [3], alterations in the metabolism of nitric oxide (NO) [4], and an increase in the production of oxygen free radicals, which in some cases may lead to oxidant stress injury [5]. Several strategies including the use of steroids [6], use of aprotinin [7], heparin-coated CPB circuits [8], and hemofiltration [9] have been reported to reduce the inflammatory reaction induced by CPB and its consequences.

A more radical and effective way of counteracting the effects of the inflammatory reaction and oxidative stress may be the omission of CPB itself. Previous studies have reported of a reduction in postoperative morbidity in patients undergoing cardiac operations without CPB [10]. Recent studies in patients undergoing bypass graft operations have reported a reduction in the inflammatory reaction when CPB was not used [11, 12]. However, in these studies the control group was comprised of patients subject to global ischemia and cardioplegia in addition to hypothermic CPB, and it was therefore not possible to separate the effects attributable to CPB alone, and those due to variations in temperature and global ischemia. Furthermore, the effect of off-pump operations on oxidative stress was not studied.

The present study was designed to investigate the effect of off-pump bypass operations on the oxidative stress and the inflammatory reaction that is commonly observed in patients undergoing operation under CPB. A cardiac stabilizer (Octopus Tissue Stabilizer; Medtronic Inc, Minneapolis, MN) was used to perform coronary anastomoses on the beating heart without the need for global ischemia and cardioplegia. Hence, all patients in the study were operated under identical conditions, and the only variable of the study was the use or not of CPB.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 References
 
Twenty patients with single or double vessel coronary disease and undergoing elective bypass graft operations were randomized to be operated with or without CPB (n = 10 per group). Patients with diabetes, diseases of the circumflex and the left main stem, valvular diseases, ventricular aneurysm, heart failure, and poor left ventricular function were excluded from the study. The study protocol was approved by the local Medical Ethics Committee, and informed consent was obtained from all the patients. Administration of aspirin was discontinued at least 7 days before the operation and no patient was given preoperative heparin.

Anesthesia
All the patients received morphine (10 mg) and prochlerperazine (12.5 mg) administered intramuscularly at least 1 hour before operation. Central venous and radial artery cannulas were inserted under local anesthesia (1% lignocaine) and midazolam sedation (3 to 4 mg intravenously). Anesthesia was induced with an infusion of propofol (8 mg · kg-1 · h-1 intravenously) and with fentanyl (1 mg intravenously) and pancuronium (12 mg intravenously). Anesthesia was then maintained by continuous infusion of propofol (4 mg · kg-1 · h-1 intravenously. Hypotension was controlled by intravenous infusion of fluids or 0.5 mg metaraminol with increments where appropriate. Heparin (300 IU/kg) was administered in all study patients, just before aortic cross-clamping or the initiation of the coronary anastomoses, to achieve an activated clotting time greater than 450 seconds. A further 5000 IU heparin were given in the bypass prime for the CPB group. Heparin was reversed after bypass, and following the termination of coronary anastomoses by the administration of 3 mg/kg of protamine.

Patients were sedated, while in intensive care, with propofol (1.5 to 2 mg · kg-1 · h-1 intravenously) until their core temperature was higher than 36° C, and bleeding through the chest drains was less than 50 mL/hour. Then if blood pO2 level was greater than 10 kPa and the FiO2 was 40% or less, the rate of propofol infusion was reduced. When the patient was able to respond coherently to simple instructions, propofol was discontinued and the trachea extubated. Morphine (5 mg intravenously) and diclofenac (100 mg intravenously), a nonsteroidal, antiinflammatory nonnarcotic drug, were given to treat pain when required and if not contraindicated.

Surgical procedure
In both groups, median sternotomy and harvesting of internal mammary artery and/or saphenous vein grafts was followed by full exposure of the coronary artery branches to be revascularized by utilizing a local cardiac wall restraining device (Octopus Tissue Stabilizer) on the normothermic, beating heart. Two endoOctopus tentacles were engaged in a dual holder, in a parallel fashion, employing a 400 mm Hg vacuum source as described before [13]. Suction was applied, after which the system was firmly attached to the epicardium. Clamps were then placed on the coronary artery proximally and distally to the chosen site of anastomosis. Following the completion of the surgical anastomoses, the two clamps were removed to allow reperfusion.

A standard CPB technique and median sternotomy were used in the CPB group. The CPB circuit was composed of a roller pump (Stöckert Instrumentation, Munich, Germany), a hollow fiber polypropylene oxygenator with an incorporated cardiotomy reservoir (Cobe CML; Cobe Laboratories, Gloucester, UK), and plasticized polyvinyl chloride tubing. The pump was primed with 1.4 L Hartmann’s solution. A flow rate of 2.4 L · min-1 · m2 body surface area and a normothermic temperature were maintained throughout the operation.

Blood sampling
Blood samples were collected at different time points: before induction of anesthesia, before initiation of CPB and coronary artery clamping (time 0), and 0.5, 1, 2, 4, 8, 24 and 48 hours thereafter. Blood samples were collected into sterile tubes in three aliquots containing trisodium citrate anticoagulant or potassium EDTA. An aliquot of blood in EDTA tubes was used for total leukocyte counts. Circulating leukocytes were counted by an automatic cell counter (Cell-Dyn 610; Sequoia-Turner, Mountain View, CA). The other aliquots were centrifuged immediately at 1500 g for 12 minutes at 4°C. The resultant plasma was aliquoted and stored at -80°C until analysis.

Assessment of the inflammatory reaction
The inflammatory response was assessed by examining the blood leukocyte count, the degree of activation of complement and neutrophils, and the plasma levels of the proinflammatory cytokines TNF-{alpha} and IL-8. Activation of complement was determined by the measurements in plasma of complement C3a, using a sandwich enzyme linked immunosorbent assay (ELISA) (Quidel, San Diego, CA). Activation of neutrophils was determined by the measurements of plasma elastase in complex with {alpha}1-proteinase using an ELISA as described before [14]. An interassay and intrassay variability of less than 8% was considered acceptable. The plasma levels of TNF-{alpha} and IL-8 were quantified using commercially available sandwich ELISA assays (Pharmingen, San Diego, CA).

Assessment of oxidative stress
Lipid hydroperoxides, protein carbonyls, and nitrotyrosine levels were determined in plasma, and served as indices of oxidative stress. Plasma lipid hydroperoxides were determined as described in a method based upon the selective oxidation of ferrous to ferric ions by hydroperoxides under acidic conditions (FOX-2 method) [15]. Ferric ions generated by lipid hydroperoxides in the assay are complexed by the ferric ion indicator xylenol orange [0-cresolsulfonpthalein-3’,3''-bis(methyliminodiacetic acid) sodium salt], resulting in a blue complex with a spectrophotometric extinction coefficient of 1.5 x 104 · M-1 · cm-1 at 560 nm.

Protein carbonyls were determined by colorimetric carbonyl assay that measures binding of dinitrophenylhydrazine to carbonyl-bearing amino acid residues in proteins [16].

Protein nitrotyrosine was determined by an ELISA method described by Khan and associates [17], and all the kit reagents were obtained from TCS Biologicals Ltd, (Buckingham, UK).

Assessment of endothelial activation and endothelial injury
sE-selectin and thrombomodulin in plasma were determined as markers of endothelial activation and endothelial injury respectively [18, 19]. Sandwich ELISA assays were used for the measurements of sE-selectin (Amersham International Inc, Amersham, UK) and thrombomodulin (Diagnostica Stago, Asniéres-Sur-Seine, France).

Clinical outcome
The time of mechanical ventilatory support, postoperative blood loss and transfusion requirements, the presence of postoperative fever, changes in body weight and the need for diuretics, and length of hospitalization were recorded. Patients were discharged from hospital when they were apyrexial, in an overall satisfactory stable condition, and able to perform basic routine tasks.

Statistical analysis
All data were presented as mean ± standard error of the mean. The statistical analysis was performed by using the Statistical Package for Social Sciences Software (SPSS Inc, Chicago, IL). Analysis of variance for repeated measurements was used to compare changes in time between both patient groups. The significances of changes within groups were determined by the Mann-Whitney test. Differences were considered significant at p values less than 0.05.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 References
 
Data from all the patients included in the study were analyzed. The clinical characteristics and operative data are shown in Tables 1 and 2 .


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Table 1. Patient Clinical Characteristics

 

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Table 2. Perioperative Data

 
Perioperative clinical outcome
There was no hospital mortality, no neurologic accidents, pulmonary insufficiency or incidences of myocardial infarction in the two study groups. All the patients were extubated between 3 and 12 hours after their arrival in the intensive care unit; however, the mean time of mechanical ventilatory support was 9.4 ± 1.1 hours in the CPB group, whereas it was only 5.5 ± 0.4 hours in the non CPB group (p < 0.05).

The total postoperative blood lost through the chest drains was 557 ± 52 mL in the CPB group and 445 ± 35 mL in the non CPB group, and this modest difference achieved statistical significance. Blood transfusion was performed when hemoglobin values fell below 10 mg/dL, and this was required in 3 cases in the CPB group but none in the non CPB group. Postoperative fever, defined as a timpanic temperature greater than or equal to 37.5° C and maintained for greater than or equal to 24 hours, occurred in 7 patients in the CPB group and only in 2 patients of the non CPB group (p < 0.05).

In addition, patients in the CPB group exhibited a mean body weight gain of 0.4 ± 0.9 kg by the second postoperative day while those in the non CPB had in fact lost 0.6 ± 0.6 kg. This difference in body weight, which may be a reflection of the state of vascular permeability, did not achieve statistical significance. However, it should be noted that an early use of diuretics was required in all patients in the CPB group whereas only 2 patients received the treatment in the non CPB group (p < 0.05).

In spite of the above differences, patients were discharged from hospital around the fourth postoperative day in the two study groups (4.6 ± 0.3 days in the CPB group vs 4.0 ± 0.3 days in the non CPB group; p = not significant [NS]).

Inflammatory factors
Figure 1 A shows that the plasma levels of complement C3a, a product of complement activation, were not significantly affected by anesthesia and surgical trauma, however they were rapidly elevated upon the institution of CPB. C3a levels rapidly increased after the initiation of CPB and at 0.5 hours, values were 12 times greater than those observed before anesthesia. Following this, and over the next 2 hours, the C3a levels remained elevated but to a lesser degree, and by 4 hours C3a levels were similar to the values seen before anesthesia. This rise in complement activation was almost completely abolished in the non CPB group patients. In this group, the C3a levels were slightly but significantly elevated 1 and 2 hours after the initiation of bypass grafting, coincidental with the reversion of heparin with protamine.



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Fig 1. Changes in plasma levels of complement (A) C3a, (B) elastase, and (C) blood leukocyte count before, during, and after coronary bypass graft operation, performed with or without cardiopulmonary bypass (CPB). Values are expressed as means of 10 patients per group, and bars represent the standard error of mean. *p less than 0.05 versus prior anesthesia (PA) values within the same group. {dagger}p less than 0.05 versus the corresponding value of the non CPB group.

 
The mean values on plasma elastase, an index of neutrophil activation, are shown in Figure 1B. This shows that CPB induces a rapid and considerable increase in elastase, peaking at 1 hour after the start of CPB, and then followed by a gradual decrease, so that by 24 and 48 hours values were similar to those before anesthesia. This response was markedly blunted in patients operated without CPB.

The blood leukocyte count is shown in Figure 1C. In contrast with the striking effects of CPB on complement C3a and plasma elastase, only a modest increase in leukocyte count was observed in the CPB group on the second and fourth postoperative day when compared with prior anesthesia values and values in the non CPB group. However, it should be noted that in all instances the leukocyte counts were within the physiologic range in the two study groups.

The mean IL-8 plasma levels are shown in Figure 2 A. They demonstrate a significant increase in plasma IL-8 levels between 1 and 4 hours after the start of CPB, with maximal elevation at 2 hours. Thereafter, values were similar to those seen before anesthesia. In contrast, values were not significantly altered in the non CPB group from the initial levels during the entire study period.



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Fig 2. Changes in plasma levels of (A) IL-8 and (B) TNF-{alpha} before, during, and after coronary bypass graft surgery performed with or without cardiopulmonary bypass (CPB). Values are expressed as means of 10 patients per group, and bars represent the standard error of mean. *p less than 0.05 versus prior anesthesia (PA) values within the same group. {dagger}p less than 0.05 versus the corresponding value of the non CPB group.

 
The mean values in TNF-{alpha}, as shown in Figure 2B, were not significantly altered during the initial 8 hours after the initiation of CPB, however a statistically significant increase was observed at 24 and 48 hours. Again, this increase in TNF-{alpha} levels was completely abolished in the patients without CPB.

Oxidative stress
The results on the plasma levels of lipid hydroperoxides and protein carbonyls, indices of oxidant stress on lipids and proteins, are shown in Figures 3A and 3B, respectively. Lipid hydroperoxide levels were not affected by anesthesia and the surgical trauma, but were significantly elevated between 1 and 4 hours after the initiation of CPB. Thereafter, values decreased to within the prior anesthesia range for the rest of the study period. Protein carbonyls were also unaffected by anesthesia and surgical trauma, but were rapidly elevated upon the initiation of CPB, so that by half an hour of CPB, mean values were more than 2.5 times those seen before anesthesia. This elevation was confined to the first hour after the initiation of CPB, and after this time values were similar to those seen before anesthesia. Importantly, operation without CPB abolished the increase of both lipid hydroperoxides and protein carbonyls.



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Fig 3. Changes in plasma levels of (A) lipid hydroperoxides, (B) protein carbonyls, and (C) protein nitrotyrosine before, during, and after coronary bypass graft operation with or without cardiopulmonary bypass (CPB). Values are expressed as means of 10 patients per group, and bars represent the standard error of mean. *p less than 0.05 versus prior anesthesia (PA) values within the same group. {dagger}p less than 0.05 versus the corresponding value of the non CPB group.

 
The results on plasma nitrotyrosine, an index of peroxynitrite formation, are shown in Figure 3C. In contrast with the above results, nitrotyrosine was already significantly elevated after the induction of anesthesia and the surgical trauma, and it was further increased by CPB. As seen for protein carbonyls, the increase in nitrotyrosine was limited to the first hour after the initiation of CPB. Values decreased by 2 hours, a time when CPB was already terminated, and then maintained within the prior anesthesia range values for the rest of the study period. Again operation without CPB significantly blunted the increase in nitrotyrosine.

Endothelial activation and endothelial injury
The results of the plasma levels of sE-selectin, an index of endothelial activation, are shown in Figure 4 A. They indicate that sE-selectin is not significantly increased 1 hour after the initiation of CPB, although by 8 hours there was a modest but significant increase, and that by 24 hours values were again within the prior anesthesia range. Once more, the greater sE-selectin levels, as seen at 8 hours during CPB, were markedly reduced when operation was performed without CPB.



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Fig 4. Changes in plasma levels of (A) soluble E-selectin and (B) thrombomodulin concentrations before, during, and after coronary bypass graft operation with or without cardiopulmonary bypass (CPB). Values are expressed as means of 10 patients per group, and bars represent the standard error of mean. *p less than 0.05 versus prior anesthesia (PA) values within the same group.

 
Interestingly, the thrombomodulin plasma levels shown in Figure 4B, an index of endothelial damage, were unaffected by anesthesia, surgical trauma and even by CPB during the entire study period.


    Comment
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 References
 
This study has shown for the first time that off-pump bypass graft operation on the beating heart significantly reduces oxidative stress increase, and that in addition it blunts the inflammatory reaction observed in patients operated with CPB. These two major findings were associated with a significant reduction in postoperative blood loss and in the need for blood transfusion, a shortening of time required for mechanical ventilatory support, a lower incidence of postoperative fever, and have less vascular permeability, as indicated by the decrease in the need for diuretics than that observed in patients operated with the use of CPB. The importance of these findings are discussed in detail below.

Reduction of the inflammatory reaction
The present study has demonstrated that operating off-pump in the beating heart can blunt the inflammatory reaction. The only exception was a modest but statistically significant increase in the complement activation product C3a, 1 and 2 hours after coronary anastomoses. However, this increase in C3a had no influence on other inflammatory factors and occurred at a time when protamine was administered to reverse the action of heparin. It is well known that protamine-heparin complex [20] may induce complement activation and this may be an explanation for this isolated event.

Bypass graft operation in the beating heart was possible by using a cardiac stabilizer (Octopus Tissue Stabilizer) and by direct occlusion of the coronary artery at the site of the anastomoses, in order to achieve a standstill and bloodless field. In this way, induction of global cardiac ischemia and the use of cardioplegia were unnecessary. Although the assessment of the extent of myocardial injury was not an aim of this study, our results suggest that operation in the beating heart does not induce significant myocardial injury because of the absence of an increase in inflammatory factors. Furthermore, with the exception of 2 patients (1 in each group exhibiting ST-segment elevation), electrocardiographic ischemic changes were not detected during coronary occlusion, this is probably due to the existence of good collateral flow. In the 2 patients exhibiting ST-segment elevation, the electrical abnormality was immediately reversed upon removal of the coronary occlusion, and there were no hemodynamic consequences. In addition, the inflammatory reaction in the 2 patients was of the same order of magnitude as that observed in the other patients in each group. Certainly, the occlusion of a coronary artery exhibiting obstructive disease may not itself cause ischemia, if collateral circulation is well developed and myocardial demand is not further increased. It seems therefore, that the inflammatory reaction depicted in the present study was induced solely by CPB.

The present study has shown that the activation of complement and neutrophils are the initial events of the inflammatory response, followed by the production of proinflammatory cytokines such as IL-8, an observation consistent with earlier reports [21]. sE-selectin release was a delayed event, as was the production of TNF-{alpha}. TNF-{alpha} and sE-selectin may activate a number of other inflammatory mechanisms, but in this case no further activation of complement and neutrophils was observed at the time of their release.

It is worth noting that a similar increase in plasma IL-6 levels was reported by Fransen and colleagues [12] in patients with or without CPB; however, the temporal pattern of release was different with an earlier rise in the CPB group. The explanation for these results is unclear, but one possibility is that anesthesia and surgical trauma may be the principal contributors for the rise in IL-6, a thesis that is supported by the demonstration that leukocytes release IL-6 in response to a variety of stimuli, including infection, major operation, and thermal injury [22].

Reduction in oxidative stress
There is evidence in the literature that free radicals are produced during cardiac operation under CPB [5], and that the reduction in the observed oxidative stress may be alleviated by the use of antioxidants [23]. A major finding of the present study is that oxidative stress can also be significantly reduced if graft operation is performed in the beating heart without CPB.

The early activation of neutrophils, as demonstrated by the profile of plasma elastase, and the late activation of the endothelium, as shown by the small and delayed increase of sE-selectin 8 hours after the initiation of CPB, suggest that the most likely source of free radicals during CPB are leukocytes. However, the early increase in the formation of nitrotyrosine, a byproduct of the nitration of tyrosine by peroxynitrite, even before the initiation of CPB at a time when neutrophils are not yet activated, may suggest the existence of other potential sources of NO and superoxide anion, possibly the endothelium. Evidence from the literature has shown that heparin [24] can cause an increase in NO production by the endothelium, and therefore result in the early increase in nitrotyrosine levels in both CPB and off-pump patients. This therefore indicates that although CPB is the most important instigator of oxidative stress, anesthetic agents and surgical trauma may play some role.

Limitations of the study and clinical implications
This study was performed in a small number of selected patients, and although operation in the beating heart without the use of CPB was shown to be advantageous, it may not have revealed the entire usefulness of this approach. Thus, for example, high-risk patients such as those with renal failure, respiratory insufficiency, advanced age, cerebrovascular complications, and other systemic diseases may benefit most from a reduction of oxidative stress and inflammatory factors.

In terms of postoperative blood losses, it appears that they were slightly greater in our study than those reported by Gu and coworkers [11]. The reason for this difference may be attributed to the higher levels of heparin used in our study (300 IU/kg) than in their study (100 IU/kg). The anticoagulation regimen used in this study was adopted in order to standardize the patient’s treatment, but it should be emphasized that lower doses of heparin may still be safe when CPB is not used, and that such an approach may result in reduction on the postoperative bleeding and the need for blood transfusion.

Several therapeutic strategies, including the use of heparin-coated CPB circuits, [8] hemofiltration, [9] and the administration of steroids [6], among others, have been proposed to combat the inflammatory reaction induced by CPB and the associated complications. A more effective and radical approach to abolish the inflammatory reaction is the removal of CPB as demonstrated in this study. The present study was carried out in patients with single and double vessel coronary artery disease involving the left anterior descending artery and the right coronary artery, however the beating heart technique without CPB may also be successfully applied in patients with triple vessel coronary artery disease by using additional maneuvers for the exposure of arteries in the circumflex territory [25].

Myocardial ischemic injury is more frequently observed when long ischemic periods are required and in patients with poor cardiac contractile function. Graft operation in the beating heart avoids global ischemia, and by preventing myocardial ischemic injury the oxidative stress and inflammatory reaction may be ameliorated, and the clinical outcome improved. Undoubtedly, more clinical studies are required to define the beneficial effects of graft operation in the beating heart with or without CPB.


    Acknowledgments
 
This study was partially supported by grants from The Glenfield Hospital NHS Trust, Leicester, The University of Leicester, Heart Link Trust and Medtronics Inc. We wish to acknowledge Dr David Duthie, Department of Anesthesia, Glenfield Hospital for the help with the anesthesia of patients and Dr Yana Podinovskaia, Medical Statistician, Division of Cardiology, Glenfield Hospital for the advice on the statistical analysis. We would also like to thank Dr Alan Fowler for the help in preparing the manuscript.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 References
 

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Accepted for publication August 27, 1999.




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Eur. J. Cardiothorac. Surg.Home page
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I Kutschka, J Skorpil, A El Essawi, T Hajek, and W Harringer
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T. N. Hoel, V. Videm, T. E. Mollnes, K. Saatvedt, F. Brosstad, A. E. Fiane, E. Fosse, and J. L. Svennevig
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Effects of N-3 Fatty Acids on Postoperative Atrial Fibrillation Following Coronary Artery Bypass Surgery
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