Ann Thorac Surg 2006;81:1256-1261
© 2006 The Society of Thoracic Surgeons
Original article: Cardiovascular
Reduced Cytokines Release and Myocardial Damage in Coronary Artery Bypass Patients Due to L-Arginine Cardioplegia Supplementation
Luisa Colagrande, MD, PhD
a
,
*
,
Francesco Formica, MD
a
,
Fabiano Porta, MD
a
,
Antonello Martino, MD
a
,
Fabio Sangalli, MD
b
,
Leonello Avalli, MD
b
,
Giovanni Paolini, MD, PhD
a
a Cardiac Surgery Clinic, Department of Surgical Science and Intensive Care, University of Milano-Bicocca, San Gerardo Hospital, Monza, Milan, Italy
b Cardio-Anaesthesia Service, Department of Surgical Science and Intensive Care, University of Milano-Bicocca, San Gerardo Hospital, Monza, Milan, Italy
Accepted for publication October 6, 2005.
* Address correspondence to Dr Colagrande, Cardiac Surgery, San Gerardo Hospital, Via Donizetti 106, Monza 20052, Milan, Italy (Email: lcolagrande{at}yahoo.it).
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Abstract
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BACKGROUND: Recently, L-arginine has been added to cardioplegia to limit myocardial ischemic damage. The mechanism of action is related to the production of nitric oxide, with vasodilatation and reduction of endothelial dysfunction. Our prospective randomized study on coronary artery bypass patients investigates the effect of L-arginine on myocardial stress as expressed by myocardial cytokines release and myocardial ischemia in terms of troponin T concentration.
METHODS: Coronary artery surgery patients were randomly assigned to receive 7.5 g L-arginine in 500 mL of cardioplegic solution (group A). Group B was used as control. Cold blood 4:1 anterograde and retrograde cardioplegia with warm induction was administered. Blood samples were collected from the retrograde coronary sinus catheter to determine interleukin-2 receptor, interleukin-6, and tumor necrosis factor levels. Serum samples at different time points were also analyzed to measure myocardial ischemia markers. Hemodynamic and echocardiographic evaluations were obtained perioperatively.
RESULTS: Sixty-five patients were enrolled (group A, treated with L-arginine, n = 33; group B, control, n = 32). Wedge pressure and intensive care unit stay were significantly reduced in group A (p = 0.023 and p = 0.03, respectively). Cytokines levels were lower in group A, with a significance for interleukin-6 (p = 0.026); troponin T was reduced in treated patients (0.33 versus 0.57 ng/mL at 18 hours: p = 0.009).
CONCLUSIONS: Coronary artery surgery patients benefit from L-arginine cardioplegia supplementation in terms of reduced inflammatory reaction, limitation of myocardial ischemia, and better hemodynamic performance. Moreover, a clinical advantage is evident in terms of a shorter intensive care unit stay in patients treated with L-arginine.
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Introduction
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Recent advances in cardiac surgery have focused upon the optimization of myocardial protection, and the preservation of cardiac function is the goal of more of experimental and clinical trials.
Nitric oxide (NO) has been studied for a long time because of its vasodilatatory effects and preservation of endothelial function playing a role in protection against ischemia-reperfusion damage [1–3]. Recently, the first clinical studies about the use of L-arginine, a donor of NO, in addition to cardioplegia, demonstrated the efficacy in reducing postoperative troponin T and creatinine kinase–myocardial band (CK-MB) levels in coronary artery bypass graft operations [4, 5]. Moreover, the inflammatory reaction to cardiopulmonary bypass and to surgical trauma involves activation of leukocytes and endothelium, with secretion of cytokines that contribute to the systemic inflammatory response syndrome, multiple organ failure, and postoperative myocardial motion abnormalities that are frequently observed after cardiac surgery [6, 7].
A prospective, double-blind, randomized study was carried out on coronary artery bypass surgery patients to verify, firstly, if L-arginine was able to reduce myocardial cytokines release as expression of a reduction in myocardial stress, and secondly, to determine the effect on ischemic damage extension in terms of troponin T concentration modification. Finally, we wanted to prove the feasibility and safety of L-arginine added to antegrade-retrograde cold intermittent blood cardioplegia. With this purpose, we observed echocardiographic and hemodynamic consequences of the administration of this amino acid.
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Patients and Methods
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We included in this study all primary coronary artery bypass graft (CABG) surgery patients operated on by the same surgeon with extracorporeal circulation between May 2003 and February 2004. We excluded all patients younger than 45 years and older than 80 years. All patients in need of other surgical procedures or urgent operation were excluded. Patients with recent myocardial infarction (less than 10 days) or perioperative myocardial infarction were also excluded.
After informed written consent and after the study was approved by the institutional Ethics Committee (March 2003), all patients enrolled were randomly assigned to two groups according to a computer-generated random code: group A, receiving L-arginine enriched cardioplegia; and group B, used as control. The L-arginine was added by one technician, and all operating room staff and analysts were blinded to the study group.
For all patients, we used the protocol of cardioplegic administration proposed by Buckberg [8]. Warm induction of cardioplegic arrest was used to let the NO synthase work to produce NO. Cardioplegia was infused through a double-lumen needle in the ascending aorta for antegrade administration and through a coronary sinus catheter for retrograde infusion. Solution A was used for induction and for maintaining phases, and solution B for the reperfusion phase (Table 1). After aortic cross-clamping, cardioplegic arrest was obtained with blood 4:1 high-potassium solution infused antegradely at 37°C with a flow rate of 300 to 330 mL/min for 2 minutes and 30 seconds, and with a retrograde infusion at a flow rate of 200 to 250 mL/min for the next 2 minutes and 30 seconds. The correct position of the retrograde cannula was verified indirectly measuring infusion pressure that was maintained between 25 and 40 mm Hg. The cold phase (4°C) of induction began with 4:1 low-potassium solution infused for 1 minute and 30 seconds antegrade at a flow rate of 150 to 200 mL/min followed by a 30-second retrograde phase at the same flow rate. Diastolic arrest was maintained after each distal anastomosis or every 18 to 20 minutes by 1 minute of cold antegrade and 1 minute of retrograde infusion of low-potassium solution. For reperfusion, a 37°C low-potassium 4:1 solution was administrated for 2 minutes antegrade and 1 minute retrograde. Afterward, whole blood was infused retrograde for 3 minutes to obtain a final "wash-out." Group A received a dose of 7.5 g L-Arginine in 500 mL of cardioplegia. Group B was used as the control group.
A total intravenous balanced anesthesia was used. In all patients, a Swan-Ganz flotation catheter (Edwards 131 HF7; Edwards Lifesciences LLC, Irvine, CA) through the internal jugular vein was used for hemodynamic assessments. Electrocardiograph leads D2 and V5 were continuously recorded during the surgical procedure and in the intensive care unit.
All patients were operated on using the same strategy of myocardial revascularization. The left internal mammary artery was used to perform the anastomosis on the left anterior descending coronary artery in all patients. Saphenous vein was used to perform other grafts. A complete arterial revascularization, using both the mammary arteries and the radial artery or the right gastroepiploic artery, was used in patients younger than 70 years, without insulin-dependent diabetes mellitus, and not affected by obesity or chronic obstructive lung disease.
Preoperative, Operative, and Postoperative Data
The following data were collected: age, extension of coronary disease, left main stenosis of 50% or more, hypertension, previous stroke, insulin-dependent diabetes mellitus, previous acute myocardial infarction, recent acute myocardial infarction (less than 30 days), chronic obstructive lung disease, unstable angina, peripheral vascular disease. Among operative findings, we evaluated extracorporeal circulation time and aortic cross-clamping time, the total amount of cardioplegic solution administered and the number of doses, the minimum core temperature reached, the mean number of grafts performed, and the use of double mammary arteries in the two groups.
We studied the outcome of patients in terms of electrocardiograph lead modifications, postoperative inotropic drug use, mediastinal blood loss in the first 24 hours, atrial fibrillation, ventricular arrhythmias and other postoperative complications, mechanical ventilation time, intensive care unit stay, and hospital stay.
We did not consider as inotropic treatment the use of dopamine lower than 4 
· kg–1
· min–1.
Echographic, Hemodynamic, and Chemical Analysis
All patients underwent a transesophageal echocardiographic study before aortic cannulation and before protamine administration to assess myocardial contractility expressed by ejection fraction and shortening fraction and to evaluate left ventricular end-diastolic and systolic diameters and volumes.
Hemodynamic evaluations were performed before sternum opening (time 0), at sternum closure (time 1), and 1 hour after the arrival of the patient in the intensive care unit (time 2). A Swan-Ganz catheter was used to measure the cardiac index, indexed systemic vascular resistance, indexed pulmonary vascular resistance, systolic pulmonary artery pressure, and pulmonary capillary wedge pressure.
Blood samples were collected from the retrograde coronary sinus cannula, and these were submitted to evaluation of lactate, tumor necrosis factor alpha (TNF-
), interleukin-2 receptor (IL-2R), and interleukin 6 (IL-6) level measures before aortic clamping (time 0), at aortic clamping removal (time 1), and 15 minutes after (time 2).
Serum samples from systemic blood vessels were also analyzed at four different times: preoperatively, and 2, 18, and 42 hours after aortic clamp removal, to measure creatine-phosphokinase (CPK), CPK-MB fraction mass, cardiac troponin T, platelets, and leukocytes.
Statistical Analysis
Sample size was computed calculated on the basis of our primary endpoints (troponin T and IL-6 levels) to obtain a ß level greater than 85% and an level less than 5%. All continuous variables are expressed as mean ± SD, whereas discrete variables are presented as frequencies and percentages. Analysis of categorical variables was performed with the 
2 test. Analysis of continuous variables was performed with Student's t test and with the multivariate analysis of variance test. Comparisons between the two groups and within each group were analyzed using a two-way analysis of variance for repeated measures followed by a Bonferroni test for multiple comparisons as post-hoc analysis (SPSS 12.1 for Windows; SPSS, Chicago, Illinois). A p value of 0.05 or less was considered statistically significant.
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Results
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Sixty-five consecutive patients were enrolled in this prospective randomized study (33 in group A and 32 in group B). Regarding preoperative characteristics, the two groups were homogeneous for hypertension, cerebral disease, peripheral vascular disease, diabetes, previous acute myocardial infarction, chronic obstructive pulmonary disease (p = not significant). Concerning operative data, there was no statistical difference between groups. There was no statistically significant difference in cardioplegia infusion between groups in terms of doses and amount of solution administered (3.1 ± 0.9 doses in group A versus 3.4 ± 0.8 in group B; 725 ± 135 mL solution in group A versus 730 ± 114 mL in group B: p = ns). Moreover, no difference was found in postoperative data and in complications except for intensive care unit stay, which was shorter in group A (26 versus 37 hours; p = 0.03; Table 2). Bleeding, assisted ventilation time, and atrial fibrillation incidence were similar (p = not significant).
Regarding hemodynamic evaluation, pulmonary wedge pressure was significantly reduced in group A (p = 0.023; Fig 1). Other hemodynamic measurements showed a better trend but not significant results for cardiac index (2.4 ± 0.5 L · min–1
· m–2 in group A versus 2.2 ± 0.5 in group B at time 2, 1 hour after arriving in the intensive care unit), indexed systemic vascular resistance (2,624 ± 585 dyne/m2 in group A versus 2,920 ± 861 dyne/m2 in group B at time 2), indexed pulmonary vascular resistance (236 ± 94 dyne/m2 in group A versus 216 ± 95 dyne/m2 in group B at time 2), and systolic pulmonary artery pressure (14.1 ± 5.5 mm Hg in group A versus 17.4 ± 5.9 mm Hg in group B at sternal closure, time 1).
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Fig 1. Wedge pressure levels in the two groups (p = 0.023) at different time points. (Data are expressed as mean ± SE.) (Group A = triangles; group b = squares; Pre = preoperative; Post = postoperative; ICU = intensive care unit.)
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Echocardiographic findings did not show any statistical differences between groups in terms of myocardial contractility and ventricular volumes modifications (Table 3).
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Table 3. Echocardiographic Data in the Two Groups Before Aortic Cannulation (Preoperative) and Before Protamine Administration (Postoperative)
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Regarding blood samples taken from the coronary sinus cannula, all evaluated cytokines (Table 4) were reduced in group A, and the only statistical difference was for IL-6 (p = 0.026; Fig 2). There were no statistical differences between groups in terms of platelets count and leukocytes levels.
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Fig 2. Interleukin-6 (IL-6) levels in the two groups before aortic clamping (time 0), at aortic clamping removal (time 1), and 15 minutes after (time 2; p = 0.025). (Data are expressed as mean ± SE.) (Group A = triangles; group b = squares.)
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There were no statistical differences between the two groups in terms of release of CPK and CPK-MB mass, whereas troponin T, as shown in Figure 3, was significantly higher in group B (p = 0.009).
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Fig 3. Troponin T levels in the two groups preoperatively (Preop.), and at 2, 18, and 42 hours (h). (Data are expressed as mean ± SE). (Group A = triangles; group b = squares.)
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In group A, 1 patient was excluded for perioperative acute inferior myocardial infarction: the patient had three-vessel coronary disease with a 90% stenotic posterior interventricular coronary artery, which was not grafted because of heavy calcification. In group A, 1 patient had postoperative bronchoconstriction, 2 patients were reoperated on for bleeding, and 1 patient was reoperated on for sternal dehiscence. In group B, 1 patient presented with prolonged bronchoconstriction, 1 patient had low output syndrome, 1 patient experienced pulmonary collapse, and 1 patient had tracheal bleeding (p = not significant).
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Comment
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Only two clinical studies have described the use of L-arginine as an additive to cardioplegia in cardiac surgery [4, 5], and demonstrated that this amino acid is able to reduce myocardial ischemic damage, expressed by a reduction of troponin and CPK-MB levels. The study by Carrier and colleagues [4] reported lessened values of troponin T using 7.5 g L-arginine added to the first 500 mL solution, with best results when warm (33°C) cardioplegia was infused. Previously, a lower dosage (1 g) of L-arginine had been tested by the same group, with results indicating it to be safe but not efficacious [9]. We checked the efficacy of L-arginine in reducing myocardial cytokines and troponin T release when added to our anterograde-retrograde blood cardioplegia protocol. We obtained a significant reduction in troponin T release in group A; nevertheless, CPK-MB mass was not significantly reduced, using a warm induction to let the enzyme nitric oxide work better in normothermia and adding L-arginine to all cardioplegia doses except for the retrograde phase of "wash out" completed with whole blood. Our study confirms the protective capacity of L-arginine against the ischemia-reperfusion damage due to the cardioplegic arrest in coronary surgery and the previously demonstrated higher sensivity of troponin T with respect to MB mass measurement [10]. As previously reported, this protective effect should be related to the NO release during cardioplegia administration, with consequently reduced endothelial activation and reduced no-reflow phenomenon, with an increase of coronary flow and better myocardial preservation during arrest and reperfusion time.
Proinflammatory cytokines play an emerging role as responsible factors for initiating and maintaining myocardial response to environmental stress. The short-term expression of cytokines seems to let the myocardium rapidly react to injury and to activate protective homeostatic responses to damage. However, most recent experimental studies documented the negative immediate inotropic effect on heart function, mainly in case of high levels of TNF, IL-2, and IL-6 [11]. Moreover, the implication of proinflammatory cytokines in cardiac surgery and the relationship to inflammatory reaction due to cardiopulmonary bypass, myocardial arrest, and surgical trauma has been demonstrated [6, 7]. We first investigated the effects of L-arginine on myocardial stress as expressed by local cytokines. The significant decrease of IL-6 levels, and the concomitant reduction of IL-2R and TNF levels, should have the effect of a decrease in myocardial cell ischemic insult due to NO local production. Myocardial cytokines reduction should be related to L-arginine protective effects against inflammatory response, with less activation of complement leukocytes and endothelial cells, and decreased generation of oxygen-derived free radicals by polymorphonuclear neutrophils.
We observed a reduction in intensive care unit stay in patients treated with L-arginine, and that may reflect a better clinical outcome as a direct consequence of a limitation of the extension of ischemic damage. Recently, Kiziltepe and associates [5] reported the reduction in CPK-MB release caused by L-arginine added to cardioplegia with a lower intensive care unit stay in the treated group, which demonstrates the same encouraging effect described by us. Moreover, it has been demonstrated that elevated postoperative cardiac troponin T concentrations predict a prolonged intensive care unit stay, confirming that a reduction in "minor myocardial damage" can lead to evidence of a clinical benefit [12].
Wedge pressure was significantly reduced in group A patients. A systemic vasodilation due to NO release clarifies the reason why we obtained this result. A better hemodynamic performance could contribute to the shorter intensive care unit outcome. Ogilvie and Zborowska-Sluis [13] demonstrated an improved cardiac output with a decrease in pulmonary vascular resistances using L-arginine in dogs. Our results could be related to this effect, although we obtained an improved postoperative cardiac index in group A (2.4 ± 0.5 versus 2.2 ± 0.5 L · min–1
· m–2 at time 2), although not statistically significant.
Our research verifies the safety of the use of L-arginine in cardiac surgery when locally administered, and we can affirm that it does not lead to any harmful effect. We tested this amino acid for all the intermittent doses of cardioplegia, but we avoided the reperfusion phase, according to the results of experimental work published in 1996 by Engelman and colleagues [14], which documented a beneficial effect of L-arginine when given before cardioplegic arrest, a positive effect during arrest, and a detrimental effect during reperfusion as expressed by myocardial function and lactic dehydrogenase release.
In conclusion, we affirm that L-arginine is able to reduce myocardial stress expressed by a reduction in local cytokines release; its protective effects are demonstrated also by a significant reduction in troponin T release, and this supports other authors' results. Moreover, our study determined that the use of this amino acid added to cardioplegia solution is safe as assessed by echocardiographic data, Swan-Ganz measurements, and laboratory findings. The efficacy of L-arginine for critically ill patients with either unstable angina or acute myocardial infarction, or with low ejection fraction, needs to be further investigated.
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Notice From the American Board of Thoracic Surgery
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The 2006 Part I (written) examination will be held on Monday, December 4, 2006. It is planned that the examination will be given at multiple sites throughout the United States using an electronic format. The closing date for registration is August 1, 2006. Those wishing to be considered for examination must apply online at www.abts.org.
To be admissible to the Part II (oral) examination, a candidate must have successfully completed the Part I (written) examination.
A candidate applying for admission to the certifying examination must fulfill all the requirements of the Board in force at the time the application is received.
Please address all communications to the American Board of Thoracic Surgery, 6333 N St. Clair St, Suite 2320, Chicago, IL 60611; telephone: (312) 202-5900; fax: (312) 202-5960; e-mail: mailto:info{at}abts.org.
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Acknowledgments
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We wish to thank Dr Giorgia Pavan for her help in the preparation and correction of the manuscript, and Dr Nicolò Patroniti for his statistical support.
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References
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- Hoshida S, Yamashita N, Igarashi J, et al. Nitric oxide synthase protects the heart against ischemia-reperfusion injury in rabbits J Pharmacol Exp Ther 1995;274:413-418.[Abstract/Free Full Text]
- Pernow J, Uriuda Y, Wang QD, Li XS, Nordlander R, Rydeen L. The protective effect of L-Arginine on myocardial injury and endothelial function following ischemia and reperfusion in the pig Eur Heart J 1994;15:1712-1719.[Abstract/Free Full Text]
- Carrier M, Pellerin M, Perrault LP, et al. Cardioplegic arrest with L-arginine improves myocardial protectionresults of a prospective randomized clinical trial. Ann Thorac Surg 2002;73:837-842.[Abstract/Free Full Text]
- Kiziltepe U, Tuncan B, Eyeleten ZB, et al. Efficiency of L-arginine enriched cardioplegia and non-cardioplegic reperfusion in ischemic hearts Int J Cardiol 2004;97:93-100.[Medline]
- Prondzinsky R, Knupfer A, Loppnow H, et al. Surgical trauma affects the proinflammatory status after cardiac surgery to a higher degree than cardiopulmonary bypass J Thorac Cardiovasc Surg 2005;129:760-766.[Abstract/Free Full Text]
- Hennein HA, Ebba H, Rodriguez JL, et al. Relationship of the proinflammatory cytokines to myocardial ischemia and dysfunction after uncomplicated coronary revascularization J Thorac Cardiovasc Surg 1994;108:626-635.[Abstract/Free Full Text]
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- Carrier M, Pellerin M, Pagè P, et al. Can L-Arginine improve myocardial protection during cardioplegic arrest? Results of a phase 1 pilot study Ann Thorac Surg 1998;66:108-112.[Abstract/Free Full Text]
- Katus HA, Schoeppenthau M, Tanzeem A, et al. Noninvasive assessment of perioperative myocardial cell damage by circulation cardiac troponin T Br Heart J 1991;65:259-264.[Abstract/Free Full Text]
- Mann DL. Stress activated cytokines and the heartfrom adaptation to maladaptation. Annu Rev Physiol 2003;65:81-101.[Medline]
- Baggish AL, MacGillivray TE, Hoffman W, et al. Postoperative troponin-T predicts prolonged intensive care unit length of stay following cardiac surgery Crit Care Med 2004;32:1866-1871.[Medline]
- Ogilvie RI, Zborowska-Sluis D. Acute effect of L-arginine on hemodynamics and vascular capacitance in the canine pacing model of heart failure Cardiovasc Pharmacol 1995;26:407-413.[Medline]
- Engelman DT, Watanabe M, Maulik N, et al. Critical timing of nitric oxide supplementation in cardioplegic arrest and reperfusion Circulation 1996;94(Suppl 9):II407-II411.
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Abstract
Introduction
