Effect of cardiopulmonary bypass on tissue injury markers and endothelial activation during coronary artery bypass graft surgeryS Nair1, K Iqbal2, M Phadke3, UE Jadhav2, J Khandekar2, JMS Khandeparkar2
1 Department of Biochemistry, LTMG Hospital and LTMM College, Sion, India
2 Department of Cardiovascular and Thoracic Surgery, LTMG Hospital and LTMM College, Sion, India
3 Department of Biochemistry, Seth GS Medical College and K.E.M Hospital, Parel, Mumbai, India
Background: Coronary artery bypass grafting (CABG) is done either using cardiopulmonary bypass (CPB) or without using CPB (OPCAB). But, recently, reports have shown that CPB is associated with increased postoperative morbidity because of the involvement of many systems. Aims: The aim of this prospective study was to evaluate the influence of the technique of surgery on various tissue injury markers and the extent of endothelial activation in patients undergoing CABG and OPCAB coronary revascularization. Settings and Design: This study was conducted at a tertiary healthcare center during the period May 2008 to December 2009. Materials and Methods: This was a prospective nonrandomized blinded study. The activities of Creatine Phosphokinase (CK) and its isoenzyme CK-MB, Lactate dehydrogenase (LDH), levels of cardiac Troponin I, soluble vascular cell adhesion molecule-1 (sVCAM-I) and systemic nitric oxide production were assessed. Statistical analysis: All the results were expressed as Mean±SD. P value ≤0.05 was considered significant. The statistical analysis was carried out using SPSS Version 11.5-computer software (SPSS Inc., Chicago, IL, USA). Results: The surgical trauma had elevated CK, CK-MB and Troponin I in both the groups and further elevation was seen in the CABG group in comparison to OPCAB (P<0.001). The Troponin I concentrations showed an increase from 0.11±0.02 preoperatively to 6.59±0.59 (ng/ml) at 24 h (P<0.001) compared to the OPCAB group. Mean serum levels of sVCAM-1 increased significantly after surgery in both the groups (P<0.02). To determine serum nitric oxide (NO) production, NO2− and NO3− (stable end products of NO oxidation) were analyzed which also increased significantly at 24 h in both the groups. But the increase was not significant at 48 h in both the groups compared to the preoperative value in our study. Conclusion: The present study indicates that, despite comparable surgical trauma, the OPCAB significantly reduces tissue injury. The overall pattern of endothelial activation after OPCAB is significantly lower than that after CABG. This may contribute to improved organ function, and improved postoperative recovery.
Keywords: Cardiopulmonary bypass, coronary artery bypass grafting, myocardial injury, nitric oxide, troponin
Coronary-artery bypass grafting performed with cardiopulmonary bypass (CPB) and cardiac arrest provides a motionless, bloodless surgical field, allowing optimal conditions for the construction of coronary anastomosis. , CPB is associated with systemic inflammatory response  and increased postoperative morbidity  including neuropsychological impairment.  Off-pump coronary artery bypass (OPCAB) may decrease the incidence of myocardial injury  and renal failure  associated with CPB. The avoidance of cardiopulmonary bypass appears to render Coronary artery bypass grafting (CABG) less invasive.  The revival of interest in surgery without cardiopulmonary bypass reflects an endeavor to avoid the morbidity related with cardiopulmonary bypass.
The aim of this prospective study was to evaluate the influence of the technique of surgery by measuring the markers of myocardial ischemia including the activities of serum cardiac enzymes and extent of endothelial activation as reflected in the serum after coronary artery revascularization by CABG and OPCAB techniques.
This prospective nonrandomized blinded study was conducted at a tertiary healthcare center during the period May 2008 to December 2009. The patients were categorized into the two study groups:
Group 1: Those undergoing CABG by off-pump beating heart technique (OPCAB).
Group 2: Those undergoing CABG by on-pump i.e. CPB with moderate hypothermia and multidose cold sanguinous hyperkalamic cardioplegic arrested heart technique.
The option to go for CABG or OPCAB technique was entirely the surgeon's decision at the time of surgery, considering the patient's condition. The patients undergoing emergency CABG, pregnant females, patients with infective endocarditis and concomitant non-cardiac inflammatory, infective and neoplastic diseases were excluded from the study.
This study was carried out after obtaining the institutional ethics committee approval and informed consent from the patients.
Blood samples were collected preoperatively, 24 and 48 h postoperatively. The following parameters were studied - Total Creatine kinase (CK) and its isoenzyme CK-MB, Lactate dehydrogenase (LDH), cardiac Troponin I (c-Tn I), soluble vascular cell adhesion molecule-1 (s VCAM-I) and systemic nitric oxide production.
The activities of Creatine kinase (CK) and its isoenzyme CK-MB were determined using a commercial kit (Accurex, India) at 37 C; the results reported as IU/L. Lactate dehydrogenase (LDH) activity was determined on an automated analyzer using Agappe Diagnostics Pvt. Ltd., India kit. The levels of cardiac Troponin I (Diagnostic Automation, Calabasas) and soluble vascular cell adhesion molecule-1 (s vcam-1) (Bender MedSystems, Europe) were determined using specific commercially available enzyme-linked immunosorbent assay (ELISA) kits. To determine serum nitric oxide (NO) levels, NO 2− and NO 3− (stable end products of NO oxidation) were estimated by Greiss reaction.
Group differences in clinical variables between CABG and OPCAB were assessed by analysis of variance, χ2 , or Fisher exact tests when indicated. Values are expressed as mean±SD and a value of P less than or equal to 0.05 was considered statistically significant. Assuming a power of 90% and Type I error of 5%, the sample size required for the study was determined by power analysis. The primary outcome measure was the extent of myocardial damage expressed by serum levels of cTnI. The statistical analysis was carried out using SPSS Version 11.5-computer software (SPSS Inc., Chicago, IL, USA).
On the day of surgery, the patients were premedicated with morphine (0.2 mg/kg) and promethazine (0.5 mg/kg) or fentanyl (3 μg/kg) about 30-45 min prior to induction of anesthesia. Anesthesia was induced with a combination of opiod, fentanyl (1-2 μg/kg) and inhalational agent (sevoflurane, 1-2%). Vercuronium was used to accomplish endotracheal intubation and anesthesia was maintained with 50% oxygen and 50% nitrous oxide (N 2 O). Adequate cerebroprotective and cardioprotective agents were used at the initiation and throughout the procedure.
Arterial blood gas and serum glucose levels were monitored half-hourly and serum glucose levels maintained at less than 130 mg/dL by intermittent intravenous insulin injections or insulin infusion. Activated clotting time (ACT) was maintained between 450 and 500. During CPB, arterial partial oxygen pressure was kept between 150 and 250 mm Hg, and alpha-sat pH monitoring was used. Mean blood pressure was maintained between 50 and 80 mm Hg using conventional vasoactive medications.
Off-pump coronary artery bypass
Hemodynamic stabilization was maintained using ionotropes. Mechanical stabilizers like Octopus, Sea-urchin, star-fish were used for optimal positioning during different coronary revascularization. Intracoronary shunts were used for maintaining distal flow after arteriotomy and during anastomosis. ACT was maintained in the range of 250 to 300.
Coronary artery bypass graft surgery
On standard total CPB. ACT is usually maintained in the range of 450 to 500. Moderate hypothermia (28 to 32C) was maintained throughout the procedure. Antegrade, root, cold, hyperkalamic, and sanguinous cardioplegia was administered after application of cross clamp and repeated at intervals of 15 to 20 min. Coronary anastomosis was completed. Cross clamp was removed and patient rewarmed at the end of the procedure. The rest of the surgical technique was similar in both the groups.
The intraoperative characteristics of the patients in the two groups are shown in [Table 1]. Fifty-eight consecutive patients (41 males and 17 females) undergoing CABG were enrolled in this study. The OPCAB group comprised thirty patients with a mean age of 52.25±9.6 years and the CABG group comprised 28 with a mean age of 54.5±6.63 years. Each marker displayed a typical laboratory pattern. The mean values rose from a baseline concentration preoperatively to a postoperative peak, indicating myocardial injury of a certain degree in all cases. In the CABG group, CPB time was 2.25±0.18 h, and the aortic cross-clamp time was 1.75±0.18 h. The length of stay in the intensive care unit (ICU) was significantly (P<0.05) longer for the CABG patients.
The activities of CK before surgery were similar in both the groups. After surgery, the value increased in both the groups. But the CK values after CABG were significantly (P<0.001) higher compared to the OPCAB group. And the values peaked at 24 h in both the cases. The total amount of CK-MB released was higher in the CABG group than in the OPCAB group (130.3±12.6 vs. 55.11±8.3 IU/L, at 24 h P<0.001), i.e. for each sample drawn, CK-MB concentrations were significantly higher in the CABG group than in the OPCAB group at 24 and 48 h [Figure 1] and [Figure 2].
There were no major inter-group differences in baseline values of Troponin I in OPCAB group and CABG group patients. The Troponin I concentration was significantly elevated in the CABG group from 0.11±0.02 ng/ml preoperatively to 6.59±0.59 ng/ml at 24 h (P<0.001) as compared to the OPCAB group [Figure 3]. By 48 h the levels showed a decreasing trend towards baseline. Serum LDH levels also showed a similar trend [Figure 4]. The LDH level was 406.6±95.75 vs. 488.1±107.8 IU/L preoperatively in OPCAB vs. CABG. By 24 h the values were seen to increase to 681.1±111.7 IU/L in the OPCAB cases and to 899±170.2 IU/L in the CABG cases (P<0.01).
The sVCAM levels were also seen to increase significantly postoperatively in both the groups (P<0.02) as compared to the preoperative value [Figure 5]. The analysis of variance showed a significant effect of time on all these variables. The systemic NO level was lower in the OPCAB group compared to the CABG group at 24 h. The end products of nitric oxide, plasma NO2 +NO3 level was 46.03±11.7 (μmol/L) and 49.19±15.9 (μmol/L) before operation and showed a marked and significant rise to 74.19±15.89 (μmol/L) and 130.165±22.18 (μmol/L) 24 h postoperatively in the OPCAB and CABG group of patients respectively (P<0.01). But the difference was not significant at 48 h between the groups as well as compared to the preoperative value [Figure 6].
Postoperative cardiac failure due to myocardial injury still remains a major complication in cardiac surgical procedures and is the main cause of increased morbidity and mortality. Significant myocardial injuries associated with cardiac surgery result in significant increases in the complication rates.  OPCAB has gained increasing popularity due to its potential to avoid the damage induced by cardiopulmonary bypass and cross-clamping.
In our study we found that the hospital stay was significantly longer in patients undergoing CABG. Postoperative release of cardiac enzymes may influence postoperative outcome because of increased myocardial damage.
Several factors can influence the release of cardiac enzymes during a surgical myocardial revascularization, in CABG, the length of cardioplegic arrest, the duration of CPB, duration of cross-clamp, the efficacy of myocardial protection; during OPCAB, number of grafts , the time required for completing each anastomosis, the use of intracoronary shunts  and perioperative hemodynamics. However, independent of the techniques used, graft failure and incomplete revascularization are the most probable causes of postoperative release of cardiac enzymes. 
Conventional CABG with cardioplegic arrest induces global ischemic-reperfusion injury  whereas OPCAB leads only to local ischemia. A higher release of markers could be expected from a globally ischemic heart than during OPCAB. CK, CK-MB and LDH , are well-known cardiac tissue injury markers. Cardiac Troponin I (cTn-I) is also a very good predictor  and is highly specific for myocardial tissue, with no cross-reactivity with the skeletal muscle isoforms.  Elevated cTn-I is associated with ischemic electrocardiogram changes after CABG, and a poor overall prognosis.  Cardiac surgery itself induces an increase in plasma cTn-I, even in the absence of ischemic damage. Dissection of the myocardium to expose the intramyocardial arteries, manipulation of the heart, placement of the purse string sutures for cannulation, etc., cause myocardial injury, which may explain why cTn-I increases early after cardiac surgery, even in the absence of ischemic myocardial damage. 
The patients with the OPCAB technique continuously showed lower serum Troponin I and CK and CK-MB and LDH concentrations than those with the CABG technique. Our findings are compatible with those reported by others as well ,, Significantly greater release of the markers in the CABG group may be due to reperfusion of the heart following global cardiac arrest, oxidative stress and inflammation.  Different works have showed different patterns of cardiac markers' release  with respect to time as compared to what is presented in this study. The complexities of the operations and methods of detection of these markers in different laboratories may be the reason for these variations between different studies.  Our study found all three markers to be equally sensitive to the extent of injury. Different types of cardiac surgeries may also produce different release patterns of myocardial damage markers.  The different release patterns of the markers in the perioperative period may be caused not only by the surgery itself, but also by myocardial cell injury due to different causes.  Etievent et al.,  showed that increases in cardiac-specific serum markers of myocardial injury during coronary procedures might be used as an indicator of the efficiency of cardioprotective procedures.
The vascular endothelium has a pivotal role in the systemic host response to injury and therefore, the systemic injury that follows CPB.  Cardiac operations using CPB have been reported to be associated with the release of various cytokines, activation of the coagulation cascade, and increased levels of circulating adhesion molecules.  In addition, there is convincing evidence that ischemia and reperfusion injury may be an important trigger for cytokine release during CPB, and this release enhances the risk of subsequent endothelial dysfunction.  Anesthesia appears to modulate the release of some cytokines including interleukin-6 and interleukin-1-beta, thus possibly influencing endothelial function and subsequently, concentration of circulating adhesion molecules. 
An increase in circulating adhesion molecules results either from increased expression of activated endothelial cells or from increased proteolytic cleavage of endothelial-bound forms, secondary to endothelial cell damage.  Thus, increased concentrations of soluble vascular cell adhesion molecules may serve as markers for activated or damaged endothelium. Clinically, sVCAM-1 seemed to be the best-suited marker for endothelial cell activation, because it was associated only with aortic cross-clamping and heparin and protamine doses. 
The fact that plasma levels of circulating adhesion molecules were increased only temporarily in the patients having elective CABG may imply that endothelial activation or dysfunction was also of only short duration. Whether monitoring soluble adhesion molecules will be a precious tool for the early diagnosis of endothelial damage and the prognosis for the risk of development of multiple-organ failure, must be elucidated in further controlled studies.
NO formerly known as endothelium-derived relaxing factor is produced by many cells in the body; however, its production by the vascular endothelium is particularly important in the regulation of blood flow. Because of its importance in vascular function, abnormal production of NO can adversely affect blood flow and other vascular functions. NO has a half-life of only a few seconds, when it reacts with superoxide anion or to the heme moiety of hemoglobin or the heme moiety of the enzyme guanylyl cyclase. Superoxide anion has a high affinity for NO (both molecules have an unpaired electron making them highly reactive). Therefore, superoxide anion reduces NO bioavailability. NO inhibits calcium influx via cGMP-dependent inhibition of L-type Ca 2+ channels, desensitizes the myofilaments to Ca 2+ , and subsequently decreases contractile force. It has furthermore been recently suggested that cytokine-induced negative inotropic effects may be due to the formation of peroxynitrite, which is generated via interaction of superoxide and NO in the heart.  The systemic NO levels were lower in the OPCAB cases in this study compared to the CABG group.
These higher releases of serum markers showing myocardial injury during CABG with global cold cardioplegic arrest may be due to inadequate perfusion of the subendocardium and the remaining ungrafted ischemic areas, unexpected aortic regurgitation, reperfusion through the bypass grafts after unclamping, or due to direct trauma to the myocardium. , On the other hand, OPCAB is considered to be associated with a potential risk for ischemic myocardial tissue damage due to normothermic, metabolically active myocardium during the occlusion of the target coronary artery.  OPCAB is also technically more demanding for the surgeon and may require emergency conversion to CABG technique in case of hemodynamic collapse. Secondly, patients with severe left ventricular dysfunction may not be suitable for OPCAB.
In conclusion, OPCAB causes less myocardial injury than that resulting from CABG with cardioplegic arrest, as demonstrated by changes in the serum levels of CK, CK-MB, cTnI and LDH. Also , cardiac surgery procedure resulted in the increased serum levels of both soluble VCAM-1 adhesion molecules and NO, implying endothelial activation after coronary artery bypass grafting with cardiopulmonary bypass.
However, larger studies with the more generalized population are necessary to determine the effect of endothelial activation on patients' outcome after cardiac surgery.
We thank The Dean, LTM Medical College and LTMG Hospital, Sion, for granting us permission to carry out and publish this research work.
This work was supported by grants from the Department Development Fund of the Department of Cardiovascular and Thoracic Surgery, LTM Medical College and LTMG Hospital, Sion, Mumbai.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]