The Population Study: A total of 26 patients undergoing on-pump bypass operation (5 women, 21 men) were included in the study. All the patients undergoing the elective cardiopulmonary bypass were included in this study. They were informed about the study and an informed consent form stating that they were voluntary to participate in the study was signed by them. The present study’s protocol was reviewed and approved by the ethics committee of Fırat University (Reg. No. 2015015).
In the present study, blood was drawn 3 times from each patient: before the surgical intervention (preoperative period), at the end of the surgical intervention (peroperative period), and approximately 24 hours after the surgical intervention (postoperative period). Nitric oxide (NO) level and arginase activity, which were the biochemical parameters, were examined in these blood samples.
Surgical Technique: All patients were managed by the same surgical and anesthetic team in the same operating room. Six-channel electrocardiogram (ECG) and non-invasive arterial pressure monitoring were applied to the patient taken to the operating table. Before anesthesia induction, the radial artery catheter was inserted under local anesthesia, preoperative blood samples were taken together with the initial blood gas, and an invasive pressure monitoring was performed. Anesthesia induction was performed by using 100 mg lidocaine intravenous (iv), 300 mg magnesium iv, 100 μg fentanyl iv, 0.60–1.2 mg/kg esmeron iv, and 2 mg/kg propofol iv. Central venous cannula and urine catheters were inserted in the post-anesthesia period. Maintenance of general anesthesia was performed by adding 20 mg esmeron and 100 μgr fentanyl iv in the oxygenator reservoir every 30 minutes.
While propofol infusion (%1) was performed as 20 mL/hour iv out of CPB, it was reduced to the infusion dose of 10 mL/hour iv during CPB. A median sternotomy was performed to all the patients. Heparin of 350-400 unit/kg was administered to left internal mammary artery (LIMA) prior to cannulation, which was followed by routine aortic and right atrial cannulation. Membrane oxygenators and moderate systemic hypothermia were used to carry out the cardiopulmonary bypass (CPB). Myocardial protection was achieved by using antegrade mild hypothermic blood cardioplegia (32 ºC) and repeated every 20 minutes. Cold blood cardioplegia was prepared by adding 2 mmol/L magnesium sulphate, 5 mmol/L potassium chloride, and 1.6 g/1000 cc sodium bicarbonate in every 1000 cc blood taken from the reservoir. Activated clotting time was maintained for >400 sec during the procedure. During the procedure, the mean blood pressure was kept at 60 mmHg and over. All the proximal saphenous vein anastomoses were performed via the cross clamp by using a single clamp technique. Air was discharged from the proximal anastomoses and the cross clamp was removed. After sufficient cardiac performance was provided, pump flow was reduced and CPB was ended. Heparin was neutralized with protamine at the ratio of 1:1.3 for 10 minutes after CPB.
After the operation, all patients were followed up in the intensive care unit. Second (peroperative) blood samples were taken from the radial artery catheter together with the blood gas right after the patient arrived to the intensive care unit. Third (postoperative) blood samples were taken approximately 24 hours after the surgery while the intensive care follow-up of the patient was ongoing. All the samples were sent to the laboratory as soon as possible and they were properly prepared and kept.
Sample Collection: Once blood samples were taken in two heparin-containing test tubes by means of the cannula inserted in radial artery, they were taken to the laboratory for invasive blood pressure monitoring. While one of the heparinized bloods was used as the full blood, the other heparinized blood was centrifuged at 3000 rpm for 5 minutes and its plasma was separated and then washed three times by using physiological saline solution. Then, it was kept at the deep-freezer at –80 ºC before biochemical analyses.
Arginase activity and NO levels: Arginase activity was measured by determining the increase of the amount of urea (the reaction product) 7. One unit (U) of enzymatic activity was defined as μmol of the product formed per hour at 37 ºC. The results were given as units/mg of protein.
The NO level of the samples was assayed according to the method of Griess 8. To determine the effect of several compounds on NO levels, treated with glutamate (1 mM) or co-treated with L-citrulline, L-arginine, and/or taurine (20 mM). At 24 h after the treatment, 100 μL of culture supernatant was collected from each sample and added to a 96-well micro-plate. Samples were then incubated with 100 μL of modified Griess reagent with 1% sulfanilamide in 5% phosphoric acid at room temperature for 7 min. Finally, 0.1 N-1- napthylethylenediamine dihydrochloride in water was added followed by incubation at room temperature for 7 min. Absorbance at wave length of 550 nm was measured.
The protein content of the samples was assayed according to the method of Lowry et al. 9. Bovine serum albumin was used as the standard.
Statistical Analysis: Statistical analysis was carried out using the SPSS package program (15.0 for Windows). Once the difference between the groups was compared using Kruskal Wallis Test, which group was different was determined in pairs by applying to Mann Whitney-U test. All of the results were shown as mean ± standard error mean (SEM).