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Ann Thorac Surg 1999;67:1659-1663
© 1999 The Society of Thoracic Surgeons

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Original Articles

Elevated coronary endothelin-1 but not Nitric Oxide in diabetics during CABG

^a Departments of Department of Surgery, University of Illinois College of Medicine at Chicago, Chicago, Illinois, USA
^b Department of Physiology, University of Illinois College of Medicine at Chicago, Chicago, Illinois, USA
^c Department of Biophysics, University of Illinois College of Medicine at Chicago, Chicago, Illinois, USA

Accepted for publication December 15, 1998.

Address reprint requests to Dr Law, Department of Surgery M/C 958, University of Illinois at Chicago, 840 S. Wood St, Chicago, IL 60612
e-mail: wrlaw{at}uic.edu

	Abstract

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Background. After coronary artery bypass grafting procedures, a higher incidence of morbidity and mortality has been reported in diabetic patients. We tested whether coronary artery bypass grafting in diabetics affects the endothelin-1 and nitric oxide coronary effluent profile during reperfusion.

Methods. Twenty-one consecutive patients (9 with type II diabetes mellitus, 12 non-diabetics) underwent coronary artery bypass grafting by one surgeon. The two groups did not differ in preoperative ejection fraction, Parsonnet score, number of vessels bypassed, or cross-clamp time. Each patient was treated in the same intraoperative manner with single atrial, aortic, and antegrade and retrograde cardioplegia (CPL) cannulas. Cold CPL arrest was by antegrade and retrograde infusion of modified Buckberg CPL solution. Warm CPL solution was infused before reperfusion. Coronary sinus blood samples were obtained for estimation of endothelin-1 and nitrite plus nitrate before CPL arrest and at 1 and 15 minutes after each of 2 reperfusion periods.

Results. In diabetics, endothelin-1 was significantly increased at all reperfusion times as compared with non-diabetics. Nitrite plus nitrate levels were significantly higher in patients with diabetes than in those without, but did not change with time in either of the groups.

Conclusions. Reperfusion after CPL during coronary artery bypass grafting procedure can trigger the release of endothelin-1 in patients with diabetes mellitus. This may favor increased vascular tone or positive inotropic responses after coronary artery bypass grafting and may contribute to significant cardiovascular consequences in diabetic patients.

	Introduction

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The endothelium, which is a key regulator of vascular tone, has been implicated in the pathogenesis of diabetic vascular disease [1, 2]. Endothelins (ETs), produced by the vascular endothelium, are important mediators of vascular tone [3]. Besides ET’s own independent cardiovascular effects, ET production is closely associated with changes in nitric oxide (NO) under various cardiovascular pathologies including myocardial ischemia, hemorrhagic shock, and sepsis [4–7].

In clinical and experimental conditions, ischemia and reperfusion of the myocardium triggers the production of ET, NO, and other vasoactive substances, which can contribute to the morbidity and mortality associated with this condition [8–12]. An altered concentration of ET and NO can result in a shift in coronary vascular tone and altered myocardial perfusion. Brunner [8] demonstrated an increase in ET-1 levels after ischemia and reperfusion in isolated perfused rat hearts. Also, augmentation of NO production after cardioplegic arrest in neonatal lambs by the administration of L-arginine was demonstrated to improve myocardial function by Hiramatsu and coworkers [13]. Coronary artery bypass grafting (CABG), by its nature, is a procedure that involves ischemia and reperfusion to the myocardium. Thus, an increase in ET-1, unopposed by NO, may be associated with graft or native coronary artery vasoconstriction, potentiating myocardial ischemia. Because diabetic patients display altered endothelial dysfunction, we tested the hypothesis that diabetic patients undergoing CABG would display different profiles of ET and NO than non-diabetic patients.

	Material and methods

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This study was reviewed and approved by the University of Illinois at Chicago Institutional Review Board (IRB No. H-96-476). All procedures followed guidelines set forth in the NIH OPRR Protecting Human Research Subjects IRB Guidebook for the protection of the human subjects used in this study.

Operative protocol
Twenty-one consecutive patients undergoing nonemergent CABG at the University of Illinois at Chicago formed the basis of the current study. Nine patients had type II diabetes mellitus and 12 were non-diabetics. All subjects had preoperative cardiac catheterization for angiography and to measure left ventricular ejection fraction. General anesthesia was achieved by administration of forane and fentanyl. Each patient was monitored with a radial arterial catheter and a Swan-Ganz pulmonary arterial catheter. All patients were treated with the same preoperative technique by one attending surgeon (W.T.V.). Anticoagulation was achieved by systemic administration of sodium heparin (300 U/kg), and additional heparin was administered when activated clotting time was less than 480 seconds. Patients were cannulated with single right atrial, aortic, and antegrade and retrograde cardioplegia cannulas. Before commencement of the cardiopulmonary bypass, a coronary sinus venous blood sample was obtained from the retrograde cardioplegia catheter for ET-1 and NO estimations. The cardiopulmonary bypass circuit included a Terumo Caprio CX-SX18R membrane oxygenator (Terumo Corp, Tokyo, Japan) and a Sarnes 9000 roller pump (Sarnes Corp, Ann Arbor, MI). The bypass machine was primed with 2 L of Ringer’s lactate solution, 5,000 U sodium heparin, 50 mEq NaHCO₃, 20 mg furosemide, and 12.5 g mannitol. Pump flow rate was maintained at 1.8 to 2.5 L · m^-2 · min^-1, depending on the temperature. During cardiopulmonary bypass, body temperature was lowered to 30°C. Myocardial arrest was achieved with antegrade and retrograde infusions of cold blood cardioplegic solution administered via the BLD Advanced Blood Cardioplegia system (Sorin Biomedical, Irvine, CA; arresting dose 400 mL antegrade, 300 mL retrograde). The composition of cardioplegic solution was 4:1 of blood prime to crystalloid (tromethamine [Tham, 0.3 mol/L] 200 mL/L, KCl 60 mEq/L, citric acid 0.15 g/L, sodium citrate 1.32 g/L, monobasic sodium phosphate 0.11 g/L, dextrose 1.28 g/L, sodium chloride 550 mg/L, and made up to 1 L with 5% dextrose in water). Additional infusions of both antegrade and retrograde cardioplegia were administered after each distal anastomosis (200 mL antegrade, 200 mL retrograde). Distal anastomoses were performed during myocardial arrest. After the last distal anastomosis, warm blood cardioplegic solution (34°C) was infused. The composition of the warm cardioplegic solution was 4:1 of blood prime to crystalloid (Tham [0.3 mol/L] 114 ml/L, KCl 15 mEq/L, CPD solution [consisting of citric acid 0.34 g/L, sodium citrate 3.00 g/L, monobasic sodium phosphate 0.25 g/L, dextrose 18.91 g/L] 114 mL/L, monosodium L-glutamate 5.35 g/L, monosodium L-aspartate 4.90 g/L, and sterile water 206 ml/L). All patients had a left internal mammary artery graft to the left anterior descending coronary artery. The remainder of the bypass grafts used a reversed greater saphenous vein for conduit. Aortic cross-clamp was removed after all distal anastomosis and the native coronary vasculature and the left internal mammary artery graft were reperfused (reperfusion period A). During this reperfusion period A, a side-clamp was placed on the ascending aorta to facilitate proximal venous anastomoses of the grafts. After all proximal anastomoses were performed, the saphenous vein grafts were reperfused (reperfusion period B). During reperfusion periods A and B, coronary sinus samples were obtained at 1 and 15 minutes after initiation of the respective reperfusions for estimation of ET-1 and NO byproducts (NOx). The time between the initiation of reperfusion A and B ranged from 25 to 40 minutes.

Functional measurements
Baseline hemodynamic measurements (heart rate, arterial and central venous blood pressures, and cardiac output [by thermodilution]) were obtained before the induction of cardioplegic arrest. These measurements were repeated postoperatively at 3, 6, 12, and 24 hours after myocardial reperfusion.

Collection of samples
Coronary sinus blood samples for ET-1 and NOx estimations were collected in ice-chilled ethylenediaminetetraacetic acid glass tubes or heparinized plastic tubes, respectively. The samples were centrifuged at 3,000g for 5 minutes at 4°C, and the plasma was collected and stored at -70°C until concentrations of ET-1 or NOx were determined.

Estimation of endothelin-1
Each plasma sample was acidified by adding an equal volume of 20% acetic acid. Endothelin-1 and related peptides were extracted using Sep-columns [6]. The recovery of ET-1 from plasma was approximately 87%. Immunoassay was performed using an immunoassay kit for ET-1 (R and D Systems, Minneapolis, MN). The assay was performed in microtiter plates coated with a rat antibody to human ET-1. Diluted anti–ET-1 horseradish peroxidase conjugate (100 µL; ET-1 conjugated to horseradish peroxidase) was added in each well. Standards (0.25 to 65 pg ET-1), parameter control (24.5 pg/mL ET-1) or sample extract (100 µL, each) were added. The plates were covered with plate sealers and incubated for 1 hour at room temperature. The contents of each well were aspirated and washed using wash buffer provided with the kit. After the last wash, contents of each well were decanted and 100 µL tetramethylbenzidine was added. After 30 minutes, a stop solution (100 µL of 1 mol/L HCl) was added. The optical density of each well was counted within 30 minutes using a microtiter plate reader at 450 and 620 nm separately. A standard curve was created, and the concentration of ET-1 of each sample calculated and expressed as picograms per milliliter of plasma.

Estimation of total nitrate and nitrite
Plasma NOx concentrations were determined by the Greiss reaction [14]. Six microliters of plasma was mixed with 44 µL of distilled H₂O, 20 µL of 0.31 mol/L phosphate buffer (pH 7.5), and 10 µL each of 0.86 mmol/L reduced nicotinamide-adenine dinucleotide phosphate (Sigma, St. Louis, MO), 0.11 mmol/L flavin adenine dinucleotide, and 1.0 U/mL of nitrate reductase. Nitrate (NO₃) was converted to NO₂ by nitrate reductase (Boehringer Mannheim, Indianapolis, IN). Unknown samples were run in duplicate. The samples were allowed to incubate for 1 hour at room temperature in the dark. Two hundred microliters of Greiss reagent (1:1 mixture of 1% sulfanilamide in 5% H₃PO₃ and 0.1% N-(1-naphthyl)ethyl-enediamine) were added to each well, and the plates were incubated for an additional 10 minutes at room temperature. Absorbance was measured at 540 nm using a plate reader and converted to NOx concentration using a nitrate standard curve and calculated as micromolar concentration in plasma. In our laboratory setting, recovery of NOx was observed to be greater than 95%, and the assay results were not found to be affected by the presence of plasma proteins.

Statistical analysis
All values are expressed as mean ± standard error of the mean. After homogeneity of variance was assured, group or time differences were determined using two-way analysis of variance. When the F value indicated significance, differences were isolated using the Student-Neuman-Keuls test. A p value less than or equal to 0.05 was considered significant for all comparisons.

	Results

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Clinical demographics and intraoperative characteristics
Patients in both diabetic and non-diabetic groups possessed similar cardiovascular disease profiles. Preoperative and perioperative characteristics of diabetic and non-diabetic patients are shown in Table 1. There were no significant differences between the two groups with respect to age, left ventricular ejection fraction, Parsonnet score for risk factors, number of vessels bypassed, cross-clamp time, or perioperative therapy.

View larger version (44K): [in this window] [in a new window]   Fig 1. Effect of coronary artery bypass grafting procedure on the concentration of endothelin-1 (ET-1) in the coronary effluent at 1 and 15 minutes after initiation of reperfusion A and B (details of reperfusion A and B are provided in Material and Methods) in non-diabetic (n = 12) and diabetic (n = 9) patients. (*p < 0.05 compared with preoperative values.)

View larger version (45K): [in this window] [in a new window]   Fig 2. Effect of coronary artery bypass grafting procedure on the concentration of nitric oxide byproducts (NOx) in the coronary effluent at 1 and 15 minutes after initiation of reperfusion A and B (details of reperfusion A and B are provided in Material and Methods) in non-diabetic (n = 12) and diabetic (n = 9) patients. (*p < 0.05 compared with preoperative values.)

	Comment

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Other investigators have examined plasma ET-1 and NO concentrations immediately after CABG, but none of these studies specifically examined diabetes as a separate factor. Consistent with our findings in non-diabetic patients, Mair and colleagues [15] did not find any increase in coronary ET-1 after releasing the aortic cross-clamp in patients undergoing CABG. Similarly, others have reported unaltered systemic plasma profiles of ET-1 during cardiac revascularization procedures, including CABG and percutaneous coronary angioplasty procedures [12, 16, 17]. However, diabetes in man, and in animal models of the disease, has been shown to be associated with increased production of endothelin [18, 19]. Coronary endothelial cells from diabetic patients demonstrate increased production of vasoactive mediators including ET-1 and NO [1, 20]. In streptozotocin-treated rats an increase in the levels of ET-1 has been reported [19, 21]. Evidence also indicates that non–insulin-dependent diabetic patients show increased plasma ET-1 levels [18, 22], although some findings to the contrary exist [23]. With these findings in mind, we thought it prudent to examine diabetic patients undergoing CABG separately, regarding ET-1 concentrations on reperfusion. The novel finding from our study is that diabetic patients appear to differ significantly from the non-diabetic population in that there is a significant increase in coronary effluent ET-1 during reperfusion periods after CABG without concomitant increases in NOx concentrations.

It has been reported that systemic ET-1 concentrations can be elevated in diabetic patients and animal models, compared with non-diabetic patients [18, 22]. We found no difference in preoperative systemic concentrations of ET-1 between non-diabetic and diabetic patients. This, however, is consistent with previous reports of the effects of coronary artery disease alone. Similar to our findings, Donatelli and associates [24] found that non-diabetic patients with coronary artery disease had ET-1 concentrations that were not different from diabetic patients with coronary artery disease.

Nitric oxide has been widely accepted as one of the potent vasoactive mediators implicated in cardiovascular and diabetic pathophysiology [1, 20, 25]. Concentrations of NOx were consistently higher in diabetic patients but did not change significantly during the course of the protocol in either group. Importantly, elevated ET-1 levels in diabetic patients occurred during reperfusion without concomitant increases in NO levels, suggesting that NO synthase activity remained unaltered. However, the unchanged levels of NO we observed during reperfusion in CABG are not likely to be related to the influence of pharmacologic interventions used during surgical procedure (eg, anesthesia, nitroglycerin) or the therapeutic regimen that diabetic and non-diabetic patients followed. The therapeutic regimen for all patients, including the anesthetic management, was similar, except for the use of oral hypoglycemic agents in diabetic patients.

We believe our findings have important implications for the diabetic population. In this study there was no cardiovascular mortality observed in either group. However, diabetes is a primary risk factor for cardiovascular disease, and these patients generally demonstrate higher morbidity and mortality after CABG. Thus, understanding how the response in this patient population differs from that of non-diabetic patients is vitally important for designing appropriate therapeutic interventions.

	Acknowledgments

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This work was supported by NIH grants T32 HL07692 and RO1 48219 (W. R. Law and J. L. Ferguson), a supplement to R01 48219 (A. D. Sam), a VA Merit Review Grant (W. R. Law), and a grant from the Living Institute of Surgical Studies (A. D. Sam). Doctors Sam and Nawas were Eleanor B. Pillsbury fellows.

	References

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