IL-10 and TNF-β gene polymorphisms have no major influence on lactate levels after cardiac surgery☆

The utilization of the cardiopulmonary bypass (CPB) during cardiac surgery induces systemic inflammatory response syndrome (SIRS) characterized by alterations in cardiopulmonary functions [1]. This syndrome has many different clinical manifestations ranging from mild organ dysfunction to multiorgan failure with lactic acidosis, systemic vascular collapse, and respiratory distress syndrome. Increased lactate is a common standard marker for the occurrence of anaerobic metabolism due to tissue hypoxia or ischemia. During SIRS, increased lactate levels result from excessive cytokine production [2] despite normal oxygen delivery and carbohydrate metabolism [3]. The lactate generation is modulated by cytokines, e.g. tumor necrosis factor (TNF). TNF-α produced by activated monocytes and TNF-β produced by activated lymphocytes are similar molecules active at TNF receptors [4]. TNF is known to inhibit pyruvate dehydrogenase with the increase in lactate levels [5]. Also, other proinflammatory cytokines (interleukin (IL)-6 and IL-8) and anti-inflammatory cytokines (mainly IL-10, an immunomodulatory cytokine produced by lymphocytes, monocytes, and macrophages) have some effects on the development of SIRS [6,7]. Plasmatic levels of these molecules are modulated also through transcriptional regulation—the genetic polymorphisms in the regulatory parts of the cytokine genes could play an important role in these processes [8,9]. It has been shown that lactic acidosis after cardiac surgery is associated with polymorphism in regulatory parts of IL-10 (−1082G/A) and TNF-β (+252G/A) genes [8,10]. It is possible that lactate levels and the time course of their changes after cardiac surgery in low-risk patients without the left ventricle dysfunction are influenced by these genetic polymorphisms too. We conducted a study to test the hypothesis. During the 7-month period between September 2002 and March 2003, after IRB approval, we recruited 168 consenting adult patients (131 males and 37 females) for prospective observational study. The inclusion criteria were coronary artery disease with angina pectoris grades I–III according to Canadian Cardiovascular Society, age ≤ 70 years, left ventricle ejection fraction ≥45%, and elective coronary artery bypass grafting (CABG). Patients with unstable angina, insulin-dependent diabetes mellitus, history of hepatic disease, and preoperative therapy with steroids or nonsteroidal anti-inflammatory drugs were excluded due to the possible influence of these states on postoperative lactate levels [11]. For the same reason, inotropic support with epinephrine was not used in any case. All operations were performed by consultant grade surgeons through midline sternotomy approach. CPB circuit was primed with 1000 ml of Ringer's solution with 250 ml of 20% mannitol, and non-pulsatile flow of 2.4 l/min/m2 was established using a membrane oxygenator D703 (Dideco, Mirandola, Italy). Cardiac arrest and myocardial protection were obtained by administration of cold crystalloid (St. Thomas) cardioplegia. Patients were cooled to 33 °C applying α-stat acid–base management. Perfusion pressure was maintained between 40 and 70 mmHg. Induction and maintenance of general anesthesia with endotracheal intubation were standardized in all the patients (etomidate, sufentanil, midazolam, pancuronium, isoflurane in oxygen with air). Aprotinin and steroids were not administered in any patient throughout the perioperative period. After the surgery, the patients were transported to the cardiovascular intensive care unit (ICU). The intensive care was also standardized with implemented fast-track protocol and intravenous analgesia with morphine and ketoprofen. Extubation was performed in circulatory stable patients after a period of gradual weaning from mechanical ventilatory support. The control of blood glucose was achieved with continuous insulin infusion as needed to maintain plasmatic glycaemia in the range of 5.5–8.0 mmol/l. In all the patients, the oxygen delivery was well maintained through the surgical procedure and postoperative course. For each patient arterial blood samples were obtained through an arterial catheter. The first blood sample was drawn after the induction of anesthesia for genotyping. The other blood samples were obtained at five different time points. The first sample was taken 10 min after the beginning of CPB, the second 40 min after CPB termination, the third 30 min after arrival in cardiac surgical intensive care unit (ICU), the fourth 8 h after arrival in ICU, and the fifth 16 h after arrival in ICU. Arterial lactic acid level was measured with the analyser ABL615 (Radiometer A/S, Copenhagen, Denmark). Additional perioperative parameters were followed for each patient: preoperative left ventricle ejection fraction (LVEF), duration of CPB, duration of aortic cross-clamping (AXC), the number of aortocoronary bypasses (distal anastomosis), chest tube drainage during the first 12 postoperative hours, total chest tube drainage, the length of mechanical ventilatory support, the length of ICU stay, and 30-day mortality. For comparison, 250 healthy probands representatively selected from the same population (aged 30–65 years) were genotyped. This group worked as the control group for the comparison of genotypic distributions and allelic frequencies. DNA was isolated by standard method. To genotype the polymorphism of the IL-10 gene, oppositely oriented oligonucleotides IL10F 5′ TCT GAA GAA GTC CTG ATG TC and IL10R 5′ CTC TTA CCT ATC CCT ATC TCC; and TNFF 5′ CCC TCC TGC ACC TGC TGC CTG G and TNFR 5′ AGA GGG GTG GAT GCT TGG TTC were used. A 10 μl of PCR product was digested in a total volume of 25 μl with 5 U of restriction enzyme MnlI (IL-10 gene promoter polymorphism −1082G/A) or HinfI (TNF-β intron 1 polymorphism +252G/A) at 37 °C overnight in the buffer provided by the manufacturer (Fermentas GmbH, Germany). The restriction fragments were analyzed by 15% polyacrylamide microtiter array diagonal gel electrophoresis [12], stained with ethidium bromide and visualized on a UV transilluminator. The alleles of TNF-β intron 1 polymorphism +252G/A (G-to-A transition at position +252 in the first intron of the TNF-β gene) are by some authors marked as B1 (A) and B2 (G), e.g. genotype B1B1 is equal to AA in our notation. All the laboratory measurements (genotyping and lactate level analysis) were performed in a blinded manner. Data are expressed as means (±standard error of mean, SEM). Continuous variables were evaluated by means of Student's t-test or nonparametric tests where appropriate. Categorical data were analyzed by χ2-test or Fisher's exact test. The influence of cytokine genotypes on the time course of lactate levels was evaluated by analysis of variance (ANOVA) using a repeated measures design. Results were considered statistically significant at P < 0.05. The genotypic distribution and allelic frequencies of IL-10 and TNF-β genes did not differ between the study and control groups (Table 1). Postoperative course was uneventful in all the patients with zero 30-day mortality. There was no statistically significant association between individual polymorphism in IL-10 (−1082G/A) or TNF-β (+252G/A) genes and lactate levels and their change in time. To further study the relationship between cytokine genetic polymorphism and lactic acid levels, the patients were divided into four groups depending on the combination of IL-10/TNF-β genotypes: GG/+A, GG/GG, +A/+A, and +A/GG. Among these groups there were no statistically significant differences in documented perioperative parameters (P > 0.05), i.e. preoperative LVEF, duration of CPB, duration of aortic cross-clamping, the number of distal anastomosis, chest tube drainage during the first 12 postoperative hours, total chest tube drainage, the length of mechanical ventilatory support, and the length of ICU stay (Table 2). Only 11 of the 168 patients (3.6%) needed inotropic support with dobutamine (the dose ≤4.5 μg/kg/min for ≤5.6 h). Liver markers elevation (total bilirubin, AST, ALT) of more than 1.5 of normal values was not documented in any patient. Genotypic distribution and allelic frequencies of IL-10 and TNF-β genes Perioperative parameters of the study group The statistically significant difference was observed in lactate level changes in time. Data are summarized in Table 3 according to the IL-10/TNF-β genotype. The factors with possible influence on lactate levels were examined in detail. No association was found between the duration of aortic cross-clamping or CPB and lactate levels (P > 0.05). The time course of lactic acid levels was consistent in all the groups – increase till the eighth hour of ICU stay, and then gradual decrease – with two exceptions (Fig. 1). The first exception was decrease in lactate level after 30 min of ICU stay in the patients with IL-10/TNF-β genotype GG/+A, but this change was not statistically significant. The second exception was lactate level decrease after 8 h of ICU stay in the carriers of IL-10/TNF-β genotype +A/GG (P = 0.011); the carriers of other genotype combinations behave differently with increase in lactate levels at 8 h after arrival in ICU. Lactate levels (mmol/l) in different time points according to the IL-10/TNF-β genotype The time course of lactate levels according to different IL-10 and TNF-β genotypes. Despite all the progress in cardiovascular medicine, inflammatory response still remains the important consequence of CPB usage during cardiac surgery, which is associated with subsequent morbidity and mortality [6,7]. It is widely accepted that CPB-induced inflammation has multifactorial origin with genetic predisposition – mainly cytokine genetic polymorphism – being one of the important factors. In clinical practice it is possible to use several strategies to decrease CPB-induced inflammatory response. Some heart centers employ heparin-coated CPB circuits [13]. The pharmacological strategies include application of aprotinin, which is known to decrease blood loss [14] and general inflammation produced by CPB [15], and use of glucocorticoids. Another approach is to perform surgical myocardial revascularization without CPB (off-pump coronary artery bypass) but it is possible in selected subgroups of cardiac surgery patients only. With the ability to preoperatively identify genetic predisposing factors, it would be possible to employ goal-directed approach to markedly reduce CPB-induced inflammatory response in the risk population, and thus improve clinical outcomes in cardiac surgery. The present prospective observational study was designed to investigate whether genetic polymorphism in the regulatory parts of cytokine IL-10 and TNF-β genes (−1082G/A and +252G/A) might influence lactate levels in low-risk cardiac surgery patients without the left ventricle dysfunction. The study was based on the premise that genetic polymorphism in the regulatory parts of inflammatory cytokine genes influences the inflammatory response to CPB [7,8], which could be reflected by different parameters including lactate levels [2]. Other possible causes increasing lactate levels, e.g. diabetes or epinephrine infusion, were eliminated. It has been shown that both studied gene polymorphisms have no measurable influence on the occurrence of coronary artery disease or myocardial infarction in angiographically evaluated patients [16]. The key finding of the present study was that patients with IL-10/TNF-β genotype combination +A/GG had significantly different change of lactate levels in time after cardiac surgery with the decrease in lactate level after 8 h of ICU stay. In the same time, the patients with other genotype combinations experienced the increase in lactate level. This different time course of changes in lactic acid levels was not accompanied by any statistically significant differences in the followed perioperative clinical parameters. To our knowledge, there are only two similar studies which examined the influence of IL-10 and TNF-β genetic polymorphisms on the systematic inflammatory response in cardiac surgery. The similar study of Ryan et al. [10] was the first to analyze the association between lactate levels and the presence of cytokine genetic polymorphism in cardiac surgery patients. In comparison with the present study, Ryan et al. used case–control design with study group composed of patients who experienced lactic acidosis (lactate levels > 4 mmol/l) during the first 24 h after cardiac surgery with CPB. Total number of patients in the study by Ryan et al. was smaller (51 vs 168), but the number of patients in the study and control groups (21 and 30 patients) was comparable to the sample size of our study (31, 23, 59, and 55 patients, respectively). The allelic frequencies in our patients were in accordance with the Ryan's study. The CPB time between the groups in the Ryan's investigation was significantly different. The study results showed that carriers of TNF-β (+252G/A) genotype AA and IL-10 (−1082G/A) genotype GA or AA (i.e., +A) were prone to have increased lactic acid levels during the postoperative course with the maximum recorded difference of 5.22 mmol/l between the groups. We did not find any significant lactate level difference among different genotype groups. The other study performed by Tomasdottir et al. [8] investigated the relationship between TNF-β gene polymorphism (+252G/A) and the release of proinflammatory cytokines (TNF-α and IL-6) and postoperative cardiopulmonary morbidity. In comparison with the present study, the authors included coronary artery bypass grafting and valve surgery patients together; preoperative LVEF was not noted. They concluded that patients homozygous for the TNF-β B2 allele (i.e., TNF-β genotype GG) were at risk for development of enhanced SIRS, and had also increased morbidity in the terms of left ventricular and postoperative pulmonary dysfunctions with the tendency for longer stay in ICU. In our study, the difference in any followed postoperative parameters including the length of ICU stay among different genotypes was not found. Our study has potential pitfalls. First, the lactate levels were not overly high. This was probably due to inclusion of low-risk patients only and relatively short CPB in comparison with another cardiac surgical procedures. Second, the differences among recorded lactate levels were relatively low. The likely reason is truly homogenous study population. These pitfalls were in our opinion overcome by the number of patients included (168 vs 95 and 51 in before mentioned studies), and by using the combination of two genes polymorphism (of proinflammatory and anti-inflammatory cytokines) for analysis. The postoperative course of all the patients was uneventful thus minimizing other proinflammatory factors which could play a role in the increase of lactic acid. It is not possible to determine from this study whether other inflammatory cytokine gene polymorphisms or their combinations may exert effects on lactate levels after cardiac surgery. Our findings imply that IL-10 and TNF-β gene polymorphisms have a little, yet measurable influence on the time course of changes in lactate levels after cardiac surgery, and that IL-10/TNF-β genotype combination +A/GG seems to be protective against CPB-induced inflammation in the means of lower lactate levels in the patients without the left ventricle dysfunction. This work was supported by the Ministry of Health of the Czech Republic within the research programme MZO 00023001 ‘Research on cardiovascular diseases, diabetes mellitus and transplantation of vital organs’. The authors gratefully acknowledge the assistance of Dr Vera Lanska with statistical portion of the study.

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