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Heart, Lung and Circulation

Relationships Between Systemic Inflammation, Intestinal Damage and Postoperative Organ Dysfunction in Adults Undergoing Low-Risk Cardiac Surgery

Open AccessPublished:January 06, 2023DOI:https://doi.org/10.1016/j.hlc.2022.12.006

      Background

      Approximately half of patients who undergo cardiac surgery develop systemic inflammatory response syndrome. Extracorporeal circulation and intestinal injury may play a role in this inflammatory response, although their relative contributions remain elusive. Moreover, it is largely unknown to what extent these factors contribute to cardiac surgery-induced postoperative organ dysfunction.

      Method

      In this secondary analysis, we measured circulating levels of the intestinal damage marker intestinal fatty acid binding protein (I-FABP) and of the inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, IL-10, IL-1RA, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and MIP-1β in 180 patients undergoing on-pump cardiac surgery. The average Z-score of levels of the different cytokines was used as an integral measure of the cytokine response. Relationships between duration of extracorporeal circulation, extent of intestinal injury, inflammation, and postoperative organ dysfunction were explored.

      Results

      Plasma I-FABP levels increased during surgery, with peak levels observed at the end of cardiopulmonary bypass (CPB). Except for TNF-α, the levels of all cytokines increased during surgery, with peak levels observed either 2 (MCP-1, MIP-1α, and MIP-1β), 4 (IL-6, IL-8, and IL-1RA) or 6 (IL-10) hours after the end of CPB. While the duration of CPB significantly correlated with cytokine Z-score (r=0.544, p<0.05), no relationship with I-FABP levels was found. Furthermore, no significant correlations between I-FABP and cytokine levels were observed. The duration of CPB correlated with a deterioration in postoperative kidney function (estimated glomerular filtration rate [eGFR]) and troponin levels. Cytokine Z-score was associated with postoperative troponin levels, fluid administration, inotropic score, pulmonary alveolar–arterial gradient on the first postoperative morning, and deterioration of kidney function (eGFR). I-FABP levels did not correlate with any of the cardiovascular, pulmonary, or renal parameters.

      Conclusions

      In patients undergoing low-risk cardiac surgery, the duration of CPB represents an important determinant of the systemic cytokine response, whereas both the CPB duration and the systemic inflammatory response contribute to subsequent organ dysfunction. Intestinal damage does not appear to play a relevant role in the postoperative inflammatory response and development of postoperative organ dysfunction in these patients.

      Keywords

      Introduction

      Following cardiac surgery, approximately half of patients [
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      Prevalence and clinical impact of systemic inflammatory reaction after cardiac surgery.
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      ] develop systemic inflammatory response syndrome (SIRS). This inflammatory response, which is reflected by increased levels of inflammatory mediators [
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      ], is associated with the degree of postoperative distributive shock [
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      Cardiac vasoplegia syndrome: pathophysiology, risk factors and treatment.
      ] and organ dysfunction [
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      • Redmond J.M.
      Understanding the inflammatory response to cardiac surgery.
      ]. For example, the proinflammatory cytokines interleukin (IL)-6, IL-8, and tumour necrosis factor (TNF)-α have negative inotropic effects [
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      Relationship of the proinflammatory cytokines to myocardial ischemia and dysfunction after uncomplicated coronary revascularization.
      ,
      • te Velthuis H.
      • Jansen P.G.
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      • Sturk A.
      • Eijsman L.
      • Wildevuur C.R.
      Myocardial performance in elderly patients after cardiopulmonary bypass is suppressed by tumor necrosis factor.
      ], and the extent of the systemic inflammatory response is related to the development of postoperative acute kidney injury (AKI) [
      • Ball L.
      • Costantino F.
      • Pelosi P.
      Postoperative complications of patients undergoing cardiac surgery.
      ,
      • Ortega-Loubon C.
      • Fernandez-Molina M.
      • Carrascal-Hinojal Y.
      • Fulquet-Carreras E.
      Cardiac surgery-associated acute kidney injury.
      ]. Several mechanisms may contribute to systemic inflammation following cardiac surgery. Firstly, exposure of blood to the extracorporeal circulation circuit activates leukocytes and endothelial cells [
      • Edmunds Jr., L.H.
      Inflammatory response to cardiopulmonary bypass.
      ], resulting in increased production of proinflammatory cytokines [
      • Paparella D.
      • Yau T.M.
      • Young E.
      Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update.
      ]. Secondly, release of the aortic cross-clamp induces ischaemia–reperfusion injury through increased production of free oxygen radicals. In turn, this also results in the generation of inflammatory cytokines and leukocyte activation [
      • Wei M.
      • Kuukasjarvi P.
      • Laurikka J.
      • Pehkonen E.
      • Kaukinen S.
      • Laine S.
      • et al.
      Cytokine responses in patients undergoing coronary artery bypass surgery after ischemic preconditioning.
      ]. Of special interest is the gut, as barrier dysfunction in the gut may represent both a cause and a consequence of the inflammatory response. Normal gut barrier function is maintained by a single layer of epithelial cells, mainly enterocytes, that cover the villi of the small bowel mucosa, interconnected by large proteins called tight junctions [
      • Otani S.
      • Coopersmith C.M.
      Gut integrity in critical illness.
      ]. Attenuated mesenteric blood flow results in loss of enterocyte integrity and breakdown of tight junctions [
      • Thuijls G.
      • Derikx J.P.
      • de Haan J.J.
      • Grootjans J.
      • de Bruine A.
      • Masclee A.A.
      • et al.
      Urine-based detection of intestinal tight junction loss.
      ]. The subsequent gut barrier function loss and translocation of bacteria and/or bacterial products from the intestinal lumen have been suggested as central events in the development of organ failure in critically ill patients, as these events may amplify and perpetuate the systemic inflammatory response [
      • Mittal R.
      • Coopersmith C.M.
      Redefining the gut as the motor of critical illness.
      ,
      • Derikx J.P.
      • Poeze M.
      • van Bijnen A.A.
      • Buurman W.A.
      • Heineman E.
      Evidence for intestinal and liver epithelial cell injury in the early phase of sepsis.
      ]. Cardiac surgery also leads to a significant decrease in mesenteric blood flow as a result of extracorporeal circulation [
      • Straub U.
      • Winning J.
      • Greilach P.
      • Isringhaus H.
      • Kalweit G.
      • Huwer H.
      Alterations of mesenteric blood flow after cardiopulmonary bypass: a Doppler sonographic study.
      ] and permissive hypotension. Various studies have illustrated the occurrence of mucosal injury during cardiac surgery, reflected by elevated levels of intestinal fatty acid binding protein (I-FABP), a small protein present in enterocytes that is released into the circulation upon enterocyte injury [
      • Camkiran A.
      • Donmez A.
      • Aldemir D.
      • Isguzar R.A.
      • Gultekin B.
      Clinical significance of intestinal type fatty acid binding protein in patients undergoing coronary artery bypass surgery.
      ,
      • Holmes JHt
      • Lieberman J.M.
      • Probert C.B.
      • Marks W.H.
      • Hill M.E.
      • Paull D.L.
      • et al.
      Elevated intestinal fatty acid binding protein and gastrointestinal complications following cardiopulmonary bypass: a preliminary analysis.
      ]; it is cleared renally, with a half-life of 11 minutes [
      • Evennett N.J.
      • Petrov M.S.
      • Mittal A.
      • Windsor J.A.
      Systematic review and pooled estimates for the diagnostic accuracy of serological markers for intestinal ischemia.
      ]. Furthermore, intraoperative mesenteral hypoperfusion with subsequent gut barrier function loss can result in translocation of bacterial endotoxin from the gastrointestinal tract into the circulation [
      • Adamik B.
      • Kubler A.
      • Gozdzik A.
      • Gozdzik W.
      Prolonged cardiopulmonary bypass is a risk factor for intestinal ischaemic damage and endotoxaemia.
      ], in turn triggering a systemic immune response [
      • Jansen N.J.
      • van Oeveren W.
      • Gu Y.J.
      • van Vliet M.H.
      • Eijsman L.
      • Wildevuur C.R.
      Endotoxin release and tumor necrosis factor formation during cardiopulmonary bypass.
      ]. However, no evident relationship between the degree of gut permeability and endotoxemia has previously been found [
      • Riddington D.W.
      • Venkatesh B.
      • Boivin C.M.
      • Bonser R.S.
      • Elliott T.S.
      • Marshall T.
      • et al.
      Intestinal permeability, gastric intramucosal pH, and systemic endotoxemia in patients undergoing cardiopulmonary bypass.
      ]. Moreover, no relationships between plasma I-FABP levels and levels of TNF-α, IL-6, IL-8, or IL-10 were found in cardiac surgery patients [
      • Habes Q.L.M.
      • Linssen V.
      • Nooijen S.
      • Kiers D.
      • Gerretsen J.
      • Pickkers P.
      • et al.
      Markers of intestinal damage and their relation to cytokine levels in cardiac surgery patients.
      ]. These findings suggest that intestinal injury appears not to be a causative factor for the postoperative inflammatory response. Additionally, to what extent enterocyte damage is related to clinical postoperative parameters remains unclear, as some [
      • Holmes JHt
      • Lieberman J.M.
      • Probert C.B.
      • Marks W.H.
      • Hill M.E.
      • Paull D.L.
      • et al.
      Elevated intestinal fatty acid binding protein and gastrointestinal complications following cardiopulmonary bypass: a preliminary analysis.
      ,
      • Zou L.
      • Song X.
      • Hong L.
      • Shen X.
      • Sun J.
      • Zhang C.
      • et al.
      Intestinal fatty acid-binding protein as a predictor of prognosis in postoperative cardiac surgery patients.
      ] but not all [
      • Habes Q.L.M.
      • Linssen V.
      • Nooijen S.
      • Kiers D.
      • Gerretsen J.
      • Pickkers P.
      • et al.
      Markers of intestinal damage and their relation to cytokine levels in cardiac surgery patients.
      ] studies show an association with postoperative organ dysfunction and/or (gastrointestinal) complications. Therefore, the question remains whether or not intestinal damage plays a relevant role in the postoperative inflammatory response and development of postoperative organ dysfunction. In the present study, we further investigated the relationships between the duration of extracorporeal circulation, extent of intestinal injury, inflammatory response, and cardiac surgery-induced postoperative organ dysfunction.

      Materials and Methods

      Patients

      Data were obtained from a total of 180 patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) who participated in a randomised controlled trial [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ,
      • van Groenendael R.
      • Beunders R.
      • Hofland J.
      • Morshuis W.J.
      • Kox M.
      • van Eijk L.T.
      • et al.
      The safety, tolerability, and effects on the systemic inflammatory response and renal function of the human chorionic gonadotropin hormone-derivative EA-230 following on-pump cardiac surgery (the EASI study): protocol for a randomized, double-blind, placebo-controlled phase 2 study.
      ]. In this trial, both male and female adult patients scheduled for elective coronary artery bypass grafting (CABG), with or without valve surgery, with use of CPB were randomised to receive either the drug EA-230 or placebo. For safety reasons, the study was conducted in two phases. After the inclusion of 60 low-risk patients in the first phase, an independent interim safety analysis by the Data Safety Management Board was performed. In the second phase, both low- and high-risk patients were included [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ]. Inclusion criteria are provided in the online supplementary material of the previously published study [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ]. All patients provided written informed consent before the start of any study-related procedures. The study was approved by the local ethics committee (CMO Arnhem-Nijmegen, registration number 2015-005600-28) and conducted in accordance with the Declaration of Helsinki and in compliance with the International Conference on Harmonization E6 Guideline for Good Clinical Practice (CPMP/ICH/135/95), the Dutch Medical Research Involving Human Subjects Act, and the European Directive (2001/20/CE). The trial was registered at ClinicalTrials.gov (NCT03145220).

      Surgical Procedure

      Detailed descriptions of surgical procedures are provided elsewhere [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ,
      • van Groenendael R.
      • Beunders R.
      • Hofland J.
      • Morshuis W.J.
      • Kox M.
      • van Eijk L.T.
      • et al.
      The safety, tolerability, and effects on the systemic inflammatory response and renal function of the human chorionic gonadotropin hormone-derivative EA-230 following on-pump cardiac surgery (the EASI study): protocol for a randomized, double-blind, placebo-controlled phase 2 study.
      ]. Briefly, all patients underwent elective CABG, with or without valve surgery. Surgery was carried out using a standardised protocol, which included median sternotomy and cross-clamping of the aorta to facilitate CPB in all patients. Either crystalloid or blood-based cardioplegia fluids were used. For anaesthetic management, midazolam and sufentanil were used for induction, followed by maintenance with continuous sufentanil and sevoflurane administration. Blood pressure was continuously monitored using a 20 G arterial catheter. Rocuronium was used as muscle-relaxing agent; no corticosteroids were administered. Postoperatively, patients were admitted to the cardiothoracic intensive care unit (CICU).

      Sample Collection

      At various time points (Figure 1), blood was drawn from the arterial line, and urine samples were collected from the urinary catheter. Ethylenediaminetetraacetic acid (EDTA)- and lithium heparin-containing vacutainers were used for blood sampling, which were centrifuged within 60 minutes of collection (2000 g for 10 minutes at 4°C), after which plasma was stored at –80°C until analysis.
      Figure thumbnail gr1
      Figure 1Overview of sampling time points: 1: day before surgery; 2: preoperative 3: start cardiopulmonary bypass (CPB); 4: stop CPB; 5: 2 hours after CPB; 6: 4 hours after CPB; 7: 6 hours after CPB; 8: first postoperative day. Plasma samples were obtained at every time point (red drops). Yellow drops indicate time-points where urine was also collected.

      Data Collection

      Baseline characteristics (age, sex, weight, length, body mass index, preoperative plasma creatinine, and type of surgery) were collected from the electronic health records (Epic Hyperspace). Duration of CBP, clamping time, and surgery were recorded in case report forms. Estimated glomerular filtration rate (eGFR) was calculated on the day before surgery and day 1 postoperatively. The eGFR was estimated using the Modification of Diet in Renal Disease (MDRD) equation and standardised plasma creatinine values. Heart rate (electrocardiography), blood pressure (intra-arterial cannula), volume of administered fluids, urine output, and administration of inotropic and vasoactive medications were monitored intraoperatively and in CICU. Furthermore, during CICU admission arterial blood gas analysis was performed at least once daily, the fraction of inspired oxygen was monitored during mechanical ventilation, drain production was observed, and Sequential Organ Failure Assessment (SOFA) score was calculated. Furthermore, the highest postoperative value of troponin and pulmonary alveolar–arterial [A-a] gradient on the first postoperative morning were obtained. Routine laboratory parameters were determined in the hospital’s central clinical chemical laboratory.

      Analysis of Intestinal Fatty Acid Binding Protein and Inflammatory Cytokines

      For analysis of I-FABP in EDTA plasma, enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA) were used. Analysis of IL-6, IL-8, IL-10, and TNF-α was performed at the certified laboratory of University Medical Center Utrecht (Utrecht, the Netherlands). Plasma concentrations of cytokines were measured in EDTA plasma using an ISO9001-certified multiplex immunoassay based on Luminex technology (xMAP; Luminex Austin, TX, USA). The assay was performed as described previously [
      • Scholman R.C.
      • Giovannone B.
      • Hiddingh S.
      • Meerding J.M.
      • Malvar Fernandez B.
      • van Dijk M.E.A.
      • et al.
      Effect of anticoagulants on 162 circulating immune related proteins in healthy subjects.
      ]. The cytokine IL-1RA and the chemokines monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and MIP-1β were measured in EDTA plasma with a Milliplex HCYTOMAG-60k assay (Merck Chemicals, Amsterdam, the Netherlands) according to the manufacturer’s protocol.

      Treatment Effect of EA-230

      EA-230 is a peptide similar to the human chorionic gonadotropin produced in pregnant women, which is believed to be responsible for the immunotolerance seen during pregnancy. Because data were obtained from the intervention study in which patients were randomised to receive either treatment with EA-230 or placebo, we determined whether EA-230 affected I-FABP levels, cytokine levels, and the extent of organ dysfunction. No differences in I-FABP levels were found between the treatment and placebo groups (data not shown). Furthermore, as described previously [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ], no effects of EA-230 on any of the circulating inflammatory mediators, vasopressor and/or inotropic therapy requirement, and A-a O2 gradient (change from CICU admission to postoperative morning) were observed [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ]. However, a statistically significant difference between the treatment and placebo groups was found in eGFR-MDRD from baseline until the first postoperative day [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ]. Therefore, we added “treatment” as a covariate in the multiple linear regression analysis of eGFR-MDRD.

      Data Analysis

      Continuous data are presented as median (interquartile range [IQR]) or mean ± standard error of the mean (SEM), according to their distribution (determined using the Kolmogorov–Smirnov normality test). Dichotomous data are presented as number (percentage). Continuous variables were analysed using Kruskal–Wallis tests followed by Dunn’s post-hoc tests or paired t-tests, as appropriate. As cytokine and I-FABP levels show a monophasic pattern (i.e., an increase followed by a return to baseline levels), we calculated the area under curve (AUC) levels of I-FABP and cytokines [
      • Matthews J.N.
      • Altman D.G.
      • Campbell M.J.
      • Royston P.
      Analysis of serial measurements in medical research.
      ]. Samples were drawn at fixed time points (start CPB, stop CPB, +2 hours, +4 hours, etc., after the end of CPB) for several reasons: as the variation in duration of CPB is small and AUC levels cytokines are mostly defined by the levels after CPB is stopped, the effect of considering the duration of CPB for the AUC calculation would not have a relevant impact. Secondly, if the AUC calculation was weighted for each patient’s exposure time to CPB and variation in CPB would be substantial, this would likely have resulted in an association between the duration of CPB and AUC levels of I-FABP and cytokines, as time on CPB becomes a major driver of the AUC. By using fixed time points, any association with duration of CPB would not be the result of a higher AUC, but would show that duration is intrinsically a driver of the inflammatory response. Furthermore, we made the decision that if an in-between value was missing we would interpolate the value. However, there were no missing values in this study.
      The 2-hourly doses of inotropic and vasoactive medications were recorded for the first 24 hours after postoperative admission to the CICU: dopamine, dobutamine, epinephrine, norepinephrine, milrinone, and vasopressin. To quantify the amount of cardiovascular support, we calculated the CICU inotropic score [
      • Gaies M.G.
      • Gurney J.G.
      • Yen A.H.
      • Napoli M.L.
      • Gajarski R.J.
      • Ohye R.G.
      • et al.
      Vasoactive-inotropic score as a predictor of morbidity and mortality in infants after cardiopulmonary bypass.
      ,
      • Wernovsky G.
      • Wypij D.
      • Jonas R.A.
      • Mayer Jr., J.E.
      • Hanley F.L.
      • Hickey P.R.
      • et al.
      Postoperative course and hemodynamic profile after the arterial switch operation in neonates and infants. A comparison of low-flow cardiopulmonary bypass and circulatory arrest.
      ]. This score has been used in clinical research as a measure of illness severity in various patient groups [
      • Gaies M.G.
      • Gurney J.G.
      • Yen A.H.
      • Napoli M.L.
      • Gajarski R.J.
      • Ohye R.G.
      • et al.
      Vasoactive-inotropic score as a predictor of morbidity and mortality in infants after cardiopulmonary bypass.
      ,
      • Cruz D.N.
      • Antonelli M.
      • Fumagalli R.
      • Foltran F.
      • Brienza N.
      • Donati A.
      • et al.
      Early use of polymyxin B hemoperfusion in abdominal septic shock: the EUPHAS randomized controlled trial.
      ].
      Pearson correlation analysis on log-transformed data was used for bivariate correlation analyses. For each of the six cytokines that significantly correlated with each other (TNF-α, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β), cytokine standard scores (Z-scores) were calculated and the average Z-score of all these related cytokines was used as a composite measure of systemic inflammation. Multiple univariate linear regression analyses were performed to evaluate associations between CPB duration, intestinal damage, and the inflammatory response (independent variables) and postoperative organ dysfunction (dependent variables: change in GFR from baseline to the first postoperative day; highest postoperative troponin level; amount of fluid administration; inotropic score; and A-a gradient at the first postoperative morning). For this analysis, data were log-transformed in case of non-normally distributed data. Statistical analyses were preformed using GraphPad Prism 5.03 (GraphPad Software, La Jolla, CA, USA), IBM SPSS statistics 25 (IBM, Armonk, NY, USA), and R 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria). GraphPad Prism, R, and BioRender were used for visualisation.

      Results

      Patient Characteristics

      One hundred and eighty (180) patients were included, with one secondary exclusion after the surgeon decided to perform an off-pump procedure after sternotomy was performed. Baseline demographics and perioperative patient data are provided in Table 1. Patients included in this study had a relatively short duration of CPB and low EuroSCORE II, and a relatively small percentage of patients underwent valve surgery (Table 1).
      Table 1Baseline and perioperative characteristics of 179 patients who underwent cardiac surgery.
      Variablen=179
      Age (yr)68 (61–74)
      Sex
       Male157 (87.7)
       Female22 (12.3)
      Weight (kg)86±14.2
      Height (cm)176±8.2
      BMI (kg/m2)28±3.8
      Duration of CPB (min)84 (70–107)
      Duration of aortic clamping (min)50 (41–68)
      Kidney function
       GFR with the MDRD equation (mL/min/1.73 m2)79 (66–90)
       Plasma creatinine (μmol/L)82 (73–95)
      Subgroup stratification
      Valve surgery
       Yes28 (15.6)
       No151 (84.3)
      Preoperative eGFR (mL/min/1.73 m2)
       ≤304 (2.2)
       31–90127 (70.9)
       >9048 (26.8)
      EuroSCORE II
       ≥49 (5.0)
       <4170 (95.0)
      Postoperative urine output (mL)
       0–6 hr718 (555–949)
       6–12 hr408 (328–620)
       12–18 hr310 (245–471)
       18–24 hr255 (194–341)
      SOFA score
       Baseline0 (0–0)
       First postoperative day4 (3–5)
       Second postoperative day3 (1–4)
      Data are presented as n (%), median (interquartile range), or mean ± standard error of the mean.
      Abbreviations: BMI, body mass index; CPB, cardiopulmonary bypass; GFR, glomerular filtration rate; MDRD: Modification of Diet in Renal Disease; eGFR, estimated GRF, SOFA, Sequential Organ Failure Assessment.

      Intestinal Fatty Acid Binding Protein, Cytokines, and Chemokines

      Plasma I-FABP levels increased during surgery, with peak levels observed at the end of CPB (Figure 2). Six hours after the end of CPB, I-FABP returned to baseline levels. Levels of IL-6, IL-8, IL-10 (Figure 2), and IL-1RA (IL-1R; Supplementary Figure 1) increased intraoperatively, with peak levels observed between 4 hours (IL-6, IL-8, and IL-1RA) and 6 hours after the end of CBP (IL-10). No increase in TNF-α was observed (data not shown). Levels of the MCP-1, MIP-1α, and MIP-1β also increased during surgery, peaking 2 hours after the end of CPB (Supplementary Figure 1). Median AUC values of the cytokines, chemokines, and I-FABP are shown in Supplementary Table 1. Except for IL-1RA and IL-10, all cytokines were significantly correlated to each other (Figure 3).
      Figure thumbnail gr2
      Figure 2Levels of intestinal fatty acid binding protein (I-FABP) and cytokine in patients undergoing cardiac surgery. (A) Plasma I-FABP levels. (B) Plasma interleukin (IL)-6 levels. (C) Plasma IL-8 levels. (D) Plasma IL-10 levels. Data are presented as median and interquartile range. P value was calculated using the Kruskal–Wallis test. ∗p<0.05 (Dunn’s post-hoc test).
      Abbreviations: OR, operating room; CPB, cardiopulmonary bypass; POD, postoperative day.
      Figure thumbnail gr3
      Figure 3Correlations between area under the curve (AUC) plasma cytokine levels. Pearson correlation analysis on log-transformed AUC values was used to determine r values. All r values displayed in a red circle are significant with a p value <0.05. The size of the circles corresponds with the degree of significance, with larger circles indicating lower p-values.
      Abbreviations: IL, interleukin; MCP, monocyte chemoattractant protein; MIP, monocyte inflammatory protein.

      Cardiopulmonary Bypass

      To investigate the relationship of CBP duration and the observed increase in I-FABP and cytokine concentrations, the association between CBP duration, AUC I-FABP plasma concentrations, and AUC plasma cytokine levels, as well as the AUC cytokine Z-score (combination of all correlated cytokines), were determined. While the duration of CPB significantly correlated with AUC IL-6, IL-8, IL-10, IL-1RA, MCP-1, MIP-α, and MIP-1β levels (r=0.517, p<0.05; r=0.317, p<0.05; r=0.499, p<0.05; r=0.211, p<0.05; r=0.365, p<0.05; r=0.375, p<0.05; and r=0.273, p<0.05, respectively) and the cytokine Z-score (r=0.544, p<0.05), no relationship between duration of CBP and AUC I-FABP levels was found (Figure 4). Furthermore, no significant correlations between AUC I-FABP and AUC cytokine levels or Z-score were found.
      Figure thumbnail gr4
      Figure 4Correlation of duration of cardiopulmonary bypass (CBP) and intestinal fatty acid binding protein (I-FABP) and cytokine levels. Pearson correlation analysis on log-transformed data was used.
      Abbreviations: IL, interleukin; AUC, area under the curve; MCP, monocyte chemoattractant protein; MIP, monocyte inflammatory protein.

      Organ Dysfunction

      A significant increase in eGFR-MDRD from baseline until the first postoperative day was observed for the total patient group (from 78.5 ± 1.5 to 82.7 ± 1.8 mL/min/1.73 m2; p<0.0001). Nevertheless, 71 patients (40%) displayed a postoperative decrease in eGFR-MDRD (Supplementary Figure 2A). Previously, a statistically significant difference between patients receiving treatment with EA-230 and patients receiving placebo was found in eGFR-MDRD from baseline until the first postoperative day (increase in eGFR-MDRD of 6 ± 1 vs 2 ± 1 mL/min/1.73 m2; p=0.01) [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ], so treatment was added as an additional independent variable in the regression analyses, with eGFR-MDRD as a dependent variable. Overall, patients received a median of 3.4 L (IQR 2.9–4.0 L) crystalloids in the first 24 hours after CICU admission (Supplementary Figure 2B). Fifty patients (28%) did not require any vasopressor and/or inotropic therapy (Supplementary Figure 2C). Postoperative troponin level increased to 292 ng/L (IQR 194–534 ng/L; Supplementary Figure 2D). Pulmonary A-a gradient was 9.2 kPa (IQR 4.3–14.9 kPa) on the first postoperative morning (Supplementary Figure 2E).
      Duration of CPB correlated with the decrease in eGFR-MDRD from baseline until the first postoperative day and with the highest postoperative troponin level but not with the other cardiovascular or pulmonary parameters (Figure 5). The cytokine Z-score was associated with the highest postoperative troponin level, the amount of fluids administered 24 hours after CICU admission, the inotropic score, the pulmonary A-a at the first postoperative morning, and with the decrease in eGFR-MDRD from baseline until the first postoperative day (Figure 5). I-FABP levels did not correlate with any of the cardiovascular, pulmonary, or renal parameters.
      Figure thumbnail gr5
      Figure 5Schematic of the contribution of cardiopulmonary bypass (CBP) duration, the inflammatory response, and intestinal damage to clinical sequelae following cardiac surgery. Lines reflect statistically significant associations, whereas the dotted lines represent tested associations that were not statistically significant. R values and corresponding p values were calculated using Pearson correlation on log-transformed data. β-values and corresponding p values were calculated using multiple univariate linear regression analysis on log-transformed data. Overall, CPB duration is directly related to the extent of the inflammatory response and decrease in estimated glomerular filtration rate and increase in troponin. The inflammatory response by itself was related to the increase in troponin, inotropic score, amount of fluid needed, and pulmonary alveolar–arterial (A-a) gradient on the first postoperative morning. Duration of CPB was not related to intestinal fatty acid binding protein (I-FABP) concentration, and I-FABP was not associated with the inflammatory response or any organ dysfunction.

      Discussion

      The present study confirmed that I-FABP, as a measure of enterocyte injury, increases during surgery. However, it also demonstrates that this increase in I-FABP does not relate to the subsequent inflammatory response and development of organ dysfunction. In contrast, duration of CPB is associated with the inflammatory response, cardiac damage (troponin level), and deterioration of renal function (eGFR), while increased cytokine levels by themselves are independently associated with postoperative troponin levels, the amount of fluids administered, inotropic score, the pulmonary A-a gradient on the first postoperative morning, and deterioration of renal function. These findings suggest that CBP duration and the subsequent inflammatory response are the main driving factors for the development of postoperative organ dysfunction. While I-FABP levels increased in these patients, the lack of an association with the inflammatory response, as well as the development of organ dysfunction, argue against a relevant role of intestinal damage (as determined by I-FABP in this study) in this low-risk cohort.
      The increase in I-FABP and cytokine levels during and following cardiac surgery observed in the present study is in line with previous work [
      • Holmes JHt
      • Lieberman J.M.
      • Probert C.B.
      • Marks W.H.
      • Hill M.E.
      • Paull D.L.
      • et al.
      Elevated intestinal fatty acid binding protein and gastrointestinal complications following cardiopulmonary bypass: a preliminary analysis.
      ,
      • Adamik B.
      • Kubler A.
      • Gozdzik A.
      • Gozdzik W.
      Prolonged cardiopulmonary bypass is a risk factor for intestinal ischaemic damage and endotoxaemia.
      ,
      • Habes Q.L.M.
      • Linssen V.
      • Nooijen S.
      • Kiers D.
      • Gerretsen J.
      • Pickkers P.
      • et al.
      Markers of intestinal damage and their relation to cytokine levels in cardiac surgery patients.
      ], and illustrates that cardiac surgery results in intestinal injury and elicits an inflammatory response. Based on the kinetics of these two processes, first an increase in I-FABP and later increases in cytokine concentrations, it was assumed that intestinal injury played an important role in the initiation and perpetuation of the inflammatory response in cardiac surgery patients. In a small study performed in 22 patients, the extent of intestinal injury was shown to correlate with systemic inflammation after thoracic or thoracoabdominal aortic aneurysm repair [
      • Hanssen S.J.
      • Derikx J.P.
      • Vermeulen Windsant I.C.
      • Heijmans J.H.
      • Koeppel T.A.
      • Schurink G.W.
      • et al.
      Visceral injury and systemic inflammation in patients undergoing extracorporeal circulation during aortic surgery.
      ], but this was not confirmed in another study in 72 cardiac surgery patients [
      • Habes Q.L.M.
      • Linssen V.
      • Nooijen S.
      • Kiers D.
      • Gerretsen J.
      • Pickkers P.
      • et al.
      Markers of intestinal damage and their relation to cytokine levels in cardiac surgery patients.
      ]. In another study conducted in 34 patients undergoing cardiac surgery, CPB time correlated positively with I-FABP concentrations measured 24 hours after surgery and with circulating endotoxin levels [
      • Adamik B.
      • Kubler A.
      • Gozdzik A.
      • Gozdzik W.
      Prolonged cardiopulmonary bypass is a risk factor for intestinal ischaemic damage and endotoxaemia.
      ]. However, no correlation between CPB duration and the inflammatory response was found, likely related to the variability in the cytokine concentrations and limited statistical power because of the relatively small group size. We used AUC cytokine and I-FABP levels for our correlation analyses and identified a relationship between the duration of CPB and inflammation in a more than five times larger group of patients. However, we found no correlation between I-FABP levels and inflammation or organ dysfunction. This strongly suggests that intestinal damage is not the driver of inflammation and does not result in organ dysfunction in these patients. Our finding that enterocyte damage appears not to be a causative factor for the cardiac surgery-induced cytokine response may be explained by earlier work showing the lack of an association between splanchnic hypoperfusion and either endotoxemia or intestinal permeability during cardiac surgery with CPB [
      • Riddington D.W.
      • Venkatesh B.
      • Boivin C.M.
      • Bonser R.S.
      • Elliott T.S.
      • Marshall T.
      • et al.
      Intestinal permeability, gastric intramucosal pH, and systemic endotoxemia in patients undergoing cardiopulmonary bypass.
      ]. This may also explain why selective gut decontamination resulted in an extensive decrease in Gram-negative bacteria in the rectal swabs of cardiac surgery patients but did not affect the occurrence of perioperative endotoxemia or postoperative circulating cytokine concentrations, and failed to demonstrate clinically beneficial effects [
      • Bouter H.
      • Schippers E.F.
      • Luelmo S.A.
      • Versteegh M.I.
      • Ros P.
      • Guiot H.F.
      • et al.
      No effect of preoperative selective gut decontamination on endotoxemia and cytokine activation during cardiopulmonary bypass: a randomized, placebo-controlled study.
      ].
      Postoperative cardiovascular, pulmonary, and renal complications after cardiac surgery have a high incidence and are associated with longer-term adverse clinical outcomes [
      • Ball L.
      • Costantino F.
      • Pelosi P.
      Postoperative complications of patients undergoing cardiac surgery.
      ]. Aorta cross-clamping and extracorporeal circulation are thought to exert detrimental effects on the myocardium and the kidneys [
      • Ball L.
      • Costantino F.
      • Pelosi P.
      Postoperative complications of patients undergoing cardiac surgery.
      ] through induction of ischemia-reperfusion damage and reduced mean arterial blood pressure [
      • Adluri R.K.
      • Singh A.V.
      • Skoyles J.
      • Hitch T.
      • Robins A.
      • Baker M.
      • et al.
      Effect of increased pump flow on hepatic blood flow and systemic inflammatory response following on-pump coronary artery bypass grafting.
      ]. The systemic inflammatory response itself may also contribute to organ dysfunction by inducing distributive shock with subsequent reduced tissue perfusion [
      • Omar S.
      • Zedan A.
      • Nugent K.
      Cardiac vasoplegia syndrome: pathophysiology, risk factors and treatment.
      ,
      • Watarida S.
      • Mori A.
      • Onoe M.
      • Tabata R.
      • Shiraishi S.
      • Sugita T.
      • et al.
      A clinical study on the effects of pulsatile cardiopulmonary bypass on the blood endotoxin levels.
      ] and through direct detrimental effects of cytokines on cardiac [
      • Hennein H.A.
      • Ebba H.
      • Rodriguez J.L.
      • Merrick S.H.
      • Keith F.M.
      • Bronstein M.H.
      • et al.
      Relationship of the proinflammatory cytokines to myocardial ischemia and dysfunction after uncomplicated coronary revascularization.
      ,
      • te Velthuis H.
      • Jansen P.G.
      • Oudemans-van Straaten H.M.
      • Sturk A.
      • Eijsman L.
      • Wildevuur C.R.
      Myocardial performance in elderly patients after cardiopulmonary bypass is suppressed by tumor necrosis factor.
      ] and renal function [
      • Gomez H.
      • Ince C.
      • De Backer D.
      • Pickkers P.
      • Payen D.
      • Hotchkiss J.
      • et al.
      A unified theory of sepsis-induced acute kidney injury: inflammation, microcirculatory dysfunction, bioenergetics, and the tubular cell adaptation to injury.
      ].
      However, it has not been investigated to what extent the duration of CPB, intestinal injury, and the inflammatory response contribute to the pathophysiological pathways resulting in postoperative organ dysfunction. To the best of our knowledge, we are the first to report that CPB duration and the systemic inflammatory response represent important determinants in the development of postoperative organ dysfunction, whereas intestinal damage appears not to play a relevant role in the postoperative inflammatory response and development of postoperative organ dysfunction. In line with our results, a previous study showed that the extent of the inflammatory response correlated well with the degree of postoperative organ dysfunction in patients with infective endocarditis undergoing valve surgery [
      • Diab M.
      • Tasar R.
      • Sponholz C.
      • Lehmann T.
      • Pletz M.W.
      • Bauer M.
      • et al.
      Changes in inflammatory and vasoactive mediator profiles during valvular surgery with or without infective endocarditis: a case control pilot study.
      ]. Although it has been suggested that I-FABP may be used to identify patients at risk for gastrointestinal complications [
      • Holmes JHt
      • Lieberman J.M.
      • Probert C.B.
      • Marks W.H.
      • Hill M.E.
      • Paull D.L.
      • et al.
      Elevated intestinal fatty acid binding protein and gastrointestinal complications following cardiopulmonary bypass: a preliminary analysis.
      ], this appears not to be the case for renal, pulmonary, or cardiac dysfunction. Of interest, renal function, reflected by eGFR-MDRD, was augmented in 60% of patients on the first postoperative day. This increase in GFR, known as hyperfiltration, is a phenomenon that may also be observed in patients in the early phase of sepsis and is attributed to increased renal blood flow as a result of a high cardiac output [
      • Langenberg C.
      • Bellomo R.
      • May C.
      • Wan L.
      • Egi M.
      • Morgera S.
      Renal blood flow in sepsis.
      ]. However, cardiac output is usually low in patients on CPB during cardiac surgery, so it is unlikely that a high cardiac output accounts for an increase in renal perfusion in these patients, in contrast to patients with sepsis. Potentially, the inflammatory process itself directly influences GFR, independent of haemodynamic changes. Interestingly, during experimental human endotoxaemia, a model of short-lived systemic inflammation in humans in vivo, increases in inflammatory mediators were shown to be associated with increases in GFR [
      • Beunders R.
      • Schutz M.J.
      • van Groenendael R.
      • Leijte G.P.
      • Kox M.
      • van Eijk L.T.
      • et al.
      Endotoxemia-induced release of pro-inflammatory mediators are associated with increased glomerular filtration rate in humans in vivo.
      ]. It may therefore be hypothesised that a mild and short-lived systemic inflammatory response may result in an increase in GFR, in contrast to the situation observed in most patients with sepsis, who display an excessive and prolonged inflammatory response and may develop AKI.
      There are several limitations of this study. Firstly, data were obtained from an intervention study in which patients were randomised to receive either EA-230 or placebo. This may have influenced our results. However, no effects of EA-230 on circulating inflammatory mediators were observed [
      • van Groenendael R.
      • Beunders R.
      • Hemelaar P.
      • Hofland J.
      • Morshuis W.J.
      • van der Hoeven J.G.
      • et al.
      Safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients: a randomized double-blind placebo-controlled study.
      ]. Furthermore, in the event that the parameters used in our analyses were affected by the treatment (only applicable to eGFR-MDRD), we corrected for it by including it in the regression analysis. Secondly, this study describes correlations between different parameters. This limits the interpretation of cause-and-effect relationships, although the kinetics of the different processes and multivariable analyses strongly suggest that such a cause-and-effect relationship is present. One could argue that other types of analysis, such as causal mediation analysis [
      • Zhang Z.
      • Zheng C.
      • Kim C.
      • Van Poucke S.
      • Lin S.
      • Lan P.
      Causal mediation analysis in the context of clinical research.
      ], are more appropriate. However, these types of analyses are mainly geared towards treatment studies to identify and study the mechanisms by which a single intervention (i.e., independent variable) achieves its effect (i.e., dependent variable). In our study, we explored relationships between multiple independent and multiple dependent variables. Finally, patients included in this study had relatively short duration of the CPB and low EuroSCORE II, and only a small percentage of the patients underwent valve surgery. Therefore, the results may be less representative for high-risk patients undergoing extensive cardiac surgery.
      In conclusion, our study has revealed that, in low-risk cardiac surgery patients, duration of CPB represents the primary determinant of the systemic cytokine response and that both CPB and the cytokine response are involved in subsequent organ dysfunction. Although this type of surgery induces intestinal damage, illustrated by an increase in circulating I-FABP concentrations, this increase is not associated with the postoperative inflammatory response or organ dysfunction in this low-risk cohort.

      Funding Sources

      The study drug and placebo treatment were provided by the sponsor (Exponential Biotherapies Inc.). The sponsor had no role in the study design, data collection, analysis, or reporting.

      Conflicts of Interest

      There are no conflicts of interest to disclose.

      Acknowledgements

      We would like to thank our colleagues at the research department of the intensive care unit for their assistance with the study, and Aron Jansen for creating the heatmap in Figure 3.

      Appendices

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