Endotoxin Release and Tumor Necrosis Factor Formation During Cardiopulmonary Bypass Nicolaas J. G. Jansen, MD, PhD, Willem van Oeveren, PhD, Y. J. Gu, MD, Marilijn H. van Vliet, MD, Leon Eijsman, MD, PhD, and Charles R. H. Wildevuur, MD, PhD Department of Cardiopulmonary Surgery Research Division, University Hospital Groningen, The Netherlands, and Department of Anesthesiology and Cardio-Pulmonary Surgery, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands Endotoxin, when released into the systemic circulation during cardiopulmonary bypass (CPB), might induce activation of plasmatic systems and blood cells during CPB, in addition to a material-dependent blood activation during CPB. However, the role of endotoxin in the development of this so-called whole-body inflammatory reaction in CPB is still unclear. We investigated the release of endotoxin into the systemic circulation in relation with the activation of the complement system and in particular the formation of tumor necrosis factor in 10 patients undergoing CPB. Immediately after the start of CPB the endotoxin concentrations increased sig-
P
atients undergoing cardiopulmonary bypass (CPB)are at risk for development of a postperfusion syndrome. In its most pronounced form this consists of pulmonary and renal dysfunction and hemodynamic instability in the postoperative period [l]. These events are considered to be caused by the whole-body inflammatory reaction due to extensive blood activation by the material of the extracorporeal circuit [2]. The activation of the complement system is one of the major pathways leading to the whole body inflammatory reaction, by activation of polymorphonuclear leukocytes [3]. The activation of plasmatic systems and blood cells leading to the whole-body inflammatory reaction resembles the activation by endotoxin in gram-negative sepsis [4,51. Recent studies showed that endotoxin can be present during CPB [6, 71. Tumor necrosis factor (TNF) is released in response to endotoxin and gram-negative bacterial products and is considered to be the prime mediator involved in the pathophysiology of gram-negative sepsis [8, 91. In a recent study we showed that formation of TNF during CPB is correlated with the clinical hemodynamic instability after CPB [lo]. It might therefore be assumed that endotoxin, when it is released into the systemic circulation, could play an important role in the whole-body inflammatory reaction, especially when this is accompanied by TNF formation. In a prospective study we invesAccepted for publication Feb 21, 1992. Dr Jansen’s present address is Department of Pediatrics, University Hospital, Maastricht, The Netherlands. Address reprint requests to Dr van Oeveren, Department of Cardiopulmonary Surgery Research Division, University Hospital, Oostersingel 59, 9713 EZ Groningen, The Netherlands.
0 1992 by The Society of Thoracic Surgeons
nificantly ( p < 0.01), accompanied by increases in C3a concentration ( p < 0.05). After release of the aortic cross-clamp, there was a second increase in endotoxin followed by a continuous increase in tumor necrosis factor, reaching a peak concentration 1 hour after the end of CPB ( p < 0.01). These observations demonstrate a release of endotoxin into the systemic circulation associated with tumor necrosis factor formation, which contributes to the whole-body inflammatory reaction associated with CPB.
(Ann Tkorac Surg 1992;54:744-8)
tigated 10 patients undergoing CPB and measured endotoxin, complement activation, and TNF concentrations during and immediately after CPB.
Patients and Methods Ten patients undergoing elective primary coronary artery bypass grafting procedures were studied. All patients gave their informed consent. Patients with insulindependent diabetes mellitus, preoperative use of corticosteroids, or chronic obstructive disease were not included. The average age of the patients ( 2 standard error of the mean) was 63 +- 2 years. They had an average body surface area of 1.9 2 0.1 m2 and a mean body weight of 72 +- 3 kg. These patients were in bypass for 109 2 10 minutes and had an average cross-clamp time of 66 & 7 minutes. The extracorporeal circuit consisted of a Bard hollow fiber membrane oxygenator (Model HF 4000, Bard Inc, William Harvey, Santa Ana, CA), a Bard cardiotomy reservoir with filter (Model H-705 F), and a Bard arterial line filter (Model H-625). Polyvinyl chloride tubing was used except for the pump tubing, which was silicon rubber. The extracorporeal circuit was primed with 2.2 L of Ringer’s lactate, 0.2 L of 20% human albumin (CLB, Amsterdam, The Netherlands), and 50 mg of heparin (Leo, Emmen, The Netherlands). Heparin (300 IUikg) was given intravenously before cannulation of the aorta, and this was repeated in a dosage of 25 mg whenever the activated clotting time was shorter than 400 seconds. Heparin was neutralized by an intravenous injection of 300 IU of protamine sulfate within 5 minutes after the end of perfusion. A nonpulsa0003-4975/92/$5.00
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Ann Thorac Surg 1992;547448
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tile flow of 2.0 to 2.4 L/m2 min was used at a moderate hypothermia of 28" to 30°C. When the distal coronary artery anastomosis was established, the aorta was cross-clamped and cardioplegia was achieved with a 0°C isotonic solution containing 15 mmol/L K+, 15 mmol/L Mg2+ and 1.5 g/L procaine hydrochloride. Cardioplegia was repeated every 20 minutes. An average of about 2 L cardioplegic fluid was used.
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Anesthetic Technique Anesthesia was started with a bolus of alfentanil (50 pg * kg-' min-') and etomidate (0.1 mg/kg) followed by pancuronium bromide (0.1 mg/kg). At termination of CPB inotropic drugs were given if necessary. All patients were ventilated to normocapnia (carbon dioxide tension, 4.5 to 5.0 kPa) with 50% oxygen in air.
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Hematology Blood samples were taken from the radial artery or arterial line of the extracorporeal circuit after induction of anesthesia, 5 minutes before CPB, 5 and 30 minutes after the start of CPB, at the end of CPB, and 30 minutes after protamine sulfate administration. In addition, blood was collected 5 minutes before and 5 minutes after release of the aortic cross-clamp. Two milliliters of blood, anticoagulated with 0.318% citrate, was collected sterile and put in pyrogen-free tubes for determination of the endotoxin concentration. After collection blood was centrifuged at low speed (100 g) to obtain platelet-rich plasma, which was stored in pyrogenfree tubes. All further procedures were done aseptic to exclude contamination; only pyrogen-free tubes and reagent waters were used. In this platelet-rich plasma the endotoxin concentration was measured by activation of a proenzyme present in limulus amoebocyte lysate (Coatest-Endotoxin; Kabivitrum Diagnostica, Stockholm, Sweden) and by conversion of the substrate S 2423, which was measured in a spectrophotometer (Uvikon 710; Kontrom, Zurich, Switzerland) at 405 nm [ll]. Three milliliters of blood, anticoagulated with EDTA (0.01 mol/L), was centrifuged and stored at -70°C for C3a assays. C3a des arg concentrations were determined by radioimmunoassay (Upjohn Co, Kalamazoo, MI). One milliliter of blood, anticoagulated with EDTA (0.01 mol/L), was used for determination of TNF using a radioimmunoassay (TNFa IRMA; Medgenix, Brussels, Belgium). This assay is based on oligoclonal antibodies against different epitopes of TNFa, which are bound to the tubes, and subsequent binding of iodine-labeled antibodies to the bound TNFa. Plasma samples and antibodies are simultaneously incubated for 18 to 20 hours, which renders a high sensitivity of the assay.
Statistics A repeated-measures analysis of variance (Statview Software; Brainpower Inc, Calabasas, CA) was performed to inspect the differences among different sampling points. Significant differences between the specific time points were determined by the paired Student's t test. The data
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Fig 1. Endotoxin concentration in platelet-rich plasma during and after cardiopulmonary bypass (CPB). Immediately after the start of CPB there is a significant increase in endotoxin concentration (**p< 0.01 versus 5 minutes before the start of CPB), with a second significant increase 5 minutes after release of the aortic cross-clamp (Xclamp). (*p < 0.05 versus 5 minutes before the clamp release.)
are presented as means rt standard error of the mean. A p value less than 0.05 was considered to be significant.
Results Endotoxin At induction of anesthesia endotoxin was detectable, reaching lower concentrations before the start of CPB. Immediately after the start of CPB a significant increase in endotoxin was observed ( p < 0.01), after which the concentrations decreased. After release of the aortic crossclamp a second significant increase of endotoxin was observed ( p < 0.05), and thereafter the endotoxin concentration remained elevated until 30 minutes after protamine administration (Fig 1).
Complement Activation After the start of CPB the C3a concentration increased significantly ( p < 0.05). Upon release of the cross-clamp a second, not statistically significant, increase was seen, with a maximum of 2,268 ng/mL and a range of 1,040 to 4,400 ng/mL. Thereafter the C3a concentration decreased and remained stable until 30 minutes after protamine administration (Fig 2).
Tumor Necrosis Factor Tumor necrosis factor could be measured after release of the cross-clamp and increased significantly to a maximum of 11 pg/mL, with a range of 4 to 27 pg/mL, at 30 minutes after protamine administration ( p < 0.01) (Fig 3).
Comment The endotoxin concentration increased significantly 5 minutes after the start of CPB and after release of the
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aortic cross-clamp and remained elevated thereafter. The high level of endotoxin seen after induction of anesthesia and after the start of bypass could be due to pyrogens present in the fluids administered to the patients and to endotoxin contamination of the perfusion circuit, the cardiac suction lines, and cardioplegic fluids [7]. It has been discussed that this kind of "environmental" endotoxins are less pathogenic than endotoxin derived from gram-negative bacteria [12]. At least all patients were in good clinical condition before operation, and all blood cultures taken intraoperatively were negative. The increase in endotoxin after release of the aortic cross-clamp might be explained by the preceding hypoperfusion of the splachnic bed by the reduced peak arterial pressure associated with the nonpulsatile flow of the pump during CPB [6]. This hypoperfusion leads to an injury of the mucosal barrier between the gut and blood vessel wall [13], and large quantities of endotoxin may enter the portal vein [14]. Endotoxin, in turn, promotes bacterial translocation, a process in which bacteria, normally confined to the gut, will cross the intestinal mucosal barrier and appear in the mesenteric lymph nodes and other systemic organs [15, 161. Endotoxins, passing the mucosal barrier of the gut either by hypoperfusion or by translocation, can reach the portal or the lymph circulation. Under normal conditions a systemic endotoxemia is prevented by removal of endotoxin by the Kupffer cells of the liver [17]. However, during CPB the Kupffer cell function can be suppressed by an overloading of the reticuloendothelial cells by cellular debris and aggregated proteins, and so a variable amount of endotoxin can enter the systemic circulation [6, 181. The decrease in endotoxin concentration seen in the initial period of CPB suggests that clearance still occurs at that time. After release of the cross-clamp, however, the heart is allowed to eject normally again, which will increase the flow through the splachnic bed and could flush out more endotoxin into the 2500
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Fig 2. Complement C3a concentration in plasma during and after cardiopulmona y bypass (CPB). C3a concentration increased steadily after start of CPB, with a second small increase after release of the aortic cross-clamp (X-clamp). (*p < 0.05 versus 5 minutes before the start of CPB.)
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portal circulation, which might explain the increase in endotoxin on release of the cross-clamp. Due to the impaired function of the Kupffer cells the concentration of endotoxin remains high afterward and can lead to ongoing activation [7]. Complement activation was shown by a steep increase in C3a after the start of CPB. This increase can be explained by complement activation either by the materials of the extracorporeal circuit [19] or by endotoxin released into the systemic circulation [20]. However, the contribution of both cannot be distinguished. The second increase in complement activation, after release of the cross-clamp, is in accordance with other studies and is thought to be due to the reperfusion of heart, lungs, and other poorly perfused tissues [21,22]. It can be postulated that the release of endotoxin, at that same moment, is responsible for the increase in C3a, although no statistical evidence could be obtained for this hypothesis in the present study. Because endotoxin is known to activate the complement cascade through both the classic and the alternative pathway [20], it could also explain the observed activation of the classic pathway at the end of CPB in other studies [23]. The endotoxin release in the systemic circulation is thought to be responsible for the significant TNF formation seen at the end of CPB. In vivo studies showed that endotoxin induces TNF formation by activation of macrophages and monocytes with a peak concentration after 1 to 2 hours [24], which is in accordance with our results. We did not observe an increase in TNF after heparinization of the patient, whereas in vitro heparin seems to induce TNF production [25]. Also, all our samples were collected in EDTA, which prevents such artifacts. Tumor necrosis factor is a mediator of general inflammation and induces fever, tachycardia, and hypotension [8, 261, which is also commonly observed in the postop-
Ann Thorac Surg 1992;54:7448
erative course after CPB. In a recent study we demonstrated a significant increase in TNF during reperfusion after release of the aortic cross-clamp, which correlated with the hemodynamic instability after CPB [lo]. These observations suggest that the release of endotoxin, induced by release of the cross-clamp, as discussed above, is responsible for the TNF formation in CPB and so can play an important role in the development of the postperfusion syndrome. This study shows that the release of endotoxin, associated with the formation of TNF, plays an additional role in the development of the whole-body inflammatory reaction, independent of the material activation. As TNF is known to be a strong mediator in sepsis and endothelial injury, the release of endotoxin could play an important role in the generation of the adverse systemic effects of CPB. Therefore it seems important to focus on prevention and treatment of endotoxemia during CPB to improve the clinical course after CPB. We thank the anesthetists and perfusionists of the Onze Lieve Vrouwe Gasthuis for their cooperation during the time of our study. Furthermore we thank J. Haan for his technical assistance in performing the assays. We especially thank Prof Dr H. S. A. Heijmans for critically reading the manuscript.
References 1. Westaby S. Organ dysfunction after cardiopulmonary bypass. A systemic inflammatory reaction by the extracorporeal circuit. Intensive Care Med 1987;13:89-95. 2. Kirklin JK, Westaby S, Blackstone EH, et al. Complement and the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1983;86:84557. 3. Chenoweth DE, Cooper SW, Hugli TE, et al. Complement activation during cardiopulmonary bypass. N Engl J Med 1981;304:497-503. 4. Hale DJ, Robinson JA, Loeb HS, Gunnar RM. Pathophysiology of endotoxin shock in man. In: Proctor RA, ed. Handbook of endotoxin. Vol 4. Amsterdam: Elsevier Science Publishing, 1986:l-17. 5. Morisson DC, Ryan JL. Endotoxins and disease mechanisms. Am Rev Med 1987;38:417-32. 6. Rocke DA, Gaffin SL, Wells MT, et al. Endotoxemia associated with cardiopulmonary bypass. J Thorac Cardiovasc Surg 1987;93:832-7. 7. Andersen LW, Baek L, Degn H, Lehd J, Krasnik M, Rasmussen JP. Presence of circulating endotoxins during cardiac operations. J Thorac Cardiovasc Surg 1987;93:1159. 8. Beutler B, Cerami A. Cachectin: more than a tumor necrosis factor. N Engl J Med 1987;316:379-85. 9. Tracey KJ, Lowry SF, Fahey TJ 111, et al. Cachectin/tumor necrosis factor induces fetal shock and stress hormone responses in the dog. Surg Gynecol Obstet 1987;164:415-22.
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10. Jansen NJG, van Oeveren W, van der Broek, et al. Inhibition by dexamethasone of the reperfusion phenomena in cardiopulmonary bypass. J Thorac Cardiovasc Surg 1991;102: 515-25. 11. Sturk A, Joop K, ten Cate JW, Thomas LM. Optimalization of a chromogenic assay for endotoxin in blood. In: ten Cate JW, Buller HR, Sturk A, Levin J, eds. Bacterial endotoxins: structure, biomedical significance, and detection with the limulus amebocyte lysate test. New York: Alan R. Liss, 1985:117-36. 12. Pearson FC, Wedry ME, Bohon J, Dabbah R. Relative potency of “environmental” endotoxin as measured by the limulus amoebocyte lysate test and the USP rabbit pyrogen test. New York: Alan R. Liss, 1982:6!%77. 13. Fiddian-Green RG, Baker S. Predictive value of the stomach wall pH for complications after cardiac operations: comparison with other monitoring. Crit Care Med 1987;15:153-6. 14. Garthiram P, Gaffin SL, Wells MT, Brock-Utne JG. Superior mesenteric artery occlusion shock in cats: modification of the endotoxemia by antilipopolysaccharides antibodies. Circ Shock 1986;19:231-7. 15. Deitch EA, Berg R, Specian R. Endotoxin promotes the translocation of bacteria from the gut. Arch Surg 1987;122: 185-90. 16. Deitch EA, Ma WJ, Ma L, Berg R, Specian RD. Endotoxininduced bacterial translocation: a study of mechanisms. Surgery 1989;106:292-9. 17. Nolan JP. Endotoxin, reticuloendothelial function, and liver injury. Hepatology 1981;1:45%65. 18. Subramanian V, McLeod J, Gans H. Effect of extracorporeal circulation on reticuloendothelial function. I. Experimental evidence for impaired reticuloendothelial function following cardiopulmonary bypass in rats. Surgery 1968;64:775-84. 19. Kazatchkine MD, Nydegger UE. The human alternative pathway. Biology and immunopathology of activation and regulation. Prog Allergy 1982;30:193-234. 20. Vukajlovitch SW, Hoffman J, Morrison DC. Activation of human serum complement by bacterial lipopolysaccharides: structural requirements for antibody independent activation of the classical and alternative pathways. Mol Immunol 1987;24:319-32. 21 Engler RL, Schmid-Schonbein GW, Pavelec RS. Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol 1983;111:98-111. 22, Wildevuur ChRH, van Oeveren W. Blood interactions in extracorporeal circulation: tests to evaluate the activation of proteins and formed blood elements. Life Support Systems 1987;5:8591. 23, Bonser RS, Dave JR, John L, et al. Complement activation before, during and after cardiopulmonary bypass. Eur J Cardiothorac Surg 1990;4:2914. 24, Beutler 8, Milsark IW, Cerami A. Cachectinhmor necrosis factor: production, distribution, and metabolic fate in vivo. J Immunol 1985;135:3972-7. 25, Freeman R, Wheeler J, Robertson H, Paes ML, Laidler J. In-vitro production of TNF-a in blood samples. Lancet 1990; 336:312-3. 26. Starnes HF Jr, Warren RS, Jeevanandam M, et al. Tumor necrosis factor and the acute metabolic response to tissue injury in man. J Clin Invest 1988;82:1321-5. I
INVITED COMMENTARY The current support system of cardiopulmonary bypass (CPB) is known to induce a generalized inflammatory response, which in its severe form may induce multiorgan dysfunction as the “postperfusion syndrome.” Based on the observations from this and a previous paper [lo], Dr Jansen and colleagues proposed that the appearance of endotoxin in the circulation of patients on
CPB results in the release of tumor necrosis factor (TNF), which then plays an important role in the mechanism of the inflammatory-type responses during and after exposure to CPB. A number of recently published studies have indicated that cytokines may be a significant component of the physiologic response to CPB. This article proposes that
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one of the initiating events of cytokine production is exposure to endotoxins that appear before and after CPB. This hypothesis is consistent with data generated in several experimental systems that have shown that exposure of monocytes and macrophages to endotoxin results in the production of various cytokines including interleukin-1, interleukin-8, and TNF. Dr Jansen and colleagues discuss several possible mechanisms for the sources and kinetics of endotoxin release during CPB. It should be noted that these mechanisms, including the proposal that endotoxin results in TNF release, are speculative until a direct causal relationship is established. At this time, we only know that there is an association between the appearance of endotoxin, C3a, and TNF. The hypothesis that Dr Jansen and colleagues present and their speculations on the mechanisms of endotoxin release are readily testable with the appropriate reagents. One possible experiment is the systematic administration of endotoxins in an animal model of CPB. If their hypothesis is correct, then it would be expected that an increase in TNF release would correlate with the amount of endotoxin administered. Furthermore, the model would predict that increasing levels of endotoxin would result in adverse systemic effects of the type seen
Ann Thorac Surg 1992:54:744-8
in postperfusion syndrome. The role of TNF in the chain of events could be directly tested by the administration of monoclonal antibodies to TNF before, during, and after the exposure to endotoxin. If the role of TNF is as important as Dr Jansen and colleagues suggest, then blocking the effects of TNF with antibody should significantly change the pathology that can follow CPB. We believe that the importance of establishing direct relationships cannot be overemphasized because they provide insights into the mechanisms of postperfusion syndrome that cannot be attained by other means. Thus, although the hypothesis proposed here is interesting, the details of the mechanisms need to be tested to determine its validity.
James F . George, PkD James K . Kirklin, M D Division of Cardiotkoracic Surgery Department of Surgery Rm 739 ZRB U A B Station University of Alabama at Birmingham Birmingham, A L 45239
P
atients undergoing cardiopulmonary bypass (CPB)are at risk for development of a postperfusion syndrome. In its most pronounced form this consists of pulmonary and renal dysfunction and hemodynamic instability in the postoperative period [l]. These events are considered to be caused by the whole-body inflammatory reaction due to extensive blood activation by the material of the extracorporeal circuit [2]. The activation of the complement system is one of the major pathways leading to the whole body inflammatory reaction, by activation of polymorphonuclear leukocytes [3]. The activation of plasmatic systems and blood cells leading to the whole-body inflammatory reaction resembles the activation by endotoxin in gram-negative sepsis [4,51. Recent studies showed that endotoxin can be present during CPB [6, 71. Tumor necrosis factor (TNF) is released in response to endotoxin and gram-negative bacterial products and is considered to be the prime mediator involved in the pathophysiology of gram-negative sepsis [8, 91. In a recent study we showed that formation of TNF during CPB is correlated with the clinical hemodynamic instability after CPB [lo]. It might therefore be assumed that endotoxin, when it is released into the systemic circulation, could play an important role in the whole-body inflammatory reaction, especially when this is accompanied by TNF formation. In a prospective study we invesAccepted for publication Feb 21, 1992. Dr Jansen’s present address is Department of Pediatrics, University Hospital, Maastricht, The Netherlands. Address reprint requests to Dr van Oeveren, Department of Cardiopulmonary Surgery Research Division, University Hospital, Oostersingel 59, 9713 EZ Groningen, The Netherlands.
0 1992 by The Society of Thoracic Surgeons
nificantly ( p < 0.01), accompanied by increases in C3a concentration ( p < 0.05). After release of the aortic cross-clamp, there was a second increase in endotoxin followed by a continuous increase in tumor necrosis factor, reaching a peak concentration 1 hour after the end of CPB ( p < 0.01). These observations demonstrate a release of endotoxin into the systemic circulation associated with tumor necrosis factor formation, which contributes to the whole-body inflammatory reaction associated with CPB.
(Ann Tkorac Surg 1992;54:744-8)
tigated 10 patients undergoing CPB and measured endotoxin, complement activation, and TNF concentrations during and immediately after CPB.
Patients and Methods Ten patients undergoing elective primary coronary artery bypass grafting procedures were studied. All patients gave their informed consent. Patients with insulindependent diabetes mellitus, preoperative use of corticosteroids, or chronic obstructive disease were not included. The average age of the patients ( 2 standard error of the mean) was 63 +- 2 years. They had an average body surface area of 1.9 2 0.1 m2 and a mean body weight of 72 +- 3 kg. These patients were in bypass for 109 2 10 minutes and had an average cross-clamp time of 66 & 7 minutes. The extracorporeal circuit consisted of a Bard hollow fiber membrane oxygenator (Model HF 4000, Bard Inc, William Harvey, Santa Ana, CA), a Bard cardiotomy reservoir with filter (Model H-705 F), and a Bard arterial line filter (Model H-625). Polyvinyl chloride tubing was used except for the pump tubing, which was silicon rubber. The extracorporeal circuit was primed with 2.2 L of Ringer’s lactate, 0.2 L of 20% human albumin (CLB, Amsterdam, The Netherlands), and 50 mg of heparin (Leo, Emmen, The Netherlands). Heparin (300 IUikg) was given intravenously before cannulation of the aorta, and this was repeated in a dosage of 25 mg whenever the activated clotting time was shorter than 400 seconds. Heparin was neutralized by an intravenous injection of 300 IU of protamine sulfate within 5 minutes after the end of perfusion. A nonpulsa0003-4975/92/$5.00
JANSEN ET AL BLOOD ACTIVATION BY ENDOTOXIN
Ann Thorac Surg 1992;547448
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tile flow of 2.0 to 2.4 L/m2 min was used at a moderate hypothermia of 28" to 30°C. When the distal coronary artery anastomosis was established, the aorta was cross-clamped and cardioplegia was achieved with a 0°C isotonic solution containing 15 mmol/L K+, 15 mmol/L Mg2+ and 1.5 g/L procaine hydrochloride. Cardioplegia was repeated every 20 minutes. An average of about 2 L cardioplegic fluid was used.
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Anesthetic Technique Anesthesia was started with a bolus of alfentanil (50 pg * kg-' min-') and etomidate (0.1 mg/kg) followed by pancuronium bromide (0.1 mg/kg). At termination of CPB inotropic drugs were given if necessary. All patients were ventilated to normocapnia (carbon dioxide tension, 4.5 to 5.0 kPa) with 50% oxygen in air.
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Hematology Blood samples were taken from the radial artery or arterial line of the extracorporeal circuit after induction of anesthesia, 5 minutes before CPB, 5 and 30 minutes after the start of CPB, at the end of CPB, and 30 minutes after protamine sulfate administration. In addition, blood was collected 5 minutes before and 5 minutes after release of the aortic cross-clamp. Two milliliters of blood, anticoagulated with 0.318% citrate, was collected sterile and put in pyrogen-free tubes for determination of the endotoxin concentration. After collection blood was centrifuged at low speed (100 g) to obtain platelet-rich plasma, which was stored in pyrogenfree tubes. All further procedures were done aseptic to exclude contamination; only pyrogen-free tubes and reagent waters were used. In this platelet-rich plasma the endotoxin concentration was measured by activation of a proenzyme present in limulus amoebocyte lysate (Coatest-Endotoxin; Kabivitrum Diagnostica, Stockholm, Sweden) and by conversion of the substrate S 2423, which was measured in a spectrophotometer (Uvikon 710; Kontrom, Zurich, Switzerland) at 405 nm [ll]. Three milliliters of blood, anticoagulated with EDTA (0.01 mol/L), was centrifuged and stored at -70°C for C3a assays. C3a des arg concentrations were determined by radioimmunoassay (Upjohn Co, Kalamazoo, MI). One milliliter of blood, anticoagulated with EDTA (0.01 mol/L), was used for determination of TNF using a radioimmunoassay (TNFa IRMA; Medgenix, Brussels, Belgium). This assay is based on oligoclonal antibodies against different epitopes of TNFa, which are bound to the tubes, and subsequent binding of iodine-labeled antibodies to the bound TNFa. Plasma samples and antibodies are simultaneously incubated for 18 to 20 hours, which renders a high sensitivity of the assay.
Statistics A repeated-measures analysis of variance (Statview Software; Brainpower Inc, Calabasas, CA) was performed to inspect the differences among different sampling points. Significant differences between the specific time points were determined by the paired Student's t test. The data
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Fig 1. Endotoxin concentration in platelet-rich plasma during and after cardiopulmonary bypass (CPB). Immediately after the start of CPB there is a significant increase in endotoxin concentration (**p< 0.01 versus 5 minutes before the start of CPB), with a second significant increase 5 minutes after release of the aortic cross-clamp (Xclamp). (*p < 0.05 versus 5 minutes before the clamp release.)
are presented as means rt standard error of the mean. A p value less than 0.05 was considered to be significant.
Results Endotoxin At induction of anesthesia endotoxin was detectable, reaching lower concentrations before the start of CPB. Immediately after the start of CPB a significant increase in endotoxin was observed ( p < 0.01), after which the concentrations decreased. After release of the aortic crossclamp a second significant increase of endotoxin was observed ( p < 0.05), and thereafter the endotoxin concentration remained elevated until 30 minutes after protamine administration (Fig 1).
Complement Activation After the start of CPB the C3a concentration increased significantly ( p < 0.05). Upon release of the cross-clamp a second, not statistically significant, increase was seen, with a maximum of 2,268 ng/mL and a range of 1,040 to 4,400 ng/mL. Thereafter the C3a concentration decreased and remained stable until 30 minutes after protamine administration (Fig 2).
Tumor Necrosis Factor Tumor necrosis factor could be measured after release of the cross-clamp and increased significantly to a maximum of 11 pg/mL, with a range of 4 to 27 pg/mL, at 30 minutes after protamine administration ( p < 0.01) (Fig 3).
Comment The endotoxin concentration increased significantly 5 minutes after the start of CPB and after release of the
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aortic cross-clamp and remained elevated thereafter. The high level of endotoxin seen after induction of anesthesia and after the start of bypass could be due to pyrogens present in the fluids administered to the patients and to endotoxin contamination of the perfusion circuit, the cardiac suction lines, and cardioplegic fluids [7]. It has been discussed that this kind of "environmental" endotoxins are less pathogenic than endotoxin derived from gram-negative bacteria [12]. At least all patients were in good clinical condition before operation, and all blood cultures taken intraoperatively were negative. The increase in endotoxin after release of the aortic cross-clamp might be explained by the preceding hypoperfusion of the splachnic bed by the reduced peak arterial pressure associated with the nonpulsatile flow of the pump during CPB [6]. This hypoperfusion leads to an injury of the mucosal barrier between the gut and blood vessel wall [13], and large quantities of endotoxin may enter the portal vein [14]. Endotoxin, in turn, promotes bacterial translocation, a process in which bacteria, normally confined to the gut, will cross the intestinal mucosal barrier and appear in the mesenteric lymph nodes and other systemic organs [15, 161. Endotoxins, passing the mucosal barrier of the gut either by hypoperfusion or by translocation, can reach the portal or the lymph circulation. Under normal conditions a systemic endotoxemia is prevented by removal of endotoxin by the Kupffer cells of the liver [17]. However, during CPB the Kupffer cell function can be suppressed by an overloading of the reticuloendothelial cells by cellular debris and aggregated proteins, and so a variable amount of endotoxin can enter the systemic circulation [6, 181. The decrease in endotoxin concentration seen in the initial period of CPB suggests that clearance still occurs at that time. After release of the cross-clamp, however, the heart is allowed to eject normally again, which will increase the flow through the splachnic bed and could flush out more endotoxin into the 2500
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Fig 2. Complement C3a concentration in plasma during and after cardiopulmona y bypass (CPB). C3a concentration increased steadily after start of CPB, with a second small increase after release of the aortic cross-clamp (X-clamp). (*p < 0.05 versus 5 minutes before the start of CPB.)
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Time (min) Fig 3. Tumor necrosis factor (TNF) concentrations in plasma during and after cardiopulmonay bypass (CPB). There was a significant and steep increase in TNF concentration after release of the aortic cross-clamp (X-clamp). (**p< 0.01 versus 5 minutes before the clamp release.)
portal circulation, which might explain the increase in endotoxin on release of the cross-clamp. Due to the impaired function of the Kupffer cells the concentration of endotoxin remains high afterward and can lead to ongoing activation [7]. Complement activation was shown by a steep increase in C3a after the start of CPB. This increase can be explained by complement activation either by the materials of the extracorporeal circuit [19] or by endotoxin released into the systemic circulation [20]. However, the contribution of both cannot be distinguished. The second increase in complement activation, after release of the cross-clamp, is in accordance with other studies and is thought to be due to the reperfusion of heart, lungs, and other poorly perfused tissues [21,22]. It can be postulated that the release of endotoxin, at that same moment, is responsible for the increase in C3a, although no statistical evidence could be obtained for this hypothesis in the present study. Because endotoxin is known to activate the complement cascade through both the classic and the alternative pathway [20], it could also explain the observed activation of the classic pathway at the end of CPB in other studies [23]. The endotoxin release in the systemic circulation is thought to be responsible for the significant TNF formation seen at the end of CPB. In vivo studies showed that endotoxin induces TNF formation by activation of macrophages and monocytes with a peak concentration after 1 to 2 hours [24], which is in accordance with our results. We did not observe an increase in TNF after heparinization of the patient, whereas in vitro heparin seems to induce TNF production [25]. Also, all our samples were collected in EDTA, which prevents such artifacts. Tumor necrosis factor is a mediator of general inflammation and induces fever, tachycardia, and hypotension [8, 261, which is also commonly observed in the postop-
Ann Thorac Surg 1992;54:7448
erative course after CPB. In a recent study we demonstrated a significant increase in TNF during reperfusion after release of the aortic cross-clamp, which correlated with the hemodynamic instability after CPB [lo]. These observations suggest that the release of endotoxin, induced by release of the cross-clamp, as discussed above, is responsible for the TNF formation in CPB and so can play an important role in the development of the postperfusion syndrome. This study shows that the release of endotoxin, associated with the formation of TNF, plays an additional role in the development of the whole-body inflammatory reaction, independent of the material activation. As TNF is known to be a strong mediator in sepsis and endothelial injury, the release of endotoxin could play an important role in the generation of the adverse systemic effects of CPB. Therefore it seems important to focus on prevention and treatment of endotoxemia during CPB to improve the clinical course after CPB. We thank the anesthetists and perfusionists of the Onze Lieve Vrouwe Gasthuis for their cooperation during the time of our study. Furthermore we thank J. Haan for his technical assistance in performing the assays. We especially thank Prof Dr H. S. A. Heijmans for critically reading the manuscript.
References 1. Westaby S. Organ dysfunction after cardiopulmonary bypass. A systemic inflammatory reaction by the extracorporeal circuit. Intensive Care Med 1987;13:89-95. 2. Kirklin JK, Westaby S, Blackstone EH, et al. Complement and the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1983;86:84557. 3. Chenoweth DE, Cooper SW, Hugli TE, et al. Complement activation during cardiopulmonary bypass. N Engl J Med 1981;304:497-503. 4. Hale DJ, Robinson JA, Loeb HS, Gunnar RM. Pathophysiology of endotoxin shock in man. In: Proctor RA, ed. Handbook of endotoxin. Vol 4. Amsterdam: Elsevier Science Publishing, 1986:l-17. 5. Morisson DC, Ryan JL. Endotoxins and disease mechanisms. Am Rev Med 1987;38:417-32. 6. Rocke DA, Gaffin SL, Wells MT, et al. Endotoxemia associated with cardiopulmonary bypass. J Thorac Cardiovasc Surg 1987;93:832-7. 7. Andersen LW, Baek L, Degn H, Lehd J, Krasnik M, Rasmussen JP. Presence of circulating endotoxins during cardiac operations. J Thorac Cardiovasc Surg 1987;93:1159. 8. Beutler B, Cerami A. Cachectin: more than a tumor necrosis factor. N Engl J Med 1987;316:379-85. 9. Tracey KJ, Lowry SF, Fahey TJ 111, et al. Cachectin/tumor necrosis factor induces fetal shock and stress hormone responses in the dog. Surg Gynecol Obstet 1987;164:415-22.
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10. Jansen NJG, van Oeveren W, van der Broek, et al. Inhibition by dexamethasone of the reperfusion phenomena in cardiopulmonary bypass. J Thorac Cardiovasc Surg 1991;102: 515-25. 11. Sturk A, Joop K, ten Cate JW, Thomas LM. Optimalization of a chromogenic assay for endotoxin in blood. In: ten Cate JW, Buller HR, Sturk A, Levin J, eds. Bacterial endotoxins: structure, biomedical significance, and detection with the limulus amebocyte lysate test. New York: Alan R. Liss, 1985:117-36. 12. Pearson FC, Wedry ME, Bohon J, Dabbah R. Relative potency of “environmental” endotoxin as measured by the limulus amoebocyte lysate test and the USP rabbit pyrogen test. New York: Alan R. Liss, 1982:6!%77. 13. Fiddian-Green RG, Baker S. Predictive value of the stomach wall pH for complications after cardiac operations: comparison with other monitoring. Crit Care Med 1987;15:153-6. 14. Garthiram P, Gaffin SL, Wells MT, Brock-Utne JG. Superior mesenteric artery occlusion shock in cats: modification of the endotoxemia by antilipopolysaccharides antibodies. Circ Shock 1986;19:231-7. 15. Deitch EA, Berg R, Specian R. Endotoxin promotes the translocation of bacteria from the gut. Arch Surg 1987;122: 185-90. 16. Deitch EA, Ma WJ, Ma L, Berg R, Specian RD. Endotoxininduced bacterial translocation: a study of mechanisms. Surgery 1989;106:292-9. 17. Nolan JP. Endotoxin, reticuloendothelial function, and liver injury. Hepatology 1981;1:45%65. 18. Subramanian V, McLeod J, Gans H. Effect of extracorporeal circulation on reticuloendothelial function. I. Experimental evidence for impaired reticuloendothelial function following cardiopulmonary bypass in rats. Surgery 1968;64:775-84. 19. Kazatchkine MD, Nydegger UE. The human alternative pathway. Biology and immunopathology of activation and regulation. Prog Allergy 1982;30:193-234. 20. Vukajlovitch SW, Hoffman J, Morrison DC. Activation of human serum complement by bacterial lipopolysaccharides: structural requirements for antibody independent activation of the classical and alternative pathways. Mol Immunol 1987;24:319-32. 21 Engler RL, Schmid-Schonbein GW, Pavelec RS. Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol 1983;111:98-111. 22, Wildevuur ChRH, van Oeveren W. Blood interactions in extracorporeal circulation: tests to evaluate the activation of proteins and formed blood elements. Life Support Systems 1987;5:8591. 23, Bonser RS, Dave JR, John L, et al. Complement activation before, during and after cardiopulmonary bypass. Eur J Cardiothorac Surg 1990;4:2914. 24, Beutler 8, Milsark IW, Cerami A. Cachectinhmor necrosis factor: production, distribution, and metabolic fate in vivo. J Immunol 1985;135:3972-7. 25, Freeman R, Wheeler J, Robertson H, Paes ML, Laidler J. In-vitro production of TNF-a in blood samples. Lancet 1990; 336:312-3. 26. Starnes HF Jr, Warren RS, Jeevanandam M, et al. Tumor necrosis factor and the acute metabolic response to tissue injury in man. J Clin Invest 1988;82:1321-5. I
INVITED COMMENTARY The current support system of cardiopulmonary bypass (CPB) is known to induce a generalized inflammatory response, which in its severe form may induce multiorgan dysfunction as the “postperfusion syndrome.” Based on the observations from this and a previous paper [lo], Dr Jansen and colleagues proposed that the appearance of endotoxin in the circulation of patients on
CPB results in the release of tumor necrosis factor (TNF), which then plays an important role in the mechanism of the inflammatory-type responses during and after exposure to CPB. A number of recently published studies have indicated that cytokines may be a significant component of the physiologic response to CPB. This article proposes that
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one of the initiating events of cytokine production is exposure to endotoxins that appear before and after CPB. This hypothesis is consistent with data generated in several experimental systems that have shown that exposure of monocytes and macrophages to endotoxin results in the production of various cytokines including interleukin-1, interleukin-8, and TNF. Dr Jansen and colleagues discuss several possible mechanisms for the sources and kinetics of endotoxin release during CPB. It should be noted that these mechanisms, including the proposal that endotoxin results in TNF release, are speculative until a direct causal relationship is established. At this time, we only know that there is an association between the appearance of endotoxin, C3a, and TNF. The hypothesis that Dr Jansen and colleagues present and their speculations on the mechanisms of endotoxin release are readily testable with the appropriate reagents. One possible experiment is the systematic administration of endotoxins in an animal model of CPB. If their hypothesis is correct, then it would be expected that an increase in TNF release would correlate with the amount of endotoxin administered. Furthermore, the model would predict that increasing levels of endotoxin would result in adverse systemic effects of the type seen
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in postperfusion syndrome. The role of TNF in the chain of events could be directly tested by the administration of monoclonal antibodies to TNF before, during, and after the exposure to endotoxin. If the role of TNF is as important as Dr Jansen and colleagues suggest, then blocking the effects of TNF with antibody should significantly change the pathology that can follow CPB. We believe that the importance of establishing direct relationships cannot be overemphasized because they provide insights into the mechanisms of postperfusion syndrome that cannot be attained by other means. Thus, although the hypothesis proposed here is interesting, the details of the mechanisms need to be tested to determine its validity.
James F . George, PkD James K . Kirklin, M D Division of Cardiotkoracic Surgery Department of Surgery Rm 739 ZRB U A B Station University of Alabama at Birmingham Birmingham, A L 45239
