Endotoxins in cardiopulmonary bypass

J

THORAC CARDIOVASC SURG

1990;100:777-80

Endotoxins in cardiopulmonary bypass Endotoxins are biologically active substances derived from the ceU waD of degraded gram-negative bacteria. Since sterile water may also contain large amounts of endotoxins, these are easily introduced into the manufacturing processes of technical medical material, such as the extracorporeal components used in cardiopulmonary bypass. In hemodialysis, the presence of endotoxins has been related to untoward effects in patients. Using the limulus amebocyte lysate test, we determined the serum concentration of endotoxin in 42 patients undergoing coronary bypass operations. The values increased during cardiopulmonary bypass, exceeding the normal range of 0 to 20 ng/L in 10 patients with a maximum of 82 ngjL, which probably indicates endotoxin release from the extracorporeal equipment. We found no obvious relation to postoperative morbidity. The ·endotoxin levels of this study are considerably lower than those reported in two other studies of patients having cardiopulmonary bypass. This might be due to less intraoperative contamination but possibly also to differences in analytic methods. Leif Nilsson, MD,a Lena Kulander,b Sven-Olov Nystrom, MD,a and Orjan Eriksson, MD,b

Uppsa/a, Sweden

Extraco~real

circulation, such as cardiopulmonary bypass (CPB) and hemodialysis, involves exposition of blood to large surfaces of artificial materials. This causes mechanical damage to blood cells 1• 2 and activation of blood-borne biologic cascade systems such as coagulationU and complement4 • 5 systems--changes that may be related to the development of postoperative complications, "postperfusion syndrome." 1• 6 Obviously there is also a risk of contamination of blood and patient with substances released from the extracorporeal device. Endotoxins are lipopolysaccharides derived from the walls of degraded gram-negative bacteria. They have profound biologic effects with ability to activate different cascade systems such as coagulation and complement_?· 8 Different levels of endotoxernia are related to a range of clinical symptoms from headache, chills, and myalgia over fever, hypotension, and metabolic acidosis to septic shock and disseminated intravascular coagulation. 8- 10 From the Departments of Thoracic Surgery" and Clinical Chemistry,b University Hospital, Uppsala, Sweden. Supported by grants from The Swedish Association Against Heart and Chest Diseases.

Endotoxins have been found in several types of pharmaceutical and medical technical material, such as infusion and dialysis solutions, dialysis membranes and filters, and surgical gloves. 11 • 12 In hemodialysis the presence of endotoxins has been associated with untoward effects in patients. 13 There are no current pharmacopeia! specifications for endotoxins in medical technical material such as CPB oxygenators. The possible role of endotoxins in postoperative morbidity after cardiac operations motivates further investigation, particularly in view of the sparse literature on this subject. The aim of this study was therefore to monitor the presence of endotoxernia in patients undergoing cardiac operations and to attempt to correlate any changes with postoperative morbidity. Patients and methods The study was performed as part of a wider comparative investigation of bubble and membrane oxygenators, in which complement activation and the release of neutrophil granulocyte factors were also followed. Forty-two men with a mean age of 57.8 years (36 to 70), scheduled to undergo elective coronary bypass operations, were randomized to one of four oxygenator groups:

Address for reprints: Leif Nilsson, MD, Department of Thoracic Surgery, University Hospital, S-751 85 Uppsala, Sweden.

Group 1: Cobe Optiflo II bubble oxygenator, 10 patients Group 2: Cobe CML membrane oxygenator, 12 patients Group 3: Harvey HF 4000 membrane oxygenator, 10 patients Group 4: Harvey 1700 bubble oxygenator, 10 patients

12/1/17267

The Cobe oxygenators were manufactured by Cobe La bora-

Received for publication May I, 1989. Accepted for publication Sept. 28, 1989.

777

The Journal of

7 7 8 Nilsson et a/.

Thoracic and Cardiovascular Surgery

Table I. Median values and (range) of endotoxin in nanograms per liter for the four oxygenator groups and for all 42 patients; distribution of values above normal range (0-20 ngjL) Group 2 3 4 All Abnormal values

n 10 12 10 10 42 n=

Preop. 4.0 3.5 7.0 6.0 4.5

(0-12) (0-12) (3-34) (0-12) (0-34) I

tories, Lakewood, Colorado, and the Harvey oxygenators by Bard Inc., Santa Ana, California. Surgical procedure. The operations were standard routine procedures with the following main features: priming with about 2000 ml Ringer's acetate, polyvinyl chloride (PVC) tubes, roller pump, no arterial filter, nonpulsatile flow of 2.2 Ljm 2 , reduced by 25% after lowering body temperature to 30° C, and cold crystalloid cardioplegia. The average CPB time for all patients was 109 minutes (48 to 159). Sampling protocol. The blood samples were taken from an arterial line preoperatively (I), at the end of CPB (II), 3 hours after CPB (III), and the first postoperative morning (IV). The samples were collected in disposable evacuated glass tubes without additives and kept at room temperature for 30 minutes before centrifugation. Serum was transferred to a plastic tube and frozen to -70° C. Laboratory methods. For quantification of endotoxin in serum, limulus amebocyte lysate (LAL) and a chromogenic substrate were employed, with use of a modification of the technique described by Friberger. 14 Lysate from M.A. Bioproducts, Walkersville, Maryland, and chromogenic substrate were supplied by Kabi, Stockholm, Sweden. Escherichia coil 0 Ill :84 was used as a standard, and the activity of 1 ng was equivalent to 12 endotoxin units (EU) compared with Food and Drug Administration standard EC-5/ USP lot F. All glassware was covered with aluminum foil and heated in a hot-air oven at 200° C for 4 hours. From the stock solution of the endotoxin standard, serial dilutions were made to label the serum. The serial dilutions covered the concentration range of 10 to 100 ng/L. The sample and the labeled sample were diluted I: I 0 with sterile endotoxinfree water and heat-treated for 10 minutes at 75° C to destroy inhibitors. They were then kept at room temperature for 15 minutes. LAL was reconstituted in 1.4 ml of sterile endotoxin-free water; I 00 ~!lysate was incubated with 100 ~I sample or labeled sample in a block heater at 37° C (step I). After 25 minutes 200 ~I substrate was added--one volume of chromogenic substrate (2.0 mmol/L) mixed with one volume of TRIS buffer (50 mmoljL, pH 9.0) (step 2). Five minutes later the reaction was stopped by adding 200 ~I of 20% acetic acid. The yellow color was read at 405 nm. There was a linearity of extinction between 5 and I 00 ng/L. Statistics. Statistical analysis was performed with the use of nonparametric tests (Wilcoxon for comparison, Spearman for correlation), because endotoxin concentrations were not normally distributed.

EndCPB

3 hr post-CPB

6.5 9.0 5.0 6.5 7.0

7.0 7.0 4.0 10.5 7.0

(2-24) (2-82) (2-29) (3-15) (2-82) 5

(4-51) (0-27) (1-18) (0-36) (0-51) 3

First postop. morning 5.5 6.0 3.0 4.0 4.0

(0-15) (0-36) (0-7) (0-36) (0-36) 2

The study was approved by the institutional review board. Informed consent was obtained from each patient.

Results

Endotoxin concentrations are presented in Table I. There are no statistically significant differences between groups or between sampling periods. The normal range of endotoxin in our laboratory is 0 to 20 ng/L. This value was exceeded on 11 occasions in 10 patients, with a maximum value of 82 ng/L recorded in one patient at the end of CPB. We did not see any correlation with postoperative morbidity, although this factor was rather carefully analyzed in the main study, in terms of circulatory, respiratory, renal, and cerebral dysfunction. Endotoxin levels at the end of CPB had no significant relation to the duration of CPB. During CPB there is considerable hemodilution caused by the addition of priming and cardioplegic solutions. The hematocrit value at the end of CPB was in our patients 23.8 ± 0.4 (mean ± standard error of the mean). When changes of endotoxin during CPB are being calculated, this dilution factor should be considered and concentration values should be corrected. We had no true plasma dilution marker available. Using hematocrit for dilution correction, which underestimates plasma dilution, we obtained a corrected median endotoxin value of 13.0 (3 to 138) ng/L at the end of CPB for the whole series of 42 patients. This value is significantly higher than the preoperative value (p < 0.001), which clearly demonstrates a release of endotoxin to the circulation during CPB. There were no differences between oxygenator groups. Fig. 1 shows the changes of white blood cell count and endotoxin levels in serum. There was a significant correlation between these two parameters preoperatively (r = 0.4l4,p = 0.006) and at the end ofCPB (r = 0.432, p = 0.004). The rise in white blood cell count that occurs during CPB, despite the great hemodilution, might therefore partially be related to endotoxemia.

Volume 100 Number 5 November 1990

Discussion There are many possible sources of endotoxin in cardiac operations. Apart from the components of the extracorporeal setup, infusion solutions, drugs, and surgical material such as instruments and gloves must also be considered. There is also a possibility of self-contamination from the patient's own gram-negative intestinal bacteria, 15 • 16 an effect that might be enhanced by reduced splanchnic circulation during CPB. To our knowledge there are only two other reports on endotoxin and CPB in the literature, both published in this JouRNAL Andersen and colleagues 17 stated that all patients (n = 10) were "free of endotoxins" preoperatively but had rising concentrations to 64 ng/L (16 to 250) at the end of CPB, peaking at a median level of 95 ng/L (16 to 250) at 90 minutes after CPB. Rocke and colleagues 18 'studied nine patients with coronary disease. Preoperatively the mean endotoxin concentration was 128 ngjL, increasing during CPB to a mean peak value of 556 ngjL. As seen, these two studies report considerably higher values than we do. The differences may be true, reflecting different levels of intraoperative contamination. AnQther possibility is different analytic techniques. Ander~en and colleagues 17 used a modified LAL technique, including a rocket immunOelectrophoresis, for blood samples. Since the ordinary LAL technique cannot be used with blood samples, the results are not fully comparabl~;: to ours. . Rocke and colleagues 18 used the LAL technique with plasma from heparinized blood. The very high levels of endotoxin, also seen preoperatively, and the lack of concomitant clinical symptoms raise the suspicion of false high values. We would only like to point to the possibility of contaminated heparin in the test tubes. In our experience, pharmaceutical heparin, a biologic product, very often contains significant amounts of endotoxin (unpublished results). Rocke and colleagues used "heparinized pyrogen-free tubes." It is not clear whether pyrogen testing was done after addition of heparin. Furthermore, the biologic pyrogen tests are highly nonspecific and unreliable regarding demonstration of endotoxin. 19 Even if the levels should be falsely high, there was a remarkable increase during bypass, indicating a substantial intraoperative contamination. None of the studies referred to 17• 18 report any observations of clinically harmful effects of endotoxin. Elin and colleagues9 studied effects of endotoxin injection in healthy volunteers. Doses corresponding to serum/plasma levels of 2.5 to 12.5 ng/L elicited fever, but no other symptoms. Subjects injected with a dose corresponding to 100 ng/L complained of headache, chills, and myalgia. In

Endotoxins in CPB

12

12

WBC

Etx

WBC 109 /1

ng/1

10

10

8

8

6

6

4

4

2

2

Preop

end CPB

779

~

3 hrs

post CPB

postop

morning

Fig. 1. Changes of white blood cell count (WBC) and serum concentrations of endotoxin (Etx) during and after operation. The two parameters correlate significantly preoperatively and at the end of CPB.

septic shock, levels of more than 3000 ng/L have been reported. 19 Endotoxin-related symptoms, anticipated at the levels found in our study and in that of Andersen and colleagues, are easily masked and overlooked in the early postoperative course after cardiac operations. Our findings indicate that the endotoxin levels generally are low in the patients undergoing CPB at our clinic. This does not rule out the possibility that, on a large basis, there may be some patients with clinically important endotoxemia. The affinity of endotoxin to plastic material was studied by Sawada and co-workers. 20 They found that a microporous polyethylene hollow fiber membrane adsorbed practically all endotoxin when perfused with Japanese tap water, which, like tap water from most countries, is very rich in endotoxin. Rinsing of the membrane with pure water for 120 minutes removed only 5.6% of the endotoxin, whereas the same treatment with 70% ethanol or 70% ethanol +0.1 N sodium hydroxide removed 43% and 78%, respectively. It is possible that the extracorporeal device used in CPB, and especially the oxygenator with its large plastic surfaces, con.tains bound endotoxin. The question is whether this firmly attached endotoxin can be harmful to the patient. We do not know this, but there is a theoretical possibility that bound endotoxin can trigger cascade systems, such as the complement system.

7 8 0 Nilsson et a/. Conclusion

During CPB there is a release of endotoxin into the patient's circulation. The magnitude, however, differs considerably between different reports, which might be due to different analysis techniques or different levels of intraoperative contamination. In the small series hitherto published, it has not been possible to find any relation to postoperative morbidity. There are, however, good reasons to believe that endotoxemia is negative for the postoperative condition of the patient and should therefore be minimized. Endotoxin contamination of CPB devices should be analyzed with special techniques to reveal bound endotoxin. REFERENCES

l. Dungen van den JJAM, Karliczek G F, Brenken U, Homan van der Heide JN, Wildevuur CRH. Clinical study of blood trauma during perfusion with membrane and bubble oxygenators. J THORAC CARDIOVASC SURG 1982;83:108-16. 2. van Oeveren W, Kazatchkine MD, Descamps-Latscha B, eta!. Deleterious effects of cardiopulmonary bypass: a prospective study of bubble versus membrane oxygenation. J THORAC CARDIOVASC SURG 1985;89:888-99. 3. Bagge L, Lilienberg G, Nystrom SO, Tyden H. Coagulation, fibrinolysis and bleeding after open-heart surgery. Scand J Thorac Cardiovasc Surg 1986;20: 151-60. 4. Chenoweth DE, Cooper SW, Hugli TE, Stewart RW, Blackstone EH, Kirklin JW. Complement activation during cardiopulmonary bypass. N Eng! J Med 1981 ;304:497503. 5. Nilsson L, Brunnkvist S, Nilsson U, eta!. Activation of inflammatory systems during cardiopulmonary bypass. Scand J Thorac Cardiovasc Surg 1988;22:51-3. 6. Kirklin JK, Westaby S, Blackstone EH, Kirklin JW, Chenoweth DE, Pacifico AD. Complement and the damaging effects of cardiopulmonary bypass. J THORAC CARDIOVASC SURG 1983;86:845-57. 7. Morrison DC, Raziuddin S. Lipopolysaccharides and endotoxin. In: Sirois P, Rola-Pleszczynski M, eds. Immunopharmacology. Amsterdam: Elsevier Biomedical, 1982; 169-99. 8. Morrison DC. Endotoxins and disease mechanisms: Annu Rev Med 1987;38:417-32.

The Journal of Thoracic and Cardiovascular Surgery

9. Elin RJ, WolffSM, McAdam KPWJ, eta!. Properties of reference Escherichia coli endotoxin and its phthalylated derivative in humans. J Infect Dis 1981;144:329-36. 10. Harthug S, Bjorvatn B, Osterud B. Quantitation of endotoxin in blood from patients with meningococcal disease, using a limulus lysate test in combination with a chromogenic substrate. Infection 1983;11 :192-6. 11. Petersen NJ, Carson LA, Favero MS. Bacterial endotoxin in new and reused hemodialyzers: a potential cause of endotoxemia. Trans Am Soc Artif Intern Organs 1981; 27:155-60. 12. Shmunes E, Darby T. Contact dermatitis due to endotoxin in irradiated latex gloves. Contact Dermatitis 1984;10: 240-4. 13. Nisbeth U, Hallgren R, Eriksson 6, Danielsson BG. Endotoxemia in chronic renal failure. Nephron 1987;45: 93-7. 14. Friberger P. The design of a reliable endotoxin test. In: ten Cate JW, Bueller HR, Sturk A, Levin J, eds. Bacterial endotoxins: structure, biomedical significance and detection with limulus amebocyte lysate test. New York: Alan R Liss, 1985;139-49. 15. Gathiram P, Gaffin SL, Wells MT, Brock-Utine JG. Superior mesenteric artery occlusion shock in cats: modification of the endotoxemia by antilipopolysaccharide antibodies (anti-LPS). Circ Shock 1986;19:231-7. 16. Rush BF, Sori AJ, Murphy TF, SmithS, Flanagan JJ, Machiedo GW. Endotoxemia and bacteremia during hemorrhagic shock. Ann Surg 1988;207:549-54. 17. Andersen LW, Baek L, Degn H, LehdJ, Krasnik M, Rasmussen JP. Presence of circulating endotoxi.ns during cardiac operations. J THORAC CARDIOV ASC "SURG 1987; 93:115-9. 18. Rocke DA, Gaffin SL, Wells MT, Koen Y, Brock-Utine J G. Endotoxemia associated with cardiopulmonary bypass. J THORAC CARDIOVASC SURG 1987;93:832-7. 19. Levin J. The limulus test and bacterial endotoxins: some perspectives. In: Watson SW, Levin J, Novitsky TJ, eds. Endotoxins and their detection with the limulus amebocyte test. New York: Alan R Liss, 1982;7-24. 20. Sawada Y, Fujii R, Igami I, Kawai A, Kamiki T, Niwa M. Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane. Appl Environ Microbiol1986;51:813-20.