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Ann Thorac Surg 1995;59:713-716
© 1995 The Society of Thoracic Surgeons

Eosinophil Granule Proteins in Cardiopulmonary Bypass With and Without Heparin Coating

Departments of Thoracic Surgery and Clinical Chemistry, University Hospital, Uppsala, Sweden

Accepted for publication December 5, 1994.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Extracorporeal circulation with exposure of blood to foreign surfaces causes activation of different defense systems, eg, white cells. Several potent mediators are released into plasma, capable of causing harmful effects to different organs, contributing to postoperative morbidity after operations using cardiopulmonary bypass. The eosinophil granulocyte has not previously been investigated in this respect. We studied two of its activation products, eosinophil cationic protein and eosinophil protein X in coronary bypass patients. In 17 control patients, plasma levels of eosinophil cationic protein and eosinophil protein X increased considerably during cardiopulmonary bypass. In 19 patients with heparin-coated cardiopulmonary bypass equipment the levels were significantly reduced, indicating improved biocompatibility of the cardiopulmonary bypass circuit. The heparin-coated surface causes less activation of eosinophils; also, released eosinophil cationic protein is bound to the heparinized surface.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
The exposure of blood to artificial surfaces during extracorporeal circulation causes a general inflammatory response in the organism. During cardiopulmonary bypass (CPB) there is a considerable degree of complement activation [1, 2] and neutrophil degranulation [2, 3] taking place. The clinical significance is not fully clear, although there is good evidence that the high levels of inflammatory mediators may be harmful and contribute to postoperative morbidity [1, 4, 5]. The role of the eosinophil granulocyte in inflammation has attracted increasing interest during the past years. Physiologically this cell is important for the defense against parasites. In pathophysiologic processes; however, it may be injurious, causing destruction to tissues in different organs in different inflammatory diseases such as asthma [6]. In a small study we demonstrated an increase in eosinophil cationic protein (ECP) in plasma during CPB [7]. We also demonstrated previously a reduced activation of neutrophils during CPB [8] when using heparin-coated CPB circuits (Carmeda BioActive Surface; Carmeda, Stockholm, Sweden). The aim of this investigation was to study the effect of CPB, with and without heparin-coated circuits, on plasma levels of ECP and the eosinophil protein X (EPX). Because plasma levels may be affected by binding of these proteins to the heparin-coated surfaces, we also studied their affinity to that surface.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Patients
The study was performed in two steps with subsequent reduction of the systemic heparinization. In the first part 16 male coronary bypass patients were randomized into two groups. Nine patients were operated on using heparin-coated CPB circuits and a 75% heparin dose whereas 7 control patients had a full heparin dose and identical equipment without heparin coating. The second part comprised 20 randomized patients: 10 controls and 10 with 50% heparin dose and heparin-coated circuits. All patients were ordinary elective coronary cases with two- or three-vessel disease without diabetes, neurologic disease, renal insufficiency, or coagulation defects.

The study protocol was approved by the Ethical Committee of the Medical Faculty. Informed consent was obtained from the patients.

Anesthesia
Premedication was given with morphine and scopolamine. Anesthesia was induced with fentanyl and thiopental and maintained with fentanyl and isoflurane. Muscle relaxation was obtained with pancuronium.

Cardiopulmonary Bypass
A Stöckert heart-lung machine with a roller pump was used. The CPB circuit consisted of a Maxima membrane oxygenator with a collapsible soft venous reservoir and a 40-µm Intercept arterial filter, all of it manufactured by Medtronic Inc, Minneapolis, MN. The tubings were made of polyvinylchloride. The heparin coating of all internal surfaces was made by Carmeda, Stockholm, Sweden, according to the Carmeda BioActive Surface method with end-linkage binding of heparin molecules to the artificial surface, which first is pretreated with an amination procedure [9]. A Cell Saver (Haemonetics Corp, Braintree, MA) was used instead of coronary suction. Priming was done with 2,000 mL of Ringer's acetate. Control patients received an initial heparin dose of 300 IU/kg body weight and additional heparin was given when the activated clotting time (ACT) fell to less than 400 seconds. For safety reasons the systemic heparinization was reduced in two steps in the study groups. In the first part of the study the initial heparin dose was 225 IU/kg and ACT was allowed to fall to 300 seconds before addition of heparin. In part two the heparin bolus was 150 IU/kg and supplement administered at ACT values less than 250 seconds. Reversal with protamine was started immediately after CPB with doses adjusted for heparin dose and ACT value. The flow was nonpulsatile and initially 2.2 L/m². After cooling to 30°C it was reduced by 25%.

A modified St. Thomas' solution was employed for cardioplegia. Cardiopulmonary bypass times and aortic cross-clamping times did not differ between the groups.

Protocol
Blood samples in EDTA were taken on five occasions: before anesthesia, after 45 minutes of CPB, at the end of CPB, 3 hours after protamine administration, and in the morning of the first postoperative day. The samples were immediately centrifuged, and plasma was removed and frozen to -20°C.

Biochemical Analysis
Levels of EPX and ECP in EDTA plasma were analyzed with radioimmunoassay methods commercially available from Pharmacia Diagnostics, Uppsala, Sweden.

For the investigation of the affinity of the eosinophil proteins to the heparinized surface the following technique was used. Sephadex G 15 gel (Pharmacia Fine Chemicals, Uppsala, Sweden) was aminated and then coated with heparin according to the Carmeda BioActive Surface method. Pasteur pipettes were packed with 5 cm of aminated or heparin-coated Sephadex G 15, respectively. Three different sera with known concentrations of ECP and EPX were filtered through the columns with a PBS buffer. Five hundred microliters of test serum was added to the column and then four consecutive fractions of about 500 µL each were taken out and analyzed. The aminated columns served as controls, and the recovery percentage was calculated.

Statistics
Student's t test was used.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
All patients had an uneventful intraoperative and postoperative course. The low heparin and ACT regimen caused no problems. The risk of clotting in stagnant blood made us, however, refrain from using the cardiotomy suction. There was no heparin-coated cardiotomy reservoir available at that time. The control patients in the two parts of the study, 7 and 10 patients, respectively, were treated in an identical manner and are reported as one group of 17 control patients. The 9 patients with heparin-coated circuits and 75% heparin bolus and the 10 patients with heparin-coated circuits and 50% heparin bolus are considered separately.

Changes in EPX levels are shown in Figure 1. In the control group there was a threefold increase during CPB with a decrease after bypass down to normalized values 3 hours after CPB. In the 75% and 50% heparin groups with heparin-coated circuits the EPX levels were significantly lower, increasing only about 50%.

View larger version (13K): [in this window] [in a new window]   Fig 1. . Changes in eosinophil protein X level (mean values ± standard error of the mean) in plasma during and after cardiopulmonary bypass (CPB) using standard equipment (), heparin-coated device with 50% systemic heparin bolus (), and heparin-coated device with 75% systemic heparin bolus dose ().

View larger version (11K): [in this window] [in a new window]   Fig 2. . Changes in eosinophil cationic protein level (mean values ± standard error of the mean) in plasma during and after cardiopulmonary bypass (CPB) using standard equipment (), heparin-coated device with 50% systemic heparin bolus (), and heparin-coated device with 75% systemic heparin bolus dose ().

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

Can granulocyte activation during CPB be reduced? We do not know the exact mechanism of activation. Hällgren and associates [11] found increased levels of serum ECP during hemodialysis. Finding no decreased content of ECP in circulating granulocytes, they postulated a local degranulation on the surfaces of the dialyzer. Activating properties of the surface, ie, the biocompatibility, should be important. Heparin coating (Carmeda BioActive Surface) reduces plasma levels of the neutrophil granule proteins lactoferrin and myeloperoxidase [8]. The question was raised if the plasma level reduction could be due to binding of released proteins to the heparinized surface. For myeloperoxidase this seems not to be true as no affinity to the Carmeda BioActive Surface was found [12]. Heparin's capacity to bind and neutralize ECP was reported previously by Fredens and associates [13]. Hence systemic heparinization per se probably will reduce ECP levels during CPB. In our study we confirmed a high degree of affinity of ECP to the heparinized surface, whereas there was no clear evidence of EPX binding.

It seems reasonable to conclude that the dramatic reduction of the levels of eosinophil degranulation products found in plasma during CPB using a heparin-coated device compared with the routine method is a combined effect of reduced eosinophil activation and clearance from plasma. The biocompatibility of the CPB circuit would be improved although the clinical benefits remain to be proved. The affinity of ECP to heparinized surfaces might be used as an indicator in quality control tests.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Address reprint requests to Dr Nilsson, Department of Thoracic Surgery, University Hospital, S-751 85 Uppsala, Sweden.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

1. 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;86:845–7.[Abstract]
2. Nilsson L, Nilsson U, Venge P, et al. Inflammatory system activation during cardiopulmonary bypass as an indicator of biocompatibility: a randomized comparison of bubble and membrane oxygenators. Scand J Thorac Cardiovasc Surg 1990;24:53–8.[Medline]
3. Riegel W, Spillner G, Schlosser V, Hörl WH. Plasma levels of main granulocyte components during cardiopulmonary bypass. J Thorac Cardiovasc Surg 1988;95:1014–9.[Abstract]
4. Van Oeveren W, Kazatchkine MD, Descamps-Latscha B, et al. Deleterious effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1985;89:888–99.[Abstract]
5. Nilsson L, Tydén H, Johansson O, et al. Bubble and membrane oxygenators-a comparison of postoperative organ dysfunction with special reference to inflammatory activity. Scand J Thorac Cardiovasc Surg 1990;24:59–64.[Medline]
6. Venge P. Human eosinophil granule proteins: structure, function and release. In: Smith H, Cook RM, eds. Immunopharmacology of eosinophils. London: Academic Press, 1993:43–55.
7. Peterson CGB, Enander I, Nystrand J, Anderson AS, Nilsson L, Venge P. Radioimmunoassay of human eosinophil cationic protein (ECP) by an improved method. Establishment of normal levels in serum and turnover in vivo. Clin Exp Allergy 1991;21:561–7.[Medline]
8. Borowiec J, Thelin S, Bagge L, Nilsson L, Venge P, Hansson HE. Heparin-coated circuits reduce activation of granulocytes during cardiopulmonary bypass. A clinical study. J Thorac Cardiovasc Surg 1992;104:642–7.[Abstract]
9. Larm O, Larsson R, Olsson P. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue. Biomater Med Device Artif Organs 1983;11:161–73.
10. Hällgren R, Samuelsson T, Venge P, Modig J. Eosinophil activation in the lung is related to lung damage in adult respiratory distress syndrome. Am Rev Respir Dis 1987;135:639–42.[Medline]
11. Hällgren R, Venge P, Wikström B. Hemodialysis-induced increase in serum lactoferrin and serum eosinophil cationic proteins as signs of local neutrophil and eosinophil degranulation. Nephron 1981;29:233–8.[Medline]
12. Videm V, Nilsson L, Venge P, Svennevig JL. Reduced granulocyte activation with a heparin-coated device in an in vitro model of cardiopulmonary bypass. Artif Org 1991;15:90–5.[Medline]
13. Fredens K, Dahl R, Venge P. In vitro studies of the interaction between heparin and eosinophil cationic protein. Allergy 1991;46:27–9.[Medline]

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Leif Nilsson Jan W. Borowiec Stefan Thelin Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nilsson, L. Articles by Thelin, S. Search for Related Content PubMed PubMed Citation Articles by Nilsson, L. Articles by Thelin, S.