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Interact CardioVasc Thorac Surg 2008;7:373-377. doi:10.1510/icvts.2007.168302
© 2008 European Association of Cardio-Thoracic Surgery
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Work in progress report - Coronary

Biochemical markers of myocardial injury in the pericardial fluid of patients undergoing heart surgery{star}

Angel L. Fernándeza,*, José B. García-Bengocheaa, Julián Alvarezb and José R. González Juanateyc

a Department of Cardiac Surgery, University Hospital of Santiago, 15706 Santiago de Compostela, Spain
b Department of Anesthesiology, University Hospital of Santiago, 15706 Santiago de Compostela, Spain
c Department of Cardiology, University Hospital of Santiago, 15706 Santiago de Compostela, Spain

Received 16 September 2007; received in revised form 21 January 2008; accepted 22 January 2008

{star} Presented at the 21st Annual Meeting of the European Association for Cardio-thoracic Surgery, Geneva, Switzerland, September 16–19, 2007.

*Corresponding author. Tel.: +34-981-950-212; fax: +34-981-950-227.

E-mail address: alfg{at}secardiologia.es (A.L. Fernández).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Conference discussion
 References
 
The purpose of this study was to compare cardiac markers in the pericardial fluid and serum in order to evaluate preoperative myocardial injury. Thirty patients were divided into three groups. The first group (AVR; n=10) received an aortic valve replacement. The second group (SA; n=10) included patients with stable angina who underwent elective coronary artery bypass grafting (CABG). The third group (ACS; n=10) included patients with acute coronary syndrome who underwent urgent CABG. Pericardial fluid and venous samples were taken after opening the pericardium and 24 h postoperatively. Serum and pericardial concentration of troponin I (cTnI), creatine kinase (CK), its MB isoenzyme (CK-MB) and myoglobin were determined. Preoperative pericardial cTnI was significantly (P<0.01) higher than in serum in all groups. Preoperative pericardial CK, CK-MB and myoglobin were significantly (P<0.01) lower than in serum in groups AVR and SA. Preoperative pericardial and serum cTnI were significantly higher in the ACS than in AVR and SA groups (P<0.01). Postoperative pericardial concentration of all markers was significantly higher (P<0.01) than in serum in all groups. We conclude that preoperative pericardial accumulation of cTnI may reflect subclinical injury which may not be demonstrated by the usual laboratory tests.

Key Words: Pericardium; Pericardial fluid; Troponin; Myocardial ischemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Conference discussion
 References
 
Cardiac interstitial fluid diffuses to the pericardial cavity through the epicardium and is the main component of the pericardial fluid. This fluid is considered an ultrafiltrate of plasma and contains an overall protein concentration lower than plasma [1]. In addition, the mesothelial cells may secrete different substances to the pericardial cavity. The membrane characteristics of mesothelial cells favor clearance rather than accumulation of fluid. The drainage of the pericardial cavity occurs both by the thoracic duct via the parietal pericardium and by the right lymphatic duct via the right pleural cavity [2, 3].

The myocardium and the epicardial fat are known to synthesize proteins which may exert both autocrine and paracrine functions [4]. Proteins from cardiomyocytes and fat that are released into the interstitial space may reach the peripheral circulation through the capillary network and the venous system. Alternatively, the myocardial interstitial fluid containing these substances may diffuse into the pericardial cavity through the visceral epicardium, being a component of the pericardial fluid [1, 2].

It has been described that there is a greater concentration of some proteins in pericardial fluid compared with serum. These substances are not actively concentrated from the serum but are most likely released from the myocardium and pericardial fat into the interstitial space and diffused subsequently into the pericardial cavity [5, 6]. The determination of serum levels of proteins that are primarily synthesized in the heart may underestimate the modifications which occur at the cardiac cellular level [4, 7].

Cardiac troponin I (cTnI), creatine kinase (CK) and its MB isoenzyme (CK-MB) and myoglobin are proteins that can be released from the damaged cardiomyocytes into the interstitial space [8]. They reach the peripheral circulation either by crossing the wall of myocardial capillaries or by diffusing through the epicardium into the pericardial cavity [9, 11]. Serum concentrations of these biochemical markers are increased in the acute coronary syndrome (ACS) and in other conditions such as pericarditis, cardiac surgery, and renal dysfunction [12].

The objective of this study is to determine if the levels of cTnI, CK, CK-MB and myoglobin in pericardial fluid are more reliable than their concentration in serum as an indicator of the preoperative myocardial injury.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Conference discussion
 References
 
Thirty patients who underwent open heart surgery were enrolled in a prospective manner. A written informed consent was obtained according to the protocol approved by the Institutional Ethics Committee.

Patients were classified into three groups. In the first group, patients underwent elective aortic valve replacement (AVR) (Group AVR; n=10) while the second group included the patients with stable angina who underwent elective coronary artery bypass grafting (CABG) (Group SA; n=10). The third group included patients who underwent urgent CABG due to an acute coronary syndrome (Group ACS; n=10).

All surgical interventions were performed through a median sternotomy incision. Standard cardiopulmonary bypass was used. After aortic cross-clamping, myocardial protection was achieved using cold cardioplegic solution. Both the left internal mammary artery and a great saphenous vein graft were harvested in all patients who underwent an elective CABG procedure, whereas six patients who underwent urgent CABG did not receive any arterial graft. After completing graft harvesting the pericardium was opened vertically and fluid sampling was taken by using a 10 ml syringe (BD Discardit II, Becton Dickinson, Ireland) and a 14 G catheter (Abbocath-T, Abbott, Sligo, Ireland). Simultaneous peripheral venous sample was withdrawn. Samples were transferred to Vacutainer SST II Advance sterile tubes (Plymouth, UK) and immediately centrifuged at 3600 rpm for 15 min. Determination of serum and pericardial fluid level of cardiac markers was carried out afterwards.

Before closing the sternum, two chest tubes were inserted and connected to an under-water seal draining system. In patients with either aortic valve replacement or CABG revascularized with only venous grafts, both tubes were placed in the pericardial cavity. When the left internal mammary artery was harvested, the left pleura was opened and one of the draining tubes was placed inside the left pleural cavity.

Additional peripheral blood and pericardial fluid samples were taken 24 h after admission into the postoperative cardiac care unit. Pericardial samples were obtained by puncturing the rubber conduit which connects the draining tubes and the under-water seal system.

CK-MB, cTnI and myoglobin serum and pericardial levels were analyzed with an enzyme immunoassay technique (Dade Behring, Newark, DE, USA). CK was analyzed by a colorimetric method (Dade Behring, Newark, DE, USA). Normal values of these cardiac proteins and analytical sensitivities of tests used were as follows: cTnI: normal value 0.00–0.05 ng/ml; sensitivity 0.04 ng/ml. CK: normal value 21–232 IU/l; sensitivity 0.001 IU/l; CK-MB: normal value 0–3.5 ng/ml; sensitivity: 0.5 ng/ml. Myoglobin: normal value 10–92 ng/ml; sensitivity 1 ng/ml.

All results were expressed as mean±S.D. Student's paired t-test was used in assessing differences within the groups. One-way repeated measures analysis of variance was used to assess the significance of differences between the groups. Linear regression was performed to analyze the relationship between serum and pericardial marker levels. A value of P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Conference discussion
 References
 
Demographic and preoperative data are shown in Table 1. No significant differences were observed between the groups with regard to age and sex. Left ventricular ejection fraction (LVEF) was lower in the ACS group. The preoperative use of intra-aortic balloon pump and the presence of left main coronary artery disease were significantly higher in this group.


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  		Table 1 Demographic and preoperative characteristics of the patients

 
When comparing pericardial and blood concentration of myocardial markers within the groups, three main findings could be observed. Firstly, pericardial cTnI resulted higher than serum concentration and superior to the normal range in the three groups. Secondly, patients with aortic valve disease and stable angina presented lower values of pericardial than serum CK, CK-MB and myoglobin. Finally, patients with ACS presented a high variability of biochemical markers concentration without significant differences between pericardial and serum CK, CK-MB and myoglobin.

Comparison between the groups could demonstrate that serum concentrations of the four cardiac markers tested were in normal ranges in groups AVR and SA while significantly increased values were observed in group ACS. This last group included patients with a preoperative diagnosis of unstable angina (six patients) and acute non-ST segment elevation myocardial infarction (four patients).

No differences could be demonstrated between groups AVR and SA regarding the pericardial concentration of cardiac markers, while the ACS group presented a significantly higher concentration of all the markers. The preoperative pericardial and serum concentrations of cardiac markers are shown in Table 2.


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  		Table 2 Preoperative serum and pericardial fluid levels of cTnI, CK, CK-MB and myoglobin

 
The postoperative course was uneventful in all patients in groups AVR and SA. Two patients of the ACS group presented postoperative low cardiac output in absence of new Q waves and one of them died two weeks after the intervention due to sepsis-related multiorgan failure. The 24 h postoperative serum concentrations of cTnI, myoglobin, CK and CK-MB were significantly higher in group ACS. Postoperative pericardial levels of all markers were significantly higher than serum concentrations with no significant differences between the three groups. The postoperative data are shown in Table 3.


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  		Table 3 Postoperative data of patients studied

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Conference discussion
 References
 
This present study has demonstrated that, in absence of signs suggesting preoperative myocardial ischemia, cTnI is higher in the pericardial fluid than in blood while CK, CK-MB and myoglobin are lower. There are two possible explanations for this finding. The first is the relationship between ultrafiltration and molecular weight [4, 7] while the second is the preferential leak of cardiac proteins from the interstitial space to the pericardial cavity instead of to the intravascular space [1].

It is assumed that the pericardial fluid is an ultrafiltrate of plasma through the myocardium reflecting the concentrations of all permeable substances and therefore the composition of cardiac interstitium in normal and diseased hearts. During utrafiltration the fluid is enriched in molecules produced by the cardiac tissue. Diffusion of proteins from the myocardial interstitium through the epicardium into the pericardial cavity depends on the molecular weight and protein structure. Under normal conditions proteins with a molecular weight lower than 40 kDa can easily diffuse into the pericardial cavity [7, 9]. The cTnI is a 23 kDa protein and therefore it may cross freely. Conversely, CK and its cardiospecific isoenzyme CK-MB, with a molecular weight of 85 kDa, is too large for traversing the epicardium and thus its pericardial concentration is lower than that in blood [9, 10].

The relation between ultrafiltration and molecular weight does not explain the lower pericardial level of myoglobin, a small protein of 17.5 kDa, that was observed in this study, as well as in a previous report [9]. Myoglobin was expected to migrate easily to the pericardial cavity due to its small size. However, the proportion of myoglobin released from cardiomyocytes is lower than from skeletal muscle and therefore in healthy subjects the serum concentration of myoglobin is higher than in pericardium.

On the other hand, the ultrafiltration process cannot explain the higher concentration of large molecules as lactate dehydrogenase (LDH) – 140 kDa – in the pericardial fluid compared to serum. A possible answer is the continuous and preferential leak of cardiac tissue proteins from the heart interstitium to the pericardial cavity which takes place in the steady state and after myocardial injury [1]. This leak provokes accumulation of proteins of cardiac origin in the pericardial fluid in spite of their relatively large molecular weight [11].

Moreover, this study could demonstrate that patients with recent or ongoing ischemia exhibited higher preoperative cTnI, CK, CK-MB and myoglobin concentration in the pericardial fluid than in serum [9]. Possible mechanisms involved in this pericardial accumulation of large, small, cardiospecific and non-specific myocardial markers include increased leaks of cytoplasmatic proteins due to dysfunction of the cardyomyocyte membrane and augmented vascular and epicardial permeability [2, 3]. As a consequence of myocardial ischemia, the integrity of the cell membrane is compromised and cytosolic proteins may be released into the myocardial interstitium. This accounts for a preferential protein leak from the heart to the pericardial cavity for all markers. In addition, a higher capillary and epicardial permeability may increase the macromolecular exchange of tissue and plasma proteins [1].

This study has also demonstrated that the 24-h postoperative concentration of biochemical markers resulted significantly higher in pericardial fluid than in serum, independently of the preoperative clinical diagnosis and the presence of postoperative myocardial infarction. Mechanisms that may explain such a finding include increased leak of intracellular proteins from cardiomyocytes secondary to surgical ischemia-reperfusion, cardiac mechanical trauma and finally the effect of postoperative inflammatory response [8]. Transient membrane dysfunction that causes enhanced cell membrane permeability rather than permanent cellular damage may be responsible for increasing the rate of release of cardiac markers after heart surgery in the absence of permanent cellular damage [13]. A variable degree of myopericardial postoperative inflammatory response which may result in cardiac edema is frequent after open heart surgery [12, 13]. Cardiac edema is accompanied by an increase in the myocardial interstitial hydrostatic pressure which expands tissue spaces and increases the distance necessary for diffusion of oxygen and nutrients from the capillaries which may negatively affect the myocardium [2].

In a previous report, a significant correlation between postoperative serum and pericardial markers concentration has been observed, suggesting that the time course of protein release was not different between pericardial fluid and serum [11]. In the present work as well as in other articles [10], there is no correlation between the postoperative levels of biochemical markers in pericardium and serum. Surgical trauma and tissue debris may cause difficult lymphatic drainage of the pericardial cavity and disturb normal function of the mesothelium [14]. A slower turnover of the substances present in the pericardial fluid may result and, therefore, a decrease of proteins reaching the systemic circulation [15]. As a consequence, the time required for the equilibrium between protein levels in pericardium and serum may be longer.

In conclusion, it remains a matter of debate if the enhanced preoperative accumulation of cTnI in the pericardial fluid of patients with and without evidence of ischemia reflects the normal condition of the cardiac interstitium or a subclinical injury which may not be demonstrated by the usual laboratory tests. A possible slower turnover of the pericardial fluid associated to an increased release of cardiac markers due to surgical trauma justifies the high postoperative concentration of cardiac markers of injury in the pericardium.


   Conference discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Conference discussion
References
 
Dr. P.T. Sergeant (Leuven, Belgium): It is correct to refocus our attention on this issue, even if this information has been reported earlier.

Have you controlled for a procedure where intramural or intraseptal dissections were done?

Dr. Fernandez: No, all the patients were operated by me, and no intramural coronary arteries were involved.

Dr. Sergeant: We see very often that studies on Troponine release do not mention whether there was suction of the pericardial fluid or not at the end of the procedure.

Should this not be included in these studies?

Dr. Fernandez: Well, do you refer to the postoperative or intraoperative period.

Dr. Sergeant: Or at the end of surgery or postoperative?

Dr. Fernandez: Well, of course we must take in mind that infusion of pericardial fluid into the circulation may increase serum concentration of markers.

Dr. D. Chambers (London, UK): If I understood you correctly, you took your samples at 24 h after surgery?

Dr. Fernandez: Yes.

Dr. Chambers: But CK-MB and CK and myoglobin will be released much earlier than that, so you possibly missed your highest release which you could have measured in your serum samples.

CK-MB peak release will occur around 8 h after surgery.

Dr. Fernandez: Yes, well, I agree. But we can't puncture the rubber connection every 6 h as this is only just an observational study. We thought that a single sample, 24 h after surgery, was the most significant sample.

Dr. Chambers: But you may not be measuring it at the most relevant time is all I'm saying, so you may have missed seeing the effect in your serum samples.

Dr. M. Grimm (Vienna, Austria): Did you correct for the amount of bleeding when calculating the levels of the troponin, for example?

Dr. Fernandez: No. We did not take into account this.

Dr. G.K. Mani (New Delhi, India): This is not a question directly for this paper, but I've been left very confused after this session about various issues, whether calcium channel blockers should be used in radial artery, whether the heparin dose should be ACT of 400, whether aspirin should be used in conjunction with clopidogrel.

I would request the Chair to put some light on these issues because they're very current.

Dr. Sergeant: Scientific discussions are very often to identify problems and then see from different perspectives. I don't know if any of the studies allow us to present the solutions to the studies that have been presented here today. At least they have identified problems, and that's what it's all about.

I think only an in-depth analysis of the problem will help you try to find the solutions. But I think the authors have been very correct in their inference building of their manuscripts.

So, there are still some problems left over. It is clear that the problem of aspirin resistance is an existing problem, and it's clear, at least for me it's rather clear, that aspirin alone is insufficient therapy certainly after off-pump coronary surgery and certainly in the presence of aspirin resistance. If there is aspirin resistance, it makes even no sense. There are suggestions of clopidogrel. There are suggestions of low molecular weight in therapeutic doses.

I think the best thing to do as is well-known in learning curves and in operational learning is to identify for yourself how the prevalence of the events are. We run very strict databases, and we have optimized procedures through evaluation of the systems.

Dr. Mani: What about calcium channel blockers and radial artery?

Dr. Sergeant: We don't use radial arteries for the reasons that were identified in the abstracts.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Conference discussion
 References
 

  1. Ben-Horin S, Shinfeld A, Kachel E, Chetrit A, Livneh A. The composition of normal pericardial fluid and its implications for diagnosing pericardial effusions. Am J Med 2005;118:636–640.[CrossRef][Medline]
  2. Mehlhorn U, Geissler HJ, Laine GA, Allen SJ. Myocardial fluid balance. Eur J Cardiothorac Surg 2001;20:1220–1230.[Abstract/Free Full Text]
  3. Stewart RH, Rohn DA, Allen SJ, Laine GL. Basic determinants of epicardial transudation. Am J Physiol 1997;273:H1408–H1414.[Medline]
  4. Fujita M, Komeda M, Hasegawa K, Kihara Y, Nohara R, Sasayama S. Pericardial fluid as a new material for clinical heart research. Int J Cardiol 2001;77:113–118.[CrossRef][Medline]
  5. Ege T, Canbaz S, Yuksel V, Duran E. Effect of pericardial fluid pro-inflammatory cytokines on hemodynamic parameters. Cytokine 2003;23:47–51.[CrossRef][Medline]
  6. Klemola R, Tikkanen I, Vuolteenaho O, Toivonen L, Laine M. Plasma and pericardial fluid natriuretic peptide levels in post-infarction ventricular dysfunction. Eur J Heart Fail 2001;3:211–226.
  7. Tambara K, Fujita M, Miyamoto S, Doi K, Nishimura K, Komedad M. Pericardial fluid level of heart-type cytoplasmic fatty acid-binding protein (H-FABP) is an indicator of severe myocardial ischemia. Int J Cardiol 2004;93:281–284.[CrossRef][Medline]
  8. Abramov D, Abut-Tailakh M, Frieger M, Ganiel A, Tuvbin D, Wolak A. Plasma troponin levels after cadiac surgery vs. after myocardial infarction. Asian Cardiovasc Thorac Ann 2006;14:530–535.[Abstract/Free Full Text]
  9. Ege T, Hulusi M, Cikirikcioglu M, Arar C, Duran E. Analysis of C-reactive protein and biochemical parameters in pericardial fluid. Yonsey Med J 2006;47:372–376.
  10. Cihan HB, Gulca O, Hazar A, Turkoz R, Olmez E. Determination of the pericardial to serum myoglobin ratio for the early diagnosis of peri-operative myocardial infarction after coronary artery bypass grafting. Pharmacol Res 2004;50:247–251.[CrossRef][Medline]
  11. Fellahi JL, Léger P, Philippe E, Arthaud M, Riou B, Gandjbakhch I, Coriat P. Pericardial cardiac troponin I release after coronary artery bypass grafting. Anesth Analg 1999;89:829–834.[Abstract/Free Full Text]
  12. Botha P, Nagarajan DV, Lewis PS, Dunning J. Can cardiac troponins be used to diagnose a perioperative myocardial infarction post-cardiac surgery? Interact Cardiovasc Thorac Surg 2004;3:442–449.[Abstract/Free Full Text]
  13. Mahajan N, Mehta Y, Rose M, Shani J, Lichstein E. Elevated troponin level is not synonymous with myocardial infarction. Int J Cardiol 2006;111:442–449.[CrossRef][Medline]
  14. Bodega F, Zocchi L, Agostoni E. Albumin transcytosis in mesothelium. Am J Physiol 2002;282:L3–L11.
  15. Boulanger B, Yuan Z, Flessner M, Hay J, Johnston M. Pericardial fluid absortion into lymphatic vessels in sheep. Microvasc Res 1999;57:174–186.[CrossRef][Medline]




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