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Eur J Cardiothorac Surg 2001;19:321-325
© 2001 Elsevier Science NL

Investigations on the new free radical scavenger polynitroxyl-albumin to prevent ischemia and reperfusion injury after orthotopic heart transplantation in the pig model

Juergen Martina, Georg Luttera, Koppany Saraia, Mareike Senn-Grossbergera, Noriyuki Takahashic, José Bitu-Morenod, Joerg Haberstrohb, Friedhelm Beyersdorfa

a Department of Cardiovascular Surgery, Albert-Ludwigs-University, Hugstetter Strasse 55, D-79106 Freiburg, Germany
b Department of Surgical Research, Albert-Ludwigs-University, Freiburg, Germany
c Department of Cardiovascular Surgery, Sapporo University Medical Center, Sapporo, Japan
d Faculdade de Medicina de Marilia e Faculdade de Medicina de Botucatu, State University of São Paulo, São Paulo, Brazil

Received 2 August 2000; received in revised form 22 November 2000; accepted 13 December 2000.

Corresponding author. Tel.: +49-761-270-2818; fax: +49-761-270-2550
e-mail: martin{at}ch11.ukl.uni-freiburg.de


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Nitroxides have strong antioxidant capacity but their effectiveness is limited by their rapid intracellular inactivation. Polynitroxyl-Albumin (PNA) is capable of regenerating inactivated nitroxide. We tested the effect of PNA against reperfusion injury in heart transplantation. Methods: Pig hearts were transplanted orthotopically. In the control group (n=9) reperfusion was performed without reperfusion modifications. In the experimental group (n=10) 1 ml/kg PNA was given before cross-clamp release. Results: Hemodynamic performance was impaired after transplantation in both groups without significant intergroup differences. Plasma malonedialdehyde levels were significantly diminished in the PNA group as compared to the controls. CK-MB levels in both groups were increased within the first 2 h of reperfusion without significant intergroup differences. In contrast, there were found significant higher values of myocardial specific lactate dehydrogenase (LD1) in the controls versus PNA group. Conclusions: PNA was able to reduce lipid peroxidation and attenuate free radical activity. Contractile dysfunction could no be improved, indicating that (a) the radical scavenging effect was to weak or (b) other mechanisms than free oxygen radicals are responsible for myocardial damage in this experimental model.

Key Words: Reperfusion injury • Heart transplantation • Free radical scavenger • Polynitroxyl-albumin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Considerable evidence suggests that oxygen-derived free radicals are involved in ischemia/reperfusion injury [1–4]. But due to their large molecular weights most free radical scavengers, e.g. superoxide dismutase and catalase, can not cross cell membranes and are active against extracellular free radicals only [5–7].

Nitroxides such as 4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl (TEMPOL) exhibit superoxide dismutase-mimicking properties [8–14]. However, their effect is limited by rapid inactivation in the intracellular space [15,16]. The novel polynitroxyl-albumin (PNA), prepared by labeling human serum albumin with nitroxide, is membrane impermeable and distributes in the extracellular space (Fig. 1). PNA can reverse the bioreduction of TEMPOL resulting in a strikingly improved stability of the nitroxide [17,18]. PNA reduces infarct size in focal cerebral ischemia and inhibits ischemia-reperfusion induced leucocyte-endothelial cell adhesion [19,20].



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  		Fig. 1. The intracellular space is the major site of nitroxide inactivation by reduction to hydroxylamine. When PNA is present, the hydroxylamine which diffuses back into the extracellular space is regenerated by PNA to nitroxide. PNA is membrane impermeable and distributes in the vascular and extracellular space.

 
The purpose of this study was to investigate the effectiveness of PNA to attenuate reperfusion injury in a pig model of orthotopic heart transplantation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
Pigs of the German Landrace type weighing 25±4 kg were anaesthesized, intubated and mechanically ventilated. All procedures were performed in conformity with the European Convention on Animal Care and approved by the local ethics committee.

2.1. Procurement of the donor heart
One thousand five hundred millilitres of Bretschneider's HTK solution (4° C) were given via the ascending aorta over a period of 8–10 min at a pressure of 40 mmHg. The heart was excised and stored in ice-cold Bretschneider's solution for 3 h.

The heart lung machine (Stöckert, München, Germany) was primed with 1000 ml of hydroxyethyl starch. The ascending aorta and both venae cavae were cannulated. The blood flow was kept at 2.2–2.5 l/min.

Orthotopic heart transplantation was performed in a biatrial technique.

2.2. Experimental groups
In the control group (n=9) cross-clamping was released after an ischemic time of 4 h. No reperfusion modifications were performed. In the PNA group (n=10) all procedures were performed as in the control group, but reperfusion after transplantation was started with the application of PNA (B. Braun Melsungen AG, Melsungen, Germany) at a dosage of 1 ml/kg (3.26 µmol/kg) body weight (24.1 g/dl protein), a temperature of 37°C, and a pressure of 30–40 mmHg via the aortic root over a period of 1 min immediately before the aortic clamp was released.

Hemodynamics were measured before procurement of the donor heart and 2 h after cross-clamp release. Cardiac preload was increased by stepwise volume loading and Starling curves were registered. Left and right ventricular stroke work were calculated and normalized for heart weight as stroke work index:

where SWI, stroke work index (mJ/g); MAP, mean arterial pressure (mmHg); LAP, left atrial pressure (mmHg); PAP, mean pulmonary artery pressure (mmHg); CVP, central venous pressure (mmHg); CO, cardiac output (l/min); HR, heart rate (beats/min), HW, heart weight (g)

To assess the contractility in the experimental groups we used the maximal achieved left and right ventricular stroke work index (LVSWImax RVSWImax, respectively). Dp/dtmin and dp/dtmax were measured by Millar catheters (Millar, Houston, TX).

2.3. Enzymes
Myocardial fractions of creatine kinase (CK-MB) and lactate dehydrogenase (LD 1) in percent of total CK and LD were measured (REP-CK/LD-isoenzyme combo method, Helena Laboratories, Beaumont, TX).

Malondialdehyde (MDA).The plasma concentration of MDA, a marker of lipid peroxidation, was measured by a colorimetric assay kit (Lipid Peroxidation Assay Kit, Calbiochem, San Diego, CA).

2.4. Statistical analysis
Statistical analysis was performed with a statistical computer program (Prism, Graph Pad Software, San Diego, CA). The incidence of sinus rhythm and arrhythmias was analysed by Fischer's exact test. Comparison of the post-transplant values with the baselines was performed running the Wilcoxon signed rank test with a two-tail P value. Data of the experimental group were compared to the control group using the Mann–Whitney test. Repeated enzyme measurements and MDA levels were compared by Friedman test, for significance testing between the several time points Dunn's Multiple Comparison Test was used. P<0.05 was considered statistically significant. Group statistics were expressed as mean±standard deviation.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
All hearts could be weaned from CPB after 76.7±16.0 min in the control group and 77.9±24.7 min in the PNA group (P=0.90). In the control group 100% and in the PNA group 90% of the animals regained sinus rhythm (P=1.0). The incidence of supraventricular or ventricular arrhythmias was 11% in the control group and 10% in the PNA group (P=1.0). The number of postoperative defibrillations/animal was 3.4±1.9 vs. 2.7±1.1 in the control vs. the experimental group (P=0.33).

Hemodynamics (Table 1, Figs. 2 and 3) remained stable throughout the observation period. Inotropic support was necessary during weaning from CPB only and stopped at least 30 min before hemodynamic measurements were performed.


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  		Table 1. Hemodynamic parameters at standardized left ventricular preload (LAP 8 mmHg)a

 


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  		Fig. 2. Hemodynamic data measured 2 h after start of reperfusion and expressed as fraction of baseline. CO, cardiac output; HR, heart rate; CVP, central venous pressure; MAP, mean arterial pressure; PAP, mean pulmonary artery pressure. Data are given as mean±standard deviation. *P<0.05 vs. pretransplant value There were no intergroup differences found.

 


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  		Fig. 3. Hemodynamic data measured 2 h after start of reperfusion and expressed as fraction of baseline. LVSWImax, maximal left ventricular stroke work index; RVSWImax, maximal right ventricular stroke work index; LVdp/dtmax, maximum of the first derivative of left ventricular pressure; LVdp/dtmin, minimum of the first derivative of left ventricular pressure; RVdp/dtmax, maximum of the first derivative of right ventricular pressure; RVdp/dtmin, minimum of the first derivative of right ventricular pressure Data are given as mean ± standard deviation. *P<0.05 vs. pretransplant value. There were no intergroup differences found.

 
Cardiac output and maximal stroke work index of both ventricles were significantly impaired after transplantation in both groups. There were no significant intergroup differences.

Left ventricular dp/dtmax was significantly impaired after transplant in the PNA group, but not in the control group. Left ventricular dp/dtmin was significantly diminished in both groups after transplantation. Right ventricular parameters did not change significantly after transplantation.

3.1. Enzymes (Figs. 4 and 5)
CK-MB serum concentration was expressed in % of total CK. Because baseline values varied considerably among the different animals, all enzyme values were expressed as a fraction of baseline.



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  		Fig. 4. CK-MB serum levels in % of total CK expressed as fractions of baseline at 5, 15, 30, 60, and 120 min after start of reperfusion. *P<0.05 vs. baseline (Dunn's multiple comparison test).

 


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  		Fig. 5. LD1 serum levels as fractions of baseline at 5, 15, 30, 60, and 120 min after start of reperfusion. *P<0.05 vs. baseline (Dunn's multiple comparison test); {dagger}P<0.05 vs. control group.

 
In the control group CK-MB activity was significantly increased versus baseline at 30, 60, and 120 min after start of reperfusion (P<0.05). In the PNA group a significant increase of CK-MB versus baseline was found after 60 and 120 min (P<0.01). Differences of the CK-MB levels between the PNA group and the control group were not significant.

LD1 levels in the control group were significantly increased as compared to baseline after 5, 15, 30, 60, and 120 min (P<0.05). In contrast, there was only a slight increase of the enzyme release in the PNA group becoming significant after 30, 60 and 120 min (P<0.05). There were significantly decreased LD1 levels in the PNA group as compared to the controls at 5, 15, 30, 60, and 120 min.

3.2. Malondialdehyde (MDA) (Fig. 6)
MDA plasma levels were increased versus baselines at 5, 60, and 120 min after start of reperfusion in the control group (P<0.05) and after 120 min only in the PNA group (P<005). Intergroup differences were significant after 5, 15, 60 and 120 min.



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  		Fig. 6. MDA plasma levels as fractions of baseline at 5, 15, 30, 60, and 120 min after start of reperfusion. *P<0.05 vs. baseline (Dunn's multiple comparison test); {dagger}P<0.05 versus control group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The evaluation of PNA to reduce reperfusion injury in this experimental model of myocardial ischemia revealed:
  1. There was no significant difference between the two groups concerning mortality, the time for weaning from cardiopulmonary bypass, the number of defibrillations and the incidence of arrhythmias
  2. Contractility in both groups was significantly impaired after heart transplantation
  3. CK-MB showed no intergroup differences. In contrast, all post-transplant LD1 levels were significantly decreased in the PNA group versus the control group.
  4. Malondialdehyde levels after transplantation were significantly reduced in the PNA group

The significantly decreased levels of malondialdehyde after start of reperfusion indicate a reduction of lipid peroxidation due to free radical injury [21]. Free radicals are generated during early reperfusion with a burst peaking at 2–3 min after onset of reflow and abating within 20 min [4]. The administration of PNA immediately before start of reperfusion could markedly attenuate this burst of free radical generation.

The reduction of free radical lipid peroxidation by PNA was not associated with an improved contractility after heart transplantation. Furthermore, the application of PNA resulted in a diminished release of myocardial specific LD1, but was not accompanied by decreased CK-MB levels.

A possible explanation for this discrepancy could be (1) that PNA was able to reduce the burst of free radicals but could not completely prevent myocardial damage by these agents or (2) that additional mechanisms are responsible for ischemia/reperfusion injury in this experimental model.

Contractile dysfunction after heart transplantation can be explained by myocardial stunning due to cardioplegia and cold ischemia. Energy depletion, acidosis, and crucial dysfunction of ionic transport mechanisms can cause severe myocardial damage [22].

The application of PNA before reperfusion may be helpful to attenuate injury by free oxygen radicals but is not able to prevent myocardial stunning, a major problem in heart transplantation.


   Acknowledgments
 
Supported by the Clinical Research Center II of the Albert-Ludwigs-University Freiburg, grant No B4 and the B. Braun Melsungen AG, Melsungen, Germany. Dr José Bitu-Moreno was supported by the Fundacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil, grant No 97/13631-4.


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

  1. Zweier J.L., Flaherty J.T., Weisfeldt M.L. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 1987;84:1404-1407.[Abstract/Free Full Text]
  2. Bolli R. Oxygen derived free radicals and myocardial reperfusion injury: an overview. Cardiovasc Drugs Ther 1991;5:249-268.
  3. Steare S.E., Yellon D.M. The potential for endogenous myocardial antioxidants to protect the myocardium against ischaemia-reperfusion injury: refreshing the parts exogenous antioxidants cannot reach?. J Mol Cell Cardiol 1995;27:65-74.[Medline]
  4. Bolli R. Basic and clinical aspects of myocardial stunning. Progr Cardiovasc Dis 1998;40:477-516.[Medline]
  5. Jolly S.R., Kane W.J., Bilie M.B., Abrams G.D., Lucchesi B.R. Canine myocardial reperfusion injury: its reduction by superoxide dimutase but not catalase. Circ Res 1984;54:277-285.[Abstract/Free Full Text]
  6. Ambrosio G., Becker L.C., Hutchins G.M., Weisman H.F., Seisfeldt M.L. Reduction in experimental infarct size by recombinant human superoxide dismutase: insights into the pathophysiology of reperfusion injury. Circulation 1986;74:1424-1433.[Abstract/Free Full Text]
  7. Courtois M., Maupoil V., Fantini E., Durot I., Javouhey-Donzel A., Athias P., Grynberg A., Rochette L. Correlation between direct ESR spectroscopic measurements and electromechanical and biochemical assessments of exogenous free radical injury in isolated rat cardiac myocytes. Free Rad Biol Med 1998;24:121-131.[Medline]
  8. Belkin S., Mehlhorn R.J., Hideg K., Hankovsky O., Packer L. Reduction and destruction rates of nitroxide spin probes. Arch Biochem Biophys 1987;256:232-243.[Medline]
  9. Mitchell J.B., Samuni A., Krishna C.M., DeGraff W.G., Ahn M.S., Samuni U., Russo A. Biologically active metal-independent superoxide dismutase mimics. Biochemistry 1990;29:2802-2807.[Medline]
  10. Samuni A., Krishna C.M., Mitchell J.B., Collins C.R., Russo A. Superoxide reaction with nitroxides. Free Rad Res Comm 1990;9:241-249.[Medline]
  11. Mehlhorn R.J., Swanson C.E. Nitroxide-stimulated H2O2 decomposition by peroxides and pseudoperoxides. Free Rad Res Comm 1992;17:157-175.[Medline]
  12. Voest E.E., van Faasen E., Marx J.J.M. An electron paramagnetic resonance study of the antioxidant properties of the nitroxide free radical TEMPO. Free Rad Biol Med 1993;15:589-595.[Medline]
  13. Twomey P., Taira J., DeGraff W., Mitchell J.B., Russo A., Krischna M.C., Hankovsky O.H., Frank L., Hideg K. Direct evidence for in vivo nitroxide free radical production from a new antiarrhythmic drug by EPR spectroscopy. Free Rad Biol Med 1997;22:909-916.[Medline]
  14. Gelvan G., Saltman P., Powell S.R. Cardiac reperfusion damage prevented by a nitroxide free radical. Proc Natl Acad Sci USA 1991;88:4580-4584.
  15. Swartz H.M., Sentjure M., Morse P.D. Cellular metabolism of water-soluble nitroxides: effect on rate of reduction of cell/nitroxide ratio, oxygen concentrations and permeability of nitroxides. Biochim Biophys Acta 1986;888:82-90.[Medline]
  16. Chen K., Morse P.D., Swarzt H.M. Kinetics of enzyme-mediated reduction of lipid soluble nitroxide spin labels by living cells. Biochim Biophys Acta 1988;943:477-484.[Medline]
  17. Kuppusamy P., Wang P., Zweier J.L. Electron paramagnetic resonance imaging of rat heart with nitroxide and polynitroxyl-albumin. Biochemistry 1996;35:7051-7057.[Medline]
  18. Kuppusamy P., Wang P., Shankar R.A., Ma L., Trimble C.E., Hsia C.J., Zweier J.L. In vivo topical EPR spectroscopy and imaging of nitroxide free radicals and polynitroxyl-albumin. Magnet Res Med 1998;40:806-811.
  19. Beaulieu C., Busch E., Röther J., de Crespigny A., Hsia C.J.C., Moseley M.E. Polynitroxyl albumin reduces infarct size in transient focal cerebral ischemia in the rat: potential mechanisms studied by magnetic resonance imaging. J Cereb Blood Flow Metab 1998;18:1022-1031.[Medline]
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  21. Ceconi C., Cargnoni A., Pasini E., Condorelli E., Curello S., Ferrari R. Evaluation of phospholipid peroxidation as malondialdehyde during myocardial ischemia and reperfusion inury. Am J Physiol 1991;260:H1057-H1061.[Abstract/Free Full Text]
  22. Opie L.H. Reperfusion injuriy and its pharmacologic modification. Circulation 1989;80:1049-1062.[Abstract/Free Full Text]



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