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Comparison of Euro-Collins and University of Wisconsin solutions in the isolated perfused rat liver model: evaluation by proton nuclear magnetic resonance spectroscopy
Comparison of Euro-Collins and University of Wisconsin Solutions in the Isolated Perfused Rat Liver Model: Evaluation by Proton Nuclear Magnetic Resonance Spectroscopy H. Gibelin, M. Eugene, W. Hebrard, Y. Malard, M. Carretier, and T. Hauet
P
RIMARY dysfunction or nonfunction of liver graft is still an important problem in liver transplantation despite surgical progress. The determination of new markers of graft viability and metabolic activities seems to be an important challenge to assess the early graft function. Proton nuclear magnetic resonance spectroscopy (NMRp) has previously demonstrated its interest in the evaluation of renal allograft dysfunction after ischemia reperfusion in isolated perfused pig kidney.1 For the kidney, NMRp study determines specific markers of function like TMAO and citrate. The aim of this study was to assess liver graft function in an isolated perfused rat liver model after 24 hours of preservation in Euro-Collins (EC) or University of Wisconsin (UW) solution.
MATERIALS AND METHODS The animals were divided in two experimental groups; the EuroCollins preservation group (EC, n ⫽ 10) and the University of Wisconsin preservation group (UW, n ⫽ 0). Rat livers were flushed in situ via the portal vein with cold (4°C) EC and UW and preserved for 24 hours at 4°C. After preservation, livers were reperfused via the portal vein for 90 minutes at 37.4°C and at a constant flow with oxygenated Krebs-Henseleit-bicarbonate (KHB) buffer containing bovine albumin, lactates, taurocholate, benzoic acid, alanine, and alphacetoglutarate for functional evaluation. Effluent and perfusate samples were collected after 15, 30, 45, 60, 75 and 90 minutes of reperfusion. The following functional parameters were measured: pressure and resistances, bile, and transaminases production. Production of lactate, pyruvate, citrate, and succinate (Krebs cycle metabolites), b-hydroxybutyrate and acetoacetate (ketones bodies production) were measured by NMRp in the effluents. Results were analysed using the Student Newman Keuls test with P ⬍ .05 values being considered significant.
RESULTS
During reperfusion, resistances were significantly lower in UW than in EC group (0.9 ⫾ 0.03 vs 1.32 ⫾ 0.14 mm Hg/mL per minute at t15; 0.77 ⫾ 0.08 vs 1.27 ⫾ 0.9 at t45; 0.92 ⫾ 0.07 vs 1.31 ⫾ 0.1 at t90, P ⬍ .05) and correlated with a significant improvement of bile production in UW group (1.3 ⫾ 0.04 vs 0.2 ⫾ 0.2, P ⬍ .05). Transaminases production was not significantly different in these groups (data not shown). However, detection of lactates, pyruvate, succinate, citrate, aceto-acetate, and b-hydroxybutyrate by NMRp was
Fig 1. Proton NMR spectra of isolated perfused rat liver after preservation in EC or UW solutions and during normothermic reperfusion.
significantly higher (P ⬍ .05) in the UW group than in the EC group (Fig 1). DISCUSSION
UW solution is the gold standard in liver transplantation. In this study, the UW solution improved bile production and reduced resistances when compared to the EC. Transaminases activity was similar in the two groups, and this parameter was not sufficiently efficient to differentiate the experimental groups in this model. In contrast, the NMRp study was efficient to assess different metabolic pathways as the Krebs cycle and the ketogenesis. These two pathways are important for the evaluation of metabolic function and oxydative metabolism restauration after prolonged ischemia and normothermic reperfusion. In this study, the poor quality of EC preservation was highlighted by a very low metabolic activity as determined by the absence of excretion From the Laboratoire de Transplantation Experimentale, INRA Le Magneraud, Surge`res and GRTMV EA 2624, Faculte´ de Me´decine, Poitiers, France. Supported by grants from the Foundation pour la Recherche Me´dicale and Etablissement Franc¸ais des Greffes (EFG). Address reprint requests to Th Hauet, Laboratoire de Transplantation Expe´rimentale, Institut National de Recherche Agronomique Le Magneraud, BP 52, 17770 Surge`res, France.
0041-1345/00/$–see front matter PII S0041-1345(00)01888-1
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2792
Transplantation Proceedings, 32, 2792–2793 (2000)
COMPARISON OF SOLUTIONS
of different metabolites by NMRp study. In contrast in the UW group, the different parameters were detected during reperfusion. This detection indicated a better metabolic recovery after ischemia reperfusion. In conclusion, NMRp is a discriminating technique for the study of different hepatic metabolic pathways when compared to conventional biochemical methods. This evaluation of metabolic status allows an efficient evaluation of preservation quality
2793
of liver grafts and the functional recovery during reperfusion. ACKNOWLEDGMENT We thank C. Henry for the excellent technical assistance.
REFERENCES 1. Hauet T, Mothes D, Goujon JM, et al: J Urol 157:1155, 1997
P
RIMARY dysfunction or nonfunction of liver graft is still an important problem in liver transplantation despite surgical progress. The determination of new markers of graft viability and metabolic activities seems to be an important challenge to assess the early graft function. Proton nuclear magnetic resonance spectroscopy (NMRp) has previously demonstrated its interest in the evaluation of renal allograft dysfunction after ischemia reperfusion in isolated perfused pig kidney.1 For the kidney, NMRp study determines specific markers of function like TMAO and citrate. The aim of this study was to assess liver graft function in an isolated perfused rat liver model after 24 hours of preservation in Euro-Collins (EC) or University of Wisconsin (UW) solution.
MATERIALS AND METHODS The animals were divided in two experimental groups; the EuroCollins preservation group (EC, n ⫽ 10) and the University of Wisconsin preservation group (UW, n ⫽ 0). Rat livers were flushed in situ via the portal vein with cold (4°C) EC and UW and preserved for 24 hours at 4°C. After preservation, livers were reperfused via the portal vein for 90 minutes at 37.4°C and at a constant flow with oxygenated Krebs-Henseleit-bicarbonate (KHB) buffer containing bovine albumin, lactates, taurocholate, benzoic acid, alanine, and alphacetoglutarate for functional evaluation. Effluent and perfusate samples were collected after 15, 30, 45, 60, 75 and 90 minutes of reperfusion. The following functional parameters were measured: pressure and resistances, bile, and transaminases production. Production of lactate, pyruvate, citrate, and succinate (Krebs cycle metabolites), b-hydroxybutyrate and acetoacetate (ketones bodies production) were measured by NMRp in the effluents. Results were analysed using the Student Newman Keuls test with P ⬍ .05 values being considered significant.
RESULTS
During reperfusion, resistances were significantly lower in UW than in EC group (0.9 ⫾ 0.03 vs 1.32 ⫾ 0.14 mm Hg/mL per minute at t15; 0.77 ⫾ 0.08 vs 1.27 ⫾ 0.9 at t45; 0.92 ⫾ 0.07 vs 1.31 ⫾ 0.1 at t90, P ⬍ .05) and correlated with a significant improvement of bile production in UW group (1.3 ⫾ 0.04 vs 0.2 ⫾ 0.2, P ⬍ .05). Transaminases production was not significantly different in these groups (data not shown). However, detection of lactates, pyruvate, succinate, citrate, aceto-acetate, and b-hydroxybutyrate by NMRp was
Fig 1. Proton NMR spectra of isolated perfused rat liver after preservation in EC or UW solutions and during normothermic reperfusion.
significantly higher (P ⬍ .05) in the UW group than in the EC group (Fig 1). DISCUSSION
UW solution is the gold standard in liver transplantation. In this study, the UW solution improved bile production and reduced resistances when compared to the EC. Transaminases activity was similar in the two groups, and this parameter was not sufficiently efficient to differentiate the experimental groups in this model. In contrast, the NMRp study was efficient to assess different metabolic pathways as the Krebs cycle and the ketogenesis. These two pathways are important for the evaluation of metabolic function and oxydative metabolism restauration after prolonged ischemia and normothermic reperfusion. In this study, the poor quality of EC preservation was highlighted by a very low metabolic activity as determined by the absence of excretion From the Laboratoire de Transplantation Experimentale, INRA Le Magneraud, Surge`res and GRTMV EA 2624, Faculte´ de Me´decine, Poitiers, France. Supported by grants from the Foundation pour la Recherche Me´dicale and Etablissement Franc¸ais des Greffes (EFG). Address reprint requests to Th Hauet, Laboratoire de Transplantation Expe´rimentale, Institut National de Recherche Agronomique Le Magneraud, BP 52, 17770 Surge`res, France.
0041-1345/00/$–see front matter PII S0041-1345(00)01888-1
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2792
Transplantation Proceedings, 32, 2792–2793 (2000)
COMPARISON OF SOLUTIONS
of different metabolites by NMRp study. In contrast in the UW group, the different parameters were detected during reperfusion. This detection indicated a better metabolic recovery after ischemia reperfusion. In conclusion, NMRp is a discriminating technique for the study of different hepatic metabolic pathways when compared to conventional biochemical methods. This evaluation of metabolic status allows an efficient evaluation of preservation quality
2793
of liver grafts and the functional recovery during reperfusion. ACKNOWLEDGMENT We thank C. Henry for the excellent technical assistance.
REFERENCES 1. Hauet T, Mothes D, Goujon JM, et al: J Urol 157:1155, 1997