Mischinger, HJ

Murase, N

Quehenberger, F

Rao, PN

Snyder, JT

Starzl, TE

Todo, S

PubMed

D-Scholarship@Pitt

Levels of Purine Nucleoside Phosphorylase (PNP) as a Viability Marker of Nonparenchymal Cells in Cold Preserved Livers H.J. Mischinger, P.N. Aao, S. Todo, J.T. Snyder, F. Quehenberger, N. Murase, and T.E. Starzl COLD preservation of the liver with currently used solutions is limited to a few hours and is often accompanied by increased rates of postoperative graft complications.1.2 Although the University of Wisconsin (UW) solution has significantly increased the optimal pres-ervation period, primary nonfunction of the graft remains a serious problem in liver transplantation.l-S While the exact mechanism underlying storage and reperfusion injury is still unkno'W'Tl. injury to the microvasculature by toxic oxygen radicals is the most favored hypothesis.6 ,7 Pres-ently. no method is available to predict the viability of the preserved liver. and biochemical assessments of graft viability after preservation are mainly focused on enzyme release.8-'O bile production. 1 1.12 and energy status, 13,14 We had recently determined that the extent of reperfusion injury to the microvascular endothelial cell could be accu-rately predicted by levels of PNP. an enzyme localized mainly in the cytoplasm of endothelial and Kupffer cells. ,s ,'6 Therefore. this study was carried out to assess if PNP could be used to predict the extent of preservation injury to the endothelial cell and hence the viability of the preserved grafts. MATERIALS AND METHODS Male Lewis (LEW) rats (Charles River Laboratories. Wilmington. Mass). weighing 250 to 300 g were used as liver donors. All animals were induced and maintained with inhalational methoxy-fturane. Three hundred units of heparin were administered via the penile vein prior to harvesting the liver. For bile collection. the common bile duct was intubated with polyethylene tubing (Clay Adams PE·IO. 10 0.2 mm). Immediately after cannulating the abdominal ·aorta. a thoracotomy was performed and after cross-clamping the thoracic aorta the right atrium was opened. The liver was gently flushed with 20 cc ice cold UW solution via the aorta. and the portal vein intubated with a l6-gauge Teflon angiocathe-ter. Finally the suprahepatic IVC was ligated. infrahepatic IVC cannulated with a l4-gauge catheter. and the liver immediately harvested. Isolated Perfusion Technique (lPAL) The IPRL apparatus was designed to perform 2 liver perfusions simultaneously, The perfusate was circulated by a Masterflex pump controller (Cole Parmer Instruments. Chicago, I11l. in each of the perfusions circuits. The oxygenator. constructed from a single container and separated into 2 chambers. served for both perfusion systems, The oxygenation was maintained using a 95% 02 and 5% co2 mixture at a flow rate of 3 mUmin. resulting in a P02 of 450 to 5.50 mm Hg. After the oxygenator. the perfusion medium (Krebs buffer containing 2% bovine serum albumin and 0,5% glucose) passed through a microfilter. reservoir. de bubbler. and effluent collecting basins which were placed in line within each circuit. The temperature was controlled by a digital ther-mometer. and the organ was kept in a humidified atmosphere at 37"C. The pH of the perfusate was maintained at 7.4 by adding sodium bicarbonate. if necessary. Experimental Protocol This study was performed in a double blind fashion. The ditTerent storage times in experimental groups are as follows: A I (6 hours), :\2 (12 hours), A3 (24 hours). and A4 (48 hours). Control livers (e) were rinsed with 4 mL Ringer's lactate solution and immediately reperfused. Monitoring of Liver Function At the end of the preser .. ation period, the liver was weighed and washed out with perfusate to remove the preservation fluid, After an initial equilibration period of 15 min (t - 15). effluent samples were taken at 0 and every 30 min during the 2·h perfusion period, The oxygen consumption of the liver was monitored during perfusion by periodic determination (15 min) of Po: (mm Hg) of the portal vein inflow (PV) and vena cava outflow using a pH-blood gas meter (ABL 2 Acid Base Laboratol1·. Radiometer Copenhagen. Denmark)' All bile (/LUg liver/min) secreted was collected into a graduated cylinder and quantitated every IS min. 1be release of hepatoceUular enzymes (AST. ALT. and LDH) into the perfusate were determined by Technicon RA 500 analyzer using commercially available kits (Technicon), For purine nucle-oside phosphorylase (PNP) levels in the effluent. we employed the nnethod of Rao et al. 16 The increase in absorbance (293 nm) produced by uric acid foUowing the nucleoside breakdown was measured by a coupled ~pectrophotometric assay. Purine nucle· oside phosphorylase from calf spleen (>99')( pure) and xanthine o'\idase from cow's milk .... ere purchased from Boehringer Mann· heim (Indianapolis. Ind). Inosine was from Sigma Chemical Com-pany (St Louis. Mo), A TP Measurement At the end of the 1200minutes reperfusion period the liver tissue was immediately freeze clamped and stored in liquid nitrogen. The adenosine triphosphate (ATP) content was determined enzymat-ica1Jy. " From the Department of Surgery. University 01 Pittsburgh School of Medicine. Pittsburgh, Pennsylvania. Dr Hans J. Miscninger is supported by the Fond ZU' Forderung der WlSSenschatlichen Forschung. Austria (J355-Med). Address reprint requests to Thomas E. Starzl. MD. PhD. Uni-versily of Pittsburgh. 3601 Ftfth Avenue, Falk Clinic. 5C Pittsburgh, PA 15213. C 1991 by Appleton & Lange 0041-1345/911$3.00/+0 222 Transplantalion Proceedings, Vol 23. No 1 (February), 1991: pp 222-225 223 AST ALT 10000 10000 f~------------------------ f~------------------------Fig 1. Changes in hepatocel· 'ular enzyme concentrations during 120 minutes of reperlu-51011 after 6. 12. 24 and 48 hours of cold storage as com-pared to controls. Values were averaged and shown as natural logarithm of enzyme concen-trations (:-: SD) at each time point. Twenty-four and 48 h caused more enzyme release (P < .05) indicating nonviable livers. Similar to hepatocellular enzymes. levels of PNP in-creased dramatically and be-came significantly different af-ter 24 and 48 hours storage from those preserved for 6 and 12 hours. After 120 minutes reperfusion. PNP levels up to 50 U/L seemed to be a mar'Ker of reversible microvascular damage. whereas higher levels indicated irreversible damage. Statistical Analysis o 30 A 10000 10 0 ao B Storage 8J'OIIP 60 1-.me (miDuto.) LDH II T_CIIIiII_, ------ At Data is presented as mean = SD. In order to investigate differ-ences between groups. nonpardmetric analysis of variance was performed. Correlations were expressed by a Spearman regres-sion coefficient. Maximum likelihood discriminant analysis was performed with a normal distribution model. Variance was pooled if Bartlett's test on homonegeneity of variances was significant at the 5% level. Error rates were estimated by a posterior probability estimator in order to reduce their variance CSAS/ST AT PC-Program). RESULTS Transaminase Release Enzyme values are presented in Fig I. AST and LDH were the most sensitive hepatocellular enzymes in this model. No statistically relevant differences in AST and LDH release could be observed throughout the 120-minute reperfusion between livers stored for 6 (A I) or 12 (A2) 90 uo o 30 60 uo lime (miD","") PNP 1000 10 II 0 3D 60 10 nm. (mmu_l ----. A2 --- Al --A4 --CO hours. and controls. that did not undergo a preservation period. Livers stored for 24 (A3) and 48 (A4) hours leaked significantly more AST. ALT. and LDH into the perfusate indicating nonviability and ret1ecting continuing damage during reperfusion. The discriminant analysis between the viable E~f and nonviable (A3) groups was calculated from measurements during 0 to 60 minutes and 60 to 120 minutes. respectively. The error estimation for hepatocel-lular enzymes was clearly lower during the second reper-fusion period compared to the first 60 minutes (Fig 4). Bile Production Bile production commenced 5 minutes after the livers were connected to the circuit. The amounts of bile produced per gram of liver/min during the 2-hour perfusion are summa-rized in Fig 2. At the end of reperfusion the volumes of excreted bile were not statistically different from each 224 1.2 0,9 0,3 o~---+----~---+----~----~~~ co Al A2 A3 A4 WI -0- 0 min -0- 60 min -0- 120 min -+- 30 min ..... 90 min Fig 2. Effect of different storage periods on rate of bile excretion (J.Ll-g liver w weightimin) during 120 minutes reperfusion. Between the 4 storage groups only livers stored for 48 hours showed a significant reduction in bile production. other or from controls in livers stored in UW for 6. 12. and 24 hours. resulting in higher probability error rates (0.43) than cellular enzymes or PNP (Fig 4). The bile flow averaged 0.43 :!: 0.12. 0.39 :!: 0.06. and 0.37 :!: 0.04 #-LUg liver min. respectively. Forty eight hours of storage led to a distinctly depressed bile flow over 120 min (0.22 :!: 0.011 #-LUg liver/min). However. bile production was signifi-cantly lower than controls in 24-hours preserved livers after shorter periods of reperfusion up to 90 minutes. Purine Nucleoside Phosphorylase Al E~hF and A2 (l2-hl preservation in UW solution re-sulted in an increase of PNP concentrations from 33.4 :!: 3.0 and 33.8 :!: 2.9 UIL at time 0 to 49.2 :!: 8.5 and 49.3 :!: ~r--------------------------------D 0.00 Controf - A, _.02 c:::J M Fig 3. Livers stored at 4"C for 6, 12, 24 and 48 hours manifested a signiflCal'lt reduction in tissue ATP content when compared with nonischemic livers. All preserved organs were able to regenerate A TP. After 120 minutes reperfusion, recovery of ATP was not significanlly ditlerent within the 4 storage groups. MISCHINGER. RAO. TODO ET At. 0,5 0,4 0,3 0.2 0,1 o Q.6O min 60-120 min 0-120 min I R PNP ~ LDH D Bile 0 AST D ALT I Fig 4. PNP showed a distinctly reduced error rate (0.038) during the first 60 min as well as throughout the whole perfusion period (0 to 120 min) compared to conventional viability parameters (AST 0.29, AL T 0.35, LDH 0.16, and bile 0.43 probability error). In the second perfusion period (60 to 120 min) no difference could be shown except using bile flow as a viability parameter (0.42). 7.9 U/L after 120 min repenusion (Fig I). No difference could be shown between these groups throughout the whole perfusion period. Levels of PNP increased dramat-ically and became significantly different after 30 min of reperfusion between livers stored for 24 h (94.5 :!: 25 UIL) (A3) and 48 hours (152.7 :!: 31.9 UlL) (A4) following a pattern similar to that of hepatocellular enzymes. This is confirmed by a distinctly reduced error rate for the first 60 min of reperfusion (0.038). Adenosine Triphosphate CATP) The amount of A TP measured in organs at the end of cold preservation period was low. ranging from 0.04 :!: 0.02 and 0.06 :!: 0.06 J'mol/g wet weight liver. All cold preserved livers were able to regenerate ATP (Fig 3). After 120-minutes reperfusion the A TP levels were not significantly different in livers preserved for either 6. 12. 24, or 48 hours. A TP levels in livers preserved for 48 hours were significantly lower (P <.05) than control livers. DISCUSSION Recent results have established that the microvasculature is the site of repenusion injury following cold preserva-tion.· .... 20 PNP localized mainly in the cytoplasm of endo-thelial and Kupffer cells has been demonstrated to be a reliable marker to assess repenusion damage to the micro-vascular endothelial cell. 16 In the present study. no signif-icant increase in PNP levels was observed in livers pre-PNP AS A VIABILITY MARKER served for either 6 or 12 h. Viability of the livers was also confirmed by normal levels of hepatocellular enzymes. Significant increases in PNP levels were. however. ob-served when livers were reperfused following either 24 or 48 hours of cold preservation and this increase occurred within the first 60 minutes of reperfusion. Nonviability of grafts was also confirmed by significant increases in hep-atocellular enzymes. It has been determined in other studies that only grafts preserved for up to 18 h are capable of survival. '9-22 Therefore. it is apparent that PNP levels can accurately predict this. since in our studies. although 12 h of cold preservation was associated with normal levels of PNP. 24 h and more of cold preservation resulted in significant injury to the endothelial cell. Bile production is considered to be a measure of A TP generation and has traditionally been used as a marker of graft viability. Og-~S In our study. however. bile production dropped significantly only after 48 hours of cold preservation. a period when the organ is no longer viable. 19-22 Therefore. it is obvious that in this model. bile production has a limited usefulness as a marker of graft viability. Tissue ATP levels were similarly nonpre-dictive. In conclusion. PNP levels appear to be an early indica-tor of preservation injury to the microvascular endothelial cell, and could prove to be an accurate diagnostic aid of graft viability following liver transplantation. REFERENCES I. Collins GM. Br.lvo-Sugarman M. Ter.lsaki P: Lancet 2: 1219. 1969 2. Oreikom K. Horsch R. Rohl L: Euro Urol 6:211. 1980 3. Wahlberg JA. Southard R. Belzer F: Cryobiology 23:477. 1986 4. Jamieson NV. Sundberg R. Lindell S: Transplant Proc 312:158.1988 225 5. Todo S. Nery J. Yanaga K. et al: JAMA 261:711. 1989 6. McCord JM: New Engl J Med 312:158. 1985 7. Parks OA. Bulkey GB. Granger DN: Surgery 10\:566. 1987 8. Vladovic-Relja: Cryobiology 21 :511. 1989 9. Lee O. Holland RK: Transplantation 27:384. 1979 10. DeBruijn KM. Krom RAF. aeouijterd~K et al: Tr.lnsplant Proc 16: 141. 1984 II. Kamiike W. Nakahar.l M. Nakao Y. et al: Transplantation 39:50. 1985 12. Bowers BA. Branum GO. Rotolo FS. et a1: J Surg Research 42:565. 1987 13. Kamiike W. Watanabe F. Hashimoto T. et al: J Biochem 91:1349.1982 14. Marubayashi S. Takenaka M. Dohi K. et al: Tr.lnsplanta-tion 30:294. 1980 15. Rubio R. Berne RM: Am J Physiol 239:721. 1980 16. Rao PN. Walsh TR. Makowka L. et a1: Hepatology 11: 193. 1990 17. Lamprecht W. Trautschold I: In Bergmeyer HU (ed): Methods in Enzymology 2nd Ed. New York. Academic. 1974. p 2101. Vol 4 18. Otto G. Wolff H. David H: Transplant Proc 16:1247. 1984 19. McKeown CMB. Edwards V. Phillips MJ. et al: Transplan-tation 46: 178. 1988 20. Myagkaya GL. Van Veen HA. James J: Virchow Arch P 53:176.1987 21. Holloway CMB. Harvey PRC. Mullen JBM. et al: Tr.lns-plantation 25:300. 1989 22. Jamieson NV. Sundberg R. Lindell S. et al: Cryobiology 25:300. 1988 23. Kamiike W. Bude1ski M. SteinhoffG. et al: Tr.lnsplantation 45:138.1988 24. Lanir A. Jenkins RL. Caldwell C. et al: Hepatology 8:471. 1988 25. Slater TF. Delaney VB: J Biochem 303. 1978 26. lu S. Harvey PRC. Makowka L. et al: Transplantation 44:562. 1987 

Levels of purine nucleoside phosphorylase (PNP) as a viability marker of nonparenchymal cells in cold preserved livers.

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Levels of purine nucleoside phosphorylase (PNP) as a viability marker of nonparenchymal cells in cold preserved livers

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Levels of purine nucleoside phosphorylase (PNP) as a viability marker of nonparenchymal cells in cold preserved livers.

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