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β-Galactosidase as a novel marker of ischaemic injury and a mechanism for viability assessment in liver transplantation
-Galactosidase as a Novel Marker of Ischaemic Injury and a Mechanism for Viability Assessment in Liver Transplantation S. D. St. Peter, C.J. Imber, I. Lopez, J. McGuire, T. James, R. Taylor, D. Pigott, and P.J. Friend
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IVER PRESERVATION (procedures) currently do not allow the doctor to assess organ viability. An effective means of viability assessment would allow for the use of more marginal donors and minimize the risk of primary nonfunction by identifying nonviable organs. Glycohydrolases are intracellular enzymes that have been found to increase markedly in serum after porcine liver transplantation.1 A study on the effect of warm ischaemia on glycohydrolase levels in the porcine model in two groups showed a correlation with ischaemia time, and the level of glycohydrolase recovery after transplantation was different between livers that recover function and those that did not.2 The glycohydrolases most sensitive to ischaemic injury in both groups were -galactosidase and -glucuronidase. We have shown the use of a normothermic extracorporeal circuit in order to perfuse livers and can provide preservation with minimal ischaemia compared to cold storage.3 In this study, we compare the increase and recovery of -galactosidase with the levels of traditional markers of liver injury and how they relate to the level of function that livers regain on an extracorporeal circuit after being subjected to a prolonged preservation period by either ischaemic cold storage or normothermic, oxygenated perfusion.
Assessment of Function Measurements taken throughout the reperfusion phase included -galactosidase, hepatocellular enzymes, factor V, bile, blood glucose, and haemodynamics. At the end of each perfusion, each liver was sectioned and assessed histologically for evaluation of tissue quality. -Galactosidase was measured using a specific substrate, 4-methylumbellifery-galactosidase, which is digested to a fluorescent product 4-methylumbelliferone. Detection was performed using a fluorescent micro-titre plate reader: The excitation wavelength was 355 nm; the emission wavelength was 460 nm. The reaction took place in a citrate-phosphate buffer optimized pH 4.4 with a substrate concentration at 3.33 mmol/L. Specimen volume (10 L) and substrate solution (80 L) were incubated for 30 to 60 minutes at 37°C, when the reaction was terminated by the addition of 200 L of glycine/sodium hydroxide, pH 12.8. The assay was linear up to 3000 U/mL. The intra-assay coefficient of variation at 50, 502, and 2012 U/mL was 4.73, 3.14, and 3.39, respectively, and the interassay coefficient of variation at 493 U/mL was 3.84 (n ⫽ 5).
Statistical Analysis Welch’s t test (assumption of unequal variances) was used for each interval reflecting the mean of five perfusions with a P value of ⱕ .05 defining significance.
RESULTS
Perfusion Circuit
Group C displayed greater evidence of hepatocellular damage with increases in aspartate and alanine aminotransferase (AST and ALT). AST levels increased over the 24-hour reperfusion phase to a peak of 5653 IU/L reaching a significant difference over group W at 16 hours of reperfusion. ALT levels peaked at 208 IU/L and were significant over group W after 7 hours of reperfusion. -galactosidase, however, increased immediately on reperfusion in group C to a significant level over group W after just one hour of reperfusion (P ⫽.003). This increase continues for the next four hours of reperfusion to a plateau from 5 to 7 hours. Group W showed little increase during reperfusion never exceeding 138.95 IU/L. -galactosidase levels peaked at 5
The perfusion circuit consisted of a centrifugal pump, oxygenator, heat exchanger, and a soft shell reservoir. Blood was pumped actively to the hepatic artery and passively to the portal vein from the soft shell reservoir. Infusions and maintenance were as previously described.3
From the Department of Surgery, John Radcliffe Hospital, Oxford, UK. Address reprint requests to S. St Peter, John Radcliffe Hospital, Department of Surgery, Level 6, Oxford OX3 9DV, UK.
MATERIALS AND METHODS Experimental Design Porcine livers were obtained by standard technique and preserved for 24 hours using cold storage in University of Wisconsin (UW) solution (group C, n ⫽ 5), or sanguineous perfusion with whole blood at 38°C (group W, n ⫽ 5). Both groups were then flushed with 2 L of non-oxygenated, cold EC solution and left 45 minutes before being flushed with a warm plasma substitute and reperfused on the extracorporeal circuit to replicate the time of surgical anastamosis.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)02589-1
Transplantation Proceedings, 33, 3753–3755 (2001)
3753
3754
ST PETER, IMBER, LOPEZ ET AL
Fig. 1 Enzyme present in the perfusate for -Galactosidase, aspartate transaminase (AST), and alanine transaminase (ALT). The fold of increase over levels obtained at time zero show the relative amount of enzyme present in the perfusate. The increase found in -galactosidase occurs before and to a greater degree than the increases found in transaminases in the ischaemic-injured group C, showing that -galactosidase is a more sensitive marker of ischaemia-reperfusion injury.
hours of reperfusion, while ALT and AST levels were still increasing at 24 hours. Synthetic Function
Factor V production decreases throughout reperfusion in group C to 108 IU/dL at 24 hours. Group W increases toward 206 IU/dL at 24 hours (P ⫽ .02). Bile production from 10 hours onward was 1.56 mL/hr in group C and 5.66 mL/hr in group W, which was significant throughout the study. Metabolic Function
Glucose concentrations in group C remained supraphysiologic throughout perfusion while group W livers used enough glucose to maintain lower perfusate levels throughout reperfusion. This difference was significant at all time points. Haemodynamic Parameters
Total flow in group C was 1.5 L/min during reperfusion, which decreased significantly from 20 hours onward. By contrast, the mean flow in group W, remained stable at 2 L/min. Portal pressures were increased in group C above those in group W throughout reperfusion. Histology
Group C showed large (more than 30%) amounts of necrosis on multiple wedge biopsy specimens. Group W showed less than 30% sinusoidal dilatation and more than 60% vacuolization, but very little necrosis. DISCUSSION
The livers stored under ischaemic conditions (group C) resulted in poor quality organs as was evident by inferior
bile production, factor V production, and glucose utilization than livers perfused during preservation (group W). Group C livers experienced more cellular injury as seen by the differences in hepatocellular enzymes and histology. The increase in -galactosidase clearly corresponded to the amount of ischaemic injury as is found in Fig 1 with group C livers consistently following a distinctly different curve on an order of magnitude above that of the group W livers. Kupffer’s cells represent most (80% to 90%) of the body’s resident macrophages and have an enormous potential for releasing inflammatory mediators.4 They are rich in lysosomes and release their lysosomal enzymes after activation.5 -galactosidase is a lysosomal enzyme; therefore, Kupffer’s cells are thought to be the primary source of glycohydrolases increases found immediately on reperfusion of ischaemic tissue.2 Evidence suggests that Kupffer’s cells and endothelial cells are particularly sensitive to ischaemia, and are the initial cells to become activated in ischaemic injury, this preceding hepatocellular damage.6,7 Our data support this, showing increases in -galactosidase that precede an increase in hepatocellular enzymes (Fig 1). Another source of ischaemic damage occuring on reperfusion, free radicals, lead to lipid peroxidation and cellular destruction.8 Lipid peroxidation has been shown to follow the same pattern as -galactosidase in livers injured by ischaemia-reperfusion confirming that free radical reactions ensue after Kupffer’s cell activation/injury.2 In conclusion, -galactosidase is a sensitive marker of ischaemia-reperfusion injury and it correlates well with liver function. It is a more sensitive marker of ischaemia-reperfusion injury than hepatocellular enzymes. It can be used as viability marker when perfusion is the preservation. The precise levels of -galactosidase that predict survival will
VIABILITY MARKERS
require a transplant model, experiments that are currently in progress. REFERENCES 1. Chang CW, Imai K, Chang YC, et al: Enzyme 45:145, 1991 2. Liu W, Schob O, Pugmire JE, et al: Hepatology 24:157, 1996 3. Imber CJ, St Peter SD, Lopez de Cenerazzabeietia I, et al: Transplantation (in press), 2001
3755 4. Arii S, Imamura M: J Hepatobiliary Pancreat Surg 7:40, 2000 5. Caldwell-Kenkel JC, Currin RT, Tanaka Y, et al: Hepatology 13:83, 1991 6. Liu P, McGuire GM, Fisher MA, et al: Shock 3:56, 1995 7. Sido B, Datsis K, Mehrabi A, et al: Transplantation 60:462, 1995 8. Schroeder RA, Kuo PC: In Grace PA, Mathie RT (eds): Blackwell Science, 1999, 113
L
IVER PRESERVATION (procedures) currently do not allow the doctor to assess organ viability. An effective means of viability assessment would allow for the use of more marginal donors and minimize the risk of primary nonfunction by identifying nonviable organs. Glycohydrolases are intracellular enzymes that have been found to increase markedly in serum after porcine liver transplantation.1 A study on the effect of warm ischaemia on glycohydrolase levels in the porcine model in two groups showed a correlation with ischaemia time, and the level of glycohydrolase recovery after transplantation was different between livers that recover function and those that did not.2 The glycohydrolases most sensitive to ischaemic injury in both groups were -galactosidase and -glucuronidase. We have shown the use of a normothermic extracorporeal circuit in order to perfuse livers and can provide preservation with minimal ischaemia compared to cold storage.3 In this study, we compare the increase and recovery of -galactosidase with the levels of traditional markers of liver injury and how they relate to the level of function that livers regain on an extracorporeal circuit after being subjected to a prolonged preservation period by either ischaemic cold storage or normothermic, oxygenated perfusion.
Assessment of Function Measurements taken throughout the reperfusion phase included -galactosidase, hepatocellular enzymes, factor V, bile, blood glucose, and haemodynamics. At the end of each perfusion, each liver was sectioned and assessed histologically for evaluation of tissue quality. -Galactosidase was measured using a specific substrate, 4-methylumbellifery-galactosidase, which is digested to a fluorescent product 4-methylumbelliferone. Detection was performed using a fluorescent micro-titre plate reader: The excitation wavelength was 355 nm; the emission wavelength was 460 nm. The reaction took place in a citrate-phosphate buffer optimized pH 4.4 with a substrate concentration at 3.33 mmol/L. Specimen volume (10 L) and substrate solution (80 L) were incubated for 30 to 60 minutes at 37°C, when the reaction was terminated by the addition of 200 L of glycine/sodium hydroxide, pH 12.8. The assay was linear up to 3000 U/mL. The intra-assay coefficient of variation at 50, 502, and 2012 U/mL was 4.73, 3.14, and 3.39, respectively, and the interassay coefficient of variation at 493 U/mL was 3.84 (n ⫽ 5).
Statistical Analysis Welch’s t test (assumption of unequal variances) was used for each interval reflecting the mean of five perfusions with a P value of ⱕ .05 defining significance.
RESULTS
Perfusion Circuit
Group C displayed greater evidence of hepatocellular damage with increases in aspartate and alanine aminotransferase (AST and ALT). AST levels increased over the 24-hour reperfusion phase to a peak of 5653 IU/L reaching a significant difference over group W at 16 hours of reperfusion. ALT levels peaked at 208 IU/L and were significant over group W after 7 hours of reperfusion. -galactosidase, however, increased immediately on reperfusion in group C to a significant level over group W after just one hour of reperfusion (P ⫽.003). This increase continues for the next four hours of reperfusion to a plateau from 5 to 7 hours. Group W showed little increase during reperfusion never exceeding 138.95 IU/L. -galactosidase levels peaked at 5
The perfusion circuit consisted of a centrifugal pump, oxygenator, heat exchanger, and a soft shell reservoir. Blood was pumped actively to the hepatic artery and passively to the portal vein from the soft shell reservoir. Infusions and maintenance were as previously described.3
From the Department of Surgery, John Radcliffe Hospital, Oxford, UK. Address reprint requests to S. St Peter, John Radcliffe Hospital, Department of Surgery, Level 6, Oxford OX3 9DV, UK.
MATERIALS AND METHODS Experimental Design Porcine livers were obtained by standard technique and preserved for 24 hours using cold storage in University of Wisconsin (UW) solution (group C, n ⫽ 5), or sanguineous perfusion with whole blood at 38°C (group W, n ⫽ 5). Both groups were then flushed with 2 L of non-oxygenated, cold EC solution and left 45 minutes before being flushed with a warm plasma substitute and reperfused on the extracorporeal circuit to replicate the time of surgical anastamosis.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)02589-1
Transplantation Proceedings, 33, 3753–3755 (2001)
3753
3754
ST PETER, IMBER, LOPEZ ET AL
Fig. 1 Enzyme present in the perfusate for -Galactosidase, aspartate transaminase (AST), and alanine transaminase (ALT). The fold of increase over levels obtained at time zero show the relative amount of enzyme present in the perfusate. The increase found in -galactosidase occurs before and to a greater degree than the increases found in transaminases in the ischaemic-injured group C, showing that -galactosidase is a more sensitive marker of ischaemia-reperfusion injury.
hours of reperfusion, while ALT and AST levels were still increasing at 24 hours. Synthetic Function
Factor V production decreases throughout reperfusion in group C to 108 IU/dL at 24 hours. Group W increases toward 206 IU/dL at 24 hours (P ⫽ .02). Bile production from 10 hours onward was 1.56 mL/hr in group C and 5.66 mL/hr in group W, which was significant throughout the study. Metabolic Function
Glucose concentrations in group C remained supraphysiologic throughout perfusion while group W livers used enough glucose to maintain lower perfusate levels throughout reperfusion. This difference was significant at all time points. Haemodynamic Parameters
Total flow in group C was 1.5 L/min during reperfusion, which decreased significantly from 20 hours onward. By contrast, the mean flow in group W, remained stable at 2 L/min. Portal pressures were increased in group C above those in group W throughout reperfusion. Histology
Group C showed large (more than 30%) amounts of necrosis on multiple wedge biopsy specimens. Group W showed less than 30% sinusoidal dilatation and more than 60% vacuolization, but very little necrosis. DISCUSSION
The livers stored under ischaemic conditions (group C) resulted in poor quality organs as was evident by inferior
bile production, factor V production, and glucose utilization than livers perfused during preservation (group W). Group C livers experienced more cellular injury as seen by the differences in hepatocellular enzymes and histology. The increase in -galactosidase clearly corresponded to the amount of ischaemic injury as is found in Fig 1 with group C livers consistently following a distinctly different curve on an order of magnitude above that of the group W livers. Kupffer’s cells represent most (80% to 90%) of the body’s resident macrophages and have an enormous potential for releasing inflammatory mediators.4 They are rich in lysosomes and release their lysosomal enzymes after activation.5 -galactosidase is a lysosomal enzyme; therefore, Kupffer’s cells are thought to be the primary source of glycohydrolases increases found immediately on reperfusion of ischaemic tissue.2 Evidence suggests that Kupffer’s cells and endothelial cells are particularly sensitive to ischaemia, and are the initial cells to become activated in ischaemic injury, this preceding hepatocellular damage.6,7 Our data support this, showing increases in -galactosidase that precede an increase in hepatocellular enzymes (Fig 1). Another source of ischaemic damage occuring on reperfusion, free radicals, lead to lipid peroxidation and cellular destruction.8 Lipid peroxidation has been shown to follow the same pattern as -galactosidase in livers injured by ischaemia-reperfusion confirming that free radical reactions ensue after Kupffer’s cell activation/injury.2 In conclusion, -galactosidase is a sensitive marker of ischaemia-reperfusion injury and it correlates well with liver function. It is a more sensitive marker of ischaemia-reperfusion injury than hepatocellular enzymes. It can be used as viability marker when perfusion is the preservation. The precise levels of -galactosidase that predict survival will
VIABILITY MARKERS
require a transplant model, experiments that are currently in progress. REFERENCES 1. Chang CW, Imai K, Chang YC, et al: Enzyme 45:145, 1991 2. Liu W, Schob O, Pugmire JE, et al: Hepatology 24:157, 1996 3. Imber CJ, St Peter SD, Lopez de Cenerazzabeietia I, et al: Transplantation (in press), 2001
3755 4. Arii S, Imamura M: J Hepatobiliary Pancreat Surg 7:40, 2000 5. Caldwell-Kenkel JC, Currin RT, Tanaka Y, et al: Hepatology 13:83, 1991 6. Liu P, McGuire GM, Fisher MA, et al: Shock 3:56, 1995 7. Sido B, Datsis K, Mehrabi A, et al: Transplantation 60:462, 1995 8. Schroeder RA, Kuo PC: In Grace PA, Mathie RT (eds): Blackwell Science, 1999, 113