CRYOBIOLOGY
28, 143-149 (191)
Effect of Cold Storage
on Tissue and Cellular
Glutathione’
P. K. VREUGDENHIL,
F. 0. BELZER, AND J. H. SOUTHARD Department of Surgery, University of Wisconsin, Madison, Wisconsin 53792
One of the mechanisms thought to cause injury in preserved organs is the formation of oxygen free radicals. The cell is protected from oxidative stress by many defense mechanisms. A major defense mechanism involves glutathione and glutathione-dependent enzymes. During organ preservation by simple cold storage the loss of glutathione may sensitize the organ to free radical damage after transplantation. In this study we show that glutathione is depleted from the rabbit liver, kidney, and heart cold-stored (YC) for up to 72 h in the UW solution without glutathione. In the first 24 h kidney glutathione decreased to 84 2 3% of control values, liver glutathione decreased to 49 f 3% of control values, and heart glutathione decreased to 73 2 3% of control values. After 48 h of storage the kidney and liver lost an additional 30 and 20%, respectively, whereas heart glutathione changed very little. By 72 h all three organs had lost more than 50% of the glutathione found in freshly obtained tissue. To determine if glutathione added to the UW solution can effectively prevent this loss of glutathione during preservation, hepatocytes were cold-stored for up to 72 h in a preservation solution with and without glutathione. We found that adding glutathione to the preservation solution slowed the rate of loss of glutathione from the cells. These data suggest that at hypothermia the cell may be permeable to GSH. Methods to suppress the loss of glutathione during preservation of organs may be an important factor in suppressing oxygen free radical injury. Q 1991 Academic press, Inc.
Most organs used for transplantation are stored cold (YC) without continuous perfusion (i.e., cold ischemia). Once removed from the circulatory system organs begin the inexorable process of cell death and the rate of cell death is dependent upon the storage conditions. The temperature and time dependency of loss of organ viability suggest that degenerative reactions occur in the tissue that eventually cause irreversible injury. Which components of the cell degenerate during cold storage of organs and how this relates to loss of viability are not known. Glutathione (tGSH, total glutathione, = GSH (reduced) + GSSG (oxidized)), the major cellular thiol, is rapidly metabolized by most tissues and the biosynthesis of GSH requires three moles of adenosine triphosphate (ATP) per mole of GSH. In simple cold storage of organs the synthesis of ATP is limited and up to 90% of tissue
ATP is lost within 24 h of cold storage (4, 20, 22). However the pathways for the catabolism of GSH remain active in the absence of a source of energy. Glutathione serves many roles in cellular metabolism, one of which is the reduction of peroxides (hydrogen peroxide and lipid peroxides) to innocuous products (18). Thus, GSH is an important cellular antioxidant and is a major factor in protecting the cell from various forms of oxidative stress caused by the generation of cytotoxic metabolites of oxygen (hydroxyl radicals and lipid peroxides). How cold storage of organs affects the tissue concentration of glutathione is not known although it is well known that in warm ischemia there is a loss of glutathione from tissue (27). In this study we have determined the rate of loss of tGSH from organs cold-stored for up to 3 days in the UW solution, including the rabbit kidney, liver, and heart. The results show that tGSH is catabolized during cold storage of organs. The loss of tGSH Received January 10, 1990; accepted May 14, 1990. may be a factor contributing to oxygen free r This work supported by NIH Grant DK 35143. 143 001l-2240/91 $3.00 Copytisbt 6 1991 by Academic Press, Inc. All lights of reproduction in any foml reserved.
144
VREUGDENHIL,
BELZER,
radical injury in organs following transplantation. MATERIALS
AND
METHODS
Whole Tissue Kidneys, livers, and hearts were obtained from New Zealand white rabbits (2 to 3 kg) anesthetized with Nembutal (3.9 mg/kg). The organs were flushed out with cold (5°C) UW solution (31) not containing GSH. The kidney was flushed through the renal artery (60 ml), the liver through the portal vein (200 ml), and the heart through the aorta (80 ml) with the indicated volumes of the UW solution. Warm ischemia time was less than 2 min. The organs were stored at 5°C for up to 72 h. Some organs were flushed with warm (37°C) UW solution, stored warm (37°C) for 10 to 30 min, and then flushed with cold UW solution and stored for 24 h. Tissue samples (kidney cortex, liver, and left ventricle of the heart) weighing approximately 1 g were taken at 0,24,48, and 72 h. The samples were frozen in a dry ice and acetone bath, weighed, and homogenized in trichloroacetic acid (6.7%). The homogenate was centrifuged at 1600gfor 4 min and the supernatant collected. The concentration of tGSH (GSH + GSSG) in the supernatant was determined enzymatically (6).
AND
SOUTHARD
20 m&f; raffinose, 30 mM; and polyethylene glycol, 5% (MW = 8000 Da). In some experiments GSH (3 m&f) was added. The cells were allowed to settle and stored for 72 h at 5°C under N2 in sealed 50-ml polycarbonate centrifuge tubes to simulate simple cold storage of the liver (cold ischemia). Every 24 h cellular tGSH was determined by the method of Griffith (6). An aliquot of cells (1 ml) was centrifuged at 13,OOOg for 1 min (Eppendorf microcentrifuge). The supernatant was removed by aspiration and discarded. The sediment (hepatocytes) was sonicated in 1.5 ml trichloroacetic acid (3.3%) and centrifuged as above and GSH was determined enzymatically in the supernatant. RESULTS
The concentrations of tGSH in control kidney, liver, and heart were 519 + 30,2433 ? 165, and 635 ? 34 nmol per gram wet weight, respectively. These values will be used to represent 100% of tissue concentrations of tGSH in each specific organ. The concentrations of tGSH in tissue coldstored in the UW solution (without GSH) for up to 72 h is shown in Fig. 1. In the kidney, tGSH was lost at a rate of about 20% per day.
Isolated Hepatocytes Hepatocytes were prepared from Sprague Dawley rats (300 to 400 g) by a modification of Seglen’s method (28) as previously described by Marsh et al. (14). Hepatocyte protein concentration was adjusted to 6 + 1 mg/ml (Biuret method) and cell viability was determined by trypan blue dye exclusion. Preparations that showed greater than 90% exclusion of trypan blue were used for study. After determining viability, hepatocytes were suspended in the preservation solution (S‘C). The preservation solution contained: K+lactobionate, 100 mM; KH2P04,
01 0
I 24
48 Preservation
72 Time (hr)
1. Glutathione in the rabbit kidney, liver, and heart during cold storage in UW (without GSH). tGSH (GSH + GSSG) values are expressed as a percentage of control values (control = 100%). Data are given as means 2 SEM for six rabbit kidneys, five livers, and five hearts. Methods are described under Materials and Methods. FIG.
LOSS
145
OF GLUTATHIONE
The cold-stored liver lost tGSH more rapidly than the kidney and after 24 h of storage had decreased to 49 + 3% of 0 time values. After 48 and 72 h of storage liver GSH concentrations were 30 t 4 and 23 f 5% of 0 time values. In the cold-stored heart after 24 h the tGSH content was reduced to 73 + 3% and there was no significant change at 48 h. However, by 72 h the tGSH concentration was 28 + 2% of 0 time values. Thus, in all organs the tissue concentration of tGSH was reduced by 55 to 75% after 72 h of storage. In another report we showed that the composition of the storage medium did not alter the rate of breakdown of tGSH which was similar in Eurocollins solution, Ringers Lactate, or the UW solution without GSH (30). The results in Fig. 2 show how a brief period of warm ischemia affects the tGSH concentration in organs cold-stored for 24 h. After 10 min at 37°C the concentration of tGSH in the kidney was 68 ? 6% of 0 time values. After 24 h of cold storage the tGSH concentration was reduced to 36 + 4% of 0 time values and this concentration was significantly lower than the tGSH concentration in kidneys stored for 24 h without exposure to warm ischemia (84 + 3%). Ten minutes of warm ischemia had little effect on the tGSH concentration in the liver or heart. After 30 min of warm ischemia, liver and heart tGSH concentrations were 96 * 3 and 79 + 9% of 0 time values. After a subsequent period of cold storage (24 h) the concentration of tGSH in the liver was 28 * 4% (versus 49 + 3% with no warm ischemia, not significantly different). In the heart 24 h of cold storage after warm ischemia reduced the tGSH concentration to 55 + 8% (versus 73 rt 3% with no warm ischemia, not significantly different). Hepatocytes isolated from rat livers contained 8.49 + 0.51 nmol tGSH/mg protein. The effect of cold storage on tGSH concentration in rat hepatocytes is shown in Fig. 3. When stored for 24 h the tGSH concentra-
Kidney
0
30min
WI
24hr
CS
FIG. 2. Effect of warm ischemia (WI) and subsequent cold storage (CS) on the rate of loss of tGSH from cold-stored rabbit kidney, liver, and heart tissue. Bars represent mean tGSH concentrations as a percentage of control values ? SEM obtained from seven kidneys, five livers, and five hearts. Dashed lines represent means + SEM of organs cold-stored without a period of warm ischemia. Methods are described under Materials and Methods.
tion was 85 + 4 and 53 ? 2% at 48 h and 33 ? 5% after 72 h of storage. Hepatocytes were also stored in the presence of GSH (3 mM) and after sedimentation the concentration of GSH in the cells was determined.
146
VREUGDENHIL,
0
24
4.3 Preservation
BELZER,
72 Time (hr)
FIG. 3. Effect of the addition of GSH to the cold storage solution on intracellular tGSH concentrations in rat hepatocytes. Data are given as means 2 SEM for hepatocytes prepared from five rat livers. The preservation solution used is described under Materials and Methods.
The concentration of tGSH was 98 + 2, 80 + 8, and 57 + 5% of 0 time values after 24, 48, and 72 h of storage, respectively. The tGSH concentrations after each day of storage in the presence of GSH were significantly greater than in hepatocytes stored without GSH. The higher concentrations of tGSH resulting from storing hepatocytes in the presence of GSH were not due to GSH in the extracellular water in sedimented hepatocytes. We have previously shown that the amount of extracellular water in sedimented hepatocytes is 0.1 pJ/mg protein (14). This small amount of extracellular water would not contain enough GSH to elevate the tGSH of hepatocytes suspended in a solution containing 3 mM (0.3 nmoYmg protein). DISCUSSION
Simple cold storage has been the method of choice of most organ transplant centers for organ preservation. Organs are flushed out with a preservation solution and stored at 0 to 4°C without continuous perfusion. In clinical transplantation the kidney, liver, and heart can be safely preserved for about 48 h (24), 35 h (29), and 3 to 6 h (32), respectively. In the laboratory the kidney can be preserved for 72 h (25), the liver for 48 h
AND
SOUTHARD
(IO), and the heart for about 24 to 28 h (3). The quality of preservation is dependent upon many factors, one of which is the length of preservation. Longer preservation usually results in a higher incidence of delayed function of primary nonfunction and the cause for this decrease in viability of the organ is not known. Normal metabolism is a balance between biosynthetic and degradative reactions. In the absence of oxygen and a continual supply of nutrients, biosynthetic reactions are suppressed due to a lack of energy (ATP); however, many degradative reactions do not require a continual supply of energy and may continue even at hypothermia. The fact that organs stored for relatively short periods of time regain normal metabolic functions more rapidly than organs stored for longer periods of time suggests that degradative reactions occurring within the tissue limit viability. The exact nature of these degradative reactions is not clear but evidence suggests that the activity of phospholipases may play a role in the loss or organ viability after extended periods of preservation (2). Evidence suggesting that lysosomal enzymes are activated during prolonged cold storage of the kidney has also been presented (23). Suppressing the activities of some of these enzymes has been shown to improve the results of organ preservation (12). During simple cold storage of organs there also occurs a loss of metabolites including adenine nucleotides, a-tocopherol, pyridine nucleotides, and coenzyme Q (15, 16,20,22). In this study we show that tGSH is also degraded during cold storage of the kidney, liver, and heart. The loss of tGSH from preserved organs (or the inability to regenerate GSH upon reperfusion) could be a causative factor in the loss of organ viability. Many studies have shown that selective depletion of cellular tGSH sensitizes cells to various forms of oxidative injury (9, 27). Furthermore, methods used to enhance tGSH concentra-
LOSS OF GLUTATHIONE
tions in the cell have been shown to protect the cells from various forms of oxidative injury. Recently, Rush et al. (26) have shown that the addition of GSH to hepatocytes protects these cells from injury. The mechanism of protection by GSH appears to be related to the reduction of hydrogen peroxide by glutathione peroxidase. The reduction in the concentration of hydrogen peroxide in the cell (arising from the dismutation of superoxide anions) would also reduce the concentration of hydroxyl radicals formed from hydrogen peroxide and that the hydroxyl radical may be the primary toxic product of oxygen metabolism. The UW solution used for preservation of the liver, kidney, and pancreas (30) contains GSH. Glutathione was added because of its role in protecting tissues from oxidative stress. However, it is unclear if the protection by glutathione is due to its entry into the cell during cold storage. It is difficult to determine the exact tissue distribution of glutathione in a whole organ flushed out with a solution containing glutathione because much of the glutathione may reside in the extracellular spaces. However, it is simpler to determine the cellular location of glutathione in isolated hepatocytes stored in the presence of glutathione. Hepatocytes can be removed from the incubation medium by sedimentation and the cellular concentration of glutathione measured. In this study, the cellular concentration of glutathione in isolated hepatocytes decreased during hypothermic storage in the absence of added glutathione. When stored in the presence of glutathione the rate of loss of cellular glutathione was decreased. For instance, cells stored in the absence of glutathione for 48 h showed an approximate 50% decrease in the cellular concentration of glutathione. However, cells stored in the presence of glutathione lose only approximately 20% of the cellular glutathione. This suggests that glutathione may actually enter cold-stored hepatocytes. Another explanation is that glutathi-
147
one is catabolized to its amino acid constituents which serve as substrates for the regeneration of glutathione. However, since these cells were stored anoxically there would be little ATP available for the synthesis of glutathione. Thus, it is more likely that the suppression of the loss of glutathione from hepatocytes stored in the presence of glutathione was due to the entry of glutathione into the cell. Kidney cells are apparently permeable to glutathione (13, 21), although other cells, at normothermia, do not appear to be permeable to GSH (7). However, the permeability of hepatocytes to substances such as glutathione may be increased by hypothermia. We show in this study that glutathione is decreased in cold-stored rabbit organs and suggest that this decrease may increase the sensitivity of organs to reperfusion injury caused by the generation of oxygen free radicals. Studies in the past have suggested that oxygen free radicals may be a causative factor in the loss of organ viability following preservation (5,8) and one mechanism for the generation of oxygen free radicals has been related to the activity of xanthine oxidase (16, 17). The activation of this enzyme may increase the rate of generation of superoxide anions and cause oxidative stress. However, oxidative stress could result from a decrease in the cells capability to scavenge oxygen free radicals due to a loss of intracellular scavengers, such as glutathione. This could occur even without a significant increase in the rate of generation of oxygen free radicals. Thus, protection of tissues from oxidative stress may require maintenance of endogeneous free radical scavengers in the cell and methods that accomplish this may lead to improved and prolonged organ preservation. Warm ischemia prior to cold storage of the kidney caused a greater rate of loss of glutathione than in kidneys not exposed to warm ischemia. This effect was seen within as few as 10 min of warm ischemia. In the liver and heart a warm ischemic period of
148
VREUGDENHIL,
BELZER, AND SOUTHARD
4. D’Alessandro, A., Southard, J. H. Kalayoglu, M., 30 min prior to 24 h cold storage did not and Belzer, F. 0. Comparison of cold storage cause a significantly greater decrease in and perfusion of dog livers on function of tissue glutathione than just cold ischemia itself. slices. Cryobiology 23, 161-167 (1986). This might reflect the greater rate of metab- 5. Fuller, B. J., Cower, J. D., and Green, C. J. Free olism of glutathione in the kidney (50% radical damage and organ preservation: Fact or turnover time = 0.5 h) as compared to the Fiction? A review of the interrelationship between oxidative stress and physiological ion heart and liver (50% turnover time = 2 h) disbalance. Cryobiology 25, 377-393 (1988). (18, 19). Previous studies have shown that 6. Griftith, 0. W. Glutathione and glutathione disulwarm &hernia prior to cold storage sensiphide. In “Methods of Enzymatic Analysis” tizes the kidney to increased preservation (H. U. Bergmeyer, et al., Eds.,), Vol 8, pp. damage (11). One explanation is that a brief 521-529. VCH Verlagsgesellschaft, Weinheim period of warm ischemia causes a rapid (Federal Republic of Germany), 1985. degradation of essential metabolites in the 7. Hahn, R., Wendel, A., and Flohe, L. The fate of extracellular glutathione in the rat. Biochim. kidney (such as glutathione) which causes Biophys. Acta 529, 324-327 (1978). the increased sensitivity to preservation in8. Hemandez, L. A., and Granger, D. N. Role of anjury. Although warm ischemia prior to cold tioxidants in organ preservation and transplanstorage may also limit the preservation tation. Crit. Care. Med. 16, 543-549 (1988). quality of the heart and liver, in these or9. Hoberg, J., and Kristoferson, A. A correlation begans this effect may not be directly related tween glutathione levels and cellular damage in to the loss of glutathione. isolated hepatocytes. Eur. J. Biochem. 74, 7782 (1977). In summary, this study shows that cold10. Jamieson, N. V., Sundberg, R., Lindell, S., stored organs lose tGSH and the loss is Claesson, K., Moen, J., Vreugdenhil, P., more rapid in the liver than in the heart or Wight, D. G. D., Southard, J. H., and Belzer, kidney. However, after 72 h of storage all F. 0. 24-48 hour preservation of the canine organs studied had lost about 50 to 75% of liver by simple cold storage using UW lactotheir tGSH. The addition of GSH to coldbionate solution. Transplant. Proc. 21, 12921293 (1989). stored hepatocytes decreases the rate of 11. Johnson, R. W. G., Anderson, M., Morley, loss of tGSH. Another study has shown A. R., Taylor, R. M. R., and Swinney, J. that cell viability is enhanced during cold Twenty-four-hour preservation of kidneys instorage by incubation in the presence of jured by prolonged warm ischaemia. TranspfanGSH (1). The capability of the cell to regenration 13, 174-179 (1972). erate GSH following preservation and 12. Lie, T. S., Segar, R., Hong, G. S., Preissinger, transplantation may be an important factor H., and Ogawa, K. Protective effect of aprotinin on ischemic hepatocellular damage. Trunsin suppressing oxygen free radical injury on planration 48, 396-399 (1989). reperfusion. REFERENCES 1. Ametani, M., Belzer, F. O., and Southard, J. H. Importance of glutathione and adenosine in cold storage of the kidney. Transplant. Proc., in press (1990). 2. Anaise, D., Bachvaroff, R., Sato, K., Waltzer, W., Oster, Z., Atkins, H., Pollack, W., and Rapaport, F. T. A membrane stabilization approach to long-term renal preservation. Transplant. Proc. 17, 1457-1460 (1985). 3. Copeland, J. G., Jones, M., Spragg, R., and Stinson, E. B. In vitro preservation of canine hearts for 24 to 28 hours followed by successful orthotopic transplantation. Ann. Surg. 178, 687-692 (1973).
13. Liebach, F. H., Fonteles, M. C., Pillon, D., and Karow, A. M. Glutathione in the isolated perfused rabbit kidney. J. Surg. Res. 17, 228-231 (1974). 14. Marsh, D. C., Lindell, S. L., Fox, L. E., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes I. Role of cell swelling. Cryobiology 26, 520-526 (1989). 15. Marubayashi, S., Dohi, K., Ochi, K., and Kawaski, T. Role of free radicals in ischemic rat liver cell injury: Prevention of damage by u-tocopherol administration. Surgery 99, 184191 (1986). 16. Marubayashi, S., Takenake, M., Dohi, K., Ezaki, H., and Kawasaki, T. Adenine nucleotide metabolism during hepatic ischemia and subse-
LOSS
OF GLUTATHIONE
quent blood reflow periods and its relation to organ viability. Transplantation 30, 294-296 (1980). 17. McCord, J. M. Oxygen derived free radicals in postischemic tissue injury. N. Engl. .I. Med. 312, 1.59-163(1985). 18. Meister, A. Mechanism and function of glutathione. In “Glutathione” (D. Dolphin, et al., Eds.), part A, pp. 367-474. Wiley-Interscience, New York, 1989. 19. Meister, A. Metabolism and transport of glutathione and other a-glutamyl compounds. In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, Ed.), pp. l-22. Raven Press, New York, 1983. 20. Nishida, T., Koseki, M., Kamiike, W., Nakahara, M., Nakao, K., Kawashima, Y., Hashimoto, T., and Tagawa, K. Levels of purine compounds in a perfusate as a biochemical marker of iscbemic injury of cold-preserved liver. Transplantation 44, 16-18 (1987). 21. Ormstad, K., Lastbom, T., and Orrenius, S. Translocation of amino acids and glutathione studied with the perfused kidney and isolated renal cells. FEBS Lett. 112, 55-60 (1980). 22. Palombo, J. D., Hirschberg, Y., Pomposelli, J. J., Blackbum, G. L., Zeisel, S. H., and Bistrian, B. R. Decreased loss of liver adenosine triphosphate during hypothermic preservation in rats pretreated with glucose: Implications for organ 95, 1043-1049 management. Gastroenterology (1988). 23. Pavlock, G. S., Southard, J. H., Starling, J. R., and Belzer, F. 0. Lysosomal enzyme release in hypothermically perfused dog kidneys. Cryobiology 21, 521-529 (1984).
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24. Ploeg, R. J. The European multicenter trial on UW solution for liver transplantation: Preliminary results. Transplant. Proc., in press (1990). 25. Ploeg, R. J., Goosens, D., McAnulty, J. F., Southard, J. H., and Belzer, F. 0. Successful 72-hour cold storage of the dog kidney with UW solution. Transplantation 46, 191-196 (1988). 26. Rush, G. F., Gorski, J. R., Ripple, M. G., Sowinski, J., Bugelski, P., and Hewitt, W. R. Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes. Toxicol. Appl. Pharmacol. 78, 473-483 (1985). 27. Scaduto, R. C., Gattore, V. H., Grotyahann, L. W., Wertz, J., and Martin, L. F. Effect of an altered glutathione content on renal ischemic injury. Amer. J. Physiol. 255, F911-F921 (1988). 28. Seglen, P. 0. Preparation of isolated rat liver cells. Methods Cell. Biol. 13, 28-83 (1976). 29. Todo, S., Nery, J., Yanaga, K., Podesta, L., Gordon, R. D., and Starzl, T. E. Extended preservation of human liver grafts with UW solution. JAMA 261, 711-714 (1989). 30. Vreugdenhil, P. K., Evans, W., Belzer, F. O., and Southard, J. H. Glutathione depletion in cold stored organs. Transplant. Proc., in press (1990). 31. Wahlberg, J. A., Southard, J. H., and Belzer, F. 0. Development of a cold storage solution for pancreas preservation. Cryobiology 23,477482 (1986). 32. Watson, D. C., Reitz, B. A., Baumgartner, W. A., Raney, A. A., Oyer, P. E., Stinson, E. B., and Shumway, N. E. Distant heart procurement for transplantation. Surgery 86,X-59 (1979).
28, 143-149 (191)
Effect of Cold Storage
on Tissue and Cellular
Glutathione’
P. K. VREUGDENHIL,
F. 0. BELZER, AND J. H. SOUTHARD Department of Surgery, University of Wisconsin, Madison, Wisconsin 53792
One of the mechanisms thought to cause injury in preserved organs is the formation of oxygen free radicals. The cell is protected from oxidative stress by many defense mechanisms. A major defense mechanism involves glutathione and glutathione-dependent enzymes. During organ preservation by simple cold storage the loss of glutathione may sensitize the organ to free radical damage after transplantation. In this study we show that glutathione is depleted from the rabbit liver, kidney, and heart cold-stored (YC) for up to 72 h in the UW solution without glutathione. In the first 24 h kidney glutathione decreased to 84 2 3% of control values, liver glutathione decreased to 49 f 3% of control values, and heart glutathione decreased to 73 2 3% of control values. After 48 h of storage the kidney and liver lost an additional 30 and 20%, respectively, whereas heart glutathione changed very little. By 72 h all three organs had lost more than 50% of the glutathione found in freshly obtained tissue. To determine if glutathione added to the UW solution can effectively prevent this loss of glutathione during preservation, hepatocytes were cold-stored for up to 72 h in a preservation solution with and without glutathione. We found that adding glutathione to the preservation solution slowed the rate of loss of glutathione from the cells. These data suggest that at hypothermia the cell may be permeable to GSH. Methods to suppress the loss of glutathione during preservation of organs may be an important factor in suppressing oxygen free radical injury. Q 1991 Academic press, Inc.
Most organs used for transplantation are stored cold (YC) without continuous perfusion (i.e., cold ischemia). Once removed from the circulatory system organs begin the inexorable process of cell death and the rate of cell death is dependent upon the storage conditions. The temperature and time dependency of loss of organ viability suggest that degenerative reactions occur in the tissue that eventually cause irreversible injury. Which components of the cell degenerate during cold storage of organs and how this relates to loss of viability are not known. Glutathione (tGSH, total glutathione, = GSH (reduced) + GSSG (oxidized)), the major cellular thiol, is rapidly metabolized by most tissues and the biosynthesis of GSH requires three moles of adenosine triphosphate (ATP) per mole of GSH. In simple cold storage of organs the synthesis of ATP is limited and up to 90% of tissue
ATP is lost within 24 h of cold storage (4, 20, 22). However the pathways for the catabolism of GSH remain active in the absence of a source of energy. Glutathione serves many roles in cellular metabolism, one of which is the reduction of peroxides (hydrogen peroxide and lipid peroxides) to innocuous products (18). Thus, GSH is an important cellular antioxidant and is a major factor in protecting the cell from various forms of oxidative stress caused by the generation of cytotoxic metabolites of oxygen (hydroxyl radicals and lipid peroxides). How cold storage of organs affects the tissue concentration of glutathione is not known although it is well known that in warm ischemia there is a loss of glutathione from tissue (27). In this study we have determined the rate of loss of tGSH from organs cold-stored for up to 3 days in the UW solution, including the rabbit kidney, liver, and heart. The results show that tGSH is catabolized during cold storage of organs. The loss of tGSH Received January 10, 1990; accepted May 14, 1990. may be a factor contributing to oxygen free r This work supported by NIH Grant DK 35143. 143 001l-2240/91 $3.00 Copytisbt 6 1991 by Academic Press, Inc. All lights of reproduction in any foml reserved.
144
VREUGDENHIL,
BELZER,
radical injury in organs following transplantation. MATERIALS
AND
METHODS
Whole Tissue Kidneys, livers, and hearts were obtained from New Zealand white rabbits (2 to 3 kg) anesthetized with Nembutal (3.9 mg/kg). The organs were flushed out with cold (5°C) UW solution (31) not containing GSH. The kidney was flushed through the renal artery (60 ml), the liver through the portal vein (200 ml), and the heart through the aorta (80 ml) with the indicated volumes of the UW solution. Warm ischemia time was less than 2 min. The organs were stored at 5°C for up to 72 h. Some organs were flushed with warm (37°C) UW solution, stored warm (37°C) for 10 to 30 min, and then flushed with cold UW solution and stored for 24 h. Tissue samples (kidney cortex, liver, and left ventricle of the heart) weighing approximately 1 g were taken at 0,24,48, and 72 h. The samples were frozen in a dry ice and acetone bath, weighed, and homogenized in trichloroacetic acid (6.7%). The homogenate was centrifuged at 1600gfor 4 min and the supernatant collected. The concentration of tGSH (GSH + GSSG) in the supernatant was determined enzymatically (6).
AND
SOUTHARD
20 m&f; raffinose, 30 mM; and polyethylene glycol, 5% (MW = 8000 Da). In some experiments GSH (3 m&f) was added. The cells were allowed to settle and stored for 72 h at 5°C under N2 in sealed 50-ml polycarbonate centrifuge tubes to simulate simple cold storage of the liver (cold ischemia). Every 24 h cellular tGSH was determined by the method of Griffith (6). An aliquot of cells (1 ml) was centrifuged at 13,OOOg for 1 min (Eppendorf microcentrifuge). The supernatant was removed by aspiration and discarded. The sediment (hepatocytes) was sonicated in 1.5 ml trichloroacetic acid (3.3%) and centrifuged as above and GSH was determined enzymatically in the supernatant. RESULTS
The concentrations of tGSH in control kidney, liver, and heart were 519 + 30,2433 ? 165, and 635 ? 34 nmol per gram wet weight, respectively. These values will be used to represent 100% of tissue concentrations of tGSH in each specific organ. The concentrations of tGSH in tissue coldstored in the UW solution (without GSH) for up to 72 h is shown in Fig. 1. In the kidney, tGSH was lost at a rate of about 20% per day.
Isolated Hepatocytes Hepatocytes were prepared from Sprague Dawley rats (300 to 400 g) by a modification of Seglen’s method (28) as previously described by Marsh et al. (14). Hepatocyte protein concentration was adjusted to 6 + 1 mg/ml (Biuret method) and cell viability was determined by trypan blue dye exclusion. Preparations that showed greater than 90% exclusion of trypan blue were used for study. After determining viability, hepatocytes were suspended in the preservation solution (S‘C). The preservation solution contained: K+lactobionate, 100 mM; KH2P04,
01 0
I 24
48 Preservation
72 Time (hr)
1. Glutathione in the rabbit kidney, liver, and heart during cold storage in UW (without GSH). tGSH (GSH + GSSG) values are expressed as a percentage of control values (control = 100%). Data are given as means 2 SEM for six rabbit kidneys, five livers, and five hearts. Methods are described under Materials and Methods. FIG.
LOSS
145
OF GLUTATHIONE
The cold-stored liver lost tGSH more rapidly than the kidney and after 24 h of storage had decreased to 49 + 3% of 0 time values. After 48 and 72 h of storage liver GSH concentrations were 30 t 4 and 23 f 5% of 0 time values. In the cold-stored heart after 24 h the tGSH content was reduced to 73 + 3% and there was no significant change at 48 h. However, by 72 h the tGSH concentration was 28 + 2% of 0 time values. Thus, in all organs the tissue concentration of tGSH was reduced by 55 to 75% after 72 h of storage. In another report we showed that the composition of the storage medium did not alter the rate of breakdown of tGSH which was similar in Eurocollins solution, Ringers Lactate, or the UW solution without GSH (30). The results in Fig. 2 show how a brief period of warm ischemia affects the tGSH concentration in organs cold-stored for 24 h. After 10 min at 37°C the concentration of tGSH in the kidney was 68 ? 6% of 0 time values. After 24 h of cold storage the tGSH concentration was reduced to 36 + 4% of 0 time values and this concentration was significantly lower than the tGSH concentration in kidneys stored for 24 h without exposure to warm ischemia (84 + 3%). Ten minutes of warm ischemia had little effect on the tGSH concentration in the liver or heart. After 30 min of warm ischemia, liver and heart tGSH concentrations were 96 * 3 and 79 + 9% of 0 time values. After a subsequent period of cold storage (24 h) the concentration of tGSH in the liver was 28 * 4% (versus 49 + 3% with no warm ischemia, not significantly different). In the heart 24 h of cold storage after warm ischemia reduced the tGSH concentration to 55 + 8% (versus 73 rt 3% with no warm ischemia, not significantly different). Hepatocytes isolated from rat livers contained 8.49 + 0.51 nmol tGSH/mg protein. The effect of cold storage on tGSH concentration in rat hepatocytes is shown in Fig. 3. When stored for 24 h the tGSH concentra-
Kidney
0
30min
WI
24hr
CS
FIG. 2. Effect of warm ischemia (WI) and subsequent cold storage (CS) on the rate of loss of tGSH from cold-stored rabbit kidney, liver, and heart tissue. Bars represent mean tGSH concentrations as a percentage of control values ? SEM obtained from seven kidneys, five livers, and five hearts. Dashed lines represent means + SEM of organs cold-stored without a period of warm ischemia. Methods are described under Materials and Methods.
tion was 85 + 4 and 53 ? 2% at 48 h and 33 ? 5% after 72 h of storage. Hepatocytes were also stored in the presence of GSH (3 mM) and after sedimentation the concentration of GSH in the cells was determined.
146
VREUGDENHIL,
0
24
4.3 Preservation
BELZER,
72 Time (hr)
FIG. 3. Effect of the addition of GSH to the cold storage solution on intracellular tGSH concentrations in rat hepatocytes. Data are given as means 2 SEM for hepatocytes prepared from five rat livers. The preservation solution used is described under Materials and Methods.
The concentration of tGSH was 98 + 2, 80 + 8, and 57 + 5% of 0 time values after 24, 48, and 72 h of storage, respectively. The tGSH concentrations after each day of storage in the presence of GSH were significantly greater than in hepatocytes stored without GSH. The higher concentrations of tGSH resulting from storing hepatocytes in the presence of GSH were not due to GSH in the extracellular water in sedimented hepatocytes. We have previously shown that the amount of extracellular water in sedimented hepatocytes is 0.1 pJ/mg protein (14). This small amount of extracellular water would not contain enough GSH to elevate the tGSH of hepatocytes suspended in a solution containing 3 mM (0.3 nmoYmg protein). DISCUSSION
Simple cold storage has been the method of choice of most organ transplant centers for organ preservation. Organs are flushed out with a preservation solution and stored at 0 to 4°C without continuous perfusion. In clinical transplantation the kidney, liver, and heart can be safely preserved for about 48 h (24), 35 h (29), and 3 to 6 h (32), respectively. In the laboratory the kidney can be preserved for 72 h (25), the liver for 48 h
AND
SOUTHARD
(IO), and the heart for about 24 to 28 h (3). The quality of preservation is dependent upon many factors, one of which is the length of preservation. Longer preservation usually results in a higher incidence of delayed function of primary nonfunction and the cause for this decrease in viability of the organ is not known. Normal metabolism is a balance between biosynthetic and degradative reactions. In the absence of oxygen and a continual supply of nutrients, biosynthetic reactions are suppressed due to a lack of energy (ATP); however, many degradative reactions do not require a continual supply of energy and may continue even at hypothermia. The fact that organs stored for relatively short periods of time regain normal metabolic functions more rapidly than organs stored for longer periods of time suggests that degradative reactions occurring within the tissue limit viability. The exact nature of these degradative reactions is not clear but evidence suggests that the activity of phospholipases may play a role in the loss or organ viability after extended periods of preservation (2). Evidence suggesting that lysosomal enzymes are activated during prolonged cold storage of the kidney has also been presented (23). Suppressing the activities of some of these enzymes has been shown to improve the results of organ preservation (12). During simple cold storage of organs there also occurs a loss of metabolites including adenine nucleotides, a-tocopherol, pyridine nucleotides, and coenzyme Q (15, 16,20,22). In this study we show that tGSH is also degraded during cold storage of the kidney, liver, and heart. The loss of tGSH from preserved organs (or the inability to regenerate GSH upon reperfusion) could be a causative factor in the loss of organ viability. Many studies have shown that selective depletion of cellular tGSH sensitizes cells to various forms of oxidative injury (9, 27). Furthermore, methods used to enhance tGSH concentra-
LOSS OF GLUTATHIONE
tions in the cell have been shown to protect the cells from various forms of oxidative injury. Recently, Rush et al. (26) have shown that the addition of GSH to hepatocytes protects these cells from injury. The mechanism of protection by GSH appears to be related to the reduction of hydrogen peroxide by glutathione peroxidase. The reduction in the concentration of hydrogen peroxide in the cell (arising from the dismutation of superoxide anions) would also reduce the concentration of hydroxyl radicals formed from hydrogen peroxide and that the hydroxyl radical may be the primary toxic product of oxygen metabolism. The UW solution used for preservation of the liver, kidney, and pancreas (30) contains GSH. Glutathione was added because of its role in protecting tissues from oxidative stress. However, it is unclear if the protection by glutathione is due to its entry into the cell during cold storage. It is difficult to determine the exact tissue distribution of glutathione in a whole organ flushed out with a solution containing glutathione because much of the glutathione may reside in the extracellular spaces. However, it is simpler to determine the cellular location of glutathione in isolated hepatocytes stored in the presence of glutathione. Hepatocytes can be removed from the incubation medium by sedimentation and the cellular concentration of glutathione measured. In this study, the cellular concentration of glutathione in isolated hepatocytes decreased during hypothermic storage in the absence of added glutathione. When stored in the presence of glutathione the rate of loss of cellular glutathione was decreased. For instance, cells stored in the absence of glutathione for 48 h showed an approximate 50% decrease in the cellular concentration of glutathione. However, cells stored in the presence of glutathione lose only approximately 20% of the cellular glutathione. This suggests that glutathione may actually enter cold-stored hepatocytes. Another explanation is that glutathi-
147
one is catabolized to its amino acid constituents which serve as substrates for the regeneration of glutathione. However, since these cells were stored anoxically there would be little ATP available for the synthesis of glutathione. Thus, it is more likely that the suppression of the loss of glutathione from hepatocytes stored in the presence of glutathione was due to the entry of glutathione into the cell. Kidney cells are apparently permeable to glutathione (13, 21), although other cells, at normothermia, do not appear to be permeable to GSH (7). However, the permeability of hepatocytes to substances such as glutathione may be increased by hypothermia. We show in this study that glutathione is decreased in cold-stored rabbit organs and suggest that this decrease may increase the sensitivity of organs to reperfusion injury caused by the generation of oxygen free radicals. Studies in the past have suggested that oxygen free radicals may be a causative factor in the loss of organ viability following preservation (5,8) and one mechanism for the generation of oxygen free radicals has been related to the activity of xanthine oxidase (16, 17). The activation of this enzyme may increase the rate of generation of superoxide anions and cause oxidative stress. However, oxidative stress could result from a decrease in the cells capability to scavenge oxygen free radicals due to a loss of intracellular scavengers, such as glutathione. This could occur even without a significant increase in the rate of generation of oxygen free radicals. Thus, protection of tissues from oxidative stress may require maintenance of endogeneous free radical scavengers in the cell and methods that accomplish this may lead to improved and prolonged organ preservation. Warm ischemia prior to cold storage of the kidney caused a greater rate of loss of glutathione than in kidneys not exposed to warm ischemia. This effect was seen within as few as 10 min of warm ischemia. In the liver and heart a warm ischemic period of
148
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4. D’Alessandro, A., Southard, J. H. Kalayoglu, M., 30 min prior to 24 h cold storage did not and Belzer, F. 0. Comparison of cold storage cause a significantly greater decrease in and perfusion of dog livers on function of tissue glutathione than just cold ischemia itself. slices. Cryobiology 23, 161-167 (1986). This might reflect the greater rate of metab- 5. Fuller, B. J., Cower, J. D., and Green, C. J. Free olism of glutathione in the kidney (50% radical damage and organ preservation: Fact or turnover time = 0.5 h) as compared to the Fiction? A review of the interrelationship between oxidative stress and physiological ion heart and liver (50% turnover time = 2 h) disbalance. Cryobiology 25, 377-393 (1988). (18, 19). Previous studies have shown that 6. Griftith, 0. W. Glutathione and glutathione disulwarm &hernia prior to cold storage sensiphide. In “Methods of Enzymatic Analysis” tizes the kidney to increased preservation (H. U. Bergmeyer, et al., Eds.,), Vol 8, pp. damage (11). One explanation is that a brief 521-529. VCH Verlagsgesellschaft, Weinheim period of warm ischemia causes a rapid (Federal Republic of Germany), 1985. degradation of essential metabolites in the 7. Hahn, R., Wendel, A., and Flohe, L. The fate of extracellular glutathione in the rat. Biochim. kidney (such as glutathione) which causes Biophys. Acta 529, 324-327 (1978). the increased sensitivity to preservation in8. Hemandez, L. A., and Granger, D. N. Role of anjury. Although warm ischemia prior to cold tioxidants in organ preservation and transplanstorage may also limit the preservation tation. Crit. Care. Med. 16, 543-549 (1988). quality of the heart and liver, in these or9. Hoberg, J., and Kristoferson, A. A correlation begans this effect may not be directly related tween glutathione levels and cellular damage in to the loss of glutathione. isolated hepatocytes. Eur. J. Biochem. 74, 7782 (1977). In summary, this study shows that cold10. Jamieson, N. V., Sundberg, R., Lindell, S., stored organs lose tGSH and the loss is Claesson, K., Moen, J., Vreugdenhil, P., more rapid in the liver than in the heart or Wight, D. G. D., Southard, J. H., and Belzer, kidney. However, after 72 h of storage all F. 0. 24-48 hour preservation of the canine organs studied had lost about 50 to 75% of liver by simple cold storage using UW lactotheir tGSH. The addition of GSH to coldbionate solution. Transplant. Proc. 21, 12921293 (1989). stored hepatocytes decreases the rate of 11. Johnson, R. W. G., Anderson, M., Morley, loss of tGSH. Another study has shown A. R., Taylor, R. M. R., and Swinney, J. that cell viability is enhanced during cold Twenty-four-hour preservation of kidneys instorage by incubation in the presence of jured by prolonged warm ischaemia. TranspfanGSH (1). The capability of the cell to regenration 13, 174-179 (1972). erate GSH following preservation and 12. Lie, T. S., Segar, R., Hong, G. S., Preissinger, transplantation may be an important factor H., and Ogawa, K. Protective effect of aprotinin on ischemic hepatocellular damage. Trunsin suppressing oxygen free radical injury on planration 48, 396-399 (1989). reperfusion. REFERENCES 1. Ametani, M., Belzer, F. O., and Southard, J. H. Importance of glutathione and adenosine in cold storage of the kidney. Transplant. Proc., in press (1990). 2. Anaise, D., Bachvaroff, R., Sato, K., Waltzer, W., Oster, Z., Atkins, H., Pollack, W., and Rapaport, F. T. A membrane stabilization approach to long-term renal preservation. Transplant. Proc. 17, 1457-1460 (1985). 3. Copeland, J. G., Jones, M., Spragg, R., and Stinson, E. B. In vitro preservation of canine hearts for 24 to 28 hours followed by successful orthotopic transplantation. Ann. Surg. 178, 687-692 (1973).
13. Liebach, F. H., Fonteles, M. C., Pillon, D., and Karow, A. M. Glutathione in the isolated perfused rabbit kidney. J. Surg. Res. 17, 228-231 (1974). 14. Marsh, D. C., Lindell, S. L., Fox, L. E., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes I. Role of cell swelling. Cryobiology 26, 520-526 (1989). 15. Marubayashi, S., Dohi, K., Ochi, K., and Kawaski, T. Role of free radicals in ischemic rat liver cell injury: Prevention of damage by u-tocopherol administration. Surgery 99, 184191 (1986). 16. Marubayashi, S., Takenake, M., Dohi, K., Ezaki, H., and Kawasaki, T. Adenine nucleotide metabolism during hepatic ischemia and subse-
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quent blood reflow periods and its relation to organ viability. Transplantation 30, 294-296 (1980). 17. McCord, J. M. Oxygen derived free radicals in postischemic tissue injury. N. Engl. .I. Med. 312, 1.59-163(1985). 18. Meister, A. Mechanism and function of glutathione. In “Glutathione” (D. Dolphin, et al., Eds.), part A, pp. 367-474. Wiley-Interscience, New York, 1989. 19. Meister, A. Metabolism and transport of glutathione and other a-glutamyl compounds. In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, Ed.), pp. l-22. Raven Press, New York, 1983. 20. Nishida, T., Koseki, M., Kamiike, W., Nakahara, M., Nakao, K., Kawashima, Y., Hashimoto, T., and Tagawa, K. Levels of purine compounds in a perfusate as a biochemical marker of iscbemic injury of cold-preserved liver. Transplantation 44, 16-18 (1987). 21. Ormstad, K., Lastbom, T., and Orrenius, S. Translocation of amino acids and glutathione studied with the perfused kidney and isolated renal cells. FEBS Lett. 112, 55-60 (1980). 22. Palombo, J. D., Hirschberg, Y., Pomposelli, J. J., Blackbum, G. L., Zeisel, S. H., and Bistrian, B. R. Decreased loss of liver adenosine triphosphate during hypothermic preservation in rats pretreated with glucose: Implications for organ 95, 1043-1049 management. Gastroenterology (1988). 23. Pavlock, G. S., Southard, J. H., Starling, J. R., and Belzer, F. 0. Lysosomal enzyme release in hypothermically perfused dog kidneys. Cryobiology 21, 521-529 (1984).
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24. Ploeg, R. J. The European multicenter trial on UW solution for liver transplantation: Preliminary results. Transplant. Proc., in press (1990). 25. Ploeg, R. J., Goosens, D., McAnulty, J. F., Southard, J. H., and Belzer, F. 0. Successful 72-hour cold storage of the dog kidney with UW solution. Transplantation 46, 191-196 (1988). 26. Rush, G. F., Gorski, J. R., Ripple, M. G., Sowinski, J., Bugelski, P., and Hewitt, W. R. Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes. Toxicol. Appl. Pharmacol. 78, 473-483 (1985). 27. Scaduto, R. C., Gattore, V. H., Grotyahann, L. W., Wertz, J., and Martin, L. F. Effect of an altered glutathione content on renal ischemic injury. Amer. J. Physiol. 255, F911-F921 (1988). 28. Seglen, P. 0. Preparation of isolated rat liver cells. Methods Cell. Biol. 13, 28-83 (1976). 29. Todo, S., Nery, J., Yanaga, K., Podesta, L., Gordon, R. D., and Starzl, T. E. Extended preservation of human liver grafts with UW solution. JAMA 261, 711-714 (1989). 30. Vreugdenhil, P. K., Evans, W., Belzer, F. O., and Southard, J. H. Glutathione depletion in cold stored organs. Transplant. Proc., in press (1990). 31. Wahlberg, J. A., Southard, J. H., and Belzer, F. 0. Development of a cold storage solution for pancreas preservation. Cryobiology 23,477482 (1986). 32. Watson, D. C., Reitz, B. A., Baumgartner, W. A., Raney, A. A., Oyer, P. E., Stinson, E. B., and Shumway, N. E. Distant heart procurement for transplantation. Surgery 86,X-59 (1979).
