- Email: [email protected]
Calcium in pig livers following ischemia and reperfusion
Journal of Hepatology 1994; 20:714-719 Printed h7 Denmark. All rights reserved Munksgaard. Copenhagen
Copyright © Journalof Hepatology 1994 Journal of Hepatology ISSN 0168-8278
Calcium in pig livers following ischemia and reperfusion Masaaki Uchida, Yoshinari T a k e m o t o , N a o f u m i Nagasue, T a k e o K i m o t o , Dipok K u m a r D h a r and Teruhisa N a k a m u r a Second Department o f Surgery. Shimane Medical University, Izumo, Japan
(Received 29 September 1992)
Calcium concentrations in pig livers were serially estimated following ischemia-reperfusion. Ischemia was produced by clamping the hepatic artery and the portal vein for 90 min (Group 1, n=6) or for 180 min (Group 2, n=6) during temporary side-to-side portacaval shunt performed before the induction of ischemia. Although there were no significant changes in hepatic calcium concentrations during ischemia, an immediate accumulation of calcium occurred 30 min after reperfusion in both groups. After these increases, the hepatic calcium concentration decreased to near the pre-ischemic level within 20 min in all animals in Group 1. The recovery of calcium was incomplete in Group 2. When the peak was defined as the highest level of calcium and the bottom as the lowest point after peak 60 min after reperfusion, the meanpeak was 11.0_+1.3 (mean_+SEM) nmol/mg dry weight liver in Group 1 and 12.8_+1.4 nmol/mg dry weight liver in Group 2 (not significant). However, the mean-bottom in Group 1 was lower than that in Group 2 (5.5_+0.3 and 8.1_+0.8 nmol/ mg dry weight liver, respectively, p<0.05). These results indicate that hepatic calcium increases immediately after reperfusion and that recovery from this calcium accumulation seems to be a crucial factor for minimizing cellular injury. © Journal of Hepatology. Key words: Calcium; Ischemia-reperfusion; Liver; Oxygen-derived free radicals
An important and persistent problem in liver transplantation is primary non-function of the hepatic allograft, which occurs at the rate of 6.5-25.0% (1-3). One of the leading causes of this event is associated with ischemiareperfusion injury, which cannot be avoided during liver transplantation. Several mechanisms of ischemia-reperfusion injury have been postulated: a) oxygen-derived free radicals (4), b) calcium influx (5), c) adenosine triphosphate (ATP) depletion and mitochondrial dysfunction (6), d) activation of lysosomal enzymes (7), and e) disturbance of microcirculation (8). However, these factors may be interrelated. It has been suggested that excessive accumulation of intracellular calcium may cause cellular injury and death (9-11). In the present study, calcium concentrations of the liver were serially estimated during the ischemia-reperfusion period. In addition, the relationship between hepatic calcium concentration and released malondialdehyde
(MDA), a parameter of lipid peroxidation, was elucidated. Materials and Methods
All animal experiments were conducted in accordance with the Institute of Experimental Animals of Shimane Medical University guidelines for the care and use of laboratory animals. Anesthesia and operation
Twelve hybrid pigs (Land Race×Yorkshire) of both sexes, weighing 18-22 kg were fasted overnight. The animals were injected with ketamine hydrochloride (25 mg/ kg) and atropine sulfate (0.025 mg/kg) intramuscularly before intratracheal intubation. Anesthesia was maintained with an intravenous bolus injection of sodium pentobarbital (25 mg/kg) at intratracheal intubation and
Correspondence to: Masaaki Uchida, M.D., The Second Department of Surgery, Shimane Medical University, Izumo 693, Japan.
HEPATIC CALCIUM IN ISCHEMIA-REPERFUSION by adding 5 mg/kg every 2 h until the end of operation. Respiration was controlled with the Harvard respirator, which was set at 15 ml/kg of the tidal volume and at 20 times/min of the respiratory rate under room air. Ceftazidime (50 mg/kg) was administered intravenously before surgery. Lactated Ringer solution containing 5% glucose was infused at the constant rate of 10-15 ml/h per kg body weight during the operation. Body temperature and blood pressure were monitored until the end of operation. Normal body temperature (more than 35.0°C) was maintained by the infusion of warm saline into the peritoneal cavity. The liver hilus was exposed through a midline laparotomy. A side-to-side portacaval shunt was performed in all animals, before ischemia, to avoid splanchnic congestion during hepatic ischemia. The animals were randomly divided into two groups: Group 1 (n=6), 90-min ischemia group and Group 2 (n=6), 180-min ischemia group. The sex distributions of pigs were 2:4 (M:F) in Group 1 and 3:3 in Group 2, respectively. Normothermic ischemia of the liver was produced by clamping both the hepatic artery and the portal vein with vascular forceps. Reperfusion of the liver was begun after each ischemic period by removing the clamp and closing the shunt with a hemostatic clip. Systemic heparinization was not performed during the experiment.
Sample collections To measure calcium concentrations of the liver tissue, liver specimens (approximately 2 g) were serially collected by incisional biopsies from the margin of the liver. The biopsies were taken from standardized points of the liver in both groups. Liver biopsy was performed 16 times; before ischemia, at 60 min (Group 1) or 90 min (Group 2) after ischemia, just before reperfusion, and at 5, 10, 15, 20, 25, 30, 45 min and 1, 2, 3, 4, 5, 6 h after reperfusion. Cannulation of the hepatic vein in the left lateral lobe of the liver was performed through the right internal jugular vein with Seldinger's method. The site of the cannulation was confirmed intraoperatively by palpation. The hepatic venous blood was collected to assay malondialdehyde and serum aspartate aminotransferase (sAST). The abdomen was closed in two layers 6 h after reperfusion when all measurements were completed. Pigs were returned to the animal room after confirming spontaneous respiration. Pigs were investigated at 12-h intervals after reperfusion for 3 days.
715 pieces with a fine surgical knife. These pieces were immediately irrigated with 0.1%0 ethylenediaminetetraacetic acid disodium salt (EDTA) in physiological saline and then rinsed with physiological saline alone. The samples were immediately frozen in liquid nitrogen and freezedried to a constant weight for 48 h using lyophilizer (Dura Dry TM Condenser Model, FTS systems, INC, N.Y.). Approximately 80 mg of the dried aliquots were dissolved in 0.5 ml of concentrated sulfuric acid and 0.5 ml of concentrated nitric acid for 24 h in a 50°C water bath. This solution was made up to a volume of 5 ml with the addition of 1 % Lanthanum chloride (LaCI3, Sigma Chemical Co., Ltd., St. Louis), and was left at room temperature for 30 min. Calcium concentrations were estimated using atomic absorption analysis (Polarized Zeeman Atomic Absorption Spectrometer, 180-80, Hitachi Co.,Ltd., Tokyo). The coefficient of variation of the assay was 8.79_ + 1.27%. The reliable detection range was 0.50 nmol/mi - 1.25/lmol/ml. In addition, it was confirmed that the performance of the initial portacaval anastomosis did not lead to changes in hepatic calcium concentration (data not reported).
Assay of MDA The assay of serum MDA concentrations in hepatic venous blood was performed using the method described by Yagi (13). In brief, 0.05 ml of serum was put into 1.0 ml of physiological saline and centrifuged at 3000 rpm for 10 min. Then 4.0 ml of N/12 H2SO 4 and 0.5 ml of 10% phosphotungstic acid were added to 0.5 ml of this solution, and the mixture was centrifuged at 3000 rpm for 10 min. The sediment was mixed with 2.0 ml of N/12 H2SO4 and 0.3 ml of 10% phosphotungstic acid, and the mixture was centrifuged at 3000 rpm for 10 min. The sediment was suspended in 4.0 ml of distilled water, and 1.0 ml of thiobarbituric acid (TBA) reagent (0.67% TBA aqueous solution+glacial acetic acid, 1:1, v/v) was added. The reaction mixture was incubated at 95°C for 60 min. After cooling with tap water, 5.0 ml of n-butanol was added, and centrifuged at 3000 rpm for 10 min. The n-butanol layer was taken for fluorometric measurement (FTC-150, Nippon Bunko Co., Ltd., Tokyo), which was done at 515 nm excitation and 553 nm emission. The standard solution was obtained by reacting 0.5 nmol of tetraethoxypropane with TBA reagent. The above assay was performed using a kit (Lipid Peroxide-Test Wako, 271-50201, Wako Pure Chemical Industries, Ltd., Tokyo). The coefficient of variation was 3.23-+0.36%. The range of the detection limit of this measurement was 0 4 0 nmol/ml.
Assay of calcium concentrations Preparation of the biopsied liver tissues was performed according to a modification of the method described by El-Mofty et al. (12). The specimen was minced into small
Assay of sAST The activities of sAST were determined using an automated analyzer (Hitachi 736, Hitachi Co.,Ltd., Tokyo).
716
M. U C H I D A et al.
Statistical analysis Results were expressed as mean+_SEM. Statistical analyses of the data were performed using the one-way analysis of variance (ANOVA) for comparison of data in the same group. Two-way ANOVA was used for assessing the differences between the two groups. Analysis of the relationship between the hepatic calcium concentration and M D A was done using the least squares linear regression test. Means were considered significantly different if the probability of error was less than 0.05.
Results
Liver test Serum AST activity, shown in Fig. 1, increased significantly in both groups (p<0.01, one-way ANOVA), and it did not decrease within the observation time of 6 h. There was a significant difference in sAST activities between the two groups at 30 rain (p<0.01) and 1, 3, and 6 h (p<0.05, two-way ANOVA) after reperfusion. Calcium concentrations of liver tissue As shown in Fig. 2a-b, there were no marked changes in calcium concentrations during the ischemic period in any of the animals. However, a rapid accumulation of calcium in the liver tissue occurred within 30 min after reperfusion in all animals in both groups. The calcium
Animal survival The experimental procedures in all animals were tolerated well. In Group I, three of six pigs lived for 3 days after reperfusion. Three animals died within 3 days; two from liver failure at 12 and 27 h and one from intestinal obstruction 48 h after reperfusion. In Group 2, all six died within 20 h from liver failure. Death from liver failure was confirmed by high sAST activities (more than 1000 IU/I) and(or) histological finding of severe centrilobular necrosis without extrahepatic causes for death. One animal in Group 2 was excluded from further analysis, because the color of this liver did not change after reperfusion indicating no perfusion.
a Group 1 (90 min ischemia) 20Reperfusion
IO
~
2oo0 ,epeusio
60 ~
bl Group 2 (180 min ischemia) C.~
20'
Reperfusion
"i
[ ***
t
10"
90 0 before // tschemia
0
'1
'2
'3
4'
'S
'6
Hours after Reperfusion Fig. 1. Changes in the activity of sAST after reperfusion. The open circles were 90 rain ischemia (Group 1) and the closed circles were 180 min ischemia (Group 2). *: p<0.05, **: p<0.01 (the two-way ANOVA)
180 rain ischemis
'
0
180(rain)
I
ischcmia
Hours after Reperfusion Fig. 2. Changes in hepatic calcium concentration during ischemiareperfusion in each animal a) in 90 min ischemia (Group 1) and b) in 180 min ischemia (Group 2).
HEPATIC CALCIUM IN 1SCHEMIA-REPERFUSION
717
Reperfusion
~
tO
Q; U c" 0
u
E
200 peak (P)
.._..c_------o
!"
bottom ( B )
u
I schemia
I
_ i_ vla
Reperfusion
Fig. 3. The scheme of changes in hepatic calcium concentration. The peak (P) is the highest level of calcium and the bottom is the lowest level after P during first 60 min after reperfusion.
level then decreased to nearly pre-ischemic levels within 20 min in all animals in G r o u p 1 (Fig. 2a). This marked decrease did not occur in G r o u p 2 (Fig. 2b). The analysis o f calcium concentrations for each animal is illustrated in Fig. 3. The peak (P) is the highest level o f calcium concentration and the b o t t o m (B) is the lowest point after P 60 min after reperfusion. The d a t a are summarized in Table 1. This mean-P level was significantly higher than the mean preischemic level in both groups (p<0.01, one-way A N O V A ) . There was no significant difference in the mean-P level o f calcium concentration in the two groups. However, the mean-B level in G r o u p 2 was significantly higher than that in G r o u p 1 (p<0.05, two-way A N O V A ) . During the late reperfusion period, the mean calcium concentrations 3 and 6 h after reperfusion were statistically higher in G r o u p 2 than in G r o u p 1 (p<0.05, twoway A N O V A ) , as shown in Table 1. The mean calcium concentrations o f the survivors at sacrifice on day 3 was 6.0 +- 1.1 nmol/mg dry weight liver ( D W L ) and of animals which died of liver failure was 12.9+-1.5 nmol/mg D W L at autopsy. A significant difference was observed between the two values (p<0.05, one-way A N O V A ) .
befo~ Ischemla
ff 0
i 10
20
30
i 60
Fig. 4. Changes in the mean MDA concentration during first 60 min after reperfusion. The open bars were 90 min ischemia (Group 1) and closed bars were 180 min ischemia (Group 2).
M D A in hepatic venous blood As shown in Fig. 4, there was no increase in M D A in G r o u p 1. In G r o u p 2, the mean M D A level increased from 72___10 (nmol/g albumin) to 135___76 (nmol/g albumin) within 15 min after reperfusion, but this was not statistically significant. There were no differences between the two groups.
Relationship between hepatic calcium concentration and MDA A relationship between hepatic calcium concentration and M D A level in hepatic venous blood was analyzed using the least squares linear regression test in each group. The values of these two parameters were compared at the same time point after reperfusion. There was no correlation between hepatic calcium concentrations and M D A in G r o u p 1 and in G r o u p 2 (r-'=0.005 and 0.096, respectively).
Calcium concentration in pig liver tissues before ischemia and following reperfusion
90 (n=6) 180 (n=5)
SO
M i n u t e s after R e p e r f u s i o n
TABLE 1 Ischemic period (min)
i 40
Hepatic calcium concentration (nmol/mg dry weight liver) Before ischemia
Peak (P)
Bottom (B)
3h
6h
4.9---0.3 5.1 ---0.4
11.0---1.3 12.8---1.4
5.5---0.3 8.1 ---0.8*
6.8±0.4 12.2---2.3"
6.0---0.6 10.3___2.7"
Data are expressed as mean_SEM. *p<0.05 (two-way ANOVA), in comparison to the values of 90 min ischemia.
718
Discussion
Hepatic ischemia and subsequent reperfusion produces severe damage of liver cells if the period of warm ischemic time is long (14,15). Normothermic ischemia longer than 180 min is fatal in pigs without treatment (15,16). Therefore, two different periods of hepatic ischemia were used in this study; 90 and 180 min. While the former model maintains reversibility of liver cells from ischemia-reperfusion injury, the latter might be fatal. In the present study, three of six pigs lived for 3 days in Group 1 (90 min ischemia), whereas all animals in Group 2 (180 min ischemia) died from liver failure. The results support data reported by others (15,16). Although excessive accumulation of intracellular calcium has been suggested as a cause of cellular injury, there is little direct evidence for this intriguing hypothesis. Moreover, calcium influx has also been suggested as one of the main factors of ischemia-reperfusion injury (9,11). Therefore, it is important to estimate serial changes in hepatic calcium concentration during ischemia and reperfusion. Chien et al. (5) reported that the calcium concentrations in reperfused rat livers corresponded well with liver injury and animal survival. However, calcium levels were not closely measured, especially during the early period after reperfusion in that investigation. Several factors have been postulated as causes of ischemia-reperfusion injury; generation of oxygen-derived free radicals, a failure of ATP recovery, and disturbance of microcirculation (6,17,18). These changes probably occur immediately after reperfusion and are associated. Therefore, in this study calcium concentrations of the liver were serially estimated during ischemia and particularly during early reperfusion periods. In the current study, there were no marked changes in hepatic calcium concentrations in either group during the ischemic period. On the other hand, calcium accumulation occurred within 30 min after reperfusion in both groups. However, the significant difference in calcium levels was observed in the recovery pattern of calcium rather than at the peak level of calcium between both groups during the early reperfusion period. There are several explanations for the differences in hepatic calcium concentration found in the early reperfusion period between the two groups. Generally, the cell membrane has a potent function of pumping out calcium against a l0 000-fold gradient between the intracellular and extracellular space. This action is mainly maintained by Ca :+ATPase and Na + exchange systems which use energy (19). Therefore, the increase or decrease of calcium levels may be the result of competition of these mechanisms against the calcium influx. The recovery of ATP after reperfusion has been shown to be impaired in parallel with the duration of
M. UCHIDA et al. ischemia (6). Thus, the longer ischemia in Group 2 might have depleted intracellular ATP more intensely because of mitochondrial dysfunction, and this might have interfered with the integrity of the membranous pump systems. During the late reperfusion period, excessive calcium which could not be excluded by these systems might promote activation of calcium-dependent autolytic enzymes such as phospholipase, protease and nuclease, resulting in the breakdown of cell structures (20). Moreover, enzymes regulated by calcium may have failed. These factors may promote liver cell injury and lead to cell death after reperfusion. Oxygen-derived free radicals break cell membranes by lipid peroxidation. Therefore, calcium influx into the injured cells may occur through the broken pores of the membranes in accordance with the calcium gradient between intracellular and extracellular spaces. Alternatively, when cells are subjected to a calcium-containing environment after being in a calcium-free condition, intracellular accumulation of calcium occurs and results in cell injury (21). This phenomenon has been called the "calcium paradox" and is compatible with ischemia followed by reperfusion. Thus, little is known about which plays the major role in calcium influx: free radical injury or calcium paradox. To elucidate the relationship between calcium concentrations of the liver and oxidative stress, MDA concentrations in hepatic venous blood were also measured in the current study. MDA is a stable end product of lipid peroxidation, and thought to be a simple and reliable method of assessing the degree of lipid peroxidation (13,22). Therefore, it has been widely used as a parameter of generation of oxygenderived free radicals (23,24). In this study, there was no correlation between hepatic calcium and serum MDA concentrations. These results suggest that the calcium accumulation of the liver during the early reperfusion period was independent of oxidative stress. However, the above theory has several problems. The most important consideration is whether MDA is a sensitive index for oxygen-derived free radicals in pigs. Stein et al. (25) reported that MDA did not change during the reperfusion period after 45 min of warm ischemia of the rat liver, despite a decrease in glutathione, which is a scavenger of oxygen-derived free radicals. This theory must be confirmed by further investigations using more sensitive parameters of oxidative stress. Also, therapeutic trials with oxygen radical scavengers such as superoxide dismutase and catalase (4), or with allopurinol (4,26) may clarify this question. In conclusion, hepatic calcium concentrations increase immediately after reperfusion. Recovery from this calcium accumulation seemed to be a crucial factor for minimizing cellular injury. Therefore, impairment of mechanisms which exclude excessive calcium may play an
HEPATIC CALCIUM IN ISCHEMIA-REPERFUSION
important
role in the d e v e l o p m e n t o f ischemia-reper-
fusion injury o f the liver.
Acknowledgements T h i s study was s u p p o r t e d by G r a n t s (No.02454317) f r o m the J a p a n e s e M i n i s t r y o f E d u c a t i o n .
References 1. Shaw BW Jr, Gordon R.D, Iwatsuki S, Starzl TE. R.etransplantation of the liver. Semin Liver Dis 1985; 5: 394-401. 2. Burdelski M, Oellerich M, Raude E, et al. A novel approach to assessment of liver function in donors. Transplant Proc 1988; 20: 591-3. 3. D'Alessandro AM, Kalayoglu M, Sollinger HW, et al. The predictive value of donor liver biopsies for the development of primary nonfunction after orthotopic liver transplantation. Transplantation 1991; 51: 157-63. 4. Adkison D, H611warth ME, Benoit JN, Parks DA, McCord JM, Granger DN. Role of free radicals in ischemia-reperfusion injury to the liver. Acta Physiol Scand Suppl 1986; 548: 101-7. 5. Chien KR, Abrams J, Pfau RG, Farber JL. Prevention by chlorpromazine of ischemic liver cell death. Am J Pathol 1977; 88: 539-58. 6. Marubayashi S, Takenaka M, Dohi K, Ezaki H, Kawasaki T. Adenine nucleotide metabolism during hepatic ischemia and subsequent blood reflow periods and its relation to organ viability. Transplantation 1980; 30: 294-6. 7. Hayashi T, Nagasue N, Kohno H, Chang YC, Nakamura T. Beneficial effect of cyclosporine pretreatment in canine liver ischemia. Enzymatic and electromicroscopic studies. Transplantation 1991; 46: 116-21. 8. Hayashi H, Chaudry IH, Clemens MG, et al. Hepatic ischemia models for determining the effects of ATP-MgC12 treatment. J Surg Res 1986; 40: 167-75. 9. Farber JL. The role of calcium and cell death. Life Sci 1981; 29: 1289-95. 10. Trump BF, Berezesky IK. The role of calcium in cell injury and repair: a hypothesis. Surv Synth Pathol Res 1985; 4: 248-56. 11. Cheung JY, Bonventre JV, Malis CD, Leaf A. Calcium and ischemic injury. N Engl J Med 1986; 26: 1670-6.
719 12. EI-Mofty SK, Scrutton MC, Serroni A, Nicolini C, Farber JL. Early, reversible plasma membrane injury in galactosamine-induced liver cell death. Am J Pathol 1975; 79: 579-96. 13. Yagi K. A simple fluorometric assay of lipoperoxide in blood plasma. Biochem Med 1976; 15: 212. 14. Alvarez-Lopez A, de Hemptinne B, Hoebeke Y, Lambotte L. Prostaglandin E2 increases the tolerance of the rat liver to warm ischemia in absence of splanchnic congestion. Transplant Proc 1987; XIX: 4105-9. 15. Nordlinger B, Douvin D, Javaudin L, et al. An experimental study of survival after two hours of normothermic hepatic ischemia. Surg Gynecol Obstet 1980; 150: 859-64. 16. Kim YI, Kawano K, Goto S, et al. Protection of pig liver against normothermic ischemia by immunosuppressants cyclosporine and azathioprine. Transplantation 1992; 54: 182-4. 17. Roberts MJD, Young IS, Trouton TG, et al. Transient release of lipid peroxides after coronary artery balloon angioplasty. Lancet 1990; 336: 143-45. 18. Koo A, Komatsu H, Tao G, Inoue M, Guth PH, Kaplowitz N. Contribution of no-reflow phenomenon to hepatic injury after ischemia-reperfusion: evidence for a role for superoxide anion. Hepatology 1991; 15: 507-14. 19. Thomas CE, Reed DJ. Current status of calcium in hepatocellular injury. Hepatology 1989; 10: 375. 20. Nicotera P, Hartzell P, Davis G, Orrenius S. The formation of plasma membrane blebs in hepatocytes exposed to agonists that increase cytosolic Ca 2÷ is mediated by the activation of a nonlysosomal proteolytic system. FEBS Lett 1986; 209: 139-46. 21. Zimmerman ANE, Hurdsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966; 211: 646-7. 22. Slater TF. Overview of methods used for detecting lipid peroxidation. In: Packer L, ed. Oxygen Radicals in Biological Systems. Methods Enzymol 1984; 105: 283-93. 23. Dhar DK, Nagasue N, Kimoto T, Uchida M, Takemoto Y, Nakamura T. The salutary effect of FK506 in ischemia-reperfusion injury of the canine liver. Transplantation 1992; 54: 583-8. 24. Omar-R, Nomikos I, Piccorelli G, Savino J, Agarwal N. Prevention of postischemic lipid peroxidation and liver cell injury by ion chelation. Gut 1989; 30: 510-4. 25. Stein H J, Oosthuizen MMJ, Hinder RA, Lamprechts H. Effect of verapamil on hepatic ischemia/reperfusion injury. Am J Surg 1993; 165: 96-100. 26. Moorhouse PC, Grootveld M, Halliwell B, Quinlan JG, Gutteridge JMC. Allopurinol and oxypurinol are hydroxyl radical scavengers. FEBS Lett 1987; 213: 23-8.
Copyright © Journalof Hepatology 1994 Journal of Hepatology ISSN 0168-8278
Calcium in pig livers following ischemia and reperfusion Masaaki Uchida, Yoshinari T a k e m o t o , N a o f u m i Nagasue, T a k e o K i m o t o , Dipok K u m a r D h a r and Teruhisa N a k a m u r a Second Department o f Surgery. Shimane Medical University, Izumo, Japan
(Received 29 September 1992)
Calcium concentrations in pig livers were serially estimated following ischemia-reperfusion. Ischemia was produced by clamping the hepatic artery and the portal vein for 90 min (Group 1, n=6) or for 180 min (Group 2, n=6) during temporary side-to-side portacaval shunt performed before the induction of ischemia. Although there were no significant changes in hepatic calcium concentrations during ischemia, an immediate accumulation of calcium occurred 30 min after reperfusion in both groups. After these increases, the hepatic calcium concentration decreased to near the pre-ischemic level within 20 min in all animals in Group 1. The recovery of calcium was incomplete in Group 2. When the peak was defined as the highest level of calcium and the bottom as the lowest point after peak 60 min after reperfusion, the meanpeak was 11.0_+1.3 (mean_+SEM) nmol/mg dry weight liver in Group 1 and 12.8_+1.4 nmol/mg dry weight liver in Group 2 (not significant). However, the mean-bottom in Group 1 was lower than that in Group 2 (5.5_+0.3 and 8.1_+0.8 nmol/ mg dry weight liver, respectively, p<0.05). These results indicate that hepatic calcium increases immediately after reperfusion and that recovery from this calcium accumulation seems to be a crucial factor for minimizing cellular injury. © Journal of Hepatology. Key words: Calcium; Ischemia-reperfusion; Liver; Oxygen-derived free radicals
An important and persistent problem in liver transplantation is primary non-function of the hepatic allograft, which occurs at the rate of 6.5-25.0% (1-3). One of the leading causes of this event is associated with ischemiareperfusion injury, which cannot be avoided during liver transplantation. Several mechanisms of ischemia-reperfusion injury have been postulated: a) oxygen-derived free radicals (4), b) calcium influx (5), c) adenosine triphosphate (ATP) depletion and mitochondrial dysfunction (6), d) activation of lysosomal enzymes (7), and e) disturbance of microcirculation (8). However, these factors may be interrelated. It has been suggested that excessive accumulation of intracellular calcium may cause cellular injury and death (9-11). In the present study, calcium concentrations of the liver were serially estimated during the ischemia-reperfusion period. In addition, the relationship between hepatic calcium concentration and released malondialdehyde
(MDA), a parameter of lipid peroxidation, was elucidated. Materials and Methods
All animal experiments were conducted in accordance with the Institute of Experimental Animals of Shimane Medical University guidelines for the care and use of laboratory animals. Anesthesia and operation
Twelve hybrid pigs (Land Race×Yorkshire) of both sexes, weighing 18-22 kg were fasted overnight. The animals were injected with ketamine hydrochloride (25 mg/ kg) and atropine sulfate (0.025 mg/kg) intramuscularly before intratracheal intubation. Anesthesia was maintained with an intravenous bolus injection of sodium pentobarbital (25 mg/kg) at intratracheal intubation and
Correspondence to: Masaaki Uchida, M.D., The Second Department of Surgery, Shimane Medical University, Izumo 693, Japan.
HEPATIC CALCIUM IN ISCHEMIA-REPERFUSION by adding 5 mg/kg every 2 h until the end of operation. Respiration was controlled with the Harvard respirator, which was set at 15 ml/kg of the tidal volume and at 20 times/min of the respiratory rate under room air. Ceftazidime (50 mg/kg) was administered intravenously before surgery. Lactated Ringer solution containing 5% glucose was infused at the constant rate of 10-15 ml/h per kg body weight during the operation. Body temperature and blood pressure were monitored until the end of operation. Normal body temperature (more than 35.0°C) was maintained by the infusion of warm saline into the peritoneal cavity. The liver hilus was exposed through a midline laparotomy. A side-to-side portacaval shunt was performed in all animals, before ischemia, to avoid splanchnic congestion during hepatic ischemia. The animals were randomly divided into two groups: Group 1 (n=6), 90-min ischemia group and Group 2 (n=6), 180-min ischemia group. The sex distributions of pigs were 2:4 (M:F) in Group 1 and 3:3 in Group 2, respectively. Normothermic ischemia of the liver was produced by clamping both the hepatic artery and the portal vein with vascular forceps. Reperfusion of the liver was begun after each ischemic period by removing the clamp and closing the shunt with a hemostatic clip. Systemic heparinization was not performed during the experiment.
Sample collections To measure calcium concentrations of the liver tissue, liver specimens (approximately 2 g) were serially collected by incisional biopsies from the margin of the liver. The biopsies were taken from standardized points of the liver in both groups. Liver biopsy was performed 16 times; before ischemia, at 60 min (Group 1) or 90 min (Group 2) after ischemia, just before reperfusion, and at 5, 10, 15, 20, 25, 30, 45 min and 1, 2, 3, 4, 5, 6 h after reperfusion. Cannulation of the hepatic vein in the left lateral lobe of the liver was performed through the right internal jugular vein with Seldinger's method. The site of the cannulation was confirmed intraoperatively by palpation. The hepatic venous blood was collected to assay malondialdehyde and serum aspartate aminotransferase (sAST). The abdomen was closed in two layers 6 h after reperfusion when all measurements were completed. Pigs were returned to the animal room after confirming spontaneous respiration. Pigs were investigated at 12-h intervals after reperfusion for 3 days.
715 pieces with a fine surgical knife. These pieces were immediately irrigated with 0.1%0 ethylenediaminetetraacetic acid disodium salt (EDTA) in physiological saline and then rinsed with physiological saline alone. The samples were immediately frozen in liquid nitrogen and freezedried to a constant weight for 48 h using lyophilizer (Dura Dry TM Condenser Model, FTS systems, INC, N.Y.). Approximately 80 mg of the dried aliquots were dissolved in 0.5 ml of concentrated sulfuric acid and 0.5 ml of concentrated nitric acid for 24 h in a 50°C water bath. This solution was made up to a volume of 5 ml with the addition of 1 % Lanthanum chloride (LaCI3, Sigma Chemical Co., Ltd., St. Louis), and was left at room temperature for 30 min. Calcium concentrations were estimated using atomic absorption analysis (Polarized Zeeman Atomic Absorption Spectrometer, 180-80, Hitachi Co.,Ltd., Tokyo). The coefficient of variation of the assay was 8.79_ + 1.27%. The reliable detection range was 0.50 nmol/mi - 1.25/lmol/ml. In addition, it was confirmed that the performance of the initial portacaval anastomosis did not lead to changes in hepatic calcium concentration (data not reported).
Assay of MDA The assay of serum MDA concentrations in hepatic venous blood was performed using the method described by Yagi (13). In brief, 0.05 ml of serum was put into 1.0 ml of physiological saline and centrifuged at 3000 rpm for 10 min. Then 4.0 ml of N/12 H2SO 4 and 0.5 ml of 10% phosphotungstic acid were added to 0.5 ml of this solution, and the mixture was centrifuged at 3000 rpm for 10 min. The sediment was mixed with 2.0 ml of N/12 H2SO4 and 0.3 ml of 10% phosphotungstic acid, and the mixture was centrifuged at 3000 rpm for 10 min. The sediment was suspended in 4.0 ml of distilled water, and 1.0 ml of thiobarbituric acid (TBA) reagent (0.67% TBA aqueous solution+glacial acetic acid, 1:1, v/v) was added. The reaction mixture was incubated at 95°C for 60 min. After cooling with tap water, 5.0 ml of n-butanol was added, and centrifuged at 3000 rpm for 10 min. The n-butanol layer was taken for fluorometric measurement (FTC-150, Nippon Bunko Co., Ltd., Tokyo), which was done at 515 nm excitation and 553 nm emission. The standard solution was obtained by reacting 0.5 nmol of tetraethoxypropane with TBA reagent. The above assay was performed using a kit (Lipid Peroxide-Test Wako, 271-50201, Wako Pure Chemical Industries, Ltd., Tokyo). The coefficient of variation was 3.23-+0.36%. The range of the detection limit of this measurement was 0 4 0 nmol/ml.
Assay of calcium concentrations Preparation of the biopsied liver tissues was performed according to a modification of the method described by El-Mofty et al. (12). The specimen was minced into small
Assay of sAST The activities of sAST were determined using an automated analyzer (Hitachi 736, Hitachi Co.,Ltd., Tokyo).
716
M. U C H I D A et al.
Statistical analysis Results were expressed as mean+_SEM. Statistical analyses of the data were performed using the one-way analysis of variance (ANOVA) for comparison of data in the same group. Two-way ANOVA was used for assessing the differences between the two groups. Analysis of the relationship between the hepatic calcium concentration and M D A was done using the least squares linear regression test. Means were considered significantly different if the probability of error was less than 0.05.
Results
Liver test Serum AST activity, shown in Fig. 1, increased significantly in both groups (p<0.01, one-way ANOVA), and it did not decrease within the observation time of 6 h. There was a significant difference in sAST activities between the two groups at 30 rain (p<0.01) and 1, 3, and 6 h (p<0.05, two-way ANOVA) after reperfusion. Calcium concentrations of liver tissue As shown in Fig. 2a-b, there were no marked changes in calcium concentrations during the ischemic period in any of the animals. However, a rapid accumulation of calcium in the liver tissue occurred within 30 min after reperfusion in all animals in both groups. The calcium
Animal survival The experimental procedures in all animals were tolerated well. In Group I, three of six pigs lived for 3 days after reperfusion. Three animals died within 3 days; two from liver failure at 12 and 27 h and one from intestinal obstruction 48 h after reperfusion. In Group 2, all six died within 20 h from liver failure. Death from liver failure was confirmed by high sAST activities (more than 1000 IU/I) and(or) histological finding of severe centrilobular necrosis without extrahepatic causes for death. One animal in Group 2 was excluded from further analysis, because the color of this liver did not change after reperfusion indicating no perfusion.
a Group 1 (90 min ischemia) 20Reperfusion
IO
~
2oo0 ,epeusio
60 ~
bl Group 2 (180 min ischemia) C.~
20'
Reperfusion
"i
[ ***
t
10"
90 0 before // tschemia
0
'1
'2
'3
4'
'S
'6
Hours after Reperfusion Fig. 1. Changes in the activity of sAST after reperfusion. The open circles were 90 rain ischemia (Group 1) and the closed circles were 180 min ischemia (Group 2). *: p<0.05, **: p<0.01 (the two-way ANOVA)
180 rain ischemis
'
0
180(rain)
I
ischcmia
Hours after Reperfusion Fig. 2. Changes in hepatic calcium concentration during ischemiareperfusion in each animal a) in 90 min ischemia (Group 1) and b) in 180 min ischemia (Group 2).
HEPATIC CALCIUM IN 1SCHEMIA-REPERFUSION
717
Reperfusion
~
tO
Q; U c" 0
u
E
200 peak (P)
.._..c_------o
!"
bottom ( B )
u
I schemia
I
_ i_ vla
Reperfusion
Fig. 3. The scheme of changes in hepatic calcium concentration. The peak (P) is the highest level of calcium and the bottom is the lowest level after P during first 60 min after reperfusion.
level then decreased to nearly pre-ischemic levels within 20 min in all animals in G r o u p 1 (Fig. 2a). This marked decrease did not occur in G r o u p 2 (Fig. 2b). The analysis o f calcium concentrations for each animal is illustrated in Fig. 3. The peak (P) is the highest level o f calcium concentration and the b o t t o m (B) is the lowest point after P 60 min after reperfusion. The d a t a are summarized in Table 1. This mean-P level was significantly higher than the mean preischemic level in both groups (p<0.01, one-way A N O V A ) . There was no significant difference in the mean-P level o f calcium concentration in the two groups. However, the mean-B level in G r o u p 2 was significantly higher than that in G r o u p 1 (p<0.05, two-way A N O V A ) . During the late reperfusion period, the mean calcium concentrations 3 and 6 h after reperfusion were statistically higher in G r o u p 2 than in G r o u p 1 (p<0.05, twoway A N O V A ) , as shown in Table 1. The mean calcium concentrations o f the survivors at sacrifice on day 3 was 6.0 +- 1.1 nmol/mg dry weight liver ( D W L ) and of animals which died of liver failure was 12.9+-1.5 nmol/mg D W L at autopsy. A significant difference was observed between the two values (p<0.05, one-way A N O V A ) .
befo~ Ischemla
ff 0
i 10
20
30
i 60
Fig. 4. Changes in the mean MDA concentration during first 60 min after reperfusion. The open bars were 90 min ischemia (Group 1) and closed bars were 180 min ischemia (Group 2).
M D A in hepatic venous blood As shown in Fig. 4, there was no increase in M D A in G r o u p 1. In G r o u p 2, the mean M D A level increased from 72___10 (nmol/g albumin) to 135___76 (nmol/g albumin) within 15 min after reperfusion, but this was not statistically significant. There were no differences between the two groups.
Relationship between hepatic calcium concentration and MDA A relationship between hepatic calcium concentration and M D A level in hepatic venous blood was analyzed using the least squares linear regression test in each group. The values of these two parameters were compared at the same time point after reperfusion. There was no correlation between hepatic calcium concentrations and M D A in G r o u p 1 and in G r o u p 2 (r-'=0.005 and 0.096, respectively).
Calcium concentration in pig liver tissues before ischemia and following reperfusion
90 (n=6) 180 (n=5)
SO
M i n u t e s after R e p e r f u s i o n
TABLE 1 Ischemic period (min)
i 40
Hepatic calcium concentration (nmol/mg dry weight liver) Before ischemia
Peak (P)
Bottom (B)
3h
6h
4.9---0.3 5.1 ---0.4
11.0---1.3 12.8---1.4
5.5---0.3 8.1 ---0.8*
6.8±0.4 12.2---2.3"
6.0---0.6 10.3___2.7"
Data are expressed as mean_SEM. *p<0.05 (two-way ANOVA), in comparison to the values of 90 min ischemia.
718
Discussion
Hepatic ischemia and subsequent reperfusion produces severe damage of liver cells if the period of warm ischemic time is long (14,15). Normothermic ischemia longer than 180 min is fatal in pigs without treatment (15,16). Therefore, two different periods of hepatic ischemia were used in this study; 90 and 180 min. While the former model maintains reversibility of liver cells from ischemia-reperfusion injury, the latter might be fatal. In the present study, three of six pigs lived for 3 days in Group 1 (90 min ischemia), whereas all animals in Group 2 (180 min ischemia) died from liver failure. The results support data reported by others (15,16). Although excessive accumulation of intracellular calcium has been suggested as a cause of cellular injury, there is little direct evidence for this intriguing hypothesis. Moreover, calcium influx has also been suggested as one of the main factors of ischemia-reperfusion injury (9,11). Therefore, it is important to estimate serial changes in hepatic calcium concentration during ischemia and reperfusion. Chien et al. (5) reported that the calcium concentrations in reperfused rat livers corresponded well with liver injury and animal survival. However, calcium levels were not closely measured, especially during the early period after reperfusion in that investigation. Several factors have been postulated as causes of ischemia-reperfusion injury; generation of oxygen-derived free radicals, a failure of ATP recovery, and disturbance of microcirculation (6,17,18). These changes probably occur immediately after reperfusion and are associated. Therefore, in this study calcium concentrations of the liver were serially estimated during ischemia and particularly during early reperfusion periods. In the current study, there were no marked changes in hepatic calcium concentrations in either group during the ischemic period. On the other hand, calcium accumulation occurred within 30 min after reperfusion in both groups. However, the significant difference in calcium levels was observed in the recovery pattern of calcium rather than at the peak level of calcium between both groups during the early reperfusion period. There are several explanations for the differences in hepatic calcium concentration found in the early reperfusion period between the two groups. Generally, the cell membrane has a potent function of pumping out calcium against a l0 000-fold gradient between the intracellular and extracellular space. This action is mainly maintained by Ca :+ATPase and Na + exchange systems which use energy (19). Therefore, the increase or decrease of calcium levels may be the result of competition of these mechanisms against the calcium influx. The recovery of ATP after reperfusion has been shown to be impaired in parallel with the duration of
M. UCHIDA et al. ischemia (6). Thus, the longer ischemia in Group 2 might have depleted intracellular ATP more intensely because of mitochondrial dysfunction, and this might have interfered with the integrity of the membranous pump systems. During the late reperfusion period, excessive calcium which could not be excluded by these systems might promote activation of calcium-dependent autolytic enzymes such as phospholipase, protease and nuclease, resulting in the breakdown of cell structures (20). Moreover, enzymes regulated by calcium may have failed. These factors may promote liver cell injury and lead to cell death after reperfusion. Oxygen-derived free radicals break cell membranes by lipid peroxidation. Therefore, calcium influx into the injured cells may occur through the broken pores of the membranes in accordance with the calcium gradient between intracellular and extracellular spaces. Alternatively, when cells are subjected to a calcium-containing environment after being in a calcium-free condition, intracellular accumulation of calcium occurs and results in cell injury (21). This phenomenon has been called the "calcium paradox" and is compatible with ischemia followed by reperfusion. Thus, little is known about which plays the major role in calcium influx: free radical injury or calcium paradox. To elucidate the relationship between calcium concentrations of the liver and oxidative stress, MDA concentrations in hepatic venous blood were also measured in the current study. MDA is a stable end product of lipid peroxidation, and thought to be a simple and reliable method of assessing the degree of lipid peroxidation (13,22). Therefore, it has been widely used as a parameter of generation of oxygenderived free radicals (23,24). In this study, there was no correlation between hepatic calcium and serum MDA concentrations. These results suggest that the calcium accumulation of the liver during the early reperfusion period was independent of oxidative stress. However, the above theory has several problems. The most important consideration is whether MDA is a sensitive index for oxygen-derived free radicals in pigs. Stein et al. (25) reported that MDA did not change during the reperfusion period after 45 min of warm ischemia of the rat liver, despite a decrease in glutathione, which is a scavenger of oxygen-derived free radicals. This theory must be confirmed by further investigations using more sensitive parameters of oxidative stress. Also, therapeutic trials with oxygen radical scavengers such as superoxide dismutase and catalase (4), or with allopurinol (4,26) may clarify this question. In conclusion, hepatic calcium concentrations increase immediately after reperfusion. Recovery from this calcium accumulation seemed to be a crucial factor for minimizing cellular injury. Therefore, impairment of mechanisms which exclude excessive calcium may play an
HEPATIC CALCIUM IN ISCHEMIA-REPERFUSION
important
role in the d e v e l o p m e n t o f ischemia-reper-
fusion injury o f the liver.
Acknowledgements T h i s study was s u p p o r t e d by G r a n t s (No.02454317) f r o m the J a p a n e s e M i n i s t r y o f E d u c a t i o n .
References 1. Shaw BW Jr, Gordon R.D, Iwatsuki S, Starzl TE. R.etransplantation of the liver. Semin Liver Dis 1985; 5: 394-401. 2. Burdelski M, Oellerich M, Raude E, et al. A novel approach to assessment of liver function in donors. Transplant Proc 1988; 20: 591-3. 3. D'Alessandro AM, Kalayoglu M, Sollinger HW, et al. The predictive value of donor liver biopsies for the development of primary nonfunction after orthotopic liver transplantation. Transplantation 1991; 51: 157-63. 4. Adkison D, H611warth ME, Benoit JN, Parks DA, McCord JM, Granger DN. Role of free radicals in ischemia-reperfusion injury to the liver. Acta Physiol Scand Suppl 1986; 548: 101-7. 5. Chien KR, Abrams J, Pfau RG, Farber JL. Prevention by chlorpromazine of ischemic liver cell death. Am J Pathol 1977; 88: 539-58. 6. Marubayashi S, Takenaka M, Dohi K, Ezaki H, Kawasaki T. Adenine nucleotide metabolism during hepatic ischemia and subsequent blood reflow periods and its relation to organ viability. Transplantation 1980; 30: 294-6. 7. Hayashi T, Nagasue N, Kohno H, Chang YC, Nakamura T. Beneficial effect of cyclosporine pretreatment in canine liver ischemia. Enzymatic and electromicroscopic studies. Transplantation 1991; 46: 116-21. 8. Hayashi H, Chaudry IH, Clemens MG, et al. Hepatic ischemia models for determining the effects of ATP-MgC12 treatment. J Surg Res 1986; 40: 167-75. 9. Farber JL. The role of calcium and cell death. Life Sci 1981; 29: 1289-95. 10. Trump BF, Berezesky IK. The role of calcium in cell injury and repair: a hypothesis. Surv Synth Pathol Res 1985; 4: 248-56. 11. Cheung JY, Bonventre JV, Malis CD, Leaf A. Calcium and ischemic injury. N Engl J Med 1986; 26: 1670-6.
719 12. EI-Mofty SK, Scrutton MC, Serroni A, Nicolini C, Farber JL. Early, reversible plasma membrane injury in galactosamine-induced liver cell death. Am J Pathol 1975; 79: 579-96. 13. Yagi K. A simple fluorometric assay of lipoperoxide in blood plasma. Biochem Med 1976; 15: 212. 14. Alvarez-Lopez A, de Hemptinne B, Hoebeke Y, Lambotte L. Prostaglandin E2 increases the tolerance of the rat liver to warm ischemia in absence of splanchnic congestion. Transplant Proc 1987; XIX: 4105-9. 15. Nordlinger B, Douvin D, Javaudin L, et al. An experimental study of survival after two hours of normothermic hepatic ischemia. Surg Gynecol Obstet 1980; 150: 859-64. 16. Kim YI, Kawano K, Goto S, et al. Protection of pig liver against normothermic ischemia by immunosuppressants cyclosporine and azathioprine. Transplantation 1992; 54: 182-4. 17. Roberts MJD, Young IS, Trouton TG, et al. Transient release of lipid peroxides after coronary artery balloon angioplasty. Lancet 1990; 336: 143-45. 18. Koo A, Komatsu H, Tao G, Inoue M, Guth PH, Kaplowitz N. Contribution of no-reflow phenomenon to hepatic injury after ischemia-reperfusion: evidence for a role for superoxide anion. Hepatology 1991; 15: 507-14. 19. Thomas CE, Reed DJ. Current status of calcium in hepatocellular injury. Hepatology 1989; 10: 375. 20. Nicotera P, Hartzell P, Davis G, Orrenius S. The formation of plasma membrane blebs in hepatocytes exposed to agonists that increase cytosolic Ca 2÷ is mediated by the activation of a nonlysosomal proteolytic system. FEBS Lett 1986; 209: 139-46. 21. Zimmerman ANE, Hurdsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966; 211: 646-7. 22. Slater TF. Overview of methods used for detecting lipid peroxidation. In: Packer L, ed. Oxygen Radicals in Biological Systems. Methods Enzymol 1984; 105: 283-93. 23. Dhar DK, Nagasue N, Kimoto T, Uchida M, Takemoto Y, Nakamura T. The salutary effect of FK506 in ischemia-reperfusion injury of the canine liver. Transplantation 1992; 54: 583-8. 24. Omar-R, Nomikos I, Piccorelli G, Savino J, Agarwal N. Prevention of postischemic lipid peroxidation and liver cell injury by ion chelation. Gut 1989; 30: 510-4. 25. Stein H J, Oosthuizen MMJ, Hinder RA, Lamprechts H. Effect of verapamil on hepatic ischemia/reperfusion injury. Am J Surg 1993; 165: 96-100. 26. Moorhouse PC, Grootveld M, Halliwell B, Quinlan JG, Gutteridge JMC. Allopurinol and oxypurinol are hydroxyl radical scavengers. FEBS Lett 1987; 213: 23-8.