- Email: [email protected]
Effect of artificial cells on hepatic function after ischemia–reperfusion injury in liver
Effect of Artificial Cells on Hepatic Function After Ischemia–Reperfusion Injury in Liver E.J. Chang, S.H. Lee, K.C. Mun, S.I. Suh, J.H. Bae, S.P. Kim, H.J. Choi, K.B. Cho, and J.S. Hwang ABSTRACT Background: The liver suffers from ischemia/reperfusion injury during transplantation. Reactive oxygen species generated by xanthine oxidase during reperfusion of the ischemic liver may be partially responsible for the hepatic injury. Oxygen free radicals are removed by antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. Using glutaraldehyde and lysine we constructed crosslinked hemoglobin, containing SOD and catalase, and assessed its ability to protect against ischemia/ reperfusion injury during transplantation. Methods: In contrast to the sham-operated control groups, blood was exchanged using crosslinked hemoglobin (polyHb) a PolyHb–SOD– catalase (PSC) group. After ischemia/ reperfusion injury, several parameters of hepatic damage and oxygen free radicals were measured as well as microscopic examination. Results: Alanine aminotransferase, aspartate aminotransferase, superoxide production, hydrogen peroxide, and malondialdehyde levels were higher among the PolyHb group than sham-operated controls. The PolyHb group revealed a few apoptotic bodies, some acute inflammatory infiltrates in the sinusoids, nuclear fragmentations, cell shrinkage, and chromatin clumping with formation of apoptotic bodies in the apoptotic cells under microscopic examination. Alanine aminotransferase, aspartate aminotransferase, superoxide production, and hydrogen peroxide levels were lower in the PSC than the PolyHb group. Hepatic structures were well preserved in the PSC group. Conclusions: Reactive oxygen species contribute to hepatic dysfunction with morphologic changes. PSC is effective to reduce hepatic damage by lowering oxygen free radical–mediated injury after ischemia/reperfusion in the liver.
T
HE LIVER undergoes ischemia/reperfusion (I/R) injuries during transplantation.1–3 Reactive oxygen species generated by xanthine oxidase during reperfusion of the ischemic liver may be partially responsible for the hepatic injury.4 – 6 Antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, remove oxygen free radicals.7 Crosslinking hemoglobin with SOD and catalase helps to limit free radical reactivity of modified hemoglobin red blood cell substitutes. Using glutaraldehyde we prepared crosslinked hemoglobin, containing SOD and catalase, to protect against I/R injury during transplantation.
MATERIALS AND METHODS Animals Normal male Sprague-Dawley rats, weighing between 280 and 320 g, were divided into three groups. Group 1 (n ⫽ 10) underwent From the Institute for Medical Science and Chronic Disease Research Center, Keimyung University School of Medicine, Daegu, Korea. Supported by Grant R05-2002-000-00012-0 (2002) from the Basic Research Program of the Korea Science & Engineering Foundation. Address reprint requests to Dr Kyo-Cheol Mun, Department of Biochemistry, Keimyung University School of Medicine, 194 Dong San Dong, Daegu, 700-712, Korea.E-mail: [email protected]
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.08.092
Transplantation Proceedings, 36, 1959 –1961 (2004)
1959
1960
CHANG, LEE, MUN ET AL Table 1. Parameters Related to Hepatic Damage and Oxygen Free Radicals Parameters
Group 1 (n ⫽ 10)
Serum alanine aminotransferase (Karmen U/mL) Cytosolic alanine aminotransferase (Karmen U/mg protein) Serum aspartate aminotransferase (Karmen U/mL) Cytosolic aspartate aminotransferase (Karmen U/mg protein) Serum alkaline phosphatase (mol phenol per min mL⫺1) Serum ␥-glutamyltransferase (p-nitroaniline per min mL⫺1) Serum total bilirubin (mg/mL) Serum total protein (g/100 mL) Serum albumin (g/100 mL) Malondialdehyde in liver homogenate (nmol/mg protein) Superoxide anion in liver mitochondria (nmol nitroblue tetrazolium per mg protein) H2O2 in liver homogenate (nmol H2O2 per mg protein)
33 ⫾ 8 628 ⫾ 95 98 ⫾ 38 694 ⫾ 69 2.72 ⫾ 0.62 0.20 ⫾ 0.19 0.33 ⫾ 0.23 7.70 ⫾ 0.98 3.63 ⫾ 0.26 0.71 ⫾ 0.08 15.35 ⫾ 2.87 4.45 ⫾ 0.47
Group 2 (n ⫽ 6)
1417 ⫾ 719 412 ⫾ 73‡ 1445 ⫾ 812‡ 530 ⫾ 55‡ 8.34 ⫾ 3.15‡ 9.01 ⫾ 4.98‡ 0.53 ⫾ 0.24 7.42 ⫾ 0.56 3.38 ⫾ 0.32 0.76 ⫾ 0.04 18.24 ⫾ 2.28* 5.11 ⫾ 0.38* ‡
Group 3 (n ⫽ 6)
603 ⫾ 163‡§ 535 ⫾ 37‡储 509 ⫾ 208‡§ 578 ⫾ 53† 5.57 ⫾ 3.34*§ 5.11 ⫾ 3.71* 0.43 ⫾ 0.14 7.14 ⫾ 0.84 3.29 ⫾ 0.17 0.67 ⫾ 0.07§ 12.44 ⫾ 1.66†¶ 3.86 ⫾ 0.91*¶
Values expressed as mean ⫾ SD. Group 1, control group; group 2, PolyHb-treated group; group 3, PSC-treated group. *P ⬍ .05 vs group 1. † P ⬍ .01 vs group 1. ‡ P ⬍ .001 vs group 1. § P ⬍ .05 vs group 2. 储 P ⬍ .01 vs group 2. ¶ P ⬍ 0.001 vs group 2.
sham operation. Group 2 (n ⫽ 6) had clamping of the hepatic artery, portal vein, and common bile duct, using a vascular clamp (Fine Science Tools, Inc) for 30 minutes after which 4 mL of blood was exchanged with crosslinked hemoglobin (PolyHb). Group 3 (n ⫽ 6) received a PolyHb–SOD– catalase solution instead of PolyHb. After 5-hour reperfusion the rats were killed.
Biochemical Assays The PolyHb–SOD– catalase (PSC) solution was prepared according to the method of D’Agnillo and Chang.8 The final activity was about 50% of original activity of each enzyme. Serum was obtained after centrifugation at 3000 rpm for 10 minutes. The livers were homogenized with 0.25 mol/L sucrose. The homogenates were centrifuged at 25,000g for 1 hour. Cytosol and mitchondrial fraction were subjected to further analysis. ALT, AST, ALP, ␥-GTP, total protein, albumin, bilirubin, superoxide anion, H2O2, and malondialdehyde levels were measured as described previously.9,10
Statistical Analysis Values are expressed as mean ⫾ SD. Statistical evaluation of the differences between means was performed using Student’s t test; P ⬍ .05 was considered statistically significant.
RESULTS
The results are shown in Table 1. ALT, AST, ALP, and ␥-GTP activities in serum were significantly higher among the PolyHb group than the sham group. However, ALT and AST activities for cytosol were significantly lower in the polyHb group than in the controls. ALT, AST, ALP, and ␥-GTP activities in serum were significantly decreased in the PSC group with the PolyHb group. ALT and AST activities for cytosol were increased significantly in the PSC group compared with the PolyHb group. Total bilirubin, total protein, and albumin levels in serum were not significantly different. Superoxide anion and H2O2 levels were significantly increased in the PolyHb group compared with the controls. MDA, superoxide anion, and H2O2 levels were
significantly decreased in the PSC group compared with the PolyH group. The PolyHb group revealed a few apoptotic bodies, some acute inflammatory infiltrates in sinusoids, nuclear fragmentations, cell shrinkage, and chromatin clumping, with formation of apoptotic bodies in apoptotic cells on microscopic examination. Hepatic structures were well reserved in the PSC group.
DISCUSSION
The liver undergoes an I/R injury during transplantation1–3; 60% to 98% of ATP is degraded after ischemia.11–13 Reactive oxygen species generated by xanthine oxidase during reperfusion of an ischemic liver may be partially responsible for the hepatic injury.4 – 6 Most transplanted patients require transfusion. When patients need transfusion, it is helpful to give antioxidant-enriched blood to prevent injury mediated by reactive oxygen species. We administered crosslinked hemoglobin containing SOD and catalase to protect rats from injury by reactive oxygen species during liver I/R. It is well known that serum aminotransferases are elevated due to necrosis and degeneration of the liver; aminotransferases that are found in the liver enter the serum as a result of injury. Group 2 showed elevated serum ALT and AST levels as well as superoxide anion and hydrogen peroxide contents. The conversion of superoxide anion and hydrogen peroxide was impaired due to decreased levels of SOD and catalase, resulting in the increase in these oxygen free radicals. The elevated superoxide and hydrogen peroxide levels generate damage to the liver and establish a vicious cycle. Superoxide is converted to hydrogen peroxide by the action of superoxide dismutase.7 Hydrogen peroxide is disposed of by the action of glutathione peroxidase and catalase.7 In group 3, the parameters were decreased, showing a protective role against radical-mediated injury. Our results suggest that hepatic
EFFECT OF ARTIFICIAL CELLS
damage was reduced due to decreased levels of superoxide anion and hydrogen peroxide. These results show that reactive oxygen species contribute to the hepatic dysfunction with morphologic changes. PSC effectively reduces hepatic damage by diminishing oxygen free radical–mediated injury after liver IR. REFERENCES 1. Urata K, Brault A, Rocheleau B, et al: Transplant Int 13:420, 2000 2. Jassem W, Battino M, Cinti C, et al: J Surg Res 94:68, 2000 3. Yokota R, Fukai M, Shimamura T, et al: Surgery 127:661, 2000
1961 4. Ishii K, Suita S, Sumimoto H: Res Exp Med (Berl) 190:389, 1990 5. Nauta RJ, Tsimoyiannis E, Uribe M, et al: Ann Surg 213:137, 1991 6. Okabe H: Nippon Jinzo Gakkai Shi 38:577, 1996 7. Murray RK: Harper’s Biochemistry, 25th ed. London: Appleton & Lange; 2000 8. D’Agnillo F, Chang TM: Free Rad Biol Med 24:906, 1998 9. Kwak CS, Mun KC: Transplant Proc 32:2009, 2000 10. Mun KC: Transplant Proc 32:2007, 2000 11. Kobayashi H, Nonami T, Kurokawa T, et al: J Surg Res 51:240, 1991 12. Bor MV, Durmus O, Bilgihan A, et al: Int J Clin Lab Res 29:75, 1999 13. Jeon BR, Yeom DH, Lee SM: Shock 15:112, 2001
T
HE LIVER undergoes ischemia/reperfusion (I/R) injuries during transplantation.1–3 Reactive oxygen species generated by xanthine oxidase during reperfusion of the ischemic liver may be partially responsible for the hepatic injury.4 – 6 Antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, remove oxygen free radicals.7 Crosslinking hemoglobin with SOD and catalase helps to limit free radical reactivity of modified hemoglobin red blood cell substitutes. Using glutaraldehyde we prepared crosslinked hemoglobin, containing SOD and catalase, to protect against I/R injury during transplantation.
MATERIALS AND METHODS Animals Normal male Sprague-Dawley rats, weighing between 280 and 320 g, were divided into three groups. Group 1 (n ⫽ 10) underwent From the Institute for Medical Science and Chronic Disease Research Center, Keimyung University School of Medicine, Daegu, Korea. Supported by Grant R05-2002-000-00012-0 (2002) from the Basic Research Program of the Korea Science & Engineering Foundation. Address reprint requests to Dr Kyo-Cheol Mun, Department of Biochemistry, Keimyung University School of Medicine, 194 Dong San Dong, Daegu, 700-712, Korea.E-mail: [email protected]
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.08.092
Transplantation Proceedings, 36, 1959 –1961 (2004)
1959
1960
CHANG, LEE, MUN ET AL Table 1. Parameters Related to Hepatic Damage and Oxygen Free Radicals Parameters
Group 1 (n ⫽ 10)
Serum alanine aminotransferase (Karmen U/mL) Cytosolic alanine aminotransferase (Karmen U/mg protein) Serum aspartate aminotransferase (Karmen U/mL) Cytosolic aspartate aminotransferase (Karmen U/mg protein) Serum alkaline phosphatase (mol phenol per min mL⫺1) Serum ␥-glutamyltransferase (p-nitroaniline per min mL⫺1) Serum total bilirubin (mg/mL) Serum total protein (g/100 mL) Serum albumin (g/100 mL) Malondialdehyde in liver homogenate (nmol/mg protein) Superoxide anion in liver mitochondria (nmol nitroblue tetrazolium per mg protein) H2O2 in liver homogenate (nmol H2O2 per mg protein)
33 ⫾ 8 628 ⫾ 95 98 ⫾ 38 694 ⫾ 69 2.72 ⫾ 0.62 0.20 ⫾ 0.19 0.33 ⫾ 0.23 7.70 ⫾ 0.98 3.63 ⫾ 0.26 0.71 ⫾ 0.08 15.35 ⫾ 2.87 4.45 ⫾ 0.47
Group 2 (n ⫽ 6)
1417 ⫾ 719 412 ⫾ 73‡ 1445 ⫾ 812‡ 530 ⫾ 55‡ 8.34 ⫾ 3.15‡ 9.01 ⫾ 4.98‡ 0.53 ⫾ 0.24 7.42 ⫾ 0.56 3.38 ⫾ 0.32 0.76 ⫾ 0.04 18.24 ⫾ 2.28* 5.11 ⫾ 0.38* ‡
Group 3 (n ⫽ 6)
603 ⫾ 163‡§ 535 ⫾ 37‡储 509 ⫾ 208‡§ 578 ⫾ 53† 5.57 ⫾ 3.34*§ 5.11 ⫾ 3.71* 0.43 ⫾ 0.14 7.14 ⫾ 0.84 3.29 ⫾ 0.17 0.67 ⫾ 0.07§ 12.44 ⫾ 1.66†¶ 3.86 ⫾ 0.91*¶
Values expressed as mean ⫾ SD. Group 1, control group; group 2, PolyHb-treated group; group 3, PSC-treated group. *P ⬍ .05 vs group 1. † P ⬍ .01 vs group 1. ‡ P ⬍ .001 vs group 1. § P ⬍ .05 vs group 2. 储 P ⬍ .01 vs group 2. ¶ P ⬍ 0.001 vs group 2.
sham operation. Group 2 (n ⫽ 6) had clamping of the hepatic artery, portal vein, and common bile duct, using a vascular clamp (Fine Science Tools, Inc) for 30 minutes after which 4 mL of blood was exchanged with crosslinked hemoglobin (PolyHb). Group 3 (n ⫽ 6) received a PolyHb–SOD– catalase solution instead of PolyHb. After 5-hour reperfusion the rats were killed.
Biochemical Assays The PolyHb–SOD– catalase (PSC) solution was prepared according to the method of D’Agnillo and Chang.8 The final activity was about 50% of original activity of each enzyme. Serum was obtained after centrifugation at 3000 rpm for 10 minutes. The livers were homogenized with 0.25 mol/L sucrose. The homogenates were centrifuged at 25,000g for 1 hour. Cytosol and mitchondrial fraction were subjected to further analysis. ALT, AST, ALP, ␥-GTP, total protein, albumin, bilirubin, superoxide anion, H2O2, and malondialdehyde levels were measured as described previously.9,10
Statistical Analysis Values are expressed as mean ⫾ SD. Statistical evaluation of the differences between means was performed using Student’s t test; P ⬍ .05 was considered statistically significant.
RESULTS
The results are shown in Table 1. ALT, AST, ALP, and ␥-GTP activities in serum were significantly higher among the PolyHb group than the sham group. However, ALT and AST activities for cytosol were significantly lower in the polyHb group than in the controls. ALT, AST, ALP, and ␥-GTP activities in serum were significantly decreased in the PSC group with the PolyHb group. ALT and AST activities for cytosol were increased significantly in the PSC group compared with the PolyHb group. Total bilirubin, total protein, and albumin levels in serum were not significantly different. Superoxide anion and H2O2 levels were significantly increased in the PolyHb group compared with the controls. MDA, superoxide anion, and H2O2 levels were
significantly decreased in the PSC group compared with the PolyH group. The PolyHb group revealed a few apoptotic bodies, some acute inflammatory infiltrates in sinusoids, nuclear fragmentations, cell shrinkage, and chromatin clumping, with formation of apoptotic bodies in apoptotic cells on microscopic examination. Hepatic structures were well reserved in the PSC group.
DISCUSSION
The liver undergoes an I/R injury during transplantation1–3; 60% to 98% of ATP is degraded after ischemia.11–13 Reactive oxygen species generated by xanthine oxidase during reperfusion of an ischemic liver may be partially responsible for the hepatic injury.4 – 6 Most transplanted patients require transfusion. When patients need transfusion, it is helpful to give antioxidant-enriched blood to prevent injury mediated by reactive oxygen species. We administered crosslinked hemoglobin containing SOD and catalase to protect rats from injury by reactive oxygen species during liver I/R. It is well known that serum aminotransferases are elevated due to necrosis and degeneration of the liver; aminotransferases that are found in the liver enter the serum as a result of injury. Group 2 showed elevated serum ALT and AST levels as well as superoxide anion and hydrogen peroxide contents. The conversion of superoxide anion and hydrogen peroxide was impaired due to decreased levels of SOD and catalase, resulting in the increase in these oxygen free radicals. The elevated superoxide and hydrogen peroxide levels generate damage to the liver and establish a vicious cycle. Superoxide is converted to hydrogen peroxide by the action of superoxide dismutase.7 Hydrogen peroxide is disposed of by the action of glutathione peroxidase and catalase.7 In group 3, the parameters were decreased, showing a protective role against radical-mediated injury. Our results suggest that hepatic
EFFECT OF ARTIFICIAL CELLS
damage was reduced due to decreased levels of superoxide anion and hydrogen peroxide. These results show that reactive oxygen species contribute to the hepatic dysfunction with morphologic changes. PSC effectively reduces hepatic damage by diminishing oxygen free radical–mediated injury after liver IR. REFERENCES 1. Urata K, Brault A, Rocheleau B, et al: Transplant Int 13:420, 2000 2. Jassem W, Battino M, Cinti C, et al: J Surg Res 94:68, 2000 3. Yokota R, Fukai M, Shimamura T, et al: Surgery 127:661, 2000
1961 4. Ishii K, Suita S, Sumimoto H: Res Exp Med (Berl) 190:389, 1990 5. Nauta RJ, Tsimoyiannis E, Uribe M, et al: Ann Surg 213:137, 1991 6. Okabe H: Nippon Jinzo Gakkai Shi 38:577, 1996 7. Murray RK: Harper’s Biochemistry, 25th ed. London: Appleton & Lange; 2000 8. D’Agnillo F, Chang TM: Free Rad Biol Med 24:906, 1998 9. Kwak CS, Mun KC: Transplant Proc 32:2009, 2000 10. Mun KC: Transplant Proc 32:2007, 2000 11. Kobayashi H, Nonami T, Kurokawa T, et al: J Surg Res 51:240, 1991 12. Bor MV, Durmus O, Bilgihan A, et al: Int J Clin Lab Res 29:75, 1999 13. Jeon BR, Yeom DH, Lee SM: Shock 15:112, 2001