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Inhibitory effect of lidocaine on lipid peroxidation during cold preservation of pancreas
Inhibitory Effect of Lidocaine on Lipid Peroxidation During Cold Preservation of Pancreas A. Kinasiewicz, M. Juszczak, W. Michalska, W. Rowin´ski, A.P. Mazurek, and P. Fiedor
R
ECOVERY of pancreatic islets and their excellent function after transplantation depends on many important factors— donor factors, preservation and islet isolation process.1,2 Free radicals and products of arachidonic acid cascade cumulated in preserved tissue/organ are responsible for cell damage during and after reperfusion. Lidocaine (LDK) is a chemical agent that reveals inhibitory effect on phospholipase A2 (PLP-A2), the main enzyme of arachidonic acid cascade.3 Effect of LDK on lipid peroxidation and pancreatic tissue injury in simple cold stored pancreases was investigated. MATERIALS AND METHODS WAG rats (250 to 280 g, male) were used as pancreas donors. Investigational groups were established as follows: group 1, control group (CIT ⫽ 0 minutes, LDK ⫽ 0 mmol); group 2, LDK added to collagenase solution in concentration of 1 mmol, (CIT ⫽ 0 minutes); group 3, control group (CIT ⫽ 180 minutes, LDK ⫽ 0 mmol); group 4, LDK added to collagenase solution in concentration of 1 mmol (CIT ⫽ 180 minutes). In all groups, in vitro experiments were done in triplets, with double samples (or triple samples in case of glucose static test) in each. Pancreata were distended under ether anesthesia with collagenase P (Boehringer Mannheim) solution with or without addition of LDK in concentration of 1 mmol. Cold ischemia was achieved by storage pancreases at 4°C for 180 minutes on Petri dishes in 5 mL of UW solution before islet isolation. Islets were isolated using modified Lacy and Kostianovsky method, and separated by spinning on discontinuous histopaque gradients. The level of lipid peroxidation was assessed by measuring the final product of arachidonic acid cascade— malondialdehyde (MDA) in homogenized pancreatic tissue specimens, using spectrophotometric assay with thiobarbituric acid. Endocrine function was estimated by three-step glucose challenge test with glucose concentration of 1.67, 16.7, and 1.67 mmol, respectively (triple samples, 10 islets per dish/pancreas). Insulin concentration in examined samples was assessed by radioimmunoassay (RIA). Amylase and lipase activity in solutions (preservation Table 1. Group
1 2 3 4
(CIT (CIT (CIT (CIT
⫽ ⫽ ⫽ ⫽
0 minutes) 0 minutes, LDK) 180 minutes) 180 minutes, LDK)
solution and supernatant achieved after tissue mass washing) was assessed using commercially available Amylase and Lipase kits from Sigma. Statistical analyses were carried out using Student t test. Differences were considered significant at P ⬍ .05.
RESULTS AND DISCUSSION
Our previous study showed that LDK influence metabolic activity and endocrine function.4 Our data indicate that cold ischemia of 180 minutes (1) does not inhibit lipid peroxidation process in pancreatic tissue, (2) aggravates islets endocrine function, and (3) causes exocrine cells damage. LDK administered intraductally before organ cooling significantly decreases MDA concentration in pancreatic tissue (0.530 ⫾ 0.073 in group 3 vs 0.209 ⫾ 0.08 in group 4; P ⬍ .05). Concentration of MDA in groups 1, 2, and 4 were comparable and no statistically significant differences were detected. Islets isolated from pancreata exposed to 180 minutes of cold ischemia revealed impaired endocrine function when compared with control group and presented deteriorating type of reaction in glucose static test (Table 1). In group 2, slightly higher insulin secretion was observed when compared with control group; however, islet reaction in glucose static test was not affected and presented normal type of secretory profile (Table 1). Relevant exocrine cells injury, expressed as high activity of lipase and amylase, From the Department of General and Transplantation Surgery, Transplantation Institute (A.K., M.J., W.M., W.R., P.F.), Medical University of Warsaw, Warsaw, the Department of Biochemistry and Clinical Chemistry, Faculty of Pharmacy (A.K., A.P.M.), Medical University of Warsaw, Warsaw, and the Drug Institute (A.P.M.), Warsaw, Poland. Supported in part by grant Nr 4P05C0641 KBN. Address reprint requests to Dr A. Kinasiewicz, Department of General and Transplantation Surgery, Medical University of Warsaw, 59 Nowogrodzka St, Warsaw, Poland.
Glucose Static Test
1.67 mmol
16.7 mmol
1.67 mmol
118 ⫾ 14 131 ⫾ 13 227 ⫾ 15 133 ⫾ 7
226 ⫾ 18 245 ⫾ 22 138 ⫾ 12 207 ⫾ 17
110 ⫾ 12 83 ⫾ 23 71 ⫾ 11 65 ⫾ 15
Data are presented as mean ⫾ SD.
0041-1345/01/$–see front matter PII S0041-1345(00)02291-0
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
972
Transplantation Proceedings, 33, 972–973 (2001)
INHIBITORY EFFECT OF LIDOCAINE
973
Table 2. Amylase and Lipase Activity in Investigated Solutions (CIT ⫽ 180 min ⫺ groups 3 and 4) Preservation Solution Group 3
Amylase Lipase
M ⫾ SD P M ⫾ SD P
5409 ⫾ 1977
Washing Solution 1
Group 4
Group 3
7853 ⫾ 1313
20421 ⫾ 4349
NS 2238 ⫾ 327
Washing Solution 2
Group 4
Group 3
15377 ⫾ 2971
10438 ⫾ 2879
.05 2647 ⫾ 1007
NS
estimated in supernatants after cell mass washing, was observed in all groups with CIT ⫽ 180 minutes (with or without LDK) as it was previously described.5 However, activities of amylase and lipase in preservation solutions were comparable in groups 3 and 4 (Table 2). The addition of LDK to collagenase solution with attendant condition of CIT ⫽ 180 minutes, resulted in significant decrease of enzymes activity in investigated supernatants when compared with group without LDK (P ⬍ .05).
4888 ⫾ 886
Group 4
5303 ⫾ 1675 .01
2535 ⫾ 1602
1418 ⫾ 355
.029
875 ⫾ 385 .042
LDK was achieved and expressed as lower activity of amylase and lipase in supernatants from washing solutions, although enzymes activity in preservation solution was on the same level. Based on our data we can conclude that lidocaine reveals protective effect on preserved pancreas by stabilization of membrane and by inhibition of exocrine enzyme release.
REFERENCES CONCLUSIONS
Lidocaine does not cause injury of pancreatic islet cells during preservation at 4°C. The lower concentration of MDA in groups with LDK indicate that it may have a protective effect on pancreatic tissue. Protective effect of
1. Shapiro AM: N Engl J Med 27;343:230, 2000 2. Rosenberg L: Surgery 126:393, 1999 3. Makela A: Scand J Clin Lab Invest 57:402, 1997 4. Juszczak M: Transplant Proc 31:2102, 1999 5. Brazda E: Transplant Proc 30:592, 1998
R
ECOVERY of pancreatic islets and their excellent function after transplantation depends on many important factors— donor factors, preservation and islet isolation process.1,2 Free radicals and products of arachidonic acid cascade cumulated in preserved tissue/organ are responsible for cell damage during and after reperfusion. Lidocaine (LDK) is a chemical agent that reveals inhibitory effect on phospholipase A2 (PLP-A2), the main enzyme of arachidonic acid cascade.3 Effect of LDK on lipid peroxidation and pancreatic tissue injury in simple cold stored pancreases was investigated. MATERIALS AND METHODS WAG rats (250 to 280 g, male) were used as pancreas donors. Investigational groups were established as follows: group 1, control group (CIT ⫽ 0 minutes, LDK ⫽ 0 mmol); group 2, LDK added to collagenase solution in concentration of 1 mmol, (CIT ⫽ 0 minutes); group 3, control group (CIT ⫽ 180 minutes, LDK ⫽ 0 mmol); group 4, LDK added to collagenase solution in concentration of 1 mmol (CIT ⫽ 180 minutes). In all groups, in vitro experiments were done in triplets, with double samples (or triple samples in case of glucose static test) in each. Pancreata were distended under ether anesthesia with collagenase P (Boehringer Mannheim) solution with or without addition of LDK in concentration of 1 mmol. Cold ischemia was achieved by storage pancreases at 4°C for 180 minutes on Petri dishes in 5 mL of UW solution before islet isolation. Islets were isolated using modified Lacy and Kostianovsky method, and separated by spinning on discontinuous histopaque gradients. The level of lipid peroxidation was assessed by measuring the final product of arachidonic acid cascade— malondialdehyde (MDA) in homogenized pancreatic tissue specimens, using spectrophotometric assay with thiobarbituric acid. Endocrine function was estimated by three-step glucose challenge test with glucose concentration of 1.67, 16.7, and 1.67 mmol, respectively (triple samples, 10 islets per dish/pancreas). Insulin concentration in examined samples was assessed by radioimmunoassay (RIA). Amylase and lipase activity in solutions (preservation Table 1. Group
1 2 3 4
(CIT (CIT (CIT (CIT
⫽ ⫽ ⫽ ⫽
0 minutes) 0 minutes, LDK) 180 minutes) 180 minutes, LDK)
solution and supernatant achieved after tissue mass washing) was assessed using commercially available Amylase and Lipase kits from Sigma. Statistical analyses were carried out using Student t test. Differences were considered significant at P ⬍ .05.
RESULTS AND DISCUSSION
Our previous study showed that LDK influence metabolic activity and endocrine function.4 Our data indicate that cold ischemia of 180 minutes (1) does not inhibit lipid peroxidation process in pancreatic tissue, (2) aggravates islets endocrine function, and (3) causes exocrine cells damage. LDK administered intraductally before organ cooling significantly decreases MDA concentration in pancreatic tissue (0.530 ⫾ 0.073 in group 3 vs 0.209 ⫾ 0.08 in group 4; P ⬍ .05). Concentration of MDA in groups 1, 2, and 4 were comparable and no statistically significant differences were detected. Islets isolated from pancreata exposed to 180 minutes of cold ischemia revealed impaired endocrine function when compared with control group and presented deteriorating type of reaction in glucose static test (Table 1). In group 2, slightly higher insulin secretion was observed when compared with control group; however, islet reaction in glucose static test was not affected and presented normal type of secretory profile (Table 1). Relevant exocrine cells injury, expressed as high activity of lipase and amylase, From the Department of General and Transplantation Surgery, Transplantation Institute (A.K., M.J., W.M., W.R., P.F.), Medical University of Warsaw, Warsaw, the Department of Biochemistry and Clinical Chemistry, Faculty of Pharmacy (A.K., A.P.M.), Medical University of Warsaw, Warsaw, and the Drug Institute (A.P.M.), Warsaw, Poland. Supported in part by grant Nr 4P05C0641 KBN. Address reprint requests to Dr A. Kinasiewicz, Department of General and Transplantation Surgery, Medical University of Warsaw, 59 Nowogrodzka St, Warsaw, Poland.
Glucose Static Test
1.67 mmol
16.7 mmol
1.67 mmol
118 ⫾ 14 131 ⫾ 13 227 ⫾ 15 133 ⫾ 7
226 ⫾ 18 245 ⫾ 22 138 ⫾ 12 207 ⫾ 17
110 ⫾ 12 83 ⫾ 23 71 ⫾ 11 65 ⫾ 15
Data are presented as mean ⫾ SD.
0041-1345/01/$–see front matter PII S0041-1345(00)02291-0
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
972
Transplantation Proceedings, 33, 972–973 (2001)
INHIBITORY EFFECT OF LIDOCAINE
973
Table 2. Amylase and Lipase Activity in Investigated Solutions (CIT ⫽ 180 min ⫺ groups 3 and 4) Preservation Solution Group 3
Amylase Lipase
M ⫾ SD P M ⫾ SD P
5409 ⫾ 1977
Washing Solution 1
Group 4
Group 3
7853 ⫾ 1313
20421 ⫾ 4349
NS 2238 ⫾ 327
Washing Solution 2
Group 4
Group 3
15377 ⫾ 2971
10438 ⫾ 2879
.05 2647 ⫾ 1007
NS
estimated in supernatants after cell mass washing, was observed in all groups with CIT ⫽ 180 minutes (with or without LDK) as it was previously described.5 However, activities of amylase and lipase in preservation solutions were comparable in groups 3 and 4 (Table 2). The addition of LDK to collagenase solution with attendant condition of CIT ⫽ 180 minutes, resulted in significant decrease of enzymes activity in investigated supernatants when compared with group without LDK (P ⬍ .05).
4888 ⫾ 886
Group 4
5303 ⫾ 1675 .01
2535 ⫾ 1602
1418 ⫾ 355
.029
875 ⫾ 385 .042
LDK was achieved and expressed as lower activity of amylase and lipase in supernatants from washing solutions, although enzymes activity in preservation solution was on the same level. Based on our data we can conclude that lidocaine reveals protective effect on preserved pancreas by stabilization of membrane and by inhibition of exocrine enzyme release.
REFERENCES CONCLUSIONS
Lidocaine does not cause injury of pancreatic islet cells during preservation at 4°C. The lower concentration of MDA in groups with LDK indicate that it may have a protective effect on pancreatic tissue. Protective effect of
1. Shapiro AM: N Engl J Med 27;343:230, 2000 2. Rosenberg L: Surgery 126:393, 1999 3. Makela A: Scand J Clin Lab Invest 57:402, 1997 4. Juszczak M: Transplant Proc 31:2102, 1999 5. Brazda E: Transplant Proc 30:592, 1998