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The role of selenium added to pulmonary preservation solutions in isolated guinea pig lungs
The role of selenium added to pulmonary preservation solutions in isolated guinea pig lungs An experimental comparative study on isolated guinea pig lungs has been undertaken to determine the probable beneficial effects of adding selenium to pulmonary preservation solutions in lung ischemia. The isolated lungs (n = 10 in each group) previously being perfused by oxygenated Krebs-Henseleit solution were put in normothermic ischemic conditions just after the infusion of 30 mI of pulmonary preservation solution (Euro-Collins in the control group, Euro-Collins plus selenium 10- 3 mol in the experiment group). After 3 hours of normothermic ischemia the lungs were reperfused with the same buffer for 20 minutes. Pulmonary artery pressures, tissue malondialdehyde levels, and adenosine deaminase levels of the perfusate were measured before and after the ischemic period and also at the end of reperfusion. An electron microscopic analysis was performed on the lung tissues at the end of the experimental procedure. According to our data, the addition of selenium to pulmonary preservation solution showed a significant protective effect regarding both ischemic and reperfusion injury. (J THORAC CARDIOVASC SURG 1994;108:922-7)
Halim Soncul, MD, Melih Kaptanoglu, MD, Eser Oz, MD, Velit Halit, MD, Ayse Bilgehan, MD, Banu Cayci, MD, Levent Gokgoz, MD, Nurten Tiirkozan, MD, and Ali Ersoz, MD, Ankara, Turkey
h e shortage of donor organs remains a major limiting factor in the widespread application oflung and combined heart and lung transplantation. The clinical success of lung transplantation depends on effective organ preservation and protection of the lung from reperfusion injury. A more reliable preservation method, providing a longer ischemic period, would permit safer procurement oflungs from greater distances and also might permit the use of lungs currently judged inadequate because of current preservation limitations. Because these factors would increase the supply of lungs, a comprehensive study on preservation techniques is ongoing. Historically, a number of perfusates have been used experimentally for pulmonary preservation. Although high potassium concentrations are known to cause intense pulmonary vasospasm, relatively successful organ preservation has been achieved when intracellular type crystalloid solutions such as Euro-Collins solution are used.!" It is widely believed that the production of activated From the Departments of Cardiovascular Surgery and Biochemistry, Gazi University Medical Faculty, Ankara, Turkey. Received for publication Feb. 16, 1994. Accepted for publication June 16, 1994. Copyright
©
1994 by Mosby-Year Book, Inc.
0022-5223/94 $3.00 + 0 12/1/58423
922
oxygen species is responsible for ischemic and reperfusion injury. Oxygen-derived free radicals can be generated in the transplanted lung either by reintroduction' Of molecular oxygen on reperfusion or by activated neutrophils. Glutathione peroxidase is considered to be a housekeeping enzyme, functioning as an important antioxidant enzyme that protects cells from oxidative damage.v' Therefore, much interest has been focused on the trace element selenium, which is an essential prosthetic group for the enzyme glutathione peroxidase.f This enzyme reduces hydrogen peroxide to the corresponding lipid alcohols or water, thereby possibly protecting the epithelium from oxidative damage.' Glutathione peroxidase may also influence the synthesis ofprostacyclin, an agent protective against platelet aggregation." According to these data, selenium is considered to be an antioxidant trace element. In this study, we hypothesised that perfusion of lungs before the ischemic period with selenium-enhanced solutions would diminish ischemic injury. We also tested the effect of the same solution on reperfusion injury. Material and methods Animals. Lungs were obtained from male guinea pigs
(n = 20) weighing 310 to 440 gm. All animals received human
care in compliance with the "Principles of Laboratory Animal
The Journal of Thoracic and Cardiovascular Surgery Volume 108, Number 5
Soncul et al. 9 2 3
PA. pressure record
ventilation of the lungs
Perfusion with Krebssolution
t.
t
20min.of perfusion '. I'" .3. .ho.':'",,()f}..c~em~:.. '20min. of regerfusion
//~
r
/
~usion of pulinop~egia J [~~~f~~~nor~~~:~;:~~I::d Fig. 1. Experimental design and protocol. PA, Pulmonary artery.
Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). The animals were anesthetized with ether and given 200 units of heparin into the femoral vein. After insertion of a No. 16 cannula into the trachea by an open tracheostomy, a sternotomy was performed. After cannulation of the pulmonary artery via the right ventricle, the lungs and the heart were rapidly harvested. Perfusion techniques and preservation solutions. The lungs were mounted on a modified Langendorf perfusion apparatus. We inflated the lungs with room air and then began perfusion with a gassed (oxygen 95%, carbon dioxide 5%) Krebs-Henseleit solution at a rate of 10 ml/rnin at 37° C. The composition of the solution was as follows: NaHC0 3 , 25 rnmol/L; NaCl, 118 mmol/L; KHzP04, 1.2 mrnol/L; KCI, 4.8 mmol/L; MgS04, 1.2 mrnol/L; CaCh, 1.2 mrnol/L; and glucose, 11.1 mmol/L, Euro-Collins solution was used as the pulmonary preservation solution in the control group. In the experiment group, we added 10- 3 mol selenium to the Euro-Collins solution.
Protocol. At the twentieth minute of perfusion, we collected perfusate samples from the left atrium to determine adenosine deaminase concentrations and excised one of the lung segments to determine tissue malondialdehyde levels. After the perfusion had been stopped, one of the pulmonary preservation solutions (Euro-Collins in the control group, Euro-Collins plus selenium in the experiment group) was infused for 3 minutes (at a rate of 10 ml/rnin), During the ischemic period (3 hours) the lungs were kept at 37° C in an isotonic saline bath. After 3 hours of ischemia, we began reperfusion with the same buffer at 37° C. Pulmonary artery mean pressures were recorded, perfusate samples were collected, and tissue pieces were excised again at the beginning of reperfusion and also at the twentieth minute of reperfusion (Fig. I). At the end of reperfusion the lungs were prepared for electron microscopic studies. Ultrastructural analyses were done on eight randomly selected lungs (four from each group). Data and statistics. Pulmonary artery pressures (millimeters of mercury) were measured with a Datascope 200lA monitor (Datascope Corp., Montvale, N.J.) with a Viggo-Spectramed pressure monitoring kit (Viggo-Spectramed, Inc., Oxnard, Calif.). The adenosine deaminase levels (units/nil) of the perfusate was calculated with a Spectronics 3000 AutoAn-
The Journal of Thoracic and Cardiovascular Surgery November 1994
9 2 4 Soncul et al.
Percentage Changes of PA Pressure 300
!
200
~
POST-ISCHEMIC
AFTER REPERFUSION
Tissue Malondialdehyde Levels
t
2
1.5
~
I l!
0.5
POST-ISCHEMIC
AFTER REPERFUSION
Adenosine Oeaminase Levels of Perfusate 40
I:5
~o
20 10
POST·ISCHEMIC
AFTER REPERFUSION
Fig. 2. Pulmonary artery pressures (percentage change), tissue malondialdehyde levels (nmol/rng protein), and adenosine deaminase levels (units/rnl) of the perfusate. *Significant difference (p < 0.05) between the indicated value and the preischemic value of the same group. tSignificant difference (p < 0.05) between the groups. Values are mean ± standard error of the mean.
alyzer (Spectronics Corporation, Westbury, N.Y.) by the Soloman Method. The tissue malondioldehyde levels (nanomoles per milligram protein) were calculated by the Spectronics 3000 AutoAnalyzer using the technique of Beauge," Ultrastructural analyses were done with uranyl acetate-lead citrate stains at X 12,000 magnification. The results are presented as mean and standard error of the mean. Overall significance of differences between the groups was determined by t test (paired two sam-
pIe for means) using the Microsoft Excel 4.0 personal computer program (Microsoft Corporation, Redmond, Wash.).
Results Because of the changes in lung sizes in different animals and our standard perfusion volume (10 ml/rnin), the alterations of pulmonary artery pressures were calcu-
The Journal of Thoracic and Cardiovascular Surgery Volume 108, Number 5
•
Soncul et al.
925
•
Fig. 3. Electron micrograph of the alveolar capillary membrane in the control group (X12,000 magnification). Al Ep, Alveolar epithelium; K En, capillaryendothelium.
lated as the percentage change of the preischemic value in each experiment. Although the percentage change in the mean pulmonary artery pressures rose in both of the groups after the ischemic period, the difference between the groups was still significant (p = 0.032). The difference between the groups was not significant after reperfusion (Table I, Fig. 2). In the control group, the mean preischemic tissue malondialdehyde level increased noticeably at the end of reperfusion (p = 0.073). The difference between the two groups at the end of reperfusion was also statistically significant (p = 0.044) (Table I, Fig. 2). The adenosine deaminase levels of the perfusate increased significantly in both of the groups after ischemia as compared with their preischemic values (p < 0.001). The difference between the two groups after the ischemic period was again significant statistically (p < 0.041) (Table I, Fig. 2). The ultrastructural analysis of the lung tissues in the control group showed a marked separation between the capillary endothelium and the alveolar epithelium and also plenty of empty cytoplasmic vacuoles with different
Fig. 4. Electron micrograph of the alveolar capillary membrane in the selenium group (X12,000 magnification). Al Ep, Alveolar epithelium; K En, capillaryendothelium.
Table I. Effect of selenium on ischemic injury Before ischemia PA pressure (mm Hg) Euro-Collins 23.0 ± 5 Euro-Collins 25.6 ± 4 + selenium PA pressure (% change) Euro-Collins 100 Euro-Collins 100 + selenium Tissue MDA levels Euro-Collins 0.86 ± 0.10 Euro-Collins 0.98 ± 0.17 + selenium ADA levels of the perfusate 7.71 ± 1.91 Euro-Collins 4.93 ± 0.91 Euro-Collins + selenium
After ischemia
After reperfusion
56.8 ± 14 46.3 ± 7
33.1 ± 5 27.9 ± 6
247 ± 17 181 ± II
144 ± 9 109 ± 7
1.07 ± 0.18 0.77 ± 0.08
1.55 ± 0041 0.74 ± 0.06
34.12 ± 6.07 21.32 ± 3.14
8.69 ± 1.75 13.2 ± 2045
PA, Pulmonary artery; MDA, malondialdehyde; ADA, adenosine deaminase. Values are mean ± standard error of the mean.
sizes of pneumocytes. The alveolar capillary membrane and cellular structures were nearly normal in the selenium group (Figs. 3 and 4).
9 2 6 Soncul et al.
Comment The lung is the only organ to which oxygen may be supplied after its blood supply is stopped. In contrast, much more difficulty has been associated with the preservation of the lung than with the preservation of solid organs in vitro because of the lung's unique structure, especially the close apposition of blood and air compartments in the alveoli.?: 10 Several studies have suggested that oxygen-derived free radicals produced by various mechanisms playa significant role in the injury sustained by a transplanted organ. Any molecule that can react with a free radical can be termed a scavenger. The clinical benefits of such a molecule are derived from the fact that the oxygenderived free radicals react with it before they can react with cellular components and cause tissue damage. I I, 12 Selenium, as selenocysteine, is placed in the active site of the enzyme glutathione peroxidase, which reduces hydrogen peroxide to the corresponding lipid alcohols or water. Besides this hypothesized radical scavenging consequence, additional specific preventive effects have been suggested for selenium.P'P The isolated perfused lung model used for our study was previously developed and used by a number of authors as a screening technique for the many factors affecting lung preservation and reperfusion injury.l'' 17 Previous experiments had demonstrated that lung function began to deteriorate when preservation time was extended to 30 hours under hypothermic conditions. 16, 18 Because it was difficult to keep the standardized experimental environment for such long periods in our laboratory conditions, in the current experiment we preferred to use 3 hours of normothermic (37 0 C) ischemia. In our study, lipid peroxidation associated with free radical generation was assessed by measuring tissue malondialdehyde, which is a three-carbon product of lipid peroxidation. Tissue damage was assessed by measuring pulmonary artery pressure, which is a relative parameter for pulmonary vascular resistance, and also by measuring adenosine deaminase activity of the perfusates. Adenosine deaminase is an enzyme in purine catabolism that catalyzes the conversion of adenosine to inosine. Activities of this enzyme have been reported to be high in pleural effusions because of various origins. This activity reflects the severity of tissue injury because the activity of the enzyme increases during the catabolism of high-energy phosphates. 19,20 In the selenium group, the tissue malondialdehyde levels decreased markedly when compared with the control group after reperfusion. These levels also decreased slightly just after the ischemic period, although the decrease was not statistically significant, probably
The Journal of Thoracic and Cardiovascular Surgery November 1994
because of the limited number of our experiments. According to these data, it can be argued that the addition of selenium to intracellular type pulmonary preservation solutions may have a noticeable effect on reperfusion injury of the ischemic lungs. Similar results for other organs such as heart and kidneys were previously reported in other recent experimental and clinical studies. 12, 13 Although pulmonary artery pressures and adenosine deaminase activity in the perfusate returned to normal after reperfusion, both of the parameters increased substantially after the ischemic period in the control group (Fig. 2). Electron microscopic evaluation of the lungs had also demonstrated serious changes in the alveolar capillary space and in the pneumocytes in the control group at the end of the reperfusion period. In conclusion, our study suggests that addition of selenium to pulmonary preservation solutions may reduce ischemic lung injury during normothermic conditions. REFERENCES 1. Collins GM, Bravo-Shugarman M, Terasaki PI. Kidney preservation for transportation. Lancet 1969;2: 1219-22. 2. Hooper TL, Locke TJ, Fetherston G, Flecknell PA, McGregor GA. Comparison of cold flush perfusion with modified blood versus modified Euro-Collins solution for lung preservation. J Heart Transplant 1990;9:4, 429-34, 3, Haverich A, Aziz S, Scott WC, Jamieson SW, Shumway NE. Improved lung preservation using Euro-Collins solution for flush perfusion. Thorac Cardiovasc Surg 1986; 34:368-78. 4. Keshavjee SH, McRitchie DI, Vittorini T, Rostein OD, Slutsky AS, Patterson GA. Improved lung preservation with dextran 40 is not mediated by a superoxide radical scavenging mechanism. J THORAC CARDIOVASC SURG 1992;2:326-8. 5. FloheL. Free radicalsin biology. PryorWA,ed. Vo15. New York: Academic Press, 1982:223-54. 6. Rotruck J, Pope A, Ganther H, Swanson A, Hafeman D, Hoekstra W. Selenium:biochemical roleas a component of glutathione peroxidase, Science 1973; 179:588-90. 7. SchoeneNW, Morris VC, LevanderOA. Effects of selenium deficiency on aggregationand tromboxaneformation in rat platelets. Fed Proc 1984;43:477-81. 8. BeugeJA. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302-10. 9. Date A, Matsumura A, Manchester JK, CooperJM, Lowry OH, CooperJD. Changes in alveolaroxygen and carbon dioxideconcentration and oxygenconsumption duringlung preservation. J. Thorac Cardiovasc. Surg 1993;105:492501. 10. Salonen JT, Salonen R, Seppanen K, et al. Relationship of serum selenium and antioxidants to plasma lipoproteins, platelet aggregability and prevalentischemic heart disease in eastern Finnish men. Atherosclerosis. 1988;70: 155-60.
The Journal of Thoracic and Cardiovascular Surgery Volume 108, Number 5
II. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312: 159-63. 12. Hernandez LA, Granger N, Role of antioxidants in organ preservation and transplantation. Crit Care Med 1988; 16:543-9. 13. Poltronieri R, Cevese A, Sharbati A. Protective effect of selenium in cardiac ischemia and reperfusion. Cardioscience 1992;3: 155-60. 14. Gromadzinska J, Sklodowska M, Wasowics W. Glutathione peroxidase activity, lipid and selenium concentration in various rat organs. Biomed Biochim Acta 1988; 47:19-24. IS. Salonen JT, Huttunen JK. Selenium in cardiovascular diseases. Ann Clin Res 1986;18:30-5. 16. Wang LS, Yoshikawa K, M itoshi S. The effect of ischemic time and temperature on lung preservation in a simple ex vivorabbit model used for functional assessment. J THORAC CARDIOVASC SURG 1989;98:333-42.
Soncul et al.
927
17. Ercan ZS, Ilhan M, Oguz A, Turker RK. Superoxide dismutase and allopurinol prevent the pressor effects of angiotensin II and histamine in the guinea-pig isolated perfused lung exposed to hypoxia. Gen Pharmacol 1992; 23: 1149-51. 18. Miyoshi S, Shimokawa S, Schreinemakers H, et al. Comparison ofthe U niversity of Wisconsin preservation solu tion and other crystalloid perfusates in a 30-hour rabbit lung preservation model. J THORAC CARDIOVASC SURG 1992; 103:27-32. 19. Ocana I, Martinez-Vasquez JM, Segura RM. Adenosine deaminase in pleural fluids: test for diagnosis tuberculosus pleural effusions. Chest. 1983;84:51-3. 20. Wang YX, Boeker K, Reuter H6Kiem J, et al. Selenium and myocardial infarction: glutathione peroxidase in platelets. Klin Wochenschr 1981;59:818-23.
Availability of JOURNAL back issues As a service to our subscribers, copies of back issues of THEJOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY for the preceding 5 years are maintained and are available for purchase from the publisher, Mosby-Year Book, Inc., at a cost of $10.50 per issue. The following quantity discounts are available: 25% off on quantities of 12 to 23, and one third off on quantities of 24 or more. Please write to Mosby-Year Book, Inc., Subscription Services, 11830 Westline Industrial Drive, St. Louis MO 6314633I8, or call (800)453-4351 or (314)453-4351 for information on availability of particular issues. If unavailable from the publisher, photocopies of complete issues are available from University Microfilms International, 300 N. Zeeb Rd. Ann Arbor, MI 48106, (313)76 1-4700,
Halim Soncul, MD, Melih Kaptanoglu, MD, Eser Oz, MD, Velit Halit, MD, Ayse Bilgehan, MD, Banu Cayci, MD, Levent Gokgoz, MD, Nurten Tiirkozan, MD, and Ali Ersoz, MD, Ankara, Turkey
h e shortage of donor organs remains a major limiting factor in the widespread application oflung and combined heart and lung transplantation. The clinical success of lung transplantation depends on effective organ preservation and protection of the lung from reperfusion injury. A more reliable preservation method, providing a longer ischemic period, would permit safer procurement oflungs from greater distances and also might permit the use of lungs currently judged inadequate because of current preservation limitations. Because these factors would increase the supply of lungs, a comprehensive study on preservation techniques is ongoing. Historically, a number of perfusates have been used experimentally for pulmonary preservation. Although high potassium concentrations are known to cause intense pulmonary vasospasm, relatively successful organ preservation has been achieved when intracellular type crystalloid solutions such as Euro-Collins solution are used.!" It is widely believed that the production of activated From the Departments of Cardiovascular Surgery and Biochemistry, Gazi University Medical Faculty, Ankara, Turkey. Received for publication Feb. 16, 1994. Accepted for publication June 16, 1994. Copyright
©
1994 by Mosby-Year Book, Inc.
0022-5223/94 $3.00 + 0 12/1/58423
922
oxygen species is responsible for ischemic and reperfusion injury. Oxygen-derived free radicals can be generated in the transplanted lung either by reintroduction' Of molecular oxygen on reperfusion or by activated neutrophils. Glutathione peroxidase is considered to be a housekeeping enzyme, functioning as an important antioxidant enzyme that protects cells from oxidative damage.v' Therefore, much interest has been focused on the trace element selenium, which is an essential prosthetic group for the enzyme glutathione peroxidase.f This enzyme reduces hydrogen peroxide to the corresponding lipid alcohols or water, thereby possibly protecting the epithelium from oxidative damage.' Glutathione peroxidase may also influence the synthesis ofprostacyclin, an agent protective against platelet aggregation." According to these data, selenium is considered to be an antioxidant trace element. In this study, we hypothesised that perfusion of lungs before the ischemic period with selenium-enhanced solutions would diminish ischemic injury. We also tested the effect of the same solution on reperfusion injury. Material and methods Animals. Lungs were obtained from male guinea pigs
(n = 20) weighing 310 to 440 gm. All animals received human
care in compliance with the "Principles of Laboratory Animal
The Journal of Thoracic and Cardiovascular Surgery Volume 108, Number 5
Soncul et al. 9 2 3
PA. pressure record
ventilation of the lungs
Perfusion with Krebssolution
t.
t
20min.of perfusion '. I'" .3. .ho.':'",,()f}..c~em~:.. '20min. of regerfusion
//~
r
/
~usion of pulinop~egia J [~~~f~~~nor~~~:~;:~~I::d Fig. 1. Experimental design and protocol. PA, Pulmonary artery.
Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). The animals were anesthetized with ether and given 200 units of heparin into the femoral vein. After insertion of a No. 16 cannula into the trachea by an open tracheostomy, a sternotomy was performed. After cannulation of the pulmonary artery via the right ventricle, the lungs and the heart were rapidly harvested. Perfusion techniques and preservation solutions. The lungs were mounted on a modified Langendorf perfusion apparatus. We inflated the lungs with room air and then began perfusion with a gassed (oxygen 95%, carbon dioxide 5%) Krebs-Henseleit solution at a rate of 10 ml/rnin at 37° C. The composition of the solution was as follows: NaHC0 3 , 25 rnmol/L; NaCl, 118 mmol/L; KHzP04, 1.2 mrnol/L; KCI, 4.8 mmol/L; MgS04, 1.2 mrnol/L; CaCh, 1.2 mrnol/L; and glucose, 11.1 mmol/L, Euro-Collins solution was used as the pulmonary preservation solution in the control group. In the experiment group, we added 10- 3 mol selenium to the Euro-Collins solution.
Protocol. At the twentieth minute of perfusion, we collected perfusate samples from the left atrium to determine adenosine deaminase concentrations and excised one of the lung segments to determine tissue malondialdehyde levels. After the perfusion had been stopped, one of the pulmonary preservation solutions (Euro-Collins in the control group, Euro-Collins plus selenium in the experiment group) was infused for 3 minutes (at a rate of 10 ml/rnin), During the ischemic period (3 hours) the lungs were kept at 37° C in an isotonic saline bath. After 3 hours of ischemia, we began reperfusion with the same buffer at 37° C. Pulmonary artery mean pressures were recorded, perfusate samples were collected, and tissue pieces were excised again at the beginning of reperfusion and also at the twentieth minute of reperfusion (Fig. I). At the end of reperfusion the lungs were prepared for electron microscopic studies. Ultrastructural analyses were done on eight randomly selected lungs (four from each group). Data and statistics. Pulmonary artery pressures (millimeters of mercury) were measured with a Datascope 200lA monitor (Datascope Corp., Montvale, N.J.) with a Viggo-Spectramed pressure monitoring kit (Viggo-Spectramed, Inc., Oxnard, Calif.). The adenosine deaminase levels (units/nil) of the perfusate was calculated with a Spectronics 3000 AutoAn-
The Journal of Thoracic and Cardiovascular Surgery November 1994
9 2 4 Soncul et al.
Percentage Changes of PA Pressure 300
!
200
~
POST-ISCHEMIC
AFTER REPERFUSION
Tissue Malondialdehyde Levels
t
2
1.5
~
I l!
0.5
POST-ISCHEMIC
AFTER REPERFUSION
Adenosine Oeaminase Levels of Perfusate 40
I:5
~o
20 10
POST·ISCHEMIC
AFTER REPERFUSION
Fig. 2. Pulmonary artery pressures (percentage change), tissue malondialdehyde levels (nmol/rng protein), and adenosine deaminase levels (units/rnl) of the perfusate. *Significant difference (p < 0.05) between the indicated value and the preischemic value of the same group. tSignificant difference (p < 0.05) between the groups. Values are mean ± standard error of the mean.
alyzer (Spectronics Corporation, Westbury, N.Y.) by the Soloman Method. The tissue malondioldehyde levels (nanomoles per milligram protein) were calculated by the Spectronics 3000 AutoAnalyzer using the technique of Beauge," Ultrastructural analyses were done with uranyl acetate-lead citrate stains at X 12,000 magnification. The results are presented as mean and standard error of the mean. Overall significance of differences between the groups was determined by t test (paired two sam-
pIe for means) using the Microsoft Excel 4.0 personal computer program (Microsoft Corporation, Redmond, Wash.).
Results Because of the changes in lung sizes in different animals and our standard perfusion volume (10 ml/rnin), the alterations of pulmonary artery pressures were calcu-
The Journal of Thoracic and Cardiovascular Surgery Volume 108, Number 5
•
Soncul et al.
925
•
Fig. 3. Electron micrograph of the alveolar capillary membrane in the control group (X12,000 magnification). Al Ep, Alveolar epithelium; K En, capillaryendothelium.
lated as the percentage change of the preischemic value in each experiment. Although the percentage change in the mean pulmonary artery pressures rose in both of the groups after the ischemic period, the difference between the groups was still significant (p = 0.032). The difference between the groups was not significant after reperfusion (Table I, Fig. 2). In the control group, the mean preischemic tissue malondialdehyde level increased noticeably at the end of reperfusion (p = 0.073). The difference between the two groups at the end of reperfusion was also statistically significant (p = 0.044) (Table I, Fig. 2). The adenosine deaminase levels of the perfusate increased significantly in both of the groups after ischemia as compared with their preischemic values (p < 0.001). The difference between the two groups after the ischemic period was again significant statistically (p < 0.041) (Table I, Fig. 2). The ultrastructural analysis of the lung tissues in the control group showed a marked separation between the capillary endothelium and the alveolar epithelium and also plenty of empty cytoplasmic vacuoles with different
Fig. 4. Electron micrograph of the alveolar capillary membrane in the selenium group (X12,000 magnification). Al Ep, Alveolar epithelium; K En, capillaryendothelium.
Table I. Effect of selenium on ischemic injury Before ischemia PA pressure (mm Hg) Euro-Collins 23.0 ± 5 Euro-Collins 25.6 ± 4 + selenium PA pressure (% change) Euro-Collins 100 Euro-Collins 100 + selenium Tissue MDA levels Euro-Collins 0.86 ± 0.10 Euro-Collins 0.98 ± 0.17 + selenium ADA levels of the perfusate 7.71 ± 1.91 Euro-Collins 4.93 ± 0.91 Euro-Collins + selenium
After ischemia
After reperfusion
56.8 ± 14 46.3 ± 7
33.1 ± 5 27.9 ± 6
247 ± 17 181 ± II
144 ± 9 109 ± 7
1.07 ± 0.18 0.77 ± 0.08
1.55 ± 0041 0.74 ± 0.06
34.12 ± 6.07 21.32 ± 3.14
8.69 ± 1.75 13.2 ± 2045
PA, Pulmonary artery; MDA, malondialdehyde; ADA, adenosine deaminase. Values are mean ± standard error of the mean.
sizes of pneumocytes. The alveolar capillary membrane and cellular structures were nearly normal in the selenium group (Figs. 3 and 4).
9 2 6 Soncul et al.
Comment The lung is the only organ to which oxygen may be supplied after its blood supply is stopped. In contrast, much more difficulty has been associated with the preservation of the lung than with the preservation of solid organs in vitro because of the lung's unique structure, especially the close apposition of blood and air compartments in the alveoli.?: 10 Several studies have suggested that oxygen-derived free radicals produced by various mechanisms playa significant role in the injury sustained by a transplanted organ. Any molecule that can react with a free radical can be termed a scavenger. The clinical benefits of such a molecule are derived from the fact that the oxygenderived free radicals react with it before they can react with cellular components and cause tissue damage. I I, 12 Selenium, as selenocysteine, is placed in the active site of the enzyme glutathione peroxidase, which reduces hydrogen peroxide to the corresponding lipid alcohols or water. Besides this hypothesized radical scavenging consequence, additional specific preventive effects have been suggested for selenium.P'P The isolated perfused lung model used for our study was previously developed and used by a number of authors as a screening technique for the many factors affecting lung preservation and reperfusion injury.l'' 17 Previous experiments had demonstrated that lung function began to deteriorate when preservation time was extended to 30 hours under hypothermic conditions. 16, 18 Because it was difficult to keep the standardized experimental environment for such long periods in our laboratory conditions, in the current experiment we preferred to use 3 hours of normothermic (37 0 C) ischemia. In our study, lipid peroxidation associated with free radical generation was assessed by measuring tissue malondialdehyde, which is a three-carbon product of lipid peroxidation. Tissue damage was assessed by measuring pulmonary artery pressure, which is a relative parameter for pulmonary vascular resistance, and also by measuring adenosine deaminase activity of the perfusates. Adenosine deaminase is an enzyme in purine catabolism that catalyzes the conversion of adenosine to inosine. Activities of this enzyme have been reported to be high in pleural effusions because of various origins. This activity reflects the severity of tissue injury because the activity of the enzyme increases during the catabolism of high-energy phosphates. 19,20 In the selenium group, the tissue malondialdehyde levels decreased markedly when compared with the control group after reperfusion. These levels also decreased slightly just after the ischemic period, although the decrease was not statistically significant, probably
The Journal of Thoracic and Cardiovascular Surgery November 1994
because of the limited number of our experiments. According to these data, it can be argued that the addition of selenium to intracellular type pulmonary preservation solutions may have a noticeable effect on reperfusion injury of the ischemic lungs. Similar results for other organs such as heart and kidneys were previously reported in other recent experimental and clinical studies. 12, 13 Although pulmonary artery pressures and adenosine deaminase activity in the perfusate returned to normal after reperfusion, both of the parameters increased substantially after the ischemic period in the control group (Fig. 2). Electron microscopic evaluation of the lungs had also demonstrated serious changes in the alveolar capillary space and in the pneumocytes in the control group at the end of the reperfusion period. In conclusion, our study suggests that addition of selenium to pulmonary preservation solutions may reduce ischemic lung injury during normothermic conditions. REFERENCES 1. Collins GM, Bravo-Shugarman M, Terasaki PI. Kidney preservation for transportation. Lancet 1969;2: 1219-22. 2. Hooper TL, Locke TJ, Fetherston G, Flecknell PA, McGregor GA. Comparison of cold flush perfusion with modified blood versus modified Euro-Collins solution for lung preservation. J Heart Transplant 1990;9:4, 429-34, 3, Haverich A, Aziz S, Scott WC, Jamieson SW, Shumway NE. Improved lung preservation using Euro-Collins solution for flush perfusion. Thorac Cardiovasc Surg 1986; 34:368-78. 4. Keshavjee SH, McRitchie DI, Vittorini T, Rostein OD, Slutsky AS, Patterson GA. Improved lung preservation with dextran 40 is not mediated by a superoxide radical scavenging mechanism. J THORAC CARDIOVASC SURG 1992;2:326-8. 5. FloheL. Free radicalsin biology. PryorWA,ed. Vo15. New York: Academic Press, 1982:223-54. 6. Rotruck J, Pope A, Ganther H, Swanson A, Hafeman D, Hoekstra W. Selenium:biochemical roleas a component of glutathione peroxidase, Science 1973; 179:588-90. 7. SchoeneNW, Morris VC, LevanderOA. Effects of selenium deficiency on aggregationand tromboxaneformation in rat platelets. Fed Proc 1984;43:477-81. 8. BeugeJA. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302-10. 9. Date A, Matsumura A, Manchester JK, CooperJM, Lowry OH, CooperJD. Changes in alveolaroxygen and carbon dioxideconcentration and oxygenconsumption duringlung preservation. J. Thorac Cardiovasc. Surg 1993;105:492501. 10. Salonen JT, Salonen R, Seppanen K, et al. Relationship of serum selenium and antioxidants to plasma lipoproteins, platelet aggregability and prevalentischemic heart disease in eastern Finnish men. Atherosclerosis. 1988;70: 155-60.
The Journal of Thoracic and Cardiovascular Surgery Volume 108, Number 5
II. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312: 159-63. 12. Hernandez LA, Granger N, Role of antioxidants in organ preservation and transplantation. Crit Care Med 1988; 16:543-9. 13. Poltronieri R, Cevese A, Sharbati A. Protective effect of selenium in cardiac ischemia and reperfusion. Cardioscience 1992;3: 155-60. 14. Gromadzinska J, Sklodowska M, Wasowics W. Glutathione peroxidase activity, lipid and selenium concentration in various rat organs. Biomed Biochim Acta 1988; 47:19-24. IS. Salonen JT, Huttunen JK. Selenium in cardiovascular diseases. Ann Clin Res 1986;18:30-5. 16. Wang LS, Yoshikawa K, M itoshi S. The effect of ischemic time and temperature on lung preservation in a simple ex vivorabbit model used for functional assessment. J THORAC CARDIOVASC SURG 1989;98:333-42.
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