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The Effect of Simple Hypothermic Preservation with Trolox and Ascorbate on Lipid Peroxidation in Dog Kidneys
33, 217–225 (1996) 0022
CRYOBIOLOGY ARTICLE NO.
The Effect of Simple Hypothermic Preservation with Trolox and Ascorbate on Lipid Peroxidation in Dog Kidneys JONATHAN F. MCANULTY
AND
XIAO Q. HUANG
Department of Surgical Sciences, University of Wisconsin, Madison, Wisconsin 53706-1102 Dog kidneys were preserved by simple hypothermic storage in UW-lactobionate organ preservation solution for 48 h and analyzed for evidence of lipid peroxidation. The antioxidant function of the UW solution was evident in stored kidney tissues which had significant reductions in conjugated dienes, lipid peroxides, and Schiff base content compared to fresh controls without vascular flushing. Aerobic incubation of kidney cortex homogenates at 377C for 60 min resulted in increases in conjugated dienes, lipid peroxides, and Schiff bases in UW-stored kidneys. Schiff base production was markedly higher in UW-stored kidneys during warm incubation than in controls. Addition of Trolox (200 mM) to UW solution resulted in significant reductions in Schiff base production during warm aerobic incubation after preservation. In contrast, adding ascorbate (1 mM) to UW solution potentiated oxidative stress during aerobic incubation, with significant increases in conjugated dienes, lipid peroxides, and Schiff bases which were only partially reversed by further addition of Trolox. Increased oxidative stress was correlated with decreased respiratory function (decreased uncoupled respiration rates and sensitivity to oligomycin inhibition) in aerobically incubated homogenates. This study showed that although the UW solution does have an antioxidant function during hypothermic preservation there remains an increased oxidative stress during warm reoxygenation even in optimally harvested kidneys. The antioxidant effect of the UW solution after preservation can be significantly enhanced using the water-soluble vitamin E analogue Trolox. Antioxidant supplementation of UW solution may be advantageous in preserving kidneys with increased oxidative stresses obtained from suboptimal donors in clinical practice. q 1996 Academic Press, Inc. INTRODUCTION
Simple hypothermic storage methods for preserving kidneys for transplantation have markedly improved in the past decade. One important advance in hypothermic storage of organs was the development of the UW-lactobionate cold storage solution (34). The salient benefits of this solution were obtained through prevention of hypothermia-induced swelling and provision of metabolically active and antioxidant compounds (4, 30, 34). However, even with this solution the rate of dialysis-dependent delayed graft function after transplantation of cold-stored kidneys continues to be unacceptably high (25). Evidence suggests that one important factor contributing to acute tubular necrosis and delayed function of cold-stored kidneys is oxidative injury incurred during storage and early
Received July 3, 1995; accepted November 15, 1995.
reperfusion (9, 15, 16). Oxidative injury, initiated by oxygen-derived free radicals, may affect cell membranes or other vital cell components such as proteins or mitochondria (3, 14, 23). In hypothermically stored kidneys, oxidative injury is reflected in increased tissue lipid peroxides, conjugated dienes, Schiff base content, and depletion of cellular glutathione and nonglutathione thiols (4, 9, 17). The UW-lactobionate solution contains compounds which have antioxidant or free radical scavenging properties (34). Glutathione, allopurinol, and the polyol raffinose are all present in concentrations sufficient to have potential effects on radical-mediated cellular oxidations (10, 18, 24, 32, 36). In addition, lactobionate is thought to prevent damaging hydroxyl radical formation through its ability to chelate free iron (7, 28). However, the antioxidant efficacy of UW-lactobionate solution remains somewhat controversial (12, 32). Evidence supporting an antioxidant effect
217 0011-2240/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of the UW-lactobionate solution has been obtained by demonstrating reductions in lipid peroxidation (malondialdehyde) after cold storage of rat livers (12) and by increased damage in transplanted dog livers when glutathione was omitted from the solution (31). In human liver transplants, decreased plasma tocopherol uptake and conjugated diene production have been interpreted to indicate a beneficial antioxidant effect of UW solution (27). However, in simple hypothermic storage of kidneys the efficacy of UW-lactobionate solution in suppressing lipid peroxidation remains unclear. The purpose of this study was to determine if addition of the antioxidants Trolox and ascorbate to the UW solution could provide increased antioxidant efficacy in kidneys preserved by simple hypothermic storage for 48 h. Trolox is a hydrophilic analogue of vitamin E which has been reported to have greater free radical scavenging capacity than its parent compound (35). Ascorbic acid, which is an efficient free radical scavenger on its own, also interacts to regenerate the antioxidant form of vitamin E (2). These compounds, which are easily dissolved in aqueous media, may be effective antioxidant additives for the UW-lactobionate solution. In this study, lipid peroxidation in dog kidneys was assessed by measurement of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances, and Schiff base content in cortex tissue homogenates made after preservation and after warm aerobic incubation for 1 h. In addition, the respiratory function of these homogenates was assessed to determine if adding Trolox or ascorbate resulted in a functional benefit to mitochondria, one important cellular target of oxidative injury (23). MATERIALS AND METHODS
Kidney Harvest and Preservation Adult purebred beagle dogs (n Å 10) of both sexes were anesthetized with thiopental sodium and maintained on halothane with oxygen. Both kidneys were removed via a mid-
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line laparotomy and the dogs were euthanized. Kidneys were randomly divided between groups (n Å 4 kidneys/group, with no two kidneys from the same dog in one group) and flushed out with cold (27C) UW-lactobionate solution (34) modified by addition of Trolox and ascorbate. Experimental groups included (1) UW solution without additives; (2) UW solution plus Trolox (200 mM); (3) UW solution plus ascorbic acid (1.0 mM); and (4) UW solution plus Trolox (200 mM) and ascorbic acid (1.0 mM). Control kidneys were not flushed out but had cortical tissue samples immediately obtained for preparation of tissue homogenates. After flushing, kidneys were stored in covered beakers in the appropriate solution on ice for 48 h. At that time tissue homogenates (10% w/v) were made from the renal cortex in phosphate-saline as previously described (16). Paired tissue samples were simultaneously obtained for measurement of water content by oven drying (21). Homogenate samples obtained before and after incubation in open vessels in a shaking water bath (377C) for 60 min were frozen by immersion in liquid nitrogen and stored at 0767C for later analysis. Homogenates (10% w/v) for analysis of respiration were made in medium containing sucrose, 225 mM; MgCl2, 5 mM; KH2PO4, 10 mM; KCl, 20 mM; Tris, 10 mM; Hepes, 5 mM; and 0.25 g% fatty acid-free bovine serum albumin. Respiration was assayed in the same medium after homogenization and after 60 min warm incubation as described above. Respiration was assessed by polaragraphic measurement of oxygen consumption at 307C in the presence of succinate (5 mM) and rotenone (15 mM) in a water-jacketed vessel (volume 1.5 ml). ADP (200 nmol) was added to the mixture after stable basal respiration rates were obtained. After determination of ADPstimulated respiration rates, oligomycin-inhibited (10–15 mg/ml) and carbonyl cyanide 3-chlorophenylhydrazone (CCCLP-10 mM)stimulated respiration rates were measured (20). Duplicate assays were performed for each sample and the results averaged. Protein
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content of the homogenate was determined by the Biuret method (29) and respiration rates calculated as nmol oxygen consumed/min/mg protein. Lipid peroxidation measurements in tissue were made from aliquots of a 10% (w/v) homogenate in 1.15% KCl solution with 0.022 mg/ml butylated hydroxytoluene (BHT). Thiobarbituric acid-reactive substances (malondialdehyde—MDA) were determined by a previously described method (33) with modifications adapted from Asakawa and Matsushita (1). In brief, 0.5 ml tissue homogenate was added to a reaction buffer containing 3.0 ml 1% phosphoric acid, 1.0 ml of a 0.6% thiobarbituric acid solution, 0.1 ml FeCl3 solution (stock prepared as 270 mg FeCl3 –6H2O in 100 ml H2O), and 0.22 mg BHT (total volume 4.7 ml). The mixture was capped and incubated at 1007C for 45 min. After cooling to room temperature, 4.0 ml n-butanol was added, vortexed, and separated by centrifugation. MDA content was determined by the difference in absorption of the butanol layer between 535 and 520 nm when compared to a standard curve generated with known quantities of MDA. Conjugated dienes, lipid peroxides, and Schiff bases were determined by extraction of 0.5 ml of the homogenate described above in 6 ml chloroform:methanol (2:1 v/v) according to the method of Lunec and Dormandy (22). Conjugated dienes (absorbance at 234 nm) and lipid peroxides (absorbance at 270 nm) were measured in the chloroform:methanol extract using a Shimadzu UV 160U spectrophotometer. Schiff base fluorescence (exc. 355; em. 455) was measured in the aqueous phase of the extract after addition of water with a Shimadzu RU5000 fluorescence spectrophotometer (sensitivity high; exc. slit width 10 nm; em. slit width 5 nm). Values cited are means { SEM. Statistical analysis was done by Student’s t test between groups and by paired t test when assessing changes before and after warm incubation within a single group using a computerized statistical package (Sigmastat, Jandel Scien-
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tific, San Rafael, CA) Differences were considered to be significant at P õ 0.05. RESULTS
Preservation of dog kidneys for 48 h in UW-lactobionate solution resulted in significant reductions in conjugated dienes, lipid peroxides, and Schiff bases compared to control tissues (Fig. 1) demonstrating the antioxidant effect of UW-lactobionate solution. Homogenates of UW preserved kidney tissue continued to have significantly lower rates of formation of conjugated dienes and lipid peroxides than controls during warm aerobic incubation (Fig. 2). However, Schiff base production during aerobic incubation was greater in UW preserved kidney tissue homogenates than in controls (P õ 0.05). MDA did not significantly vary in UW-preserved kidneys from control tissues after preservation or subsequent aerobic incubation. The addition of Trolox to the UW solution resulted in reductions in the rates of conjugated diene and lipid peroxide formation of 65 and 70%, respectively (Fig. 3). Schiff base formation was reduced 93% (P õ 0.05) when Trolox was added to the UW solution. MDA formation was minimally affected by Trolox compared to preservation with UW solution alone. In contrast, adding ascorbate to UW solution resulted in significantly increased rates of conjugated diene, lipid peroxide, and Schiff base formation (Fig. 4). MDA formation was unaffected by adding ascorbate to UW solution. Trolox was able to suppress the ascorbate-induced increase in oxidative injury to some degree (Fig. 5). However, conjugated diene and lipid peroxide formation remained significantly higher than in UW solution alone. Ascorbate-induced increases in Schiff base formation were markedly reduced by Trolox addition (P õ 0.05) and were similar to kidney tissue preserved in UW without additives. Uncoupler-stimulated respiration rates were increased (P õ 0.05) compared to control homogenates after 48 h preservation in UW solu-
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FIG. 1. Lipid peroxidation in dog kidney cortex tissue after simple hypothermic storage in UW-lactobionate solution for 48 h. Values are means { SEM; *P õ 0.05; **P õ 0.005 compared to fresh control tissue.
tion. However, uncoupler stimulated rates decreased after 60 min aerobic incubation to levels similar to controls (38.3 { 2.1 compared to 34.9 { 1.4 nmol oxygen consumed/min/mg protein for UW-preserved and control kidney homogenates after 60 min incubation, respec-
tively). Adding Trolox to the UW solution did not affect uncoupler-stimulated respiration rates (1.1 { 2.3% increased rate compared to UW-preserved kidneys). In contrast, ascorbate caused an average 23.2 { 4.1% reduction (P õ 0.05) in uncoupler-stimulated respiration
FIG. 2. Rates of lipid peroxidation in dog kidney cortex tissue homogenates (10% w/v) during 60 min aerobic incubation at 377C. Homogenates were made from either fresh control kidneys or kidneys preserved 48 h by simple hypothermic storage in UW-lactobionate ({Trolox or ascorbate). Values shown are mean differences ({SEM) in homogenates compared to measurements obtained prior to warm aerobic incubation. *P õ 0.05 compared to control homogenates.
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FIG. 3. Effect of Trolox (200 mM) added to the UW preservation solution on lipid peroxidation in homogenates made from 48-h hypothermically preserved dog kidneys. Cortical homogenates were incubated aerobically for 60 min at 377C. Results are presented as the difference in the rate of formation of products of lipid peroxidation compared to homogenates from kidneys stored in UW-lactobionate without additives. Values are means { SEM. *P õ 0.05 compared to tissues preserved in UW without additives.
rate which was only partially reversed by combination with Trolox (9.3 { 3.9% less than kidneys preserved with UW alone). The effect of ascorbate on homogenate res-
piratory function after warm aerobic incubation was also seen in a significant loss of sensitivity of ADP-stimulated respiration to oligomycin inhibition (0.08 { 0.08% inhibition by
FIG. 4. Effect of ascorbate (1 mM) added to the UW preservation solution on lipid peroxidation in homogenates from 48-h hypothermically preserved dog kidneys. Cortical homogenates were incubated aerobically for 60 min at 377C. Results are presented as the difference in the rate of formation of products of lipid peroxidation compared to homogenates from kidneys stored in UW-lactobionate without additives. Values are means { SEM. *P õ 0.05; **P õ 0.005 compared to tissues preserved in UW without additives.
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FIG. 5. Effect of Trolox (200 mM) and ascorbate (1 mM) added in combination to the UW preservation solution on lipid peroxidation in homogenates from 48-h hypothermically preserved dog kidneys. Cortical homogenates were incubated aerobically for 60 min at 377C. Results are presented as the difference in the rate of formation of products of lipid peroxidation compared to homogenates from kidneys stored in UW without additives. Values are means { SEM. *P õ 0.05 compared to tissues preserved in UW without additives.
oligomycin after preservation with ascorbate) which was partially restored when combined with Trolox (5.9 { 1.0% inhibition by oligomycin). Control and UW-preserved tissue homogenate ADP-stimulated respiration rates were inhibited by oligomycin 16.3 { 3.4 and 10.4 { 3.2%, respectively. Adding Trolox to the UW solution did not result in significant differences in oligomycin sensitivity compared to preservation in UW without additives (7.6 { 1.0% inhibition). ADP-stimulated respiration was unaffected by preservation with either Trolox or ascorbate. ADP-stimulated respiration rates were 12.0 { 1.1 and 13.6 { 0.6 nmol oxygen/min/mg protein for control and UW-preserved kidney homogenates, respectively. DISCUSSION
This study showed that the UW-lactobionate solution exerted an antioxidant effect on kidneys preserved at hypothermia for 48 h. However, the antioxidant effect of the UW solution was diminished during aerobic incubation after preservation, as indicated by the
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increased production of Schiff bases in preserved tissue compared to controls. The kidneys used in this study were optimally harvested and would be anticipated to have a relatively low level of oxidative stress prior to warm aerobic incubation. In the clinical situation, kidneys obtained from suboptimal donors with variable periods of warm ischemia or hypoperfusion would likely have higher levels of lipid peroxidation and present a greater challenge to the antioxidant capacity of the UW solution. Thus, supplementation of the UW solution with an effective antioxidant such as Trolox may be a valuable adjunct for reducing organ damage after transplantation. Lipid peroxidation would not have been clearly demonstrated in UW-preserved kidneys if the rate of MDA formation had been the sole parameter examined. Our finding that MDA content in UW-preserved kidney homogenates did not significantly change during warm aerobic incubation was similar to results reported in UW-preserved livers (12, 32). In those studies, unchanging levels of MDA during reperfusion were interpreted
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to mean either a lack of a role for lipid peroxidation in preservation – reperfusion injury (32) or as evidence of the antioxidant efficacy of UW (12). In the current study, unchanging MDA levels were accompanied by significantly increased rates of Schiff base formation. Schiff bases form when aldehydes generated by fragmentation of lipid peroxides react with amine groups on molecules such as amino acids or phospholipids (22) and, thus, reactivity to thiobarbituric acid is lost. Increased Schiff base formation with static MDA levels may suggest that hypothermic preservation with UW increased the reactivity of membranes or amino acids to free aldehydes and prevented accumulation of thiobarbituric acid reactive compounds. Regardless, these results show that measurements of MDA as a sole indicator of lipid peroxidation in preserved organs can provide an inaccurate picture of the oxidative stresses that may be present during reoxygenation after preservation. Ascorbate functioned as a prooxidant in this study. Conjugated dienes, lipid peroxides, and Schiff bases were all significantly increased when ascorbate was added to the UW solution. This result was unexpected since ascorbate is generally considered an antioxidant (2, 6) unless free iron is present in solution (5, 13, 26). Lactobionate is an effective iron chelator, a property which has been suggested to be important in the antioxidant function of the UW solution (28). However, the prooxidant effect observed when ascorbate was added to the solution indicates that the iron chelating capacity of the UW solution is insufficient to prevent lipid peroxidation when an agent which promotes iron ion redox cycling (ascorbate) is added to the solution. An alternative explanation may be that ascorbate interacts with the iron–lactobionate complex to promote ferric/ferrous-ligand complex redox cycling with generation of hydrogen peroxide or hydroxyl radicals (28). Regardless, the oxidation chemistry of the iron–lacotobionate moiety in a complicated solution such as UW is complex and not clearly understood. Addition
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of compounds with putative antioxidant effects to the UW solution should be approached with caution and predicated on direct measurements of an antioxidant effect. In contrast, Trolox proved to be an effective antioxidant when added to the UW solution. Trolox addition resulted in superior antioxidant capacity compared to preservation with UW alone and was able to reverse much of the increase in lipid peroxidation induced by addition of ascorbate to the solution. Clearly, there were no synergistic or additive antioxidant effects, as has been previously reported when Trolox and ascorbate were combined (8, 11). Increases in lipid peroxidation were well correlated with decreased respiratory function, as indicated by decreased uncoupled respiration rates and sensitivity to oligomycin inhibition. These changes indicated injury at the level of substrate metabolism or electron transport and increased mitochondrial uncoupling, respectively. These results were similar to a previous study which identified the ubiquinone–cytochrome c oxidoreductase as a site of peroxidative injury after hypothermic preservation in rat livers (23). ADP-stimulated respiratory rates were not altered concurrently with changes in lipid peroxidation. This was probably due to the offsetting effects which increased mitochondrial uncoupling and decreased uncoupled respiration rates have on state 3 (ADP-stimulated) respiration rates. Similarly, a previous study in hypothermically preserved rat kidneys found no correlation between oxidative changes and mitochondrial respiratory function (state 3 respiration rates) (19). However, in that study respiratory parameters which would clearly reflect changes in electron transport function were not measured. The current study shows that increased lipid peroxidation was associated with deleterious changes in respiration, one important parameter of cellular function. Maintenance of mitochondrial function by prevention of lipid peroxidation may be of benefit during reperfusion when the demand for ATP synthesis for reestablishment
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of ionic homeostasis and to effect cellular repair may be maximal. In summary, lipid peroxidation was present during warm aerobic incubation of homogenates made from optimally harvested kidneys preserved by simple hypothermic storage in UW solution. Trolox was easily soluble in UW solution and provided significantly increased antioxidant capability compared to UW solution alone. Increased lipid peroxidation and decreased respiratory function were observed upon addition of ascorbate. Increasing the antioxidant capacity of the UW solution by addition of Trolox may be advantageous when harvesting kidneys from suboptimal donors where oxidative stresses are likely to be increased over those of organs obtained under optimal conditions.
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The Effect of Simple Hypothermic Preservation with Trolox and Ascorbate on Lipid Peroxidation in Dog Kidneys JONATHAN F. MCANULTY
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XIAO Q. HUANG
Department of Surgical Sciences, University of Wisconsin, Madison, Wisconsin 53706-1102 Dog kidneys were preserved by simple hypothermic storage in UW-lactobionate organ preservation solution for 48 h and analyzed for evidence of lipid peroxidation. The antioxidant function of the UW solution was evident in stored kidney tissues which had significant reductions in conjugated dienes, lipid peroxides, and Schiff base content compared to fresh controls without vascular flushing. Aerobic incubation of kidney cortex homogenates at 377C for 60 min resulted in increases in conjugated dienes, lipid peroxides, and Schiff bases in UW-stored kidneys. Schiff base production was markedly higher in UW-stored kidneys during warm incubation than in controls. Addition of Trolox (200 mM) to UW solution resulted in significant reductions in Schiff base production during warm aerobic incubation after preservation. In contrast, adding ascorbate (1 mM) to UW solution potentiated oxidative stress during aerobic incubation, with significant increases in conjugated dienes, lipid peroxides, and Schiff bases which were only partially reversed by further addition of Trolox. Increased oxidative stress was correlated with decreased respiratory function (decreased uncoupled respiration rates and sensitivity to oligomycin inhibition) in aerobically incubated homogenates. This study showed that although the UW solution does have an antioxidant function during hypothermic preservation there remains an increased oxidative stress during warm reoxygenation even in optimally harvested kidneys. The antioxidant effect of the UW solution after preservation can be significantly enhanced using the water-soluble vitamin E analogue Trolox. Antioxidant supplementation of UW solution may be advantageous in preserving kidneys with increased oxidative stresses obtained from suboptimal donors in clinical practice. q 1996 Academic Press, Inc. INTRODUCTION
Simple hypothermic storage methods for preserving kidneys for transplantation have markedly improved in the past decade. One important advance in hypothermic storage of organs was the development of the UW-lactobionate cold storage solution (34). The salient benefits of this solution were obtained through prevention of hypothermia-induced swelling and provision of metabolically active and antioxidant compounds (4, 30, 34). However, even with this solution the rate of dialysis-dependent delayed graft function after transplantation of cold-stored kidneys continues to be unacceptably high (25). Evidence suggests that one important factor contributing to acute tubular necrosis and delayed function of cold-stored kidneys is oxidative injury incurred during storage and early
Received July 3, 1995; accepted November 15, 1995.
reperfusion (9, 15, 16). Oxidative injury, initiated by oxygen-derived free radicals, may affect cell membranes or other vital cell components such as proteins or mitochondria (3, 14, 23). In hypothermically stored kidneys, oxidative injury is reflected in increased tissue lipid peroxides, conjugated dienes, Schiff base content, and depletion of cellular glutathione and nonglutathione thiols (4, 9, 17). The UW-lactobionate solution contains compounds which have antioxidant or free radical scavenging properties (34). Glutathione, allopurinol, and the polyol raffinose are all present in concentrations sufficient to have potential effects on radical-mediated cellular oxidations (10, 18, 24, 32, 36). In addition, lactobionate is thought to prevent damaging hydroxyl radical formation through its ability to chelate free iron (7, 28). However, the antioxidant efficacy of UW-lactobionate solution remains somewhat controversial (12, 32). Evidence supporting an antioxidant effect
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of the UW-lactobionate solution has been obtained by demonstrating reductions in lipid peroxidation (malondialdehyde) after cold storage of rat livers (12) and by increased damage in transplanted dog livers when glutathione was omitted from the solution (31). In human liver transplants, decreased plasma tocopherol uptake and conjugated diene production have been interpreted to indicate a beneficial antioxidant effect of UW solution (27). However, in simple hypothermic storage of kidneys the efficacy of UW-lactobionate solution in suppressing lipid peroxidation remains unclear. The purpose of this study was to determine if addition of the antioxidants Trolox and ascorbate to the UW solution could provide increased antioxidant efficacy in kidneys preserved by simple hypothermic storage for 48 h. Trolox is a hydrophilic analogue of vitamin E which has been reported to have greater free radical scavenging capacity than its parent compound (35). Ascorbic acid, which is an efficient free radical scavenger on its own, also interacts to regenerate the antioxidant form of vitamin E (2). These compounds, which are easily dissolved in aqueous media, may be effective antioxidant additives for the UW-lactobionate solution. In this study, lipid peroxidation in dog kidneys was assessed by measurement of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances, and Schiff base content in cortex tissue homogenates made after preservation and after warm aerobic incubation for 1 h. In addition, the respiratory function of these homogenates was assessed to determine if adding Trolox or ascorbate resulted in a functional benefit to mitochondria, one important cellular target of oxidative injury (23). MATERIALS AND METHODS
Kidney Harvest and Preservation Adult purebred beagle dogs (n Å 10) of both sexes were anesthetized with thiopental sodium and maintained on halothane with oxygen. Both kidneys were removed via a mid-
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line laparotomy and the dogs were euthanized. Kidneys were randomly divided between groups (n Å 4 kidneys/group, with no two kidneys from the same dog in one group) and flushed out with cold (27C) UW-lactobionate solution (34) modified by addition of Trolox and ascorbate. Experimental groups included (1) UW solution without additives; (2) UW solution plus Trolox (200 mM); (3) UW solution plus ascorbic acid (1.0 mM); and (4) UW solution plus Trolox (200 mM) and ascorbic acid (1.0 mM). Control kidneys were not flushed out but had cortical tissue samples immediately obtained for preparation of tissue homogenates. After flushing, kidneys were stored in covered beakers in the appropriate solution on ice for 48 h. At that time tissue homogenates (10% w/v) were made from the renal cortex in phosphate-saline as previously described (16). Paired tissue samples were simultaneously obtained for measurement of water content by oven drying (21). Homogenate samples obtained before and after incubation in open vessels in a shaking water bath (377C) for 60 min were frozen by immersion in liquid nitrogen and stored at 0767C for later analysis. Homogenates (10% w/v) for analysis of respiration were made in medium containing sucrose, 225 mM; MgCl2, 5 mM; KH2PO4, 10 mM; KCl, 20 mM; Tris, 10 mM; Hepes, 5 mM; and 0.25 g% fatty acid-free bovine serum albumin. Respiration was assayed in the same medium after homogenization and after 60 min warm incubation as described above. Respiration was assessed by polaragraphic measurement of oxygen consumption at 307C in the presence of succinate (5 mM) and rotenone (15 mM) in a water-jacketed vessel (volume 1.5 ml). ADP (200 nmol) was added to the mixture after stable basal respiration rates were obtained. After determination of ADPstimulated respiration rates, oligomycin-inhibited (10–15 mg/ml) and carbonyl cyanide 3-chlorophenylhydrazone (CCCLP-10 mM)stimulated respiration rates were measured (20). Duplicate assays were performed for each sample and the results averaged. Protein
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content of the homogenate was determined by the Biuret method (29) and respiration rates calculated as nmol oxygen consumed/min/mg protein. Lipid peroxidation measurements in tissue were made from aliquots of a 10% (w/v) homogenate in 1.15% KCl solution with 0.022 mg/ml butylated hydroxytoluene (BHT). Thiobarbituric acid-reactive substances (malondialdehyde—MDA) were determined by a previously described method (33) with modifications adapted from Asakawa and Matsushita (1). In brief, 0.5 ml tissue homogenate was added to a reaction buffer containing 3.0 ml 1% phosphoric acid, 1.0 ml of a 0.6% thiobarbituric acid solution, 0.1 ml FeCl3 solution (stock prepared as 270 mg FeCl3 –6H2O in 100 ml H2O), and 0.22 mg BHT (total volume 4.7 ml). The mixture was capped and incubated at 1007C for 45 min. After cooling to room temperature, 4.0 ml n-butanol was added, vortexed, and separated by centrifugation. MDA content was determined by the difference in absorption of the butanol layer between 535 and 520 nm when compared to a standard curve generated with known quantities of MDA. Conjugated dienes, lipid peroxides, and Schiff bases were determined by extraction of 0.5 ml of the homogenate described above in 6 ml chloroform:methanol (2:1 v/v) according to the method of Lunec and Dormandy (22). Conjugated dienes (absorbance at 234 nm) and lipid peroxides (absorbance at 270 nm) were measured in the chloroform:methanol extract using a Shimadzu UV 160U spectrophotometer. Schiff base fluorescence (exc. 355; em. 455) was measured in the aqueous phase of the extract after addition of water with a Shimadzu RU5000 fluorescence spectrophotometer (sensitivity high; exc. slit width 10 nm; em. slit width 5 nm). Values cited are means { SEM. Statistical analysis was done by Student’s t test between groups and by paired t test when assessing changes before and after warm incubation within a single group using a computerized statistical package (Sigmastat, Jandel Scien-
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tific, San Rafael, CA) Differences were considered to be significant at P õ 0.05. RESULTS
Preservation of dog kidneys for 48 h in UW-lactobionate solution resulted in significant reductions in conjugated dienes, lipid peroxides, and Schiff bases compared to control tissues (Fig. 1) demonstrating the antioxidant effect of UW-lactobionate solution. Homogenates of UW preserved kidney tissue continued to have significantly lower rates of formation of conjugated dienes and lipid peroxides than controls during warm aerobic incubation (Fig. 2). However, Schiff base production during aerobic incubation was greater in UW preserved kidney tissue homogenates than in controls (P õ 0.05). MDA did not significantly vary in UW-preserved kidneys from control tissues after preservation or subsequent aerobic incubation. The addition of Trolox to the UW solution resulted in reductions in the rates of conjugated diene and lipid peroxide formation of 65 and 70%, respectively (Fig. 3). Schiff base formation was reduced 93% (P õ 0.05) when Trolox was added to the UW solution. MDA formation was minimally affected by Trolox compared to preservation with UW solution alone. In contrast, adding ascorbate to UW solution resulted in significantly increased rates of conjugated diene, lipid peroxide, and Schiff base formation (Fig. 4). MDA formation was unaffected by adding ascorbate to UW solution. Trolox was able to suppress the ascorbate-induced increase in oxidative injury to some degree (Fig. 5). However, conjugated diene and lipid peroxide formation remained significantly higher than in UW solution alone. Ascorbate-induced increases in Schiff base formation were markedly reduced by Trolox addition (P õ 0.05) and were similar to kidney tissue preserved in UW without additives. Uncoupler-stimulated respiration rates were increased (P õ 0.05) compared to control homogenates after 48 h preservation in UW solu-
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FIG. 1. Lipid peroxidation in dog kidney cortex tissue after simple hypothermic storage in UW-lactobionate solution for 48 h. Values are means { SEM; *P õ 0.05; **P õ 0.005 compared to fresh control tissue.
tion. However, uncoupler stimulated rates decreased after 60 min aerobic incubation to levels similar to controls (38.3 { 2.1 compared to 34.9 { 1.4 nmol oxygen consumed/min/mg protein for UW-preserved and control kidney homogenates after 60 min incubation, respec-
tively). Adding Trolox to the UW solution did not affect uncoupler-stimulated respiration rates (1.1 { 2.3% increased rate compared to UW-preserved kidneys). In contrast, ascorbate caused an average 23.2 { 4.1% reduction (P õ 0.05) in uncoupler-stimulated respiration
FIG. 2. Rates of lipid peroxidation in dog kidney cortex tissue homogenates (10% w/v) during 60 min aerobic incubation at 377C. Homogenates were made from either fresh control kidneys or kidneys preserved 48 h by simple hypothermic storage in UW-lactobionate ({Trolox or ascorbate). Values shown are mean differences ({SEM) in homogenates compared to measurements obtained prior to warm aerobic incubation. *P õ 0.05 compared to control homogenates.
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FIG. 3. Effect of Trolox (200 mM) added to the UW preservation solution on lipid peroxidation in homogenates made from 48-h hypothermically preserved dog kidneys. Cortical homogenates were incubated aerobically for 60 min at 377C. Results are presented as the difference in the rate of formation of products of lipid peroxidation compared to homogenates from kidneys stored in UW-lactobionate without additives. Values are means { SEM. *P õ 0.05 compared to tissues preserved in UW without additives.
rate which was only partially reversed by combination with Trolox (9.3 { 3.9% less than kidneys preserved with UW alone). The effect of ascorbate on homogenate res-
piratory function after warm aerobic incubation was also seen in a significant loss of sensitivity of ADP-stimulated respiration to oligomycin inhibition (0.08 { 0.08% inhibition by
FIG. 4. Effect of ascorbate (1 mM) added to the UW preservation solution on lipid peroxidation in homogenates from 48-h hypothermically preserved dog kidneys. Cortical homogenates were incubated aerobically for 60 min at 377C. Results are presented as the difference in the rate of formation of products of lipid peroxidation compared to homogenates from kidneys stored in UW-lactobionate without additives. Values are means { SEM. *P õ 0.05; **P õ 0.005 compared to tissues preserved in UW without additives.
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FIG. 5. Effect of Trolox (200 mM) and ascorbate (1 mM) added in combination to the UW preservation solution on lipid peroxidation in homogenates from 48-h hypothermically preserved dog kidneys. Cortical homogenates were incubated aerobically for 60 min at 377C. Results are presented as the difference in the rate of formation of products of lipid peroxidation compared to homogenates from kidneys stored in UW without additives. Values are means { SEM. *P õ 0.05 compared to tissues preserved in UW without additives.
oligomycin after preservation with ascorbate) which was partially restored when combined with Trolox (5.9 { 1.0% inhibition by oligomycin). Control and UW-preserved tissue homogenate ADP-stimulated respiration rates were inhibited by oligomycin 16.3 { 3.4 and 10.4 { 3.2%, respectively. Adding Trolox to the UW solution did not result in significant differences in oligomycin sensitivity compared to preservation in UW without additives (7.6 { 1.0% inhibition). ADP-stimulated respiration was unaffected by preservation with either Trolox or ascorbate. ADP-stimulated respiration rates were 12.0 { 1.1 and 13.6 { 0.6 nmol oxygen/min/mg protein for control and UW-preserved kidney homogenates, respectively. DISCUSSION
This study showed that the UW-lactobionate solution exerted an antioxidant effect on kidneys preserved at hypothermia for 48 h. However, the antioxidant effect of the UW solution was diminished during aerobic incubation after preservation, as indicated by the
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increased production of Schiff bases in preserved tissue compared to controls. The kidneys used in this study were optimally harvested and would be anticipated to have a relatively low level of oxidative stress prior to warm aerobic incubation. In the clinical situation, kidneys obtained from suboptimal donors with variable periods of warm ischemia or hypoperfusion would likely have higher levels of lipid peroxidation and present a greater challenge to the antioxidant capacity of the UW solution. Thus, supplementation of the UW solution with an effective antioxidant such as Trolox may be a valuable adjunct for reducing organ damage after transplantation. Lipid peroxidation would not have been clearly demonstrated in UW-preserved kidneys if the rate of MDA formation had been the sole parameter examined. Our finding that MDA content in UW-preserved kidney homogenates did not significantly change during warm aerobic incubation was similar to results reported in UW-preserved livers (12, 32). In those studies, unchanging levels of MDA during reperfusion were interpreted
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to mean either a lack of a role for lipid peroxidation in preservation – reperfusion injury (32) or as evidence of the antioxidant efficacy of UW (12). In the current study, unchanging MDA levels were accompanied by significantly increased rates of Schiff base formation. Schiff bases form when aldehydes generated by fragmentation of lipid peroxides react with amine groups on molecules such as amino acids or phospholipids (22) and, thus, reactivity to thiobarbituric acid is lost. Increased Schiff base formation with static MDA levels may suggest that hypothermic preservation with UW increased the reactivity of membranes or amino acids to free aldehydes and prevented accumulation of thiobarbituric acid reactive compounds. Regardless, these results show that measurements of MDA as a sole indicator of lipid peroxidation in preserved organs can provide an inaccurate picture of the oxidative stresses that may be present during reoxygenation after preservation. Ascorbate functioned as a prooxidant in this study. Conjugated dienes, lipid peroxides, and Schiff bases were all significantly increased when ascorbate was added to the UW solution. This result was unexpected since ascorbate is generally considered an antioxidant (2, 6) unless free iron is present in solution (5, 13, 26). Lactobionate is an effective iron chelator, a property which has been suggested to be important in the antioxidant function of the UW solution (28). However, the prooxidant effect observed when ascorbate was added to the solution indicates that the iron chelating capacity of the UW solution is insufficient to prevent lipid peroxidation when an agent which promotes iron ion redox cycling (ascorbate) is added to the solution. An alternative explanation may be that ascorbate interacts with the iron–lactobionate complex to promote ferric/ferrous-ligand complex redox cycling with generation of hydrogen peroxide or hydroxyl radicals (28). Regardless, the oxidation chemistry of the iron–lacotobionate moiety in a complicated solution such as UW is complex and not clearly understood. Addition
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of compounds with putative antioxidant effects to the UW solution should be approached with caution and predicated on direct measurements of an antioxidant effect. In contrast, Trolox proved to be an effective antioxidant when added to the UW solution. Trolox addition resulted in superior antioxidant capacity compared to preservation with UW alone and was able to reverse much of the increase in lipid peroxidation induced by addition of ascorbate to the solution. Clearly, there were no synergistic or additive antioxidant effects, as has been previously reported when Trolox and ascorbate were combined (8, 11). Increases in lipid peroxidation were well correlated with decreased respiratory function, as indicated by decreased uncoupled respiration rates and sensitivity to oligomycin inhibition. These changes indicated injury at the level of substrate metabolism or electron transport and increased mitochondrial uncoupling, respectively. These results were similar to a previous study which identified the ubiquinone–cytochrome c oxidoreductase as a site of peroxidative injury after hypothermic preservation in rat livers (23). ADP-stimulated respiratory rates were not altered concurrently with changes in lipid peroxidation. This was probably due to the offsetting effects which increased mitochondrial uncoupling and decreased uncoupled respiration rates have on state 3 (ADP-stimulated) respiration rates. Similarly, a previous study in hypothermically preserved rat kidneys found no correlation between oxidative changes and mitochondrial respiratory function (state 3 respiration rates) (19). However, in that study respiratory parameters which would clearly reflect changes in electron transport function were not measured. The current study shows that increased lipid peroxidation was associated with deleterious changes in respiration, one important parameter of cellular function. Maintenance of mitochondrial function by prevention of lipid peroxidation may be of benefit during reperfusion when the demand for ATP synthesis for reestablishment
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of ionic homeostasis and to effect cellular repair may be maximal. In summary, lipid peroxidation was present during warm aerobic incubation of homogenates made from optimally harvested kidneys preserved by simple hypothermic storage in UW solution. Trolox was easily soluble in UW solution and provided significantly increased antioxidant capability compared to UW solution alone. Increased lipid peroxidation and decreased respiratory function were observed upon addition of ascorbate. Increasing the antioxidant capacity of the UW solution by addition of Trolox may be advantageous when harvesting kidneys from suboptimal donors where oxidative stresses are likely to be increased over those of organs obtained under optimal conditions.
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