0039-6109/92 $0.00 + .20
INTESTINAL ISCHEMIA
REPERFUSION INJURY Barbara J. Zimmerman, PhD, and D. Neil Granger, PhD
When a tissue is subjected to ischemia, a sequence of chemical reactions is initiated that may ultimately lead to cellular dysfunction and necrosis. Although no single process can be identified as the critical event in ischemia-induced tissue injury, most studies indicate that depletion of cellular energy stores and accumulation of toxic metabolites contribute to cell death. It is undeniable that re-establishing blood flow is necessary in rescuing ischemic tissues, as this permits both the regeneration of cell charge and the washout of toxic metabolites. However, reperfusion of ischemic tissues also leads to a sequence of events that, paradoxically, injure tissues. Several studies have demonstrated the phenomenon of reperfusion injury. Parks and Granger have shown that relatively little injury to the intestinal mucosa occurs during the ischemic period, the majority occurring upon reperfusion.:" The injury observed after 3 hours of ischemia (blood flow reduced to 20% of normal) and 1 hour of reperfusion is more severe than that observed after 4 hours of ischemia. These results demonstrate that the injury produced by reperfusion can be more severe than the injury induced by ischemia per see This observation implicates some reaction initiated by the return of oxygenated blood to the ischemic tissue as the cause of reperfusion-induced injury. Indeed, several studies have demonstrated that anoxic reperfusion of ischemic tissues results in very little damage.": 44 and it appears that the reactions initiated at reperfusion involve the formation of cytotoxic oxidants derived from molecular oxygen. Microvascular permeability to plasma proteins is a useful and sensitive index for evaluating the influence of ischemia-reperfusion (I/R) on microvascular integrity." If the cat small bowel is subjected to From the Department of Physiology, Louisiana State University Medical Center, Shreveport, Louisiana
SURGICAL CLINICS OF NORTH AMERICA VOLUME 72 • NUMBER 1 • FEBRUARY 1992
65
66
ZIMMERMAN & GRANGER
1 hour of ischemia (blood flow reduced to 20% of control) without reperfusion, a doubling (0.08 ± 0.01 versus 0.15 ± 0.03) of microvascular permeability is observed. However, the same period of ischemia followed by reperfusion results in a fivefold (0.04 ± 0.03) increase in microvascular permeability. The assumption that an oxygen-dependent mechanism accounts for the potentiation in permeability after reperfusion is supported by the observation that antioxidant agents attenuate only the component of the increased permeability that follows reperfusion. The concept that reperfusion per se is largely responsible for the injury observed in intestinal models of ischemia-reperfusion is also supported by the observation that interventions administered at the time of reperfusion are as effective in attenuating mucosal injury as agents administered before ischemia. Although reperfusion of ischemic tissues appears to exacerbate the microvascular and parenchymal injury during an ischemic insult, this phenomenon occurs only after a relatively short period of ischemia. As the ischemic period lengthens, the detrimental effect of oxygen deprivation on the tissues outweighs the effect of reperfusion, resulting in tissue necrosis and eventual cell death. Thus, under conditions of prolonged or severe ischemia, restoration of blood flow will not significantly affect the ultimate viability of the tissue.
ROLE OF REACTIVE OXYGEN METABOLITES IN REPERFUSION INJURY
The observation that reperfusion (reoxygenation) of ischemic tissues produces injury led to the concept that reperfusion injury may be mediated at least in part by the formation of reactive oxygen metabolites. Molecular oxygen can accept a total of four electrons to form water; however, it can be reduced in univalent steps to generate three oxidant species. The univalent reduction of oxygen produces the superoxide anion radical (0 2 -). The cellular toxicity associated with superoxide is usually attributed to its role as a precursor to more reactive oxygen species. Hydrogen peroxide (H 202 ) may be formed as a result of the divalent reduction of O 2 - or the dismutation of O 2 - . The spontaneous dismutation of O 2 - proceeds rapidly in aqueous solution; thus, the production of O 2 - in vivo is always accompanied by the production of H 202 • The third radical species derived from molecular oxygen is the hydroxyl radical (-OH), which is formed by the interaction of O 2 - and H202 (Haber-Weiss reaction) and is a potent oxidizing agent. The result of oxygen radical formation is damage to an entire array of biomolecules found in tissues, including nucleic acids, membrane lipids, enzymes, and receptors." Membrane-associated polyunsaturated fatty acids are readily attacked by -OH in a process that results in the peroxidation of lipids. Peroxidation of membrane lipids can disrupt membrane fluidity and cell compartmentation, which can result in cell lysis. Thus, oxygen radical-initiated lipid peroxidation and protein
REPERFUSION INJURY
67
- damage may contribute to the impaired cellular function and necrosis associated with reperfusion of ischemic tissues. Evidence for the involvement of reactive oxygen metabolites in reperfusion injury is based on the use of agents that either restrict the production of these cytotoxic oxidants or act as scavengers after the oxidants are produced. Oxidant formation has been demonstrated during reperfusion of the ischemic bowel by electron-spin resonance spectrometry" and chemiluminescence. 30, 53 These studies demonstrate that there is a burst of oxidant formation immediately after reperfusion, which lasts for 2 to 5 minutes. The enhanced photoemission observed with chemiluminescence after reperfusion is suppressed by treatment with superoxide dismutase, an enzyme that scavenges O 2 - . The concept of reperfusion-induced oxidant production is also supported by the fact that the consumption of reduced glutathione in intestinal mucosa subjected to ischemia-reperfusion is largely prevented by superoxide dismutase." There is evidence that oxidants mediate both the increased· microvascular permeability produced by 1 hour of ischemia-reperfusion and the mucosal lesions produced by 3 hours of ischemia-reperfusion (Table 1). Both superoxide dismutase and copper diisopropyl salicylate (CuDIPS), a superoxide dismutase mimetic, significantly attenuate reperfusion-induced increases in microvascular permeability. Furthermore, administration of superoxide dismutase prior to reperfusion attenuates the necrosis of mucosal villi and crypt epithelium produced by longer periods of ischemia." 37, 49 Although the aforementioned studies implicate the superoxide anion in mediating reperfusion-induced changes in intestinal integrity, there is evidence that oxidants derived from superoxide play a more important role in the injury process (Table 1). Catalase, an enzyme that catalyzes the disproportionation of H 202 to H 20 and O 2 , has proved to be protective in many models of ischemia-reperfusion." Nonenzymatic scavengers of the hydroxyl radical, including dimethyl sulfoxide (DMSO), dimethylthiourea, and mannitol, also attenuate reperfusionTable 1. MODULATION OF I/R-INDUCED INCREASE IN MICROVASCULAR PERMEABILITY Condition Control Ischemia-reperfusion (I/R) I/R + superoxide dismutase I/R + Cu-DIPS I/R + catalase I/R + dimethyl sulfoxide I/R + deferoxamine IIR + iron-loaded deferoxamine I/R + apotransferrin Values are means ± SE.
Microvascular Permeability (1 - (1)
0.08 0.41 0.14 0.19 0.19 0.19 0.15 0.44 0.17
± 0.005
± 0.02 ± 0.013 ± 0.03 ± 0.01 ± 0.02 ± 0.01 ± 0.03 ± 0.01
68
ZIMMERMAN & GRANGER
induced injury. 38, 41 Inasmuch as ·OH is formed from O 2 - and H 202 and ·OH scavengers provide levels of protection similar to that observed with superoxide dismutase or catalase, many investigators have suggested that the secondarily derived hydroxyl radical may be the primary damaging radical. The protective effects of superoxide dismutase, catalase, and hydroxyl radical scavengers are consistent with the view that the highly cytotoxic hydroxyl radical is formed during reperfusion by the Haber-Weiss reaction. It is generally assumed that the Haber-Weiss reaction occurs at too Iowa rate to be of physiological significance. However, the reaction can be greatly accelerated by the presence of transition metals (e.g., iron), which act as a catalyst. The intestinal mucosa is a rich source of iron. Intestinal iron is stored as ferritin in the ferric (Fe +3) state. However, superoxide reduces the ferritin-stored Fe+ 3 to liberate ferrous (Fe+ 2 ) iron." Thus, superoxide could release ferrous iron for reaction with hydrogen peroxide to form the hydroxyl radical during reperfusion. The role of iron in reperfusion-induced hydroxyl radical production has been assessed by determining whether deferoxamine (an iron chelator) or apotransferrin (an iron-binding protein) provides protection against the increased microvascular permeability produced by ischemiareperfusion." Pretreatment with either deferoxamine or apotransferrin significantly attenuates the increase in microvascular permeability after reperfusion. The observation that iron-loaded deferoxamine or transferrin does not protect the intestine against reperfusion injury argues against a nonspecific protective effect of these iron-binding compounds. The ability of hydroxyl radicals to initiate lipid peroxidation can result in the formation of lipid-derived free radicals such as conjugated dienes, lipid hydroperoxide radicals, and lipid hydroperoxides. Measurements of conjugated dienes and malondialdehyde are frequently used as an index of lipid peroxidation. Schoenberg and coworkers'< 57 measured mucosal concentrations of conjugated dienes in cat intestine subjected to ischemia-reperfusion. These studies indicate that conjugated diene levels in tissue are not affected by ischemia; however, the levels are doubled 10 minutes after reperfusion. The reperfusioninduced increase in conjugated dienes is significantly attenuated in animals treated with superoxide dismutase. Otamiri and Tagesson35,36 measured mucosal and plasma levels of malondialdehyde in rats after reperfusion of ischemic intestine. Malondialdehyde concentrations in both intestinal mucosa and plasma increased threefold to fourfold 5 minutes after reperfusion. These observations, along with those described earlier, suggest that the hydroxyl radical mediates the lipid peroxidation associated with reperfusion of the small intestine. XANTHINE OXIDASE AS A SOURCE OF REACTIVE OXYGEN METABOLITES
Xanthine oxidase is the rate-limiting enzyme in nucleic acid degradation through which all purines are channeled for terminal oxidation.
REPERFUSION INJURY
69
Xanthine oxidase (XO) has the ability to generate H 20 2 and O 2- during the oxidation of hypoxanthine or xanthine: XO hypoxanthine + 202 + H 20 ~ xanthine + O 2- + H 20 2 xanthine + 202 + H 20
~
uric acid + O 2 - + H 202
Xanthine oxidase normally exists in nonischemic, healthy cells predominately as an NAD+-dependent dehydrogenase (XDH).39,42 This form of the enzyme uses NAD+ instead of O 2 as the electron acceptor during oxidation of purines and does not produce O 2- or H 20 2: hypoxanthine + H 20 + NAD+
~
xanthine + NADH + H+
Xanthine dehydrogenase is converted to the oxidant-producing xanthine oxidase form during tissue ischemia. I, 42 Conversion of xanthine dehydrogenase to xanthine oxidase (D-to-O conversion) can occur by two mechanisms: (1) reversible conversion by oxidation or (2) irreversibly conversion by proteolysis. 1, 4, 54 The D-to-O conversion during ischemia appears to occur at different rates in various tissues; however, the amount of D-to-O conversion is proportional to the duration of ischemia. 42 In comparison to other tissues, the intestinal mucosa has a tremendous capacity to oxidize hypoxanthine via xanthine oxidase. Xanthine oxidase (dehydrogenase plus oxidase) activity in the intestinal mucosa is approximately 100 mU/gm wet weight in most species." In humans, liver and intestine have the highest activity, with intestine containing 29 to 56 mU/gm wet weight. Although these levels are relatively low compared with values reported for other mammals, the potential cytotoxicity of this enzyme activity is exemplified by the observation that isolated cells are injured when exposed to xanthine oxidase levels as low as 2 mU/m1.50 Intestinal xanthine oxidase activity is found almost exclusively in the mucosal layer, with an increasing gradient of activity from villus base to tip." A similar gradient of vulnerability to ischemia-reperfusion has been demonstrated. Immunolocalization studies have shown that mucosal xanthine oxidase is found in the endothelial cells lining the microvasculature in the villus core." However, histochemical studies demonstrate that the majority of the enzyme is in the epithelial cells of the mucosal layer." Furthermore, freshly isolated villus epithelium accounts for the majority of mucosal xanthine oxidase in cat ileal mucosa." The significant enzyme activity remaining in the lamina propria may be concentrated in the microvascular endothelium (Table 2). Parks et al42 have shown that xanthine oxidase represents approximately 19% of the total xanthine dehydrogenase plus xanthine oxidase activity in the normally perfused rat small intestine. This increases to 34%, 46%, and 61% after 1, 2, and 3 hours of ischemia, respectively. Roy et al46 have shown that intraperitoneal injection of soybean trypsin
70
ZIMMERMAN & GRANGER
Table 2. DISTRIBUTION OF XANTHINE OXIDASE (mU/gm) IN EPITHELIUM AND LAMINA PROPRIA OF CAT INTESTINAL MUCOSA Total mucosa Epithelium Lamina propria
143 ± 39 202 ± 15
43 ± 40
Values are means ± SE.
inhibitor (a serine protease inhibitor) in rats completely inhibits D-to-O conversion in the small intestine. Utilizing soybean trypsin inhibitor, Parks and coworkers demonstrated that the protease inhibitor attenuated the reperfusion-induced increase in vascular permeability and mucosal lesions. These observations indicate that D-to-O conversion in the ischemic feline small intestine occurs by an irreversible proteolytic mechanism" and may represent an important step in the genesis of reperfusion injury. ROLE OF XANTHINE OXIDASE IN REPERFUSION INJURY
Inasmuch as oxygen radical scavengers afford protection in models of reperfusion injury and the intestine is a rich source of the oxygen radical-producing enzyme xanthine oxidase, much attention has been devoted to assessing the role of xanthine oxidase in intestinal reperfusion injury (Fig. 1).6,8,37,38 The xanthine oxidase inhibitors allopurinol, oxypurinol, and pterin aldehyde have been widely used to assess the contribution of xanthine oxidase. All of the inhibitors dramatically attenuate both the epithelial cell necrosis and the increased microvascular permeability observed after reperfusion of the ischemic bowel, suggesting that xanthine oxidase is an important source of the oxidants produced after reperfusion.": 37, 38 Additional evidence in support of the theory that xanthine oxidase is an important source of reactive oxygen metabolites in postischemic intestine is provided by studies in which tissue xanthine oxidase was depleted by administration of a tungsten-supplemented, molybdenumdeficient diet. 6, 56 Tungsten replaces molybdenum in the active site of xanthine oxidase, rendering the enzyme inactive. Feeding cats a tungsten-supplemented diet results in a 75% reduction in mucosal xanthine oxidase activity and a corresponding attenuation of the reperfusioninduced increase in intestinal microvascular permeability. 6 Although this approach also demonstrates a role for xanthine oxidase, its interpretation is limited in that other molybdenum-containing enzymes such as aldehyde oxidase are also inactivated by this diet. A role for xanthine oxidase in reperfusion injury is also supported by the observation that local intra-arterial infusion of hypoxanthine and xanthine oxidase results in increases in intestinal vascular permeability comparable to that produced by ischemia and subsequent reperfusion."
71
REPERFUSION INJURY
Control
I/R
*
I/R
+
Allopurinol
I/R
+
pterin aldehyde
I/R
+
tungsten diet
o
0.1
0.2
0.3
0.4
0.5
Microvascular permeability (t-e)
Figure 1. Effect of xanthine oxidase inhibition (allopurinol, pterin aldehyde) or inactivation (tungsten diet) on the ischemiaJreperfusion-induced increase in microvascular permeability. *P
INTESTINAL ISCHEMIA
REPERFUSION INJURY Barbara J. Zimmerman, PhD, and D. Neil Granger, PhD
When a tissue is subjected to ischemia, a sequence of chemical reactions is initiated that may ultimately lead to cellular dysfunction and necrosis. Although no single process can be identified as the critical event in ischemia-induced tissue injury, most studies indicate that depletion of cellular energy stores and accumulation of toxic metabolites contribute to cell death. It is undeniable that re-establishing blood flow is necessary in rescuing ischemic tissues, as this permits both the regeneration of cell charge and the washout of toxic metabolites. However, reperfusion of ischemic tissues also leads to a sequence of events that, paradoxically, injure tissues. Several studies have demonstrated the phenomenon of reperfusion injury. Parks and Granger have shown that relatively little injury to the intestinal mucosa occurs during the ischemic period, the majority occurring upon reperfusion.:" The injury observed after 3 hours of ischemia (blood flow reduced to 20% of normal) and 1 hour of reperfusion is more severe than that observed after 4 hours of ischemia. These results demonstrate that the injury produced by reperfusion can be more severe than the injury induced by ischemia per see This observation implicates some reaction initiated by the return of oxygenated blood to the ischemic tissue as the cause of reperfusion-induced injury. Indeed, several studies have demonstrated that anoxic reperfusion of ischemic tissues results in very little damage.": 44 and it appears that the reactions initiated at reperfusion involve the formation of cytotoxic oxidants derived from molecular oxygen. Microvascular permeability to plasma proteins is a useful and sensitive index for evaluating the influence of ischemia-reperfusion (I/R) on microvascular integrity." If the cat small bowel is subjected to From the Department of Physiology, Louisiana State University Medical Center, Shreveport, Louisiana
SURGICAL CLINICS OF NORTH AMERICA VOLUME 72 • NUMBER 1 • FEBRUARY 1992
65
66
ZIMMERMAN & GRANGER
1 hour of ischemia (blood flow reduced to 20% of control) without reperfusion, a doubling (0.08 ± 0.01 versus 0.15 ± 0.03) of microvascular permeability is observed. However, the same period of ischemia followed by reperfusion results in a fivefold (0.04 ± 0.03) increase in microvascular permeability. The assumption that an oxygen-dependent mechanism accounts for the potentiation in permeability after reperfusion is supported by the observation that antioxidant agents attenuate only the component of the increased permeability that follows reperfusion. The concept that reperfusion per se is largely responsible for the injury observed in intestinal models of ischemia-reperfusion is also supported by the observation that interventions administered at the time of reperfusion are as effective in attenuating mucosal injury as agents administered before ischemia. Although reperfusion of ischemic tissues appears to exacerbate the microvascular and parenchymal injury during an ischemic insult, this phenomenon occurs only after a relatively short period of ischemia. As the ischemic period lengthens, the detrimental effect of oxygen deprivation on the tissues outweighs the effect of reperfusion, resulting in tissue necrosis and eventual cell death. Thus, under conditions of prolonged or severe ischemia, restoration of blood flow will not significantly affect the ultimate viability of the tissue.
ROLE OF REACTIVE OXYGEN METABOLITES IN REPERFUSION INJURY
The observation that reperfusion (reoxygenation) of ischemic tissues produces injury led to the concept that reperfusion injury may be mediated at least in part by the formation of reactive oxygen metabolites. Molecular oxygen can accept a total of four electrons to form water; however, it can be reduced in univalent steps to generate three oxidant species. The univalent reduction of oxygen produces the superoxide anion radical (0 2 -). The cellular toxicity associated with superoxide is usually attributed to its role as a precursor to more reactive oxygen species. Hydrogen peroxide (H 202 ) may be formed as a result of the divalent reduction of O 2 - or the dismutation of O 2 - . The spontaneous dismutation of O 2 - proceeds rapidly in aqueous solution; thus, the production of O 2 - in vivo is always accompanied by the production of H 202 • The third radical species derived from molecular oxygen is the hydroxyl radical (-OH), which is formed by the interaction of O 2 - and H202 (Haber-Weiss reaction) and is a potent oxidizing agent. The result of oxygen radical formation is damage to an entire array of biomolecules found in tissues, including nucleic acids, membrane lipids, enzymes, and receptors." Membrane-associated polyunsaturated fatty acids are readily attacked by -OH in a process that results in the peroxidation of lipids. Peroxidation of membrane lipids can disrupt membrane fluidity and cell compartmentation, which can result in cell lysis. Thus, oxygen radical-initiated lipid peroxidation and protein
REPERFUSION INJURY
67
- damage may contribute to the impaired cellular function and necrosis associated with reperfusion of ischemic tissues. Evidence for the involvement of reactive oxygen metabolites in reperfusion injury is based on the use of agents that either restrict the production of these cytotoxic oxidants or act as scavengers after the oxidants are produced. Oxidant formation has been demonstrated during reperfusion of the ischemic bowel by electron-spin resonance spectrometry" and chemiluminescence. 30, 53 These studies demonstrate that there is a burst of oxidant formation immediately after reperfusion, which lasts for 2 to 5 minutes. The enhanced photoemission observed with chemiluminescence after reperfusion is suppressed by treatment with superoxide dismutase, an enzyme that scavenges O 2 - . The concept of reperfusion-induced oxidant production is also supported by the fact that the consumption of reduced glutathione in intestinal mucosa subjected to ischemia-reperfusion is largely prevented by superoxide dismutase." There is evidence that oxidants mediate both the increased· microvascular permeability produced by 1 hour of ischemia-reperfusion and the mucosal lesions produced by 3 hours of ischemia-reperfusion (Table 1). Both superoxide dismutase and copper diisopropyl salicylate (CuDIPS), a superoxide dismutase mimetic, significantly attenuate reperfusion-induced increases in microvascular permeability. Furthermore, administration of superoxide dismutase prior to reperfusion attenuates the necrosis of mucosal villi and crypt epithelium produced by longer periods of ischemia." 37, 49 Although the aforementioned studies implicate the superoxide anion in mediating reperfusion-induced changes in intestinal integrity, there is evidence that oxidants derived from superoxide play a more important role in the injury process (Table 1). Catalase, an enzyme that catalyzes the disproportionation of H 202 to H 20 and O 2 , has proved to be protective in many models of ischemia-reperfusion." Nonenzymatic scavengers of the hydroxyl radical, including dimethyl sulfoxide (DMSO), dimethylthiourea, and mannitol, also attenuate reperfusionTable 1. MODULATION OF I/R-INDUCED INCREASE IN MICROVASCULAR PERMEABILITY Condition Control Ischemia-reperfusion (I/R) I/R + superoxide dismutase I/R + Cu-DIPS I/R + catalase I/R + dimethyl sulfoxide I/R + deferoxamine IIR + iron-loaded deferoxamine I/R + apotransferrin Values are means ± SE.
Microvascular Permeability (1 - (1)
0.08 0.41 0.14 0.19 0.19 0.19 0.15 0.44 0.17
± 0.005
± 0.02 ± 0.013 ± 0.03 ± 0.01 ± 0.02 ± 0.01 ± 0.03 ± 0.01
68
ZIMMERMAN & GRANGER
induced injury. 38, 41 Inasmuch as ·OH is formed from O 2 - and H 202 and ·OH scavengers provide levels of protection similar to that observed with superoxide dismutase or catalase, many investigators have suggested that the secondarily derived hydroxyl radical may be the primary damaging radical. The protective effects of superoxide dismutase, catalase, and hydroxyl radical scavengers are consistent with the view that the highly cytotoxic hydroxyl radical is formed during reperfusion by the Haber-Weiss reaction. It is generally assumed that the Haber-Weiss reaction occurs at too Iowa rate to be of physiological significance. However, the reaction can be greatly accelerated by the presence of transition metals (e.g., iron), which act as a catalyst. The intestinal mucosa is a rich source of iron. Intestinal iron is stored as ferritin in the ferric (Fe +3) state. However, superoxide reduces the ferritin-stored Fe+ 3 to liberate ferrous (Fe+ 2 ) iron." Thus, superoxide could release ferrous iron for reaction with hydrogen peroxide to form the hydroxyl radical during reperfusion. The role of iron in reperfusion-induced hydroxyl radical production has been assessed by determining whether deferoxamine (an iron chelator) or apotransferrin (an iron-binding protein) provides protection against the increased microvascular permeability produced by ischemiareperfusion." Pretreatment with either deferoxamine or apotransferrin significantly attenuates the increase in microvascular permeability after reperfusion. The observation that iron-loaded deferoxamine or transferrin does not protect the intestine against reperfusion injury argues against a nonspecific protective effect of these iron-binding compounds. The ability of hydroxyl radicals to initiate lipid peroxidation can result in the formation of lipid-derived free radicals such as conjugated dienes, lipid hydroperoxide radicals, and lipid hydroperoxides. Measurements of conjugated dienes and malondialdehyde are frequently used as an index of lipid peroxidation. Schoenberg and coworkers'< 57 measured mucosal concentrations of conjugated dienes in cat intestine subjected to ischemia-reperfusion. These studies indicate that conjugated diene levels in tissue are not affected by ischemia; however, the levels are doubled 10 minutes after reperfusion. The reperfusioninduced increase in conjugated dienes is significantly attenuated in animals treated with superoxide dismutase. Otamiri and Tagesson35,36 measured mucosal and plasma levels of malondialdehyde in rats after reperfusion of ischemic intestine. Malondialdehyde concentrations in both intestinal mucosa and plasma increased threefold to fourfold 5 minutes after reperfusion. These observations, along with those described earlier, suggest that the hydroxyl radical mediates the lipid peroxidation associated with reperfusion of the small intestine. XANTHINE OXIDASE AS A SOURCE OF REACTIVE OXYGEN METABOLITES
Xanthine oxidase is the rate-limiting enzyme in nucleic acid degradation through which all purines are channeled for terminal oxidation.
REPERFUSION INJURY
69
Xanthine oxidase (XO) has the ability to generate H 20 2 and O 2- during the oxidation of hypoxanthine or xanthine: XO hypoxanthine + 202 + H 20 ~ xanthine + O 2- + H 20 2 xanthine + 202 + H 20
~
uric acid + O 2 - + H 202
Xanthine oxidase normally exists in nonischemic, healthy cells predominately as an NAD+-dependent dehydrogenase (XDH).39,42 This form of the enzyme uses NAD+ instead of O 2 as the electron acceptor during oxidation of purines and does not produce O 2- or H 20 2: hypoxanthine + H 20 + NAD+
~
xanthine + NADH + H+
Xanthine dehydrogenase is converted to the oxidant-producing xanthine oxidase form during tissue ischemia. I, 42 Conversion of xanthine dehydrogenase to xanthine oxidase (D-to-O conversion) can occur by two mechanisms: (1) reversible conversion by oxidation or (2) irreversibly conversion by proteolysis. 1, 4, 54 The D-to-O conversion during ischemia appears to occur at different rates in various tissues; however, the amount of D-to-O conversion is proportional to the duration of ischemia. 42 In comparison to other tissues, the intestinal mucosa has a tremendous capacity to oxidize hypoxanthine via xanthine oxidase. Xanthine oxidase (dehydrogenase plus oxidase) activity in the intestinal mucosa is approximately 100 mU/gm wet weight in most species." In humans, liver and intestine have the highest activity, with intestine containing 29 to 56 mU/gm wet weight. Although these levels are relatively low compared with values reported for other mammals, the potential cytotoxicity of this enzyme activity is exemplified by the observation that isolated cells are injured when exposed to xanthine oxidase levels as low as 2 mU/m1.50 Intestinal xanthine oxidase activity is found almost exclusively in the mucosal layer, with an increasing gradient of activity from villus base to tip." A similar gradient of vulnerability to ischemia-reperfusion has been demonstrated. Immunolocalization studies have shown that mucosal xanthine oxidase is found in the endothelial cells lining the microvasculature in the villus core." However, histochemical studies demonstrate that the majority of the enzyme is in the epithelial cells of the mucosal layer." Furthermore, freshly isolated villus epithelium accounts for the majority of mucosal xanthine oxidase in cat ileal mucosa." The significant enzyme activity remaining in the lamina propria may be concentrated in the microvascular endothelium (Table 2). Parks et al42 have shown that xanthine oxidase represents approximately 19% of the total xanthine dehydrogenase plus xanthine oxidase activity in the normally perfused rat small intestine. This increases to 34%, 46%, and 61% after 1, 2, and 3 hours of ischemia, respectively. Roy et al46 have shown that intraperitoneal injection of soybean trypsin
70
ZIMMERMAN & GRANGER
Table 2. DISTRIBUTION OF XANTHINE OXIDASE (mU/gm) IN EPITHELIUM AND LAMINA PROPRIA OF CAT INTESTINAL MUCOSA Total mucosa Epithelium Lamina propria
143 ± 39 202 ± 15
43 ± 40
Values are means ± SE.
inhibitor (a serine protease inhibitor) in rats completely inhibits D-to-O conversion in the small intestine. Utilizing soybean trypsin inhibitor, Parks and coworkers demonstrated that the protease inhibitor attenuated the reperfusion-induced increase in vascular permeability and mucosal lesions. These observations indicate that D-to-O conversion in the ischemic feline small intestine occurs by an irreversible proteolytic mechanism" and may represent an important step in the genesis of reperfusion injury. ROLE OF XANTHINE OXIDASE IN REPERFUSION INJURY
Inasmuch as oxygen radical scavengers afford protection in models of reperfusion injury and the intestine is a rich source of the oxygen radical-producing enzyme xanthine oxidase, much attention has been devoted to assessing the role of xanthine oxidase in intestinal reperfusion injury (Fig. 1).6,8,37,38 The xanthine oxidase inhibitors allopurinol, oxypurinol, and pterin aldehyde have been widely used to assess the contribution of xanthine oxidase. All of the inhibitors dramatically attenuate both the epithelial cell necrosis and the increased microvascular permeability observed after reperfusion of the ischemic bowel, suggesting that xanthine oxidase is an important source of the oxidants produced after reperfusion.": 37, 38 Additional evidence in support of the theory that xanthine oxidase is an important source of reactive oxygen metabolites in postischemic intestine is provided by studies in which tissue xanthine oxidase was depleted by administration of a tungsten-supplemented, molybdenumdeficient diet. 6, 56 Tungsten replaces molybdenum in the active site of xanthine oxidase, rendering the enzyme inactive. Feeding cats a tungsten-supplemented diet results in a 75% reduction in mucosal xanthine oxidase activity and a corresponding attenuation of the reperfusioninduced increase in intestinal microvascular permeability. 6 Although this approach also demonstrates a role for xanthine oxidase, its interpretation is limited in that other molybdenum-containing enzymes such as aldehyde oxidase are also inactivated by this diet. A role for xanthine oxidase in reperfusion injury is also supported by the observation that local intra-arterial infusion of hypoxanthine and xanthine oxidase results in increases in intestinal vascular permeability comparable to that produced by ischemia and subsequent reperfusion."
71
REPERFUSION INJURY
Control
I/R
*
I/R
+
Allopurinol
I/R
+
pterin aldehyde
I/R
+
tungsten diet
o
0.1
0.2
0.3
0.4
0.5
Microvascular permeability (t-e)
Figure 1. Effect of xanthine oxidase inhibition (allopurinol, pterin aldehyde) or inactivation (tungsten diet) on the ischemiaJreperfusion-induced increase in microvascular permeability. *P
