Effects of an inhibitor of adenosine deaminase,deoxycoformycin, and of nucleoside transport,propentofylline, on post-ischemic recovery of adenine nucleotides in rat brain

The effects of an adenosine deaminase inhibitor (deoxycoformycin, 500 μg/kg) and of an inhibitor of nucleoside transport (propentofylline, 10 mg/kg) on adenosine and adenine nucleotide levels in the ischemic rat brain were investigated. The brains of the rats were microwaved before, at the end of a 20 min period of cerebral ischemia (4 vessel occlusion+hypotension), or after 5, 10, 45, and 90 min of reperfusion. Deoxycoformycin increased brain adenosine levels during both ischemia and the initial phases of reperfusion. AMP levels were elevated during ischemia and after 5 min of reperfusion. ATP levels were elevated above those in the non-treated animals after 10 and 45 min of reperfusion. ADP levels were elevated above the non-drug controls at 90 min. These increases in ATP, ADP and AMP resulted in significant increases in total adenylates during ischemia, and after 10 min and 90 min of reperfusion. Propentofylline administration resulted in enhanced AMP levels during ischemia but did not alter adenosine or adenine nucleotide levels during reperfusion in comparison with non-treated controls.     

Oxypurinol administration fails to prevent free radical-mediated lipid peroxidation during loaded breathing.

The purpose of the present study was to determine whether it is possible to alter the development of fatigue and ablate free radical-mediated lipid peroxidation of the diaphragm during loaded breathing by administering oxypurinol, a xanthine oxidase inhibitor. We studied 1) room-air-breathing decerebrate, unanesthetized rats given either saline or oxypurinol (50 mg/kg) and loaded with a large inspiratory resistance until airway pressure had fallen by 50% and 2) unloaded saline- and oxypurinol-treated room-air-breathing control animals. Additional sets of studies were performed with animals breathing 100% oxygen. Animals were killed at the conclusion of loading, and diaphragmatic samples were obtained for determination of thiobarbituric acid-reactive substances and assessment of in vitro force generation. We found that loading of saline-treated animals resulted in significant diaphragmatic fatigue and thiobarbituric acid-reactive substances formation (P < 0.01). Oxypurinol administration, however, failed to increase load trial time, reduce fatigue development, or prevent lipid peroxidation in either room-air-breathing or oxygen-breathing animals. These data suggest that xanthine oxidase-dependent pathways do not generate physiologically significant levels of free radicals during the type of inspiratory resistive loading examined in this study.     

Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion

This study investigates whether ozone could confer protection from hepatic ischemia reperfusion by modifying the accumulation of adenosine and xanthine during ischemia. A significant increase in both adenosine and xanthine accumulation was observed as a consequence of ATP degradation during hepatic ischemia. Adenosine exerts a protective effect on hepatic ischemia reperfusion injury since the elimination of endogenous adenosine accumulation with adenosine deaminase increased the hepatic injury associated with this process. On the other hand, the high xanthine levels observed after ischemia could exert deleterious effects during reperfusion due to reactive oxygen species generation from xanthine oxidase. The administration of allopurinol, an inhibitor of xanthine oxidase, attenuated the increase in reactive oxygen species and transaminase levels observed after hepatic reperfusion. Ozone treatment in liver maintained adenosine levels similar to those found after ischemia but led to a marked reduction in xanthine accumulation. In order to evaluate the role of both adenosine and xanthine, we tried to modify the protection confered by ozone, by modifying the concentrations of adenosine and xanthine. The metabolization of endogenous adenosine after ischemia abolished the protective effect conferred by ozone. When xanthine was administered previous to ozone treatment, the protection conferred by adenosine disappeared, showing both postischemic reactive oxygen species and transaminase levels similar to those found after hepatic ischemia reperfusion. Ozone would confer protection against the hepatic ischemia reperfusion injury by the accumulation of adenosine that in turns benefits the liver and by blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation.     

Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion

This study investigates whether ozone could confer protection from hepatic ischemia reperfusion by modifying the accumulation of adenosine and xanthine during ischemia. A significant increase in both adenosine and xanthine accumulation was observed as a consequence of ATP degradation during hepatic ischemia. Adenosine exerts a protective effect on hepatic ischemia reperfusion injury since the elimination of endogenous adenosine accumulation with adenosine deaminase increased the hepatic injury associated with this process. On the other hand, the high xanthine levels observed after ischemia could exert deleterious effects during reperfusion due to reactive oxygen species generation from xanthine oxidase. The administration of allopurinol, an inhibitor of xanthine oxidase, attenuated the increase in reactive oxygen species and transaminase levels observed after hepatic reperfusion. Ozone treatment in liver maintained adenosine levels similar to those found after ischemia but led to a marked reduction in xanthine accumulation. In order to evaluate the role of both adenosine and xanthine, we tried to modify the protection confered by ozone, by modifying the concentrations of adenosine and xanthine. The metabolization of endogenous adenosine after ischemia abolished the protective effect conferred by ozone. When xanthine was administered previous to ozone treatment, the protection conferred by adenosine disappeared, showing both postischemic reactive oxygen species and transaminase levels similar to those found after hepatic ischemia reperfusion. Ozone would confer protection against the hepatic ischemia reperfusion injury by the accumulation of adenosine that in turns benefits the liver and by blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation.     

Allopurinol-Enhanced Postischemic Recovery in the Isolated Pat Heart Involves Repletion of High-Energy Phosphates

The effects of allopurinol (AP) on functional and metabolic recovery of the isolated rat heart after global ischemia were studied. Hearts were subjected to aerobic perfusion (30 min), cardioplegic infusion (5 min), normothermic ischemia (37 min), and reperfusion (50 min) which was started with secondary cardioplegic infusion (10 min). AP was injected into rats (44 mg/kg body wt ip 2 h before heart excision) and added to cardioplegic solution (2 mM) prior and after ischemia. AP treatment significantly improved postischemic recovery of the function and reduced the leakage of lactate dehydrogenase from reperfused hearts. These beneficial effects were accompanied by a better preservation of tissue content of ATP, the total adenine nucleotides, phosphocreatine, and the total creatine at the end of reperfusion. Inhibition of xanthine oxidase by AP substantially decreased pre- and postischemic release of xanthine and uric acid and increased postischemic release of hypoxanthine into the coronary effluent. Despite this, AP treated hearts did not exhibit a reduction in hydroxyl radical adduct formation in the effluents at reperfusion assessed by the spin-trap measurements. The results suggest that AP may protect the heart from ischemia/reperfusion injury due to enhanced energy provision rather than by prevention of oxygen-derived free radical formation.     

Pancreatitis induces HSP72 in the lung: role of neutrophils and xanthine oxidase

The aim of this work was to evaluate the systemic Hsp72 expression in rat lung and liver in vivo in a model of acute pancreatitis and investigate the possible involvement of xanthine oxidase and neutrophils in this process. Pancreatitis was induced by intraductal administration of 5% sodium taurocholate and samples of lung and liver were obtained 1 and 3 h later. In some groups of rats circulating xanthine oxidase was inhibited with oxypurinol, and neutrophil recruitment was blocked with a monoclonal antibody against P-selectin. Hsp72 expression was assessed by means of Western blot and immunohistochemistry. Results showed Hsp72 induction in lung, but not in liver, shortly after pancreatitis. Hsp72-induced expression was located in bronchial epithelium, alveolar macrophages, infiltrating neutrophils, and blood vessels. Oxypurinol and the antibody against P-selectin prevented pancreatitis-induced lung Hsp72 overexpression suggesting that Hsp72 induction is mediated by neutrophil infiltration into the lungs.     

Late preconditioning by ethanol is initiated via an oxidant-dependent signaling pathway

Ingestion of alcoholic beverages at low to moderate levels 24 h prior to ischemia and reperfusion (I/R) prevents postischemic leukocyte/endothelial cell adhesive interactions, a phenomenon referred to as late ethanol preconditioning (EtOH-PC). The aim of this study was to determine whether oxidants act as initiators of late EtOH-PC. Ethanol was instilled into the stomachs of C57BL/6 mice as a bolus by gavage at a dose that produced a peak plasma concentration of 45 mg/dl 30 min after administration and returned to control levels 60 min after ingestion. Twenty four hours later, the superior mesenteric artery was occluded for 45 min followed by 70 min of reperfusion. The numbers of rolling and firmly adherent leukocytes were quantified in postcapillary venules of the small intestine in sham animals (no EtOH-PC, no I/R), in mice subjected to I/R alone or EtOH-PC + I/R, and in animals treated with Mn-TBAP (a cell-permeant superoxide dismutase mimetic), oxypurinol (a XO inhibitor), the NAD(P)H oxidase inhibitors PR-39 or apocynin, or oxypurinol plus PR39 during the period of EtOH-PC on Day 1 followed by I/R on Day 2. In separate groups of mice, oxypurinol or apocynin were also administered 1 h after ethanol ingestion on Day 1, with induction of I/R 24 h later. I/R induced marked increases in leukocyte rolling and adherence, effects that were completely prevented by EtOH-PC. Coincident treatment with Mn-TBAP, oxypurinol, PR-39, apocynin, or oxypurinol plus PR-39 with ethanol attenuated these anti-inflammatory actions of EtOH-PC. However, administration of oxypurinol or apocynin 1 h after ethanol ingestion failed to prevent these protective effects of EtOH-PC. Our results indicate that reactive oxygen species formed during the period of ethanol exposure on Day 1 trigger the development of an anti-inflammatory phenotype that renders the small bowel resistant to the proadhesive effects of I/R 24 h later.     

Evaluation of the role of xanthine oxidase in myocardial reperfusion injury

The free radical-generating enzyme xanthine oxidase has been hypothesized to be a central mechanism of the injury which occurs in postischemic tissues; however, its importance remains controversial. Much attention has focused on the role of this enzyme in myocardial reperfusion injury. While xanthine oxidase has been observed in ischemic tissue homogenates, the presence and importance of radical generation by the enzyme in intact tissues are unknown. Therefore, we performed electron paramagnetic resonance, nuclear magnetic resonance and hemodynamic studies to measure the presence and significance of xanthine oxidase-mediated free radical generation in the isolated rat heart. When isolated perfused rat hearts were reperfused after 30 min of global ischemia, myocardial function and coronary flow were significantly improved in the presence of the definitive xanthine oxidase blocker oxypurinol. Free radical concentrations measured by spin-trapping with 5,5'-dimethyl-1-pyrroline-N-oxide were significantly decreased by oxypurinol and the energetic state of the heart was improved as reflected by an increased recovery of phosphocreatine and a higher phosphocreatine/Pi ratio. ATP recovery, however, was not altered, indicating that the improved functional and metabolic state of the heart was not due to ATP salvage. Spectrophotometric assays for the enzyme showed an increase in the amount of xanthine oxidase relative to dehydrogenase following ischemia, and a total available xanthine oxidase pool in the rat heart of approximately 150 milliunits/g of protein. Thus, xanthine oxidase is a significant source of the oxidative injury which occurs upon reperfusion of the ischemic rat heart.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia

Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three-vessel occlusion combined with hypotension (50 mm Hg) in male Sprague-Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nucleotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 microM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.     

Purine and oxypurine production in mitochondria of ischemic and reperfused myocardium

The present study was undertaken to determine whether significant breakdown of adenine nucleotides to purine bases and oxypurines occurred in mitochondria following myocardial ischemia and ischemia followed by reperfusion, and whether allopurinol prevented this effect. The adenine nucleotides adenosine, hypoxanthine, xanthine and uric acid were measured in the mitochondria and the results suggest that breakdown did occur. Malondialdehyde concentration was determined to gauge lipid peroxidation. This substance did not increase during ischemia or reperfusion, but did so in the presence of allopurinol. Xanthine dehydrogenase was converted to xanthine oxidase during reperfusion and the activity of both enzymes were inhibited by allopurinol. The results also suggested the presence of a mitochondrial 5'-nucleotidase. We conclude that significant breakdown of adenine nucleotide took place in myocardial mitochondria during ischemia and ischemia followed by reperfusion and that allopurinol may have a protective effect.     

MDA, oxypurines, and nucleosides relate to reperfusion in short-term incomplete cerebral ischemia in the rat.

Short-term incomplete cerebral ischemia (5 min) was induced in the rat by the bilateral clamping of the common carotid arteries. Reperfusion was obtained by removing carotid clamping and was carried out for the following 10 min. Animals were sacrificed either at the end of ischemia or reperfusion. Controls were represented by a group of sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. Neutralized perchloric acid extracts of brain tissue were analyzed by a highly sensitive high-performance liquid chromatography (HPLC) method for the direct determination of malondialdehyde, oxypurines, nucleosides, nicotinic coenzymes, and high-energy phosphates. In addition, plasma concentrations of malondialdehyde, hypoxanthine, xanthine, inosine, uric acid, and adenosine were determined by the same HPLC technique. Incomplete cerebral ischemia induced the appearance of a significant amount (8.05 nmol/g w.w.; SD = 2.82) of cerebral malondialdehyde (which was undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde (53.30 nmol/g w.w.; SD = 17.77) and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia (0.050 mumol/L; SD = 0.015), significantly increased after 5 min of ischemia (0.277 mumol/L; SD = 0.056) and was strikingly augmented after 10 min of reperfusion (0.682 mumol/L; SD = 0.094). A similar trend was observed for xanthine, uric acid, inosine, and adenosine, while hypoxanthine reached its maximal concentration after 5 min of incomplete ischemia, being significantly decreased after reperfusion. From the data obtained, it can be concluded that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism, thus representing early biochemical markers of short-term incomplete brain ischemia and reperfusion in the rat. In particular, these results suggest the possibility of using the variation of malondialdehyde, oxypurines, and nucleosides in peripheral blood as a potential biochemical indicator of reperfusion damage occurring to postischemic tissues.     

Inhibition of xanthine oxidase improves myocardial contractility in patients with ischemic cardiomyopathy

Reactive oxygen species, in particular superoxide, have been closely linked to the underlying pathophysiology of ischemic cardiomyopathy: superoxide not only mediates mechanoenergetic uncoupling of the myocyte but also adversely impacts on myocardial perfusion by depleting endothelial-derived nitric oxide bioavailability. Xanthine oxidase generates superoxide upon oxidation of hypoxanthine and xanthine and has been detected in cardiac myocytes and coronary endothelial cells of patients with ischemic heart disease. Here we investigated the effects of oxypurinol, a xanthine oxidase inhibitor, on myocardial contractility in patients with ischemic cardiomyopathy. Twenty patients (19 males, 66+/-8 years) with stable coronary disease, severely suppressed systolic function (left ventricular ejection fraction 22+/-2%), and nonelevated uric acid plasma levels received a single intravenous dose of oxypurinol (400 mg). Cardiac MRI studies, performed before and 5.2+/-0.9 h after oxypurinol administration, revealed a reduction in end-systolic volumes (-9.7+/-4.2%; p=0.03) and an increase in left ventricular ejection fraction (+17.5+/-5.2%; p=0.003), whereas 6 patients (6 males, 63+/-3.8 years, ejection fraction 26+/-5%) who received vehicle only did not show significant changes in any of the parameters studied. Oxypurinol improves left ventricular function in patients with ischemic cardiomyopathy. These results underscore the significance of reactive oxygen species as important pathophysiological mediators in ischemic heart failure and point toward xanthine oxidase as an important source of reactive species that serve to modulate the myocardial redox state in this disease.     

Tourniquet shock in rats: effects of allopurinol on biochemical changes of the gastrocnemius muscle subjected to ischemia followed by reperfusion

Recent data suggest that oxygen free radicals are implicated in the pathogenesis of ischemic injury. This study evaluates the effects of allopurinol, a xanthine oxidase (XO) inhibitor, on malonaldehyde generation, free sulfhydryl levels, oxygen consumption, and water contents of rat gastrocnemius muscles of female Sprague-Dawley rats subjected to tourniquet shock and after hind-limb reperfusion. Serum lactic dehydrogenase isozyme patterns after ligature release were also examined. Our results show that the four muscle parameters were not altered during 5 hr of ischemia, but that on hind-limb reperfusion, malonaldehyde production, SH levels, O2 consumption, and water contents were significantly altered in the control animals, but not in those pretreated with allopurinol. LDH serum patterns of the untreated animals showed the presence of all five isoforms; these were much less evident in the drug-protected rats. Our data suggest that following ischemia, the affected muscles are unable to recover their normal function when reperfusion is resumed. The subsequent damage is probably due to the generation of cytotoxic superoxide radicals formed during the XO-catalyzed transformation of hypoxanthine to uric acid on tissue reoxygenation. The severity of tissue damage is related to the duration of the ischemic episode possibly due to hypoxanthine accumulation during ischemia.     

Altered Ca2+ homeostasis and impaired mitochondrial function in cardiomyopathy

This work investigates whether purine metabolism and release is related to cardioprotection with hyperkalemia and hypothermia. Langendorff guinea-pig hearts were used to either monitor metabolism during ischemia or to measure functional recovery, myocardial injury and release of purine during reperfusion. Hearts underwent 30 min ischemia using one of the following protocols: control (normothermic buffer), hyperkalaemia (high-potassium buffer), hypothermia (20°C) and hyperkalemia + hypothermia. At the end of 30 min ischemia, hyperkalemia was associated with similar metabolic changes (rise in purine and lactate and fall in adenine nucleotides) to control group. Accumulation of purine was due to a rise in inosine, xanthine and hypoxanthine and was largely prevented by hypothermia and hyperkalemia + hypothermia. Upon reperfusion, there was a time-dependent release of all purine, lactate and AMP. A fast (peak in less than 20 sec) release of inosine, xanthine, hypoxanthine and lactate was highest in control followed by hyperkalemia then hypothermia and little release in hyperkalemia + hypothermia. Adenosine and AMP release was slow (peak at 3 min), only significant in control and was likely to be due to sarcolemmal disruption as the profile followed lactate dehydrogenase release. Recovery (left ventricular developed pressure) was 63% control, 82% hyperkalemia, 77% hypothermia and 98% for hyperkalemia + hypothermia. The loss of purine during reperfusion but not their production during ischemia is related to cardioprotection with hyperkalemia. The possibility that the consequences of hyperkalemia modulate a sodium-dependent purine efflux, is discussed. The reduced loss of purine in hypothermia or in hyperkalemia + hypothermia is likely to be due to a lower metabolic activity during ischemia.     

Oxypurinol improves coronary and peripheral endothelial function in patients with coronary artery disease

Coronary endothelial dysfunction is a powerful prognostic marker in patients with coronary artery disease (CAD) that is centrally related to oxidative inhibition of nitric oxide (NO)-dependent vascular cell signaling. Xanthine oxidase (XO), which both binds to and is expressed by endothelial cells, generates superoxide and hydrogen peroxide upon oxidation of purines. Whether inhibition of xanthine oxidase activity results in improved coronary vasomotor function in patients with CAD, however, remains unknown. We assessed coronary and peripheral (brachial artery) endothelial function in 18 patients (pts; 65+/-8 years, 86% male) with angiographically documented CAD, preserved left ventricular function, and non-elevated uric acid levels (233+/-10 microM). Patients received incremental doses of intracoronary acetylcholine (ACh; 10(-7) to 10(-5) microM), and minimal lumen diameter (MLD) and coronary blood flow (CBF) were assessed before and after intravenous administration of oxypurinol (200 mg). Oxypurinol inhibited plasma XO activity 63% (0.051+/- 0.001 vs 0.019+/- 0.005 microU/mg protein; p<0.01). In pts who displayed endothelial dysfunction as evidenced by coronary vasoconstriction in response to ACh (n=13), oxypurinol markedly attenuated ACh-induced vasoconstriction (-23+/- 4 vs -15+/- 4% at ACh 10(-5) microM, p<0.05) and significantly increased CBF (16+/-17 vs 62+/-18% at ACh 10(-5) microM, p<0.05), whereas in patients with preserved coronary endothelial function, oxypurinol had no effect on ACh-dependent changes in MLD (+2.8+/- 4.2 vs 5.2+/- 0.7%, p>0.05) or CBF (135+/-75 vs 154+/-61%, p>0.05). Flow-mediated dilation of the brachial artery, assessed in eight consecutive patients, increased from 5.1+/-1.5 before to 7.6+/-1.5% after oxypurinol administration (p < 0.05). Oxypurinol inhibition of XO improves coronary vascular endothelial dysfunction, a hallmark of patients with CAD. These observations reveal that XO-derived reactive oxygen species significantly contribute to impaired coronary NO bioavailability in CAD and that XO inhibition represents an additional treatment concept for inflammatory vascular diseases that deserves further investigation.     

Incomplete cerebral ischemia in the rat provokes increase of tissue and plasma malondialdehyde

Short-term incomplete cerebral ischemia was induced in the rat by bilaterally clamping for 5 min the common carotid arteries; subsequent reperfusion of 10 min was obtained by removing carotid occlusion. At the end of ischemia or reperfusion, animals were sacrificed by decapitation. A control group was represented by sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. A highly sensitive HPLC method for the direct determination of malondialdehyde, oxypurines, and nucleosides was used on 200 μL of brain tissue and plasma extracts. Incomplete cerebral ischemia induced the, appearance of a significant amout of tissue malondialdehyde (undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia, significantly increased after 5 min of ischemia, being strikingly augmented after 10 min of reperfusion. A similar trend was observed for oxypurines and nucleosides. From these data, it can be affirmed that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism thus representing early biochemical markers of short-term incomplete brain ischemia, and reperfusion in the rat.     

Oxygen free radical induced damage during intestinal ischemia/reperfusion in normal and xanthine oxidase deficient rats

This study looks at the role of xanthine oxidase (XO) in ischemia/reperfusion (I/R) induced intestinal mucosal damage using normal and xanthine oxidase deficient rats. Tungstate feeding for 3 days depleted the intestinal mucosal XO by 80%. A ligated loop of the rat small intestine (both normal and XO-deficient) was subjected to 1 h of total ischemia followed by 5 min revascularisation. The ensuing mucosal damage was assessed by biochemical and histological studies. Ischemia or I/R increased the XO levels in normal rats without any change in XO-deficient rats. Myeloperoxidase (a neutrophil marker) level was increased in both group of rats but it was comparatively higher in the XO-deficient rats. Accumulation of peroxidation products such as malondialdehyde, conjugated diene and increased production of hydroxyl radicals by microsomes were seen after ischemia and I/R and were similar in normal and XO-deficient rats. Studies on other parameters of peroxidation showed a decrease in polyunsaturated fatty acids and alpha-tocopherol, an increase in cysteine and cystine levels after I/R and were similar in both normal and XO-deficient rats. Histological results indicated gross morphological changes in the intestinal mucosa due to ischemia and I/R, and the damage was more severe in XO-deficient rats. These observations suggest that oxygen-derived free radicals are involved in the intestinal mucosal damage during I/R and infiltrated neutrophils rather than XO may be the primary source of free radicals under these conditions.     

Oxygen radical mechanisms of brain injury following ischemia and reperfusion.

This review addresses current understanding of oxygen radical mechanisms as they relate to the brain during ischemia and reperfusion. The mechanism for radical production remains speculative in large part because of the difficulty of measuring radical species in vivo. Breakdown of lipid membranes during ischemia leads to accumulation of free fatty acids. Decreased energy stores during ischemia result in the accumulation of adenine nucleotides. During reperfusion, metabolism of free fatty acids via the cyclooxygenase pathway and metabolism of adenine nucleotides via the xanthine oxidase pathway are the most likely sources of oxygen radicals. Although leukocytes have been found to accumulate in some models of ischemia and reperfusion, their mechanistic role remains in question. Therapeutic strategies aimed at decreasing brain injury have included administration of radical scavengers at the time of reperfusion. Efficacy of traditional oxygen radical scavengers such as superoxide dismutase and catalase may be limited by their inability to cross the blood-brain barrier. Lipid-soluble antioxidants appear more efficacious because of their ability to cross the blood-brain barrier and because of their presence in membrane structures where peroxidative reactions can be halted.     

Lack of conversion of xanthine dehydrogenase to xanthine oxidase during warm renal ischemia

Irreversible transformation of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) during ischemia was determined measuring XDH and total enzyme activity in kidneys before and after 60 min of clamp of the renal pedicle. Tissue levels of adenine nucleotides, xanthine and hypoxanthine were used as indicators of ischemia. After 60 min of clamping, ATP levels decreased by 72% with respect to controls whereas xanthine and hypoxanthine progressively reached tissue concentrations of 732 +/- 49 and 979 +/- 15 nmol.g tissue-1, respectively. Both total and XDH activities in ischemic kidneys (30 +/- 15 and 19 +/- 1 nmol.min-1.g tissue-1) were significantly lower than in controls when expressed on a tissue weight basis. The fraction of enzyme in the XDH form was however unchanged indicating that the reduction of the nucleotide pool is not accompanied by induction of the type-O activity of xanthine oxidase.