1 Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain
2 Department of Endocrinology, University Clinic, Valencia, Spain
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ABSTRACT |
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INTRODUCTION |
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In this article, we propose an enzymatic mechanism to explain the increased generation of free radicals in diabetes. We confirm that there is oxidative stress (oxidation of glutathione and an increase in lipoperoxides) in human type 1 diabetes and experimental diabetes. We show that xanthine oxidase (a superoxide-generating enzyme) is increased in plasma and liver of diabetic rats. Xanthine oxidase is shed by the liver into the plasma and is bound to vascular endothelial cells. Arterial rings from diabetic rabbits (but not from control rabbits) produce superoxide in the presence of xanthine. This process is inhibited by treatment with heparin (which releases xanthine oxidase from the endothelial surface).
The determination of an enzymatic mechanism of free radical formation in diabetes suggests a mechanism (inhibition of xanthine oxidase) to prevent oxidative stress in this disease. Indeed, we have found that allopurinol, an inhibitor of xanthine oxidase widely used in clinical practice, is effective in preventing oxidation of glutathione and lipoperoxidation in experimental and human type 1 diabetes. Given the fact that oxidative stress plays a role in the development of complications in diabetes (1), the prevention of free radical formation by allopurinol may have clinical significance.
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RESEARCH DESIGN AND METHODS |
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Diabetic patients were assigned to the experimental or placebo group using a random number automatically generated by a computer program. Physicians, nurses, and laboratory technicians were unaware of the type of treatment received by each individual patient.
Patients received a capsule containing 300 mg of allopurinol (Zyloric) (experimental group) or 1 g of cellulose (placebo group) to be taken by mouth daily for 14 consecutive days.
Blood samples were obtained by venipuncture of a peripheral vein at 8:00 A.M. on day 0 and day 15 after an overnight fast of 810 h. Blood samples were processed immediately.
Study protocols were approved by the hospital ethics committee. Investigation was conducted according to the principles expressed in the Declaration of Helsinki. All patients were informed of the proceedings. Patients had a glycemia level of 183 ± 67 (n = 12) mg/dl and a glycosylated hemoglobin of 6.83 ± 0.88% (n = 12). Control subjects were age-matched patients with normal glucose metabolism (glycemia, <100 mg/dl; glycosylated hemoglobin, <6%).
Animals.
We used 6-month-old male Wistar rats made diabetic by streptozotocin administration. The protocols were approved by the animal ethics committee. A dose of 55 mg/kg body wt dissolved in 0.8 ml of 0.1 mol/l citrate buffer, pH 4.5, was administered. After streptozotocin administration, animals were placed in metabolic cages, and their food intake and diuresis were controlled. Ten days after administration of streptozotocin, rats had polyuria, polydipsia, and weight loss. All animals with a glucosuria of >20 g/l were considered diabetic; in all cases, they had a glycemia of 200 mg/dl. Control animals were injected with the same volume of citrate buffer as diabetic animals but without streptozotocin. Rabbits were used to measure superoxide formation by aortic rings, because the size of the aorta in rats makes their use impractical. New Zealand rabbits weighing 2.0 to 3.2 kg were sedated with 40 mg ketamine (Ketolar) intramuscularly. Diabetes was induced by injecting alloxan (100 mg/kg body wt; Sigma, St. Louis, MO) into the lateral ear vein. To prevent hypoglycemia, 10 ml of 5% glucose was injected intravenously and drinking water was supplemented with 10% glucose for the first 24 h after the alloxan injection. Thereafter, the animals were maintained on tap water and regular food ad libitum for 6 weeks. A second group of rabbits (2.13.0 kg) was maintained in the same conditions for the same period to serve as age-matched controls. Diabetic rabbits showed a marked increase in serum glucose and a failure to gain weight compared with controls. Glycemia of controls did not change significantly during the study and was maintained at 100 ± 3 mg/dl (n = 16). The glycemia of diabetic rabbits was 360 ± 23 mg/dl (n = 14).
Allopurinol administration.
Allopurinol was administered intraperitoneally to a group of diabetic rats 5 days after they had shown signs of diabetes. Allopurinol (8 mg) was dissolved in 350 µl dimethylsulfoxide and up to 1 ml water was added. Doses were given 30 and 6 h before decapitation.
Rat liver perfusion to measure xanthine oxidase release.
Livers were washed via the portal vein with a Krebs-Henseleit saline solution, pH 7.4, for 68 min. Subsequently, livers were quickly removed and constantly perfused for 60 min as described in Viña et al. (9). Perfusion fluid was collected after 5, 10, 15, 30, 45, and 60 min. Enzyme activities (xanthine oxidase, lactate dehydrogenase [LDH], and alanine amino transferase [AAT]) were determined in the perfusate. No changes in LDH or in AAT were found, showing that livers are not damaged in the isolation procedure.
Preparation and analysis of samples.
Rat blood was obtained by decapitation. Human blood was obtained by venous puncture and was immediately put into heparinized tubes. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined as previously described (10, 11). Briefly, blood samples were treated with 6% (vol/vol) perchloric acid containing 1 mmol/l EDTA (1:1) to determine GSH or with 6% perchloric acid containing 50 mmol/l N-ethyl-maleimide and 1 mmol/l EDTA to determine GSSG by high-performance liquid chromatography (HPLC). Samples were centrifuged for 10 min at 15000g, and the acidic supernatants were neutralized and used for determination of metabolites. Blood plasma was used to measure glucose, acetoacetate, hydroxybutyrate, and enzymatic activities (12).
Xanthine oxidase was measured as previously described (13). Briefly, isoxantopterine formation from pterine was followed by fluorimetry (excitation at 345 nm and emission at 390 nm). Malondialdehyde (MDA) levels were determined by HPLC as previously described (14).
Determination of superoxide formation by rabbit aortic rings.
Superoxide-dependent lucigenin chemiluminescence of aortic rings was measured as described by White et al. (15). Chemiluminescence was determined using a scintillation counter set to operate in single-photon count mode. The rat thoracic aorta was excised, cleansed of fat and adhering tissue, and divided in three rings of 34 mm each. In one of those rings, chemiluminescence was measured in a vial containing 3 ml PBS with 0.25 mmol/l lucigenin and 50 µmol/l xanthine. In a second ring, chemiluminescence was determined in the same conditions plus 100 mmol/l allopurinol. A third ring was preincubated with heparin (1,000 units/ ml) for 10 min, and, after washing, chemiluminescence was determined in the presence of lucigenin and xanthine.
Isolation of mitochondria.
Isolation was performed following a standard differential centrifugation protocol described by Rickwood et al. (16). After isolation, heart or liver mitochondria were suspended in ice-cold respiratory buffer (with 0.3 mol/l sucrose, 1 mmol/l EGTA, 5 mmol/l MOPS, 0.1% BSA, and 5 mmol/l KH2PO4 adjusted to pH 7.4 with KOH).
Flow cytometric analysis.
Flow cytometry was performed using an EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL). Fluorochromes were excited with an argon laser tuned at 488 nm. Forward-angle light scatter and side-angle light scatter were measured, and fluorescence was detected through a 488-nm blocking filter, a 550-nm long-pass dichroic, and a 525-nm band-pass or a 575-nm long-pass. For each sample, 10,000 mitochondria were counted.
To measure the production of peroxides, dihydrorhodamine 123 was used as fluorochrome during incubation at 37°C for 30 min and was excited with an argon laser tuned at 488 nm. Oxidation of dihydrorhodamine 123 is caused by H2O2-dependent reactions involving reactive oxygen species. Dihydrorhodamine 123 is uncharged but is oxidized to positively charged rhodamine 123 in the extramitochondrial space. The latter is taken up by mitochondria, where it accumulates.
Mitochondrial membrane potential from isolated mitochondria was measured using rhodamine 123 as described above. The gate was set on logarithm of forward and side scatter to always include mitochondria with the same morphological properties. Thus, changes in fluorescence were independent of particle size.
Statistics.
Results are expressed as means ± SD. The number of experiments is included in each figure and table. Statistical analyses were performed by the least-significant difference test, which consists of two steps. First, an ANOVA was performed. The null hypothesis was accepted for all numbers of those sets in which F was nonsignificant at the level of P 0.05. Second, the sets of data in which F was significant were examined by the modified t test using P
0.05 as the critical limit.
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RESULTS |
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Oxidative stress in experimental diabetes: oxidation of glutathione and increased lipid hydroperoxide levels in blood, liver, and heart of rats.
Experimental diabetes resulted in increased GSSG levels both in blood and in organs such as heart and liver of the rat. Figure 1 shows the GSSG-to-GSH ratio in control and diabetic animals. In all cases studied, the GSSG-to-GSH ratio in diabetic animals was 200% of the values in control animals.
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Mitochondria from liver, heart, and kidney do not directly increase levels of H2O2 in type 1 diabetes.
Mitochondria are involved in aging (1820) and in many age-associated (21) and toxicologic (22) diseases. Thus, we studied the effect of diabetes on peroxide generation by mitochondria in liver, heart, and kidney from control and diabetic rats.
Table 2 shows that mitochondria from diabetic animals have peroxide production and mitochondrial potential similar to those of control animals. Moreover, liver mitochondria from diabetic rats have an even higher mitochondrial membrane potential than their control counterparts. Figure 2 shows a representative experiment in which experimental diabetes did not cause any detectable change in peroxide production by mitochondria.
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In this study, we found that experimental diabetes caused an increase in hepatic xanthine oxidase. Table 3 shows that diabetes causes a significant increase (P < 0.01) in both hepatic xanthine oxidase and xanthine dehydrogenase activities. Xanthine oxidase activity in liver from diabetic rats is > 200% of the control value.
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Origin of plasma xanthine oxidase in diabetes: increased hepatic release.
Figure 3 shows that xanthine oxidase is released from the liver of diabetic rats, but not from control rats. This hepatic release of xanthine oxidase is not the result of a nonspecific protein leakage. In fact, other hepatic enzymes, such as AAT, are not released by the liver of either control or diabetic rats (Fig. 3).
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Allopurinol decreases oxidative stress in experimental diabetes.
An important consequence of the role of xanthine oxidase in the generation of oxidative stress in diabetes is that allopurinol should be able to prevent it. Table 4 shows that allopurinol is indeed able to prevent glutathione oxidation in blood, liver, and heart of diabetic rats. In all cases studied, allopurinol afforded complete prevention against the oxidation of the glutathione pool caused by diabetes.
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DISCUSSION |
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Early observations showed a decrease in GSH content in erythrocytes from diabetic animals and an increase in plasma lipid peroxidation. The reported effects include changes in the activity of antioxidant enzymes (3), changes in hepatic glutathione metabolism (25), and changes in free radical formation due to reactions associated with heavy metals (4). It is important, however, to determine which changes are due to diabetes itself and which are a consequence of the dietary alterations associated with diabetes (25). In our study, the patients did not have any clinical sign of dietary alteration, such as polyuria, polydipsia, or polyphagia.
Using a specific method to determine the glutathione redox ratio (10,11), we have found that there is indeed glutathione oxidation in diabetes (both type 1 human diabetes and experimental diabetes). We have also found that lipoperoxide levels increase in diabetes.
The hypothesis that there is oxidative stress in diabetes has been challenged recently (26). Those authors suggested that streptozotocin causes free radical damage per se and that diabetes itself may not cause oxidative stress. The following facts make this unlikely: 1) we and other researchers (6) find signs of oxidative stress in human diabetes (Table 1); 2) there are signs of oxidative stress in models of experimental diabetes in which streptozotocin is not used (27); and 3) we have found that there is a period of time of about a week after streptozotocin administration before diabetes is established. In this period of time, we find no signs of oxidative stress, and only when hyperglycemia and hyperketonemia occur do we find oxidation of glutathione and an increase in plasma lipoperoxide levels.
Mechanism of the generation of free radicals in diabetes: role of xanthine oxidase.
Different sources of free radicals in diabetes have been proposed, including the sorbitol pathway, the induction of NAD(P)H oxidases, and nitric oxide synthase. Cosentino et al. (28) reported that high glucose increases nitric oxide synthase expression in aortic endothelial cells. In a very interesting series of articles (6,7, 8), Jain and colleagues found that hyperketonemia is associated with free radical formation in diabetes and that acetoacetate in the presence of Fe2+ can generate superoxide in vitro.
In this article, however, we report the activation of a specific enzyme activity, xanthine oxidase, which produces oxidant species and subsequently oxidative stress in diabetes.
The role of xanthine oxidase in the vascular dysfunction that occurs in atherosclerosis was studied by White et al. (15). Using aortic rings from diabetic rabbits, we have found that superoxide formation also increases in arteries from diabetic animals. This process is inhibited by allopurinol, a xanthine oxidase inhibitor widely used in clinical practice.
Xanthine oxidase is bound to endothelial cells by sulfated glycosaminoglycans (24). Treatment with heparin releases xanthine oxidase from the vessel wall. In Fig. 4, we show that treatment with heparin decreases superoxide production by aortic rings from diabetic rabbits.
The fact that the production of such a reactive molecule as superoxide is increased in the vessel wall of diabetic animals may be relevant in explaining some of the arterial complications of diabetes and underscores the importance of xanthine oxidase in this process.
Origin of xanthine oxidase in diabetes.
The increase in xanthine oxidase activity in diabetes prompts the question of the origin of this increased activity in the diabetic animal. The tissues that express the highest activity of this enzyme are liver and intestine (29). We found that diabetes causes an increase of xanthine oxidase activity in liver. Moreover, we found that xanthine oxidase is released by the liver of diabetic animals, but not by that of controls (Fig. 3). The release of this enzyme is not the result of a nonspecific leakage from the hepatocyte. We measured the release of other enzymes that are considered markers of hepatic damage, such as aspartate amino transferase (ALAT), and found that it is not significantly higher in diabetic versus control hepatocytes. These experiments lead us to conclude that xanthine oxidase is released from the liver of diabetic animals. This is not specific to diabetes. The release of xanthine oxidase from liver has been observed in other pathological states, such as hemorrhagic shock (30).
Prevention of oxidative stress in diabetes by allopurinol: possible clinical implications.
Oxidative stress has been considered an important factor in the development of complications in diabetes (1). Our studies using aortic rings indicate that superoxide is formed in the vessel wall of diabetic animals (Fig. 4).
This article shows that xanthine oxidase plays an important role in the generation of free radicals in diabetes. An obvious conclusion is that inhibition of this enzyme should prevent oxidative stress in diabetes. Allopurinol inhibits xanthine oxidase in vivo, and it is used in clinical practice. Treatment of patients with allopurinol prevented glutathione oxidation and lipoperoxidation (Table 6) in human type 1 diabetes. Inhibition of xanthine oxidase has proved effective in improving endothelial vasodilator function in hypercholesterolemic, but not hypertensive, patients (31). Very recently, Butler et al. (32) reported that allopurinol protects against endothelial dysfunction in diabetic patients with mild hypertension. Here, we suggest that the liver in type 1 diabetes releases xanthine oxidase to the plasma. The enzyme binds with glucosaminoglycans to the blood vessel, inducing local oxidative stress and tissue damage.
As we report here, heparin is able to decrease peroxide levels in aortic rings from diabetic rabbits, possibly because of the release of xanthine oxidase from the vessel wall. Heparin plays an important role in the regulation of lipid levels during diabetes. Results reported here show a new potential use of heparin in diabetes.
Given the importance of oxidative stress in the development of complications in diabetes (1), the role of xanthine oxidase in the pathogenesis of such complicationsand the protective effect of allopurinol that we have shown heremay have clinical relevance.
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ACKNOWLEDGMENTS |
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We thank Dr. Francisco Javier Miranda and Dr. José Antonio Alabadí for providing us with diabetic rabbits and Marilyn R. Noyes for revising the manuscript.
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FOOTNOTES |
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Received for publication 18 December 2000 and accepted in revised form 12 December 2001.
AAT, alanine amino transferase; GSH, reduced glutathione; GSSG, oxidized glutathione; HPLC, high-performance liquid chromatography; LDH, lactate dehydrogenase; MDA, malondialdehyde.
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REFERENCES |
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