Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

High-performance liquid chromatographic determination of hypoxanthine and xanthine in biological fluids

A rapid and selective reversed-phase high-performance liquid chromatographic method for the simultaneous determination of hypoxanthine and xanthine in biological fluids was developed. The identification of hypoxanthine and xanthine was confirmed by xanthine oxidase reaction. This method was applied to the investigation of purine metabolism in subjects with xanthine oxidase deficiency or gout. Hypoxanthine concentrations three to ten times higher than those determined in plasma were found in erythrocyte samples from normal subjects and from patients with xanthine oxidase deficiency or hyperuricemia under allopurinol therapy.     

Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure.

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.     

Association of xanthine oxidase with the bovine milk-fat-globule membrane. Catalytic properties of the free and membrane-bound enzyme

1. The catalytic properties of xanthine oxidase in bovine milk (EC 1.2.3.2) are dependent on the state of the enzyme, i.e. whether free or bound to the fat-globule membrane. Oxidase activity of the membrane-bound enzyme towards NADH is enhanced relative to that towards xanthine. This reflects a change in the relative K(m) values and enables the ratio of xanthine to NADH oxidase activities (X/N) to be used as a parameter for the relative amounts of free and membrane-bound xanthine oxidase in milk fractions. 2. Chromatography of buttermilk on Sepharose 2B yielded an excluded fraction, BM(1), with xanthine oxidase activity. The remaining xanthine oxidase activity was eluted as a single broad peak. This was further resolved on Sephadex G-200 into an excluded fraction, BM(2), and free xanthine oxidase. Fractions BM(1) and BM(2) had X/N values in the range 45-65, which is characteristic of membrane-bound xanthine oxidase. Purified xanthine oxidase has a mean X/N value of 110.3. Addition of fraction BM(1), heated to remove associated enzyme activities, to purified xanthine oxidase progressively enhanced its NADH oxidase activity to a value where its X/N value was characteristic of membrane-bound xanthine oxidase. This was shown to be due to binding of free enzyme to heated fraction BM(1). The binding constant and stoicheiometry were determined. 4. Proteolytic digestion of fraction BM(1) liberated free xanthine oxidase from the fat-globule membrane with a corresponding alteration in X/N value.     

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.     

Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol.

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.     

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes.

1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.     

The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

Superoxide-dependent and -independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Implications for oxygen-free-radical-induced tissue destruction during ischaemia and inflammation.

Xanthine oxidase is able to mobilize iron from ferritin. This mobilization can be blocked by 70% by superoxide dismutase, indicating that part of its action is mediated by superoxide (O2-). Uric acid induced the release of ferritin iron at concentrations normally found in serum. The O2(-)-independent mobilization of ferritin iron by xanthine oxidase cannot be attributed to uric acid, because uricase did not influence the O2(-)-independent part and acetaldehyde, a substrate for xanthine oxidase, also revealed an O2(-)-independent part, although no uric acid was produced. Presumably the amount of uric acid produced by xanthine oxidase and xanthine is insufficient to release a measurable amount of iron from ferritin. The liberation of iron from ferritin by xanthine oxidase has important consequences in ischaemia and inflammation. In these circumstances xanthine oxidase, formed from xanthine dehydrogenase, will stimulate the formation of a non-protein-bound iron pool, and the O2(-)-produced by xanthine oxidase, or granulocytes, will be converted by 'free' iron into much more highly toxic oxygen species such as hydroxyl radicals (OH.), exacerbating the tissue damage.     

A rapid and simple radioassay for inosinic acid: pyrophosphate phosphoribosyltransferase in the presence of xanthine oxidase and vice versa.

A simple and rapid technique for measuring IMP:pyrophosphate phosphoribosyltransferase (HPRibTase) activity of rat intestinal homogenates, in the presence of xanthine oxidase, is described. By introducing 2.5 × 10^?5m allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine) into the reaction mixture, the [8-¹⁴C]hypoxanthine (Hx) is converted only to [8-¹⁴C]inosinic acid (IMP). The xanthine oxidase activity is completely inhibited under this condition. When xanthine oxidase is not blocked, diversion of substrate to urate can invalidate assays of HPRibTase.Using [8-¹⁴C]Hx as substrate, in the presence and absence of allopurinol, the activity of both HPRibTase and xanthine oxidase of the same tissue homogenate is determined. We have simplified the conventional chromatographic separation of the reactant products by spotting the reactant on DEAE cellulose paper followed by repeated washings with 4 mm ammonium formate solution. The unreacted radiosubstrate is washed off, and the [8-¹⁴C]IMP or [8-¹⁴C]uric acid formed remains adsorbed on the paper. The major advantages of this method are speed, reproducibility, sensitivity, ability to process many samples, and a low blank value.Our studies on the enzyme distribution along the intestinal villus have shown that while most of the HPRibTase activity is associated with rapidly multiplying crypt cells, the xanthine oxidase activity is more evenly distributed along the villus, and the activity is effected more by exongeneous effectors. The colon has the highest HPRibTase and lowest xanthine oxidase activity of all the intestinal mucosa cells. Small bowel mucosa is high both in xanthine oxidase and HPRibTase.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

A simple and sensitive method for the activity staining of xanthine oxidase

Xanthine oxidase is a commercially-important enzyme. Several biochemical compounds have been quantitated by xanthine oxidase. Xanthine oxidase has been used as an auxiliary enzyme in the staining of several enzymes or tissues, however, there is no direct staining method available for it, on polyacrylamide gels. Partially-purified xanthine oxidase from cow milk was used as the enzyme source for the development of an activity-staining method on polyacrylamide gels. Staining was very sensitive. Detection of 0.02 μU of the enzyme on polyacrylamide gels was possible. Staining of 0.05 μU takes about 1 min whereas staining of 0.5 μU will take less than 5 s. Addition of TEMED is not essential for activity staining but it did increase both the rate and the intensity of the staining. The stained gels must be washed with distilled water, extensively, in order to remove excess unoxidized nitroblue tetrazolium, and must be protected from light, for a clear background and sharp activity-band staining. This method might be useful for quality control of xanthine oxidase obtained from different sources.     

Hyperthermia enhances the cytotoxic effects of reactive oxygen species to Chinese hamster cells and bovine endothelial cells in vitro.

Hyperthermia is under intensive investigation as a treatment for tumors both alone and in combination with other therapeutic agents. Hyperthermia has a profound effect on the function and structural integrity of tumor microvasculature; this has often been cited as a reason for its effectiveness in treatment of tumors. To test the role of hyperthermia in cytotoxic effects of active oxygen species, Chinese hamster, V79, and bovine endothelial cells were treated by the active oxygens, O not equal to 2 and H2O2, generated from the hypoxanthine/purine and xanthine oxidase reactions. It was found that cytotoxicity to V79 cells depends on the concentrations of purine and xanthine oxidase. A high level of cytotoxicity may be initiated in hyperthermia-treated tumors because high xanthine oxidase activity is known to be associated with tumors and endothelial cells, and degradation processes produce high concentrations of xanthine oxidase substrates in tumors. Since the cytotoxic effect can be reduced by the xanthine oxidase inhibitor, allopurinol, and the H2O2 removal enzyme, catalase, the cytotoxic effect in this experimental system is dependent on xanthine oxidase and H2O2. Adding erythrocytes at the same time as purine and xanthine oxidase could also prevent the cytotoxicity. Elevated temperatures stimulated the reaction of purine and xanthine oxidase and resulted in an increased cytotoxic effect. A similar effect is observed in growth inhibition and colony formation in endothelial cells without adding xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of homovanillamine to homovanillic acid in guinea pig liver slices.

BACKGROUND/AIMS: Homovanillamine is a biogenic amine that it is catalyzed to homovanillyl aldehyde by monoamine oxidase A and B, but the oxidation of its aldehyde to the acid derivative is usually ascribed to aldehyde dehydrogenase and a potential contribution of aldehyde oxidase and xanthine oxidase is usually ignored. METHODS: The present investigation examines the metabolism of homovanillamine to its acid derivative by concurrent incubation with monoamine oxidase and aldehyde oxidase. In addition, the metabolism of homovanillamine in freshly prepared and cryopreserved liver slices is examined and the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity by using specific inhibitors of each oxidizing enzyme is compared. RESULTS: Homovanillamine was rapidly converted mainly to homovanillic acid when incubated with both momoamine oxidase and aldehyde oxidase. Homovanillic acid was also the main metabolite in the incubations of homovanillamine with freshly prepared or cryopreserved liver slices, via the intermediate homovanillyl aldehyde. The acid formation was 70-75 % inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent (50-55 %) and allopurinol (specific inhibitor of xanthine oxidase) had almost no effect. CONCLUSIONS: Homovanillamine is rapidly oxidized to its acid, via homovanillyl aldehyde, by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

Superoxide-independent platelet response to xanthine oxidase.

Xanthine oxidase (1--5 microgram/ml) from cow's milk induces shape change, aggregation, and the release reaction of human washed platelets. Xanthine oxidase plus xanthine produce superoxide radicals, which reduce nitro blue tetrazolium. Superoxide dismutase, allopurinol, or ommission of xanthine inhibits the reduction of nitro blue tetrazolium but has no influence on the platelet response to xanthine oxidase. In contrast, small amounts of plasma or apyrase from potatoes abolish the effect on platelets, but not the enzyme activity of xanthine oxidase. Comparison of two xanthine oxidase preparations shows that higher specific enzyme activity corresponds to a lesser effect on platelets. The results suggest that platelet and enzyme activities reside in different components of xanthine oxidase preparations.     

Distribution of xanthine oxidoreductase activity in human tissues--a histochemical and biochemical study.

Localization of the activity of both the dehydrogenase and oxidase forms of xanthine oxidoreductase were studied in biopsy and postmortem specimens of various human tissues with a recently developed histochemical method using unfixed cryostat sections, poly-(vinyl alcohol) as tissue stabilizator, 1-methoxyphenazine methosulphate as intermediate electron acceptor and Tetranitro BT as final electron acceptor. High enzyme activity was found only in the liver and jejunum, whereas all the other organs studied showed no activity. In the liver, enzyme activity was found in sinusoidal cells and both in periportal and pericentral hepatocytes. In the jejunum, enterocytes and goblet cells, as well as the lamina propria beneath the basement membrane showed activity. The oxidase activity and total dehydrogenase and oxidase activity of xanthine oxidoreductase, as determined biochemically, were found in the liver and jejunum, but not in the kidney and spleen. This confirmed the histochemical results for these organs. Autolytic rat livers several hours after death were studied to exclude artefacts due to postmortem changes in the human material. These showed loss of activity both histochemically and biochemically. However, the percentage activity of xanthine oxidase did not change significantly in these livers compared with controls. The findings are discussed with respect to the possible function of the enzyme. Furthermore, the low conversion rate of xanthine dehydrogenase into xanthine oxidase during autolysis is discussed in relation to ischemia-reperfusion injury.     

Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat

Summary Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.     

Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.