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.     

Sulfoxide reductase activity of liver aldehyde oxidase

The present study provides evidence that guinea pig and rabbit liver aldehyde oxidase (EC 1.2.3.1) in the presence of its electron donors such as aldehydes or N-heterocyclic compounds functions as a sulfoxide reductase towards sulindac and other sulfoxide compounds. In addition, the study shows that a combination of liver aldehyde oxidase and milk xanthine oxidase also exhibits sulfoxide reductase activity in the presence of xanthine, and electron donor of xanthine oxidase. Based on these facts, we propose a new electron-transfer system consisting of these two flavoenzymes.     

Human xanthine oxidase changes its substrate specificity to aldehyde oxidase type upon mutation of amino acid residues in the active site: roles of active site residues in binding and activation of purine substrate

Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 A resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

The antimicrobial action of the xanthine oxidase-xanthine system on the causative agent of cholera

As revealed in experiments on V. cholerae, the enzymatic link xanthine oxidase-xanthine produces a vibriostatic effect at the concentration of xanthine oxidase equal to 0.0125 g/l and a vibriocidal effect at the concentration of xanthine oxidase equal to 0.025 g/l in a medium with pH 7.5-7.6. In the presence of protein the antivibrionic activity of the xanthine oxidase link is decreased. The introduction of bivalent iron into the enzymatic link xanthine oxidase-xanthine enhances its vibriocidal action on V. cholerae.     

An amperometric xanthine oxidase enzyme electrode based on hydrogen peroxide electroreduction

A xanthine oxidase enzyme electrode (xanthine oxidase immobilized on electrochemically modified graphite and conveniently coated with gelatine electrode working surface) for quantitative analysis of xanthine is proposed. The detection of thus developed electrochemical system is based on the electroreduction of hydrogen peroxide generated in enzyme layer and offered L-ascorbic and uric acid reducing interference effect on the substrate determination. At a working potential -50 mV (vs. Ag/AgCl) the detection limit of 4.5 microM and the linearity of the amperometric signal up to substrate concentration of about 40 microM were found. At that working potential, the electrode is practically inert towards L-ascorbic- and uric acid present. The response time did not exceed 2 min.     

Absence of xanthine oxidase or xanthine dehydrogenase in the rabbit myocardium

We directly measured the activity of the enzymes xanthine oxidase and xanthine dehydrogenase in rabbit and rat hearts, using a sensitive radiochemical assay. Neither xanthine oxidase activity nor xanthine dehydrogenase activity was detected in the rabbit heart. In the rat heart, xanthine oxidase activity was 9.1 +/- 0.5 mIU per gram wet weight and xanthine dehydrogenase activity was 53.0 +/- 1.9 mIU per gram wet weight. These results argue against the involvement of the xanthine oxidase/xanthine dehydrogenase system as a mechanism of tissue injury in the rabbit heart, and suggest that the ability of allopurinol to protect the rabbit heart against hypoxic or ischemic damage must be due to a mechanism other than inhibition of these enzymes.     

Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress

The reversible and irreversible conversion of xanthine dehydrogenase to xanthine oxidase during ischemia/reperfusion and oxidative stress induced by hydrogen peroxide or diamide and its relationship with glutathione and protein SH groups were studied. The direct spectrophotometric measurement of the various forms of the xanthine-converting enzyme indicates that, in the fresh rat heart or after normoxic perfusion, there always is a basal level of 80% xanthine dehydrogenase and 20% of xanthine oxidase (15% irreversible and 5% reversible) that could contribute to the background production of free radicals. There is no significant increase of irreversible xanthine oxidase during ischemia nor during reperfusion. After global ischemia the reversible oxidase shows almost no increase while, when ischemia is followed by reperfusion, there is a limited increase (less then 9%) of the reversible xanthine oxidase. In the latter conditions there is a decrease of glutathione and of SH groups of about 70% and 25%, respectively. Perfusion for 1 h with oxidizing agents like hydrogen peroxide (60 microM) or diamide (100 microM) determines a marked conversion of xanthine dehydrogenase to reversible xanthine oxidase of about 40% and 60%, respectively; this oxidase activity partially reconverts to the dehydrogenase after withdrawing the oxidizing agents from the perfusion medium. The level of irreversible xanthine oxidase remains unchanged in all the conditions tested. Both hydrogen peroxide and diamide induce a strong decrease in SH groups and depletion of glutathione. The xanthine dehydrogenase----xanthine oxidase conversion thus appears to be sensitive to the redox state of thiol groups.     

Effect of tranquilizers of the 1,4-benzodiazepine series on the xanthine oxidase activity of the rat brain

Experiments in vivo and in vitro on 90 rats were made to study the influence of 1,4-benzodiazepine tranquilizers (phenazepam, nitrazepam and diazepam) on cerebral xanthine oxidase activity. Phenazepam, nitrazepam and diazepam in the dose of 5 mg per 200 g bw were shown to reduce xanthine oxidase activity by 80.4%, 64.3% and 55.8%, respectively 2 h after intraperitoneal injection. 6 h after the injection of benzodiazepines the enzyme activity grows, but control values are achieved only after nitrazepam injection. In vitro experiments revealed direct influence of the tranquilizers on xanthine oxidase. Phenazepam inhibits xanthine oxidase activity in concentration as long as 10(-10) M (to 36.6%), and practically completely in 10(-6) M concentration. Nitrazepam and diazepam inhibit xanthine oxidase activity within concentration range between 10(-8) M (to 51.5% and 33.2%, respectively), and 10(-4) M (practically completely). The inhibition of xanthine oxidase activity is shown to be caused by the competition between hypoxanthine, the reaction substrate, and tranquilizer, to bind with the active site of the enzyme.     

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.     

Ferritin oxidation in vitro: implication of iron release and degradation by the 20S proteasome

Ferritin, the major iron storage protein in mammalian cells, was treated with various concentrations of different oxidants: xanthine/xanthine oxidase, Sin-1 (3-morpholinosydnonimine, purchased from Alexis, Grunberg, Germany), DEA-NO (Diethylamine NONOate, purchased from Calblochem-Novabiochem, Schwalbach, Germany), and hydrogen peroxide. The proteolytic susceptibility towards the isolated 20S proteasome of untreated ferritin and oxidized ferritin was measured in parallel with the iron liberated by these oxidants. With increasing hydrogen peroxide, Sin-1, and xanthine oxidase concentrations, the measured proteasomal degradation of ferritin also increased. At higher oxidant concentrations, however, the proteolytic susceptibility began to decrease. The oxidation of ferritin by DEA-NO was accompanied by a lesser increase of proteolytic susceptibility in comparison with the effects of the other oxidants. Addition of DEA-NO to Sin-1 suppressed the increase in proteolytic susceptibility of ferritin, whereas adding xanthine/xanthine oxidase had no additional effect. Iron was liberated readily from ferritin as a result of the oxidation process, although the increase in proteolytic susceptibility was not always correlated to the iron release. In fact, the degradation of oxidatively damaged ferritin was not accompanied by a further increase of free iron. Therefore, we conclude that the proteasome is a secondary antioxidative defense system that degrades only nonfunctional ferritin.     

Studies on the specificity toward aldehyde substrates and steady-state kinetics of xanthine oxidase

The aldehyde specificity of xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been reinvestigated. The biogenic aldehydes and succinate semialdehyde are reasonable substrates for xanthine oxidase. Pyrophosphate, which binds to xanthine oxidase, does not seem to affect significantly the enzyme's catalytic activity. The steady-state parameters for the oxidation of several substrates by xanthine oxidase and oxygen have been determined. Formaldehyde differs from xanthine and other aldehydes in phi 2, the parameter describing the reaction with oxygen. Substrate inhibition has been studied at high concentrations of xanthine with oxygen as the electron acceptor. The inhibition is hyperbolic and uncompetitive with respect to oxygen. This is possibly due to rate-limiting product release from molybdenum(IV) being slower than from molybdenum(VI).     

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)     

Production of the carbonate radical anion during xanthine oxidase turnover in the presence of bicarbonate

Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants.     

EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol

Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.     

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.     

Oxidation of uroporphyrinogens by hydroxyl radicals. Evidence for nonporphyrin products as potential inhibitors of uroporphyrinogen decarboxylase

A hydroxyl radical-generating system, hypoxanthine/xanthine oxidase/Fe-EDTA, oxidises uroporphyrinogens to uroporphyrins and to more polar nonporphyrin products. The evidence suggests that the nonporphyrin products have inhibitory activity towards mouse liver uroporphyrinogen decarboxylase which could explain some forms of human and experimental porphyrias.     

A role for xanthine oxidase in the loss of cytochrome P-450 evoked by interferon.

It has been suggested that the loss of cytochrome P-450, which is mediated by interferon and its inducers, can result from the generation of free radical species by the enzyme xanthine oxidase. Cytochrome P-450, aminopyrine N-demethylase, and ethoxyresorufin deethylase were depressed by 35, 36, 38%, respectively, in the livers of hamsters 24 h following the administration of a synthetic interferon (IFN-alpha-Con1) which contains the most frequent amino acid sequences of the human subtypes. Interferon increased the activities of the D and O forms of xanthine oxidase by 65 and 74%, respectively, in the same animals. The induction of the D form of xanthine oxidase, which is the precursor of the O form, preceded the loss in cytochrome P-450. The protein synthesis inhibitor, actinomycin D, prevented the interferon-induced loss of drug biotransformation and the increase in xanthine oxidase. The free radical scavenger, alpha-tocopherol, and the xanthine oxidase inhibitor, allopurinol, also prevented the loss of cytochrome P-450 mediated by the interferon inducer poly rI.rC. In chickens in which xanthine oxidase cannot be formed, poly rI.rC had no effect on cytochrome P-450 levels. These results suggest that xanthine oxidase induction may play some role in the interferon-mediated loss of cytochrome P-450.     

The effect of immune antibodies and the xanthine oxidase-xanthine enzymatic link on Vibrio cholerae

As revealed in experiments on V. cholerae, highly diluted cholera antiserum enhanced the inhibitory action of the enzymatic link xanthine oxidase-xanthine-Fe2+ on the multiplication of V. cholerae, while low dilutions of the antiserum weakened this action. Normal rabbit serum produced no such effect. The antivibrionic effectiveness of the immune molecular cycle, viz. antiserum--the xanthine oxidase enzymatic link, was found to depend also on the concentration of xanthine. Immune antibodies to cholera antigens activated the bacteriostatic action of the enzymatic link at the concentration of xanthine oxidase equal to 0.0125 g/l and its bactericidal action at the concentration of xanthine oxidase equal to 0.025 g/l. In this article the values of the specificity indices of immune interaction and immunological effectiveness, characterizing the effectiveness of immune molecular cycles (antibodies--the xanthine oxidase enzymatic link), are presented.     

Enzymatic oxidation of phthalazine with guinea pig liver aldehyde oxidase and liver slices: inhibition by isovanillin

The enzymes aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are widely known for their role in the metabolism of N-heterocyclic xenobiotics where they provide a protective barrier by aiding in the detoxification of ingested nitrogen-containing heterocycles. Isovanillin has been shown to inhibit the metabolism of aromatic aldehydes by aldehyde oxidase, but its inhibition towards the heterocyclic compounds has not been studied. The present investigation examines the oxidation of phthalazine in the absence and in the presence of the inhibitor isovanillin by partially purified aldehyde oxidase from guinea pig liver. In addition, the interaction of phthalazine with freshly prepared guinea pig liver slices, both in the absence and presence of specific inhibitors of several liver oxidizing enzymes, was investigated. ldehyde oxidase rapidly converted phthalazine into 1-phthalazinone, which was completely inhibited in the presence of isovanillin (a specific inhibitor of aldehyde oxidase). In freshly prepared liver slices, phthalazine was also rapidly converted to 1-phthalazinone. The formation of 1-phthalazinone was completely inhibited by isovanillin, whereas disulfiram (a specific inhibitor of aldehyde dehydrogenase) only inhibited 1-phthalazinone formation by 24% and allopurinol (a specific inhibitor of xanthine oxidase) had little effect. Therefore, isovanillin has been proved as an inhibitor of the metabolism of heterocyclic substrates, such as phthalazine, by guinea pig liver aldehyde oxidase, since it had not been tested before. Thus it would appear from the inhibitor results that aldehyde oxidase is the predominant enzyme in the oxidation of phthalazine to 1-phthalazinone in freshly prepared guinea pig liver slices, whereas xanthine oxidase only contributes to a small extent and aldehyde dehydrogenase does not take any part.