Superoxide radical production by allopurinol and xanthine oxidase

Oxypurinol, an inhibitor of xanthine oxidase (XO), is being studied to block XO-catalyzed superoxide radical formation and thereby treat and protect failing heart tissue. Allopurinol, a prodrug that is converted to oxypurinol by xanthine oxidase, is also being studied for similar purposes. Because allopurinol, itself, may be generating superoxide radicals, we currently studied the reaction of allopurinol with xanthine oxidase and confirmed that allopurinol does produce superoxide radicals during its conversion to oxypurinol. At pH 6.8 and 25 degrees C in the presence of 0.02 U/ml of XO, 10 and 20 microM allopurinol both produced 10 microM oxypurinol and 2.8 microM superoxide radical (determined by cytochrome C reduction). The 10 microM allopurinol was completely converted to oxypurinol, while the 20 microM allopurinol required a second addition of xanthine oxidase to complete the conversion. Fourteen percent of the reducing equivalents donated from allopurinol or xanthine reacted with oxygen to form superoxide radicals. Superoxide dismutase prevented the reduction of cytochrome C by these substrates. At higher xanthine oxidase concentrations, or at lower temperatures, more of the 20 microM allopurinol was converted to oxypurinol during the initial reaction. At lower xanthine oxidase concentrations, or higher temperatures, less conversion occurred. At pH 7.8, the amount of superoxide radicals produced from allopurinol and xanthine was nearly doubled. These results indicate that allopurinol is a conventional substrate that generates superoxide radicals during its oxidation by xanthine oxidase. Oxypurinol did not produce superoxide radicals.     

Oxypurinol as an inhibitor of xanthine oxidase-catalyzed production of superoxide radical

A recent study of the mechanism by which oxypurinol inhibits uric acid generation [T. Spector, W. W. Hall and T. A. Krenitsky, Biochem. Pharmac. 35, 3109(1986)] showed that xanthine is ineffective in impeding the binding of oxypurinol to reduced xanthine oxidase. This study prompted the present hypothesis that, at elevated concentrations of substrates, oxypurinol would be superior to allopurinol as an inhibitor of the xanthine oxidase-catalyzed production of superoxide radical. It was found that the potency of allopurinol was attenuated by elevated concentrations of xanthine and hypoxanthine, whereas the potency of oxypurinol was relatively unaffected. Oxypurinol produced immediate inhibition of superoxide radical production as well as progressive inhibition with time. In contrast, allopurinol, which is also a substrate for xanthine oxidase, produced very little immediate inhibition and caused progressive inhibition only after conversion to oxypurinol. The theoretical advantages of treating ischemic tissues with oxypurinol are discussed.     

Stimulatory effect of platinum (IV) ion on the production of superoxide radical from xanthine oxidase and macrophages.

Superoxide radical (^.O^?₂) production was measured spectrophotometrically using NADH and lactate dehydrogenase (LDH) in a xanthine oxidase(XOD) plus hypoxanthine (HX) system and in an isolated guinea pig macrophages system. Sodium platinum (IV) chloride (Na₂PtCl₆: 2.5 × 10^?4?1 × 10^?3M) enhanced the production of ^.O₂^? in both systems (2–10 times). The degree of the enhancement was dependent on incubation time, basal level of ^.O₂^? production and concentration of Na₂PtCl₆. The stimulated ^.O₂^? production in the XOD system was inhibited by luminol (O-aminophtalhydrazide) and that in the macrophages was inhibited by an anti-inflammatory drug, Diclofenac sodium (Dc). These results show that platinum (IV) ion is either a potent stabilizer of ^.O₂^? or a stimulator of ^.O₂^? production as are paraquat or streptonigrin. This specific character of platinum (IV) ion may explain its bactericidal and inflammation-inducing properties.     

Inhibition of carrageenan-induced paw edema by superoxide dismutase that binds to heparan sulfates on vascular endothelial cells

The effect of heparin-binding superoxide dismutase (HB-SOD), a fusion gene product consisting of human Cu/Zn-SOD and a C-terminal basic domain with high affinity for heparin-like proteoglycans, was examined on carrageenan-induced paw edema in mice and rats. When injected intravenously to mice just before carrageenan, HB-SOD suppressed significantly paw edema. ED30 of HB-SOD (1000 units/kg) was markedly lower than that of SOD (bovine free Cu/Zn-SOD, 7000 units/kg). When HB-SOD was administered with heparin (500-2000 units/kg), edema was suppressed more markedly than by HB-SOD alone. In contrast, the suppressive action of SOD was decreased by heparin. HB-SOD also suppressed carrageenan paw edema in rats with an ED30 of 2500 units/kg which was also obtained by SOD. Heparin prolonged significantly the duration of HB-SOD suppression of edema. The inhibitory effect of HB-SOD alone disappeared within 5 hr of injection, while more than 80% of the effect remained at this time when HB-SOD has been injected with 1000 units/kg of heparin. Heparin failed to enhance the anti-inflammatory effect of SOD under any of the conditions tested and heparin alone showed no suppression up to 5 hr after injection. HB-SOD might permit studies on pathophysiological events in and around vascular endothelial cells where reactive oxygen species play critical roles.     

Role of phosphate, pyrophosphate, adenine nucleotides and sulfate in activating production of the superoxide radical by macrophages, and in formation of rat paw edema.

The presence of anions of phosphate (Pi), pyrophosphate (PPi), adenine nucleotides and sulfate greatly enhanced the production of superoxide radical (-O-2) by isolated guinea-pig macrophages. These anions, however, failed to enhance the production of -O-2 by the xanthine oxidase system, suggesting that they serve only as activators of -O-2 generating enzyme(s) located on the macrophage cell membrane. Many other common anions were ineffective in the macrophage system. In the presence of concentrations of Pi, PPi, adenine-5'-triphosphate (ATP) reported to be in the synovial fluid, -O-2 was produced efficiently and was inhibited by diclofenac sodium. These anions induced rat paw edema, maintained the swelling at least up to 6 h. The edema was suppressed partially by repeated injection of superoxide dismutase (SOD). High doses of sodium chloride and nitrate failed to maintain the swelling.     

Inhibition by reactive aldehydes of superoxide anion radical production from stimulated polymorphonuclear leukocytes and pulmonary alveolar macrophages. Effects on cellular sulfhydryl groups and NADPH oxidase activity

Alpha,beta-unsaturated aldehydes such as acrolein (ACR) and crotonaldehyde (CRO) have been shown previously in our laboratory to inhibit the production of superoxide anion radical (O2-) by stimulated phagocytic cells in vitro in a dose-related manner. Based on the known reactivity of these compounds towards cellular sulfhydryls (SH), the present studies were aimed at investigating cellular SH status in relation to O2- production. Plasma membrane surface SH groups were measured using carboxypyridinedisulfide and monitoring the resultant formation of mixed disulfides through assay of thione released into the supernatant fraction. Intracellular non-protein sulfhydryls were measured using 5,5'-dithiobis-2-nitrobenzoic acid. In both human polymorphonuclear leukocytes (PMN) and rat pulmonary alveolar macrophages (PAM) there was a dose-related decrease in surface SH and soluble SH after ACR and CRO treatment. Propionaldehyde, a three-carbon saturated aldehyde, was without effect. The decrease in surface SH was greater than the decrease in soluble SH. In addition, in PMN and PAM preincubated with 5-40 microM ACR, there was a dose-related inhibition in the rate of O2- production with no effect on the lag time as measured by cytochrome c reduction. In stimulated PMN, there was a dose-related decrease in the rate after addition of 5-40 microM ACR. These data suggest that changes in SH status by reactive aldehydes can modulate the activity of the plasma membrane NADPH oxidase responsible for O2- production.     

Activity of polyphenolic crude extracts as scavengers of superoxide radicals and inhibitors of xanthine oxidase.

In view of the pharmacological interest in phenolic substances, we have determined the total amount of anthocyanins and polyphenols present in the berries of several cultivars of Ribes, Rubus, and Vaccinium genera. The in vitro antiradical activity of the crude extracts on chemically-generated superoxide radicals as well as the inhibitory activity towards the enzyme xanthine oxidase were studied. All the crude extracts examined showed a remarkably high activity towards chemically-generated superoxide radicals. The activities were greater than those expected on the basis of the quantities of anthocyanins and polyphenols present in the samples. Furthermore, the extracts showed a certain inhibitory activity towards xanthine oxidase. Ribes nigrum extracts exhibit the highest activity, being the richest in both anthocyanins and polyphenols. On the other hand, Ribes rubrum extracts seem to contain more active substances than the other crude extracts.     

Inhibition of buttermilk xanthine oxidase by folate analogues and derivatives

Folic acid has been reported recently to be an effective agent for the treatment of hyperuricernia, although conflicting data exist. The relative inhibitory activities of this compound and its breakdown products, pterin aldehyde and 6-hydroxymethylpterin, for the enzyme xanthine oxidase have not been clear. In this study, folic acid purified from these two compounds competitively inhibited buttermilk xanthine oxidase under aerobic conditions by a mechanism kinetically distinct from that of pterin aldehyde, with an inhibition constant (K_i) of 0.12 μM. Methotrexate, leucovorin and N⁵-methylH₄folate were competitive inhibitors of the enzyme with K_i values ranging from 12 to 53 μM. MethyleneH⁴folate, H₂folate and H₄folate did not inhibit xanthine oxidase. N⁵-MethylH₄folate could not be evaluated by the reduction of cytochrome c because of the nonenzymatic oxidation of this folate derivative by cytochrome c to a compound, shown to be N⁵-methylH₂folate. Unless high intracellular concentrations of unchanged folic acid, pterin aldehyde or hydroxymethylpterin can be achieved or folic acid proves to be a more effective inhibitor of reduced than of oxidized enzyme, it is unlikely that this compound will be an effective clinical agent for the inhibition of xanthine oxidase.     

丹参二萜醌对黄嘌呤氧化酶活性的抑制作用(英文)

目的黄嘌呤氧化酶(XOD)抑制剂对痛风或其他XOD诱导的疾病有潜在的治疗作用,因此探讨了从丹参(Salvia miltiorrhiza Bge.)中分离的丹参二萜醌---隐丹参酮(CT)和次甲丹参酮(MT)对XOD的抑制作用。方法在分子氧存在的条件下,XOD催化黄嘌呤产生尿酸和超氧阴离子。在黄嘌呤/XOD的反应媒介中加入CT或MT,通过测量波长290nm处吸光度的增加测定尿酸形成速率。结果CT和MT对XOD有抑制作用,酶动力学曲线Dixon图显示抑制方式是竞争型。CT和MT的Ki值分别为17.8和25.9μmol.L-1,抑制活性与浓度正相关。CT和MT的IC50值分别为70和67μmol.L-1,阳性对照别黄嘌呤醇的IC50值为60μmol.L-1。结论CT和MT对XOD活性有抑制作用,对痛风或其他XOD诱导的疾病可能有一定的治疗作用。     

Involvement of superoxide and xanthine oxidase in neutrophil-independent rat gastric damage induced by NO donors.

1. Nitric oxide (NO) and the superoxide anion can interact to form the cytotoxic moiety, peroxynitrite. The involvement and potential source of superoxide in the gastric mucosal damage induced by local infusion of NO donors, has now been investigated in the pentobarbitone-anaesthetized rat. 2. Local intra-arterial infusion of the NO donor, sodium nitroprusside (40 micrograms kg-1 min-1) for 10 min induced macroscopically apparent gastric mucosal injury. 3. This mucosal damage was dose-dependently reduced by prior administration of a systemically acting form of superoxide dismutase conjugated with polyethylene glycol (500-2000 iu kg-1, i.v.). 4. Likewise, the mucosal damage induced by nitroprusside was dose-dependently reduced by prior administration of the xanthine oxidase inhibitor, allopurinol (20-100 mg kg-1, i.p. or 100 mg kg-1, p.o.). 5. Pretreatment with allopurinol (100 mg kg-1, i.p.) also reduced the mucosal injury induced by local intra-arterial infusion of the nitrosothiol, S-nitroso-N-acetyl-penicillamine (40 micrograms kg-1 min-1), but not that induced by local infusion of endothelin-1 (5 pmol kg-1 min-1), indicating specificity of action. 6. Prior administration (4h) of rabbit anti-rat neutrophil serum (0.4 ml kg-1, i.p.), which reduced circulating neutrophils by 90%, did not significantly protect against mucosal injury induced by nitroprusside. 7. Intravenous administration of the platelet-activating factor receptor antagonists, WEB 2086 (1 mg kg-1) or BN 52021 (10 mg kg-1), or the thromboxane synthase inhibitor, OKY 15181 (25 mg kg-1), did not modify mucosal damage induced by nitroprusside, showing lack of involvement of these neutrophil-derived mediators. 8. These findings indicate the involvement of superoxide in the injurious actions of the NO donors, implicating a cytotoxic role of peroxynitrite. Xanthine oxidase, but not neutrophils, appears to be a source of the superoxide.     

物质对人中性粒细胞超氧阴离子生成的活化作用

神经肽P物质能激活多种参与炎症和免疫反应的细胞. 然而P物质直接刺激人粒细胞产生超氧阴离子(O^÷₂)需要较高的浓度(>10 μmol·L^-1). 本实验观察了低浓度P物质对人粒细胞超氧阴离子生成的活化作用. 结果表明, P物质在低浓度时(3 μmol·L^-1)能活化人粒细胞, 使?甲酰基-甲硫氨酰基-亮氨酰基苯丙氨酸(fMLP)刺激产生的超氧阴离子明显增多. P物质的这一活化作用呈剂量和时间依赖性. 在无细胞外钙的情况下，P物质没有这种活化作用. P物质活化fMLP引起的O^÷₂生成增多作用可被神经激肽(NK)-1受体阻断剂spentide (1 μmol·L^-1), 磷脂酶C抑制剂U-73122 (10 nmol·L^-1)以及Ca²⁺通道阻断剂尼卡地平 (1 μmol·L^-1)所抑制. 这些发现提示, P物质活化人粒细胞可能是经NK-1受体偶联的磷脂酰肌?醇代谢途径, 并且是细胞外钙依赖性的.     

Priming effect of substance Ponsuper oxideanionradical production inhumanneu trophils

神经肽Ｐ物质能激活多种参与炎症和免疫反应的细胞．然而Ｐ物质直接刺激人粒细胞产生超氧阴离子（Ｏ÷２）需要较高的浓度（＞１０μｍｏｌ·Ｌ－１）．本实验观察了低浓度Ｐ物质对人粒细胞超氧阴离子生成的活化作用．结果表明，Ｐ物质在低浓度时（３μｍｏｌ·Ｌ－１）能活化人粒细胞，使甲酰基－甲硫氨酰基－亮氨酰基苯丙氨酸（ｆＭＬＰ）刺激产生的超氧阴离子明显增多．Ｐ物质的这一活化作用呈剂量和时间依赖性．在无细胞外钙的情况下，Ｐ物质没有这种活化作用．Ｐ物质活化ｆＭＬＰ引起的Ｏ÷２生成增多作用可被神经激肽（ＮＫ）－１受体阻断剂ｓｐｅｎｔｉｄｅ（１μｍｏｌ·Ｌ－１），磷脂酶Ｃ抑制剂Ｕ－７３１２２（１０ｎｍｏｌ·Ｌ－１）以及Ｃａ２＋通道阻断剂尼卡地平（１μｍｏｌ·Ｌ－１）所抑制．这些发现提示，Ｐ物质活化人粒细胞可能是经ＮＫ－１受体偶联的磷脂酰肌醇代谢途径，并且是细胞外钙依赖性的     

TEMPOL, a membrane-permeable radical scavenger, attenuates peroxynitrite- and superoxide anion-enhanced carrageenan-induced paw edema and hyperalgesia: a key role for superoxide anion

Carrageenan produces both inflammation and pain when injected in rat paws via enhancement of the formation of reactive oxygen species. We have tested the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), a membrane-permeable superoxide dismutase (SOD) mimetic in carrageenan-induced rat paw edema. Treatment of rats with TEMPOL (15, 30, and 60 mg/kg, 15 min prior to carrageenan) inhibited the paw edema. Furthermore, treatment of rats with the SOD inhibitor diethylthiocarbamate (DETCA, 100 mg/kg, 1 h before carrageenan) enhanced the carrageenan-induced paw edema. Co-administration of peroxynitrite with carrageenan produced a similar fortification of the carrageenan-induced edema. Prior treatment of rats with TEMPOL (30 mg/kg) inhibited the enhancement produced by DETCA treatment (endogenous superoxide anion stress) as well as that produced by the peroxynitrite stress. The effect of TEMPOL as well as the influence of superoxide anion and peroxynitrite stresses was also tested in carrageenan-induced hyperalgesia model. Carrageenan (500 mug/paw) produced significant hyperalgesia presented as shortening of withdrawal latency times using hot plate (52 degrees C) starting 30 min after carrageenan and lasting for 3 h. TEMPOL (60 mg/kg, injected 15 min before carrageenan) ameliorated this hyperalgesia during the first 2 h. Concurrent administration of peroxynitrite promptly intensified the carrageenan hyperalgesia. TEMPOL (60 mg/kg, 15 min before peroxynitrite-carrageenan) inhibited the peroxynitrite enhancement of carrageenan hyperalgesia when tested at 60 min after injection of the cocktail. The present investigation gives the proof for the effectiveness of TEMPOL as anti-inflammation and analgesic agents in carrageenan-induced model of inflammation and hyperalgesia. It further indicated the importance of superoxide anion and peroxynitrite in acute inflammation and inflammatory pain. This raises the chances for considering pharmacologic interventions that interrupt superoxide anion and peroxynitrite stress for putative alternative agents as anti-inflammatory analgesic new medical strategies.     

Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid

The caffeoyl conjugates of prenylhydroquinone glucoside and of quinic acid, either in the carboxyl-free or carboxymethyl forms, isolated from Phagnalon rupestre (Asteraceae), showed inhibitory activity on lipid peroxidation induced by Fe 2+/ascorbate and by CCl4/NADPH in rat liver microsomes, with IC50 values ranging from 3 to 11 microM. After having demonstrated their effect on the xanthine oxidase-regulated superoxide production, the active compounds were tested for the direct inhibition of this enzyme. Methylated dicaffeoylquinic conjugates competitively inhibited the enzyme and the highest potency was obtained for the 4,5-diester, with an IC50 value of 3.6 microM, nearly ten times lower than that of the 3,5-analogue. In conclusion, the presence of the caffeoyl moiety is essential for both the antiperoxidative and radical scavenging activities, and the methylation of the quinic carboxyl group enhances the potency on xanthine oxidase inhibitory activity.     

Anti-inflammatory effects of polyamines in serotonin and carrageenan paw edemata - possible mechanism to increase vascular permeability inhibitory protein level which is regulated by glucocorticoids and superoxide radical

Serotonin paw edema of mice and carrageenan paw edema of rats were inhibited by subcutaneously or orally administered certain polyamines. They must be given at least 2 h before serotonin challenge to get inhibitions which were blocked by the concomitant injections of cycloheximide. Thirty percent inhibitory dose (ID30) of polyamines (s.c.) 3 h before serotonin (s.c.) were: spermidine (8 mg/kg), spermine 28 mg/kg) and putrescine (55 mg/kg). Agmatine, cadaverine, ornithine, citrulline, lysine and arginine were not inhibitory even at 200 mg/kg. Three inhibitory polyamines were effective by oral administration but were not inhibitory by local administration into the paws. Intravenous injections of spermidine also required 2 h of lag period for inhibitions. Serotonin edema was inhibited by dexamethasone (1 mg/kg), prednisolone (1 mg/kg) or by superoxide dismutase (SOD, 5 mg/kg) in lag period requiring manner (s.c. and i.v.). High dose of cyclo-oxygenase inhibitors indomethacin and diclofenac sodium, lipo-oxygenase inhibitor BW755C (30 mg/kg s.c., respectively) and phospholipase A2 inhibitor quinacrine (100 mg/kg s.c.) failed to inhibit serotonin edema, suggesting that arachidonate metabolites are not participating in this model. ID30 of polyamines which were administered (s.c. and oral) to rats 3 h before carrageenan and determined at 3 h by paw weight were: spermidine (28 and 100 mg/kg), spermine (18 and 90 mg/kg) and putrescine (both greater than 200 mg/kg). Adrenalectomized rats responded to polyamines just as normal rats. Local vascular permeability, irritancy and acute toxicity were also tested in mice. Polyamines were proved to be glucocorticoid-type anti-inflammatory drugs. Polyamines may be mediators of glucocorticoids for the synthesis of the postulated vascular permeability inhibitory protein (called as 'vasoregulin' for convenience). Anti-inflammatory effect of glucocorticoid is recently explained by its capacity to induce phospholipase A2 inhibitory protein(s) (macrocortin or lipomodulin). However, this hypothesis has not yet been proved by in vivo experiment and our data suggest that there is induction by glucocorticoid of another kind of protein which does not inhibit phospholipase A2 activity.     

Inhibition of superoxide anion production in macrophages by anti-inflammatory drugs.

The inhibition by anti-inflammatory drugs of the production of Superoxide anions (O₂^?) by isolated guinea pig macrophages was studied spectrophotometrically using NADH and lactate dehydrogenase. id₅₀ values were: 4 × 10^?7M (diclophenac sodium), 1 × 10^?6M (oxyphenbutazone), 1 × 10^?5M (indomethacin), 4 × 10^?5M (phenylbutazone), 7 × 10^?5M (mefenamic acid), 8 × 10^?5 M (flufenamic acid), 8 × 10^?5M (colchicine), 3 × 10^?4M (aspirin), 3 × 10^?4M (benzydamine), 10^?3M < (dexamethasone) and 10^?3M < (gold sodium thiomalate). They seemed to block the cell membrane-associated mechanism to produce Superoxide anions, since most of them did not abolish the generation of superoxide anions from the xanthine oxidase plus hypoxanthine system. Cytochalasin B, pyrogallol, ascorbate, NEM, l-epinephrine and chlorpromazine also inhibited, the production of Superoxide anion, but many non anti-inflammatory drugs were ineffective. This technique was evaluated as a screening method in vitro for nonsteroidal anti-inflammatory drugs.