Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophore-sis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodo-nium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopwinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.     

In vitro Damage to Rat Lens by Lumazine and Xanthine Oxidase: Prevention by Superoxide Dismutase

Intact rat lenses incubated with lumazine and xanthine oxidase are physiologically damaged as evidenced by a decrease in the net accumulation of rubidium ions against a concentration gradient. Superoxide dismutase protected the tissue against this damage. These experiments, therefore, demonstrate the susceptibility of the lens tissue to O₂^?_? injury under ambient and nonphotochemical conditions, suggesting a possible implication of this radical in the tissue in vivo and eventual cataract formation. The lumazine/xanthine oxidase system which is known to cause oxygen reduction predominantly by the monovalent route, producing superoxide, appears quite suitable to evaluate the toxicity of O₂^?_? to the tissues in vitro.     

microRNA-21在大鼠心肌缺氧复氧损伤中的作用及其对细胞自噬的影响

本文应用大鼠心肌细胞缺氧/复氧损伤模型,探讨microRNA-21(miR-21)在大鼠心肌缺氧复氧损伤中的作用及其对细胞自噬的影响.缺氧复氧后,RT-PCR检测发现心肌miR-21表达上调(P0.05),流式细胞术检测表明细胞凋亡增加,RT-PCR及蛋白质印迹(Western blot)检测发现p62显著下调而beclin-1显著上调(P0.05),提示缺氧复氧诱导心肌细胞凋亡和自噬异常.脂质体转染miR-21 mimic后,细胞凋亡显著增加(P0.05),p62显著上调而beclin-1显著下调(P0.05),而转染miR-21抑制剂引起相反结果,提示miR-21在心肌缺氧复氧损伤中具有促进细胞凋亡、抑制细胞自噬的作用.生物信息学预测显示,Rab11a的3′-UTR含有miR-21的结合位点,双荧光素酶基因报告系统及Rab11a过表达实验表明Rab11a是miR-21的靶基因之一.心肌过表达Rab11a能减少缺氧复氧后miR-21介导的细胞凋亡及自噬.由此表明,在大鼠心肌缺氧复氧损伤中,miR-21可能通过负调控Rab11a促进心肌细胞凋亡,抑制心肌细胞自噬.本研究可能为预防和治疗心肌缺血再灌注损伤提供新策略.     

Novel Xanthine Oxidase (XO) inhibitory phenylindanes produced by thermal reaction of caffeic acid

The products from the thermal reaction of chlorogenic and caffeic acids, which is a model process of roasting coffee beans, exhibited xanthine oxidase (XO) inhibitory activity. From caffeic acid, six inhibitory phenylindanes were identified, and a new phenylindane displayed the highest inhibitory activity among them. The activity of these phenylindanes may contribute to XO inhibition-related functions of roasted coffee beverages.     

Screening and Inhibition Mechanism of Xanthine Oxidase Inhibitors in Ethanolic Extracts of Chimonanthus salicifolius Hu Leaves

This study aimed to evaluate the inhibition of the ethanol elutions of Chimonanthus salicifolius Hu leaves (CsHL) against xanthine oxidase (XO). The results of XO inhibition assay and enzymatic superoxide free radical scavenging assay in vitro showed that 70 % ethanol eluate (EE) had the best inhibitory effect and followed by 40 % EE. High performance liquid chromatograph analysis showed that quercetin and kaempferol were the potential active components of XO inhibition. The inhibition mechanism of quercetin and kaempferol on XO was investigated by kinetic analysis and fluorescence quenching titration assay. The molecular simulation further revealed that quercetin and kaempferol bind to XO mainly by hydrogen bonding and van der Waals, blocking the entry of substrates and leading to the inhibition of XO. In conclusion, the CsHL have inhibitory effects on XO activity, which provides a theoretical basis for relieving or preventing hyperuricemia and gout as a natural food or medicinal plant in the future.     

Allopurinol Enhances Adenine Nucleotide Levels and Improves Myocardial Function in Isolated Hypoxic Rat Heart

Allopurinol, a competitive inhibitor of xanthine oxidase, was found to have a protective effect on ischemic myocardium. Its mechanism of action is still controversial. We used Langendorff isolated rat heart preparation to test the hypothesis that allopurinol could maintain a level of the adenine nucleotide pool (ATP, ADP, and AMP) that would protect and improve the functional activity of the heart during a period of hypoxia. Hearts were initially perfused for 30 min until steady state was attained. This was followed by 20 min of experimental perfusion divided into 5 min of control perfusion followed by 15 min of hypoxic perfusion with or without allopurinol in the perfusate. Hearts were quick-frozen and enzymatically analyzed for adenine nucleotides and creatine phosphate at the end of the hypoxic period. Left ventricular pressure, heart rate, and coronary flow were measured in all preparations. Allopurinol (0.1 mM) treated hearts had greater levels of ATP (12.3 ± 0.8 vs. 9.3 ± 0.8 µmol/g dry weight; p < 0.01). This improvement occurred in the presence as well as the absence of glucose. Total adenine nucleotides improved from 17 ± 1 to 20.3 ± 2.4 µmol/g dry weight (p < 0.01). This improvement also occurred in the presence as well as in the absence of glucose in the perfusate. It also improved cell energy state significantly in the presence as well as the absence of glucose. There was insignificant change in creatine phosphate. Allopurinol improved left ventricular pressure from 38 ± 7% to 55 ± 9% (p < 0.002) in the presence of glucose and from 8 ± 3% to 27 ± 6.3% (p < 0.001) in the absence of glucose. Coronary flow improved from 110 ± 5% to 120 ± 8% (p < 0.04) in the presence of glucose. These results support the suggestion that allopurinol at 0.1 mM exerts its protective effect on rat heart during hypoxia by enhancing the adenine nucleotide pool.     

桑叶黄酮对腺嘌呤诱导大鼠高尿酸血症肾损伤的防治作用

本文研究了桑叶黄酮(mulberry leaf flavonoids,MLF)对腺嘌呤诱导大鼠高尿酸血症、肾损伤的防治作用.采用腺嘌呤灌胃法诱导SD大鼠制备高尿酸血症和肾损伤模型,MLF预防治疗3w,测定与高尿酸血症和肾衰相关的各项组织器官和血液生化指标.结果显示桑叶总黄酮可显著降低血清尿酸水平,与别嘌醇的降尿酸效果相当;并能显著降低尿酸氮、肌酐、丙二醛、甘油三酯、游离脂肪酸水平和肝脏系数、肾脏系数.上述结果表明桑叶总黄酮有干预腺嘌呤诱导高尿酸血症和肾损伤的作用.     

Monoamine Oxidase Activity of Mitochondria Prepared from Rat Liver and Rat Heart

Abstract: The effect of agents that change the respiratory state of the mitochondrion on tyramine oxidation was investigated. Neither uncoupler nor ADP and P_t in the presence of substrate produced any change in the rate of tyramine oxidation, as judged by direct measurement of tyramine oxidation or by H₂O₂ production. We conclude that previously reported depression of monoamine oxidase activity by stimulated respiration was due to oxygen depletion.     

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells: A Major Role for Xanthine Oxidase in Oxygen Radical Formation

Abstract: To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2',7'-dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 µ M glutamate pulse administered to 7-day-old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O₂^•−) production are completely inhibited by concomitant incubation of glutamate with MK-801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate-triggered, NMDA-mediated, massive Ca²⁺ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death.     

Formation of Antibodies to Native DNA in Rats after Administration of Native DNA Treated with the Xanthine-Xanthine Oxidase System

The injection of native (double-stranded) deoxyribonucleic acid treated with the xanthine-xanthine oxidase system and emulsified with complete Freund's adjuvant into rats over a prolonged period of time induces the formation of antibodies to double-Stranded DNA. The titer of antibodies was determined by an enzyme-linked immunosorbent assay (ELISA) in sera from treated animals. Control experiments using untreated native DNA or phosphate buffered saline likewise emulsified with Freund's Adjuvant showed only insignificant increases in titers of the antibody.     

三种中药提取物对黄嘌呤氧化酶的抑制作用

分别用水、60%乙醇和95%乙醇为提取溶剂,对泽泻、青风藤和白芷进行提取,通过测定黄嘌呤氧化酶(XOD)-黄嘌呤反应体系终产物尿酸的吸光度,得出各提取物对XOD的抑制率,并判断其抑制机制。实验结果显示,在一定浓度范围内,泽泻和青风藤两种醇提物对XOD的抑制率较高,泽泻60%乙醇提取物抑制率最高达86%,青风藤95%乙醇提取物抑制率最高为71.2%,白芷水提取物对XOD抑制率较高,最高为72.7%。结果表明,三种中药的提取物均具有较强的体外抑制XOD活性的作用。     

On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals

Allopurinol has been employed as a “specific” inhihitor of xanthine oxidase in studies of hypoxic/ reoxygenation injury. Pulse radiolysis was used to establish rate constants for the reactions of allopurinol and its major metabolite oxypurinol with hydroxyl radicals: values were (1.45 ± 0.241 × 10⁹ M^-1 s^-1 for allopurinol and (4.95 ± 0.84) × 10⁹ M^-1 s^-1 for oxypurinol. These rate constants show that, in view of the amounts of allopurinol that have been used in animal studies. hydroxyl radical scavenging by this molecule could contribute to its biological actions. especially if animals are pre-treated with allopurinol. so allowing oxypurinol to form. The ability of allopurinol to protect tissues not containing xanthine oxidase against reoxygenation injury may be related to radical scavenging by allopurinol and oxypurinol.     

On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals

Allopurinol has been employed as a “specific” inhihitor of xanthine oxidase in studies of hypoxic/ reoxygenation injury. Pulse radiolysis was used to establish rate constants for the reactions of allopurinol and its major metabolite oxypurinol with hydroxyl radicals: values were (1.45 ± 0.241 × 10⁹ M^-1 s^-1 for allopurinol and (4.95 ± 0.84) × 10⁹ M^-1 s^-1 for oxypurinol. These rate constants show that, in view of the amounts of allopurinol that have been used in animal studies. hydroxyl radical scavenging by this molecule could contribute to its biological actions. especially if animals are pre-treated with allopurinol. so allowing oxypurinol to form. The ability of allopurinol to protect tissues not containing xanthine oxidase against reoxygenation injury may be related to radical scavenging by allopurinol and oxypurinol.     

Confirmation of Superoxide Generation via Xanthine Oxidase in Streptozotocin-induced Diabetic Mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33?μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6?μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.     

腺苷预处理对大鼠心脏移植缺血再灌注损伤心肌的保护作用

目的探讨腺苷（adenosine,Ado）预处理在心脏移植缺血再灌注损伤的保护作用。方法健康雄性Sprague．Dawley（SD）大鼠40只,建立大鼠同系异位心脏移植模型。随机分为3组：假手术组、对照组、实验组（供体心在获取前15min给予Ado预处理后4℃改良St．Thoms液10mL。主动脉根部灌注停跳）。术后5h测定血清中丙二醛（MDA）、超氧化物歧化酶（SOD）、肌酸激酶同工酶（CK-MB）含量,电镜观测心肌细胞超微结构变化。结果实验组MDA、CK-MB明显减少（P〈0．01）,SOD明显增加（P〈0．01）,心肌超微结构改变明显减轻。实验组的移植心脏存活时间较对照组明显延长[（22．1±2．9）dw（12．0±1．8）d,P〈0．01）]。结论Ado预处理对心脏移植缺血再灌注损伤有保护作用,使受者的移植心脏存活期延长。     

Radical Formation in the Rat Small Intestine During and Following Ischemia

Oxidative loading during the reperfusion of the proximal jejunum of rats following a one hour-period of complete ischemia was demonstrated in in vivo-experiments by the increases of the GSSG: total glutathione ratio and the concentration of TBA-RS. The pretreatment of the animals with the xanthine oxidoreductase inhibitor allopurinol diminished the accumulation of GSSG and of TBA-RS. It was concluded that the purine nucleotide degradation is an important source of oxygen reduction products in reoxygenated small intestine. The tissue concentrations of nucleotides, nucleosides and nucleobases were measured by an ion-pair reversed-phase HPLC separation. There occurred fast declines of ATP and GTP concentrations during ischaemia leading to temporary increases of nucleoside mono- and diphosphate pools. The hypoxanthine concentration is increased about twenty fold during oxygen deficiency. The ATP and GTP restoration during the reperfusion was accelerated in presence of allopurinol. The shares of the beneficial allopurinol effects are not yet clarified.     

Xanthine Oxidase/Dehydrogenase Activity in Intact Cultured Cells (In Situ Analysis)

The measured ratio of xanthine oxidase activity to the total activity of xanthine oxidase and dehydrogenase showed higher values in intact cells than when similar cells were homogenized. The total activity was the same for both systems. The xanthine oxidase ratio was 90, 60, 50, 50, 60% in V79, RIF/Ha³, SCC7, KHT intact cells and freshly extracted murine peritoneal macrophages respectively while the corresponding ratios measured were 25, 40, 38, 35, 22% when the cells were lysed by homogenization. Superoxide radical 0₂ production by addition of xanthine to intact or homogenized cells to activate intracellular xanthine oxidase was higher in intact than homogenized cells. Homogenization of cells and tissues in the presence of dithioerythritol (DTE) can evidently lead to a considerable under-estimation of the xanthine oxidase ratio. The effect of hypoxia on cells has also been examined.     

Changes in Monoamine Oxidase Activity in Rat Brain During Alloxan Diabetes

Abstract: The effect of alloxan diabetes on the activity of monoamine oxidase was studied in three regions of the rat brain at various time intervals after the onset of diabetes. It was observed that monoamine oxidase activity was decreased at early time intervals after diabetes, followed by a recovery in all three regions of the brain. A reversal of the effect was observed with insulin administration to the diabetic rats.