Improved method for measurement of human plasma xanthine oxidoreductase activity

The XOR activity in human plasma was measured by quantifying the XOR-derived uric acid (UA) in plasma using the high-performance liquid chromatography (HPLC) equipped with a UV detector. Chromatographic separation consisted of the mobile phase (a mixture of 0.1% trifluoroacetic acid in Milli-Q water and 0.085% trifluoroacetic acid in acetonitrile in a mix ratio of 99:1) running through a Zorbax StableBond SB-C(18) column at a flow-rate of 1 ml/min. Deproteinization with heat-treatment of plasma samples after the reaction was used in the assay to avoid splitting of the UA and xanthine peaks caused by acid deproteinization that could interfere the accurate determination of human plasma XOR activity in our case. Based on the examination of the dependence of XOR activity on added amounts of xanthine and reaction times, the amount of xanthine and reaction time for XOR activity assay were determined to prevent the errors caused by the limiting effect of substrates and plateau phase of the reaction. Using this method, human plasma XOR activities of 25 healthy people were measured. The average human plasma XOR activity was 2.1+/-0.8 (x10(-3) U/ml).     

黄嘌呤氧化还原酶的结构、功能和作用

黄嘌呤氧化还原酶(XOR)参与人体内的嘌呤代谢,并且是这一代谢过程的限速酶。其终产物是活性氧(包括OH·、H2O2和O2-)和尿酸。这两种产物参与体内多种生理活动。从XOR基因的结构、XOR蛋白的分子结构和基本功能、控制XOR活性的多个环节以及XOR的两种催化产物活性氧和尿酸在生理和病理情况下的功能及机制进行了总结,以期对XOR的发现、研究历史及现状和有待解决的问题有一个系统的了解。     

Febuxostat inhibition of endothelial-bound XO: implications for targeting vascular ROS production

Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) that contribute to vascular inflammation. Binding of XO to vascular endothelial cell glycosaminoglycans (GAGs) results in significant resistance to inhibition by traditional pyrazolopyrimidine-based inhibitors such as allopurinol. Therefore, we compared the extent of XO inhibition (free and GAG-bound) by allopurinol to that by febuxostat, a newly approved nonpurine XO-specific inhibitor. In solution, febuxostat was 1000-fold more potent than allopurinol at inhibiting XO-dependent uric acid formation (IC₅₀ = 1.8 nM vs 2.9 μM). Association of XO with heparin-Sepharose 6B (HS6B-XO) had minimal effect on the inhibition of uric acid formation by febuxostat (IC₅₀ = 4.4 nM) while further limiting the effect of allopurinol (IC₅₀ = 64 μM). Kinetic analysis of febuxostat inhibition revealed K_i values of 0.96 (free) and 0.92 nM (HS6B-XO), confirming equivalent inhibition for both free and GAG-immobilized enzyme. When XO was bound to endothelial cell GAGs, complete enzyme inhibition was observed with 25 nM febuxostat, whereas no more than 80% inhibition was seen with either allopurinol or oxypurinol, even at concentrations above those tolerated clinically. The superior potency for inhibition of endothelium-associated XO is predictive of a significant role for febuxostat in investigating pathological states in which XO-derived ROS are contributive and traditional XO inhibitors are only slightly effective.     

The role of xanthine oxidoreductase and uric acid in metabolic syndrome

Xanthine oxidoreductase (XOR) could contribute to the pathogenesis of metabolic syndrome through the oxidative stress and the inflammatory response induced by XOR-derived reactive oxygen species and uric acid. Hyperuricemia is strongly linked to hypertension, insulin resistance, obesity and hypertriglyceridemia. The serum level of XOR is correlated to triglyceride/high density lipoprotein cholesterol ratio, fasting glycemia, fasting insulinemia and insulin resistance index. Increased activity of endothelium-linked XOR may promote hypertension. In addition, XOR is implicated in pre-adipocyte differentiation and adipogenesis. XOR and uric acid play a role in cell transformation and proliferation as well as in the progression and metastatic process. Collected evidences confirm the contribution of XOR and uric acid in metabolic syndrome. However, in some circumstances XOR and uric acid may have anti-oxidant protective outcomes. The dual-face role of both XOR and uric acid explains the contradictory results obtained with XOR inhibitors and suggests caution in their therapeutic use.     

Oxidases and peroxidases in cardiovascular and lung disease: new concepts in reactive oxygen species signaling

Reactive oxygen species (ROS) are involved in numerous physiological and pathophysiological responses. Increasing evidence implicates ROS as signaling molecules involved in the propagation of cellular pathways. The NADPH oxidase (Nox) family of enzymes is a major source of ROS in the cell and has been related to the progression of many diseases and even environmental toxicity. The complexity of this family's effects on cellular processes stems from the fact that there are seven members, each with unique tissue distribution, cellular localization, and expression. Nox proteins also differ in activation mechanisms and the major ROS detected as their product. To add to this complexity, mounting evidence suggests that other cellular oxidases or their products may be involved in Nox regulation. The overall redox and metabolic status of the cell, specifically the mitochondria, also has implications on ROS signaling. Signaling of such molecules as electrophilic fatty acids has an impact on many redox-sensitive pathologies and thus, as anti-inflammatory molecules, contributes to the complexity of ROS regulation. This review is based on the proceedings of a recent international Oxidase Signaling Symposium at the University of Pittsburgh's Vascular Medicine Institute and Department of Pharmacology and Chemical Biology and encompasses further interaction and discussion among the presenters.     

稳定表达黄嘌呤氧化还原酶的人类神经外胚层瘤PFSK细胞株的建立(简报)

黄嘌呤氧化还原酶(Xanthine Oxidoreductase,XOR)参与嘌呤类物质代谢,是嘌呤代谢的关键酶。在哺乳动物中,XOR以两种可互相转换的形式存在,即黄嘌呤脱氢酶(XDH)和黄嘌呤氧化酶(XO)。同时,由于XOR催化反应的副产物是氧自由基,因此XOR参与氧自由基产生的作用也日益受到重视。大量动物实验模型已经表明在氧化性组织损伤过程如脑缺血/再灌注等病理情况下,XOR活性增强导致的氧自由基积聚是造成组织损伤的直接原因之一。但是,人类XOR的研究却由于正常人类XOR活性和基因表达水平较低(仅