Endogenous Xanthine Oxidase Does Not Significantly Contribute to Vascular Endothelial Production of Reactive Oxygen Species

The contribution of xanthine oxidoreductase (XDH + XO) to the extracellular release of hydrogen peroxide (H2O2) and intracellular H2O2 concentration in cultured bovine aortic endothelial cells (BAEC) was determined. Intracellular H2O2 concentration was measured by the aminotriazole-mediated inactivation of catalase, while extracellular H2O2 release was measured by the horseradish peroxidase-mediated oxidation of p-hydroxyphenyl acetic acid to a fluorescent dimer. Supplementation of reaction systems with xanthine did not increase H2O2 production by cells. Inhibition of XO activity with allopurinol did not decrease either intracellular concentrations or the extracellular release of H2O2. Similarly, inactivation of XO by culture of cells with tungsten did not have any effect on intracellular levels of H2O2, while it increased extracellular release of H2O2 by 86 and 103% from cells cultured in Medium 199 (M199) and Dulbecco′s modified Eagle′s medium (DMEM), respectively. Cells cultured in DMEM had an average of 8 times greater XDH + XO specific activity, compared to M199 cultured cells, and had a threefold greater rate of release of H2O2 than M199-grown cells. However, DMEM-cultured cells did not have a greater rate of myxothiazole-resistant respiration, suggesting that this increase in H2O2 release comes from sources other than XO. These results show that cellular XO does not contribute significantly to basal H2O2 production in bovine endothelial cells. Analysis of XDH + XO activity of endothelial cells derived from vessels of various species showed a relatively low specific activity of this potential oxidant source in human-derived cells compared with cells cultured from other species such as rodents.