Hydrogen peroxide is the major oxidant product of xanthine oxidase

Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) in inflammatory disease. Focus, however, has centered almost exclusively on XO-derived superoxide (O₂^•-), whereas direct H₂O₂ production from XO has been less well investigated. Therefore, we examined the relative quantities of O₂^•- and H₂O₂ produced by XO under a range (1-21%) of O₂ tensions. At O₂ concentrations between 10 and 21%, H₂O₂ accounted for ∼75% of ROS production. As O₂ concentrations were lowered, there was a concentration-dependent increase in H₂O₂ formation, accounting for 90% of ROS production at 1% O₂. Alterations in pH between 5.5 and 7.4 did not affect the relative proportions of H₂O₂ and O₂^•- formation. Immobilization of XO, by binding to heparin-Sepharose, further enhanced relative H₂O₂ production by ∼30%, under both normoxic and hypoxic conditions. Furthermore, XO bound to glycosaminoglycans on the apical surface of bovine aortic endothelial cells demonstrated a similar ROS production profile. These data establish H₂O₂ as the dominant (70-95%) reactive product produced by XO under clinically relevant conditions and emphasize the importance of H₂O₂ as a critical factor when examining the contributory roles of XO-catalyzed ROS in inflammatory processes as well as cellular signaling.