Measurement and Characterization of Superoxide Generation from Xanthine Dehydrogenase: A Redox-Regulated Pathway of Radical Generation in Ischemic Tissues

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, •O₂⁻, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of •O₂⁻, experiments were performed to measure and characterize •O²⁻ generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of •O₂⁻. NAD⁺ and NADH inhibited the generation of •O₂⁻ from XDH in a dose-dependent manner, with NAD⁺ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD⁺ and NADH, which occur during and following tissue ischemia, enhanced the generation of •O₂⁻ from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca²⁺-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD⁺ and NADH. Thus, XDH can be an important redox-regulated source of •O₂⁻ generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury.