Generation of superoxide by xanthine oxidase can be stimulated under ischemic and aberrant calcium homeostasis. Given that patients and mice with Duchenne muscular dystrophy (DMD) are affected by ischemia and excessive calcium influx, this study tested the hypothesis that xanthine oxidase activity is elevated and contributes to disease pathology. Xanthine oxidase activity was measured by urinary isoxanthopterin in DMD patients at rest and in response to exercise. Urinary isoxanthopterin/creatinine was elevated compared to age-matched controls and Becker muscular dystrophy (BMD) patients. Concentrations were also increased after a six-minute walk test in ambulatory patients. Urinary isoxanthopterin was also measured in wildtype mice and a number of dystrophic mouse models, including the DMD mouse model (mdx) and the β-sarcoglycan deficient (Scgb-/-) mouse which represents type 2E human limb-girdle muscular dystrophy (LGMD). Mdx and Scgb-/-mice had greater urinary isoxanthopterin/creatinine than wildtype mice while mdx mice expressing dystrophin or utrophin linking the extracellular matrix to the actin cytoskeleton were not different than wildtype. Higher levels of urinary ortho-tyrosine were also measured in humans and mice deficient for dystrophin to confirm elevated oxidative stress. Downhill treadmill running caused significant increases in mdx urinary isoxanthopterin that was prevented with the xanthine oxidase inhibitor allopurinol. The data from this study suggest hyper-activity of xanthine oxidase in DMD, identifies xanthine oxidase activity as a contributing factor in eccentric contraction-induced force drop of dystrophin-deficient skeletal muscle and highlights the potential of isoxanthopterin as a noninvasive biomarker in DMD.
