Is xanthine oxidase an alternate source for the generation of nitric oxide during in vivo hypoxia

Nitric oxide production by inducible Nitric Oxide Synthase is dependent on the availability of arginine and molecular oxygen. During periods of hypoxia, molecular oxygen is limited in nitric oxide generation. Recent studies have determined that there are alternate pathways that contribute to nitric oxide generation during hypoxia in vitro. One such alternate pathway involves xanthine oxidase, a primary producer of toxic oxygen metabolites during ischemia. It has been demonstrated in other studies that xanthine oxidase can generate nitric oxide during hypoxia in vitro (Millar, et al., 1997, 1998; Godber, et al., 2000, Doel, et al. 2000). It is the purpose of the present hypothesis to determine if xanthine oxidase is a potential alternate source for nitric oxide generation in an in vivo model of hypoxia and reoxygenation.; Male Sprague-Dawley rats (275–300g) were subjected to 10% FIO ₂ for 30 minutes. Lipopolysaccharide (10mg/kg) and allopurinol (50mg/kg), an inhibitor of xanthine oxidase activity, were given to experimental animals intraperitoneally 30 minutes prior to hypoxia. A control group received lipopolysaccharide and allopurinol intraperitoneally without hypoxia. Animals were sacrificed immediately after hypoxia or after four hours in room air. Liver, proximal jejunum and spleen were harvested and flash frozen in liquid nitrogen until use. Nitric oxide concentrations were determined. Xanthine oxidase activity was determined spectrophotometrically. Reverse transcriptase polymerase chain reaction and Western blotting were performed to determine the presence or absence of inducible nitric oxide synthase. A statistical significance with a p-value ≤ 0.05 was demonstrated using the non-parametric analyses of Kruskal-Wallis ANOVA, Wilcoxin matched pairs and Spearman R sets.; Comparisons of the data indicate that, although allopurinol significantly reduced xanthine oxidase activity, it had no effect on the presence or absence of nitric oxide. These results indicate that xanthine oxidase is not an alternate source of nitric oxide generation during in vivo hypoxia. Further study is necessary to determine the sources of nitric oxide production during conditions of low oxygen tension.