Glyceryl trinitrate (GTN) and pentaerythrityl tetranitrate (PETN) are among the most known organic nitrates that are used in cardiovascular therapy as vasodilators. However, anti-ischemic therapy with organic nitrates is complicated by the induction of nitrate tolerance. When nitrates are metabolized to release nitric oxide (NO), there is considerable coproduction of superoxide radicals in vessels leading to inactivation of NO. However, nitrate-induced increase of superoxide radical formation in vivo has not been reported. In this work, the authors studied the in vivo formation of superoxide radicals induced by treatment with PETN or GTN and determined the antioxidant effect of vitamin C. The formation of superoxide radicals was determined by the oxidation of 1-hydroxy-3-carboxy-pyrrolidine (CP-H) to paramagnetic 3-carboxy-proxyl (CP) using electron spin resonance spectroscopy. CP-H (9 mg/kg intravenous bolus and 0.225 mg/kg per minute continuous intravenous GTN or PETN 130 microg/kg) were infused into anesthetized rabbits. Every 5 min, blood samples were obtained from Arteria carotis to measure the CP formation. Both PETN and GTN showed similar vasodilator effects. Formation of CP in blood after infusions of GTN and PETN were 2.0+/-0.4 microM and 0.98+/-0.23 microM, respectively. Pretreatment with 30 mg/kg vitamin C led to a significant decrease in CP formation: 0.27+/-0.14 microM (vitamin C plus GTN) and 0.34+/-0.15 microM (vitamin C plus PETN). Pretreatment of animals with superoxide dismutase (15,000 units/kg) significantly inhibited nitrate-induced nitroxide formation. Therefore, in vivo infusion of GTN or PETN in rabbits increased the formation of superoxide radicals in the vasculature. PETN provoked a minimal stimulation of superoxide radical formation without simultaneous development of nitrate tolerance. The data suggest that the formation of superoxide radicals induced by organic nitrate correlates with the development of nitrate tolerance. The effect of vitamin C on CP formation leads to the conclusion that vitamin C can be used as an effective antioxidant for protection against nitrate-induced superoxide radical formation in vivo.