Oxidative stress measurement by in vivo electron spin resonance spectroscopy in rats with streptozotocin-induced diabetes

Enhanced oxidative stress in diabetic patients may contribute to the pathogenesis of diabetic angiopathy. We have recently developed a method to determine the electron spin resonance (ESR, electron paramagnetic resonance; EPR) of reactive oxygen species and free radicals in vivo, using the nitroxide derivative, carbamoyl-PROXYL as a probe. In this study, diabetes was induced in Wistar rats by streptozotocin (STZ) injection (65 mg/kg. body weight, intravenously). Two, 4, and a weeks later, the animals received carbamoyl-PROXYL (300 nmol/g, intravenously), and ESR was measured at the upper abdominal level at a frequency of 300 MHz. The intensity of the carbamoyl-PROXYL ESR signal decreased gradually after the injection, and the spin clearance fate was determined over the first 5 min. At all time points, the spin clearance rate was significantly greater in the diabetic rats than in control rats. Moreover, the spin clearance rate in the diabetic rats was significantly correlated with urinary malondialdehyde (MDA) levels, which serve as a marker for lipid peroxidation. Daily treatment with 4 units neutral protamin Hagedorn (NPH) insulin for 4 weeks reduced the spin clearance rate in the diabetic rats. Simultaneous injection of carbamoyl-PROXYL and superoxide dismutase reduced the spin clearance rate in the diabetic rats in a dose-dependent manner. Injection of the antioxidant a-tocopherol (40 mg/kg, intraperitoneally) for 2 weeks restored the spin clearance rate in the diabetic rats without concomitant glycaemic restoration. These results suggest that a diabetic state enhances the generation of free radicals in vivo, and that both glycaemic control and antioxidant treatment can reduce this oxidative stress. Non-invasive in vivo ESR measurement may be useful for evaluating oxidative stress in diabetes.