Electron spin resonance assay of ascorbate free radicals in Vivo

This chapter describes the electron spin resonance (ESR) assay of ascorbate radicals in vivo in the rat blood circulation. The free radicals generated by ferric citrate and ascorbic acid in vivo are analyzed at room temperature without using any labeling or trapping agents. The signals of the doublet peak spectrum of ascorbate are confirmed in the following manner. First, the g value and hyperfine coupling constant (a_H) of the doublet peaks are identical with those of ascorbate itself. Second, ferric citrate alone only induces transient peaks, and third, the injection of ascorbate after ferric citrate dramatically increases the peak height, whereas large doses of ascorbate alone are able to induce similar (but weaker and transient) signals, which is consistent with in vitro observations with human serum. Moreover, ascorbate oxidase completely inhibits the signals of the radicals. In the experiment involving simultaneous injection of ferric citrate and ascorbate, frequently the animals die (death rate 50%), suggesting the toxicity of iron-induced free radicals in biological systems.