An in vivo ESR spin-trapping study: Free radical generation in rats from formate intoxication - role of the Fenton reaction

Electron spin resonance spectroscopy has been used to study free radical ge neration in rats with acute sodium formate poisoning. The in vivo spin-trap ping technique was used with alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (PO BN), which reacts with free radical metabolites to form radical adducts, wh ich were detected in the bile and urine samples from Fischer rats. The use of [C-13]-sodium formate and computer simulations of the spectra identified the 12-line spectrum as arising from the POBN/carbon dioxide anion radical adduct. The identification of POBN/(CO2-)-C-. radical adduct provides dire ct electron spin resonance spectroscopy evidence for the formation of (CO2- )-C-. radicals during acute intoxication by sodium formate, suggesting a fr ee radical metabolic pathway. To study the mechanism of free radical genera tion by formate, we tested several known inhibitors. Both allopurinol, an i nhibitor of xanthine oxidase, and aminobenzotriazole, a cytochrome P450 inh ibitor, decreased free radical formation from formate, which may imply a de pendence on hydrogen peroxide. In accord with this hypothesis, the catalase inhibitor 3-aminotriazole caused a significant increase in free radical fo rmation. The iron chelator Desferal decreased the formation of free radical s up to 2-fold. Presumably, iron plays a role in the mechanism of free radi cal generation by formate via the Fenton reaction. The detection of formate free radical metabolites generated in vivo and the key role of the Fenton reaction in this process may be important for understanding the pathogenesi s of both formate and methanol intoxication.