Sensitive quantitation of nitric oxide by EPR spectroscopy

A recent method for NO detection is electron paramagnetic resonance (EPR) with ferrous and mononitrosyl dithiocarbamate (Fe2- (DETC)2) for spin trapping [Menon, N. K., et al., J. Mol. Cell Cardiol., 23:389; 1991]. However, by this technique, we failed to detect the spectrum of the NOFe2+ (DETC)2 complex in biological systems because of the low solubility of Fe2+ (DETC)2 and rapid oxidation of the NOFe2+ (DETC)2 complex. To overcome these problems, we modified this method by adding albumin to solubilize Fe2+ (DETC)2 and Na2S2O4 as a strong reductant to increase the sensitivity and stability of the EPR spectrum of the NOFe2+ (DETC)2 complex. The optimal concentrations of these reagents were 3.3 mM of Fe2+ and DETC, 33 mg/ml albumin and 2 M Na2S2O4. The detection limit was less than 10 μmol/ml under these conditions. By this modified method, we succeeded in quantifying NO production from porcine aorta induced by forskolin.