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 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 pmol/ml under these conditions. By this modified method, we succeeded in quantifying NO production from porcine aorta induced by forskolin.
Tsuchiya
K
K
Department of Pharmacy, Tokushima University Hospital, Japan.
Takasugi
M
M
Minakuchi
K
K
Fukuzawa
K
K
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002478:Cells, Cultured; D004050:Ditiocarb / Q000031:analogs & derivatives; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004730:Endothelium, Vascular / Q000378:metabolism; D005296:Ferrous Compounds; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000032:analysis* / Q000096:biosynthesis; D013113:Spin Labels; D013552:Swine
D005296:Ferrous Compounds; D013113:Spin Labels; C085124:diethyldithiocarbamate iron(II); D009569:Nitric Oxide; D004050:Ditiocarb