Tsuchiya K, Takasugi M, Minakuchi K, Fukuzawa K
Department of Pharmacy, Tokushima University Hospital, Japan.
Free Radic Biol Med. 1996;21(5):733-7. doi: 10.1016/0891-5849(96)00221-3.
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.
一种最近用于检测一氧化氮（NO）的方法是利用亚铁和单亚硝基二硫代氨基甲酸盐（Fe2+(DETC)2）进行自旋捕获的电子顺磁共振（EPR）技术[梅农，N.K.等人，《分子与细胞心脏学杂志》，23:389；1991年]。然而，通过这种技术，我们未能在生物系统中检测到NOFe2+(DETC)2复合物的光谱，这是因为Fe2+(DETC)2的低溶解度以及NOFe2+(DETC)2复合物的快速氧化。为克服这些问题，我们对该方法进行了改进，通过添加白蛋白来溶解Fe2+(DETC)2，并添加连二亚硫酸钠（Na2S2O4）作为强还原剂，以提高NOFe2+(DETC)2复合物EPR光谱的灵敏度和稳定性。这些试剂的最佳浓度分别为3.3 mM的Fe2+和DETC、33 mg/ml白蛋白以及2 M的Na2S2O4。在这些条件下，检测限低于10 pmol/ml。通过这种改进方法，我们成功地定量检测了由福司可林诱导的猪主动脉中NO的生成量。
