Electron Spin Resonance Analysis of Superoxide Anion Radical Scavenging Activity with Spin Trapping Agent, Diphenyl-PMPO

Abstract

A given amount of oxygen is always converted into reactive oxygen species (ROS) such as superoxide anion radicals, hydrogen peroxide, or hydroxyl radicals by various enzymatic systems in the human body. The ROS are essential mechanisms against extraneous factors, but excessive amounts cause harmful effects and oxidative damages that would induce various diseases, including cancer, cardiovascular disease, and diabetes. 1,2 Therefore, the ROS scavenging capacities of antioxidative enzymes and/or natural antioxidants have attracted many researchers' attention during the past few decades.2 In particular, the superoxide anion radical scavenging activity (SOSA) has been investigated in a lot of studies, because superoxide anion radical is a precursor of hydroxyl radical which has remarkable reactivity. In order for researchers to evaluate the superoxide dismutase (SOD) activity and the SOSA of natural antioxidants, a number of assays, such as spectrophotometric and chemiluminescence assays, have been developed. Electron spin resonance (ESR) spin-trapping technique is one of them. This technique has been verified to be useful to detect the short live superoxide anion radical at room temperature, and in fact it has been widely used for the investigations of the SOD activity and the SOSA of natural antioxidants. 4,5 There are many spin-trapping agents for superoxide anion radical, including 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and α-phenyl-N-tert-butylnitrone (PBN). 6 Although DMPO is the most common agent in the ESR analysis, it has some limitations. For example, its reaction rate with superoxide anion radical is not so high and it turns yellow with time even at -20˚C in a sealed tube, that is, DMPO is unstable. 7 In addition, its spin adduct with superoxide anion radical decomposes rapidly. 8 In order to overcome the defects of the conventional spin-trapping agents, Shioji et al. synthesized a new spin-trapping agent, 2-(diphenylphosphinoyl)-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (Diphenyl-PMPO, Fig. 1). 9This compound has two phenyl groups attached to phosphoryl-DMPO structure, and such structure is able to enhance the stability of its spin adduct. Indeed the half life of Diphenyl-PMPO spin adduct with superoxide anion radical (Diphenyl-PMPO-OOH, Fig. 1) was 8.3 times longer than that of DMPO. 10 It was also revealed to have a larger rate constant with superoxide anion radical. In addition, the partition coefficient of Diphenyl-PMPO in 1-octanal/aqueous solution was larger than those of DMPO and DEPMPO, suggesting that Diphenyl-PMPO has a high lipophilicity and is useful for the assay of lipophilic antioxidants. 10 Besides, Diphenyl-PMPO is available in solid state and can be stored at room temperature.9 These characteristics of Diphenyl-PMPO are very attractive in the ESR analysis. While Diphenyl-PMPO was already applied to the detection of hydroxyl radical in human erythrocyte ghosts, 11 there is no information on the evaluation of SOSA of antioxidat...

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