Limitations of Electron Spin Resonance (ESR) Spectroscopy in the Superoxide Dismutase Assay of Blood
We have employed the electron spin resonance spin-trapping method to determine the superoxide dismutase (SOD) activity in plasma and erythrocytes, and investigated various possible obstacles associated with detection of the superoxide radical derived from the hypoxanthinexanthine oxidase system. With the exception of SOD itself, ascorbate, present in plasma at 40-140 μM, diminished the peak for the DMPO-OOH spin adduct most remarkably. Its 50% inhibitory concentration was 115 μM. Cysteine, reduced glutathione (GSH), urate, NADH and 5'-ATP also showed remarkable diminishing effects, although their concentrations in blood were too low to affect the DMPO-OOH spin adduct. Sugars had little or no effect. Hemoglobin and serum albumin showed a significant effect above a concentration of 1.5 and 5 mg/ml, respectively. In addition, ascorbate and albumin each showed a dose-dependent diminishing effect when 1.5 units/ml of SOD was used simultaneously with ascorbate or albumin. Therefore, the presence of ascorbate and albumin was not negligible when a slight amount of SOD activity in plasma was determined directly. The effect of hemoglobin, however, was overcome by dilution because the activity in erythrocytes was sufficiently high.
