[A simple method of measuring superoxide dismutase activity using photochemiluminescence]

At present, photochemical detection of SOD-activity is used in most cases, where superoxide production is based on the NADPH oxidase activity or autooxidation of some substances, for instance, adrenaline. The shortcomings of these assays are the requirement of stable pH and temperature and need long-term experiments and sophisticated preliminary work. For this reason a novel method to determine SOD activity have been proposed, based on the photochemiluminescence (PCL), where riboflavine is used as a photosensitizer and lucigenin as a free-radical detector. Minimal concentrations of the system components have been selected, at which PCL intensity became virtually independent on the concentration and was sufficient for proper measurements: 25 nmole/ml of riboflavine, 3 nmole/ml of lucigenin and 250 nmole/ml of methionine. Under this conditions, the amount of SOD causing two-fold inhibition of PCL was 10 +/- 0.3 ng of SOD (Sigma, USA). Antioxidants occurring in the blood changed the PCL intensity by 5% at following concentrations: ascorbic acid--above 3.6 microM, uric acid--above 0.3 microM, glutathione--above 4.0 microM. Taking into account the amount of these antioxidants in whole blood and erythrocytes, it has been calculated that the antioxidants containing in the sample do not influence the results of SOD assay, even without erythrocytes washing. The proposed method was compared with a procedure offered by Calbiochem. The amounts of SOD causing two-fold decrease of PCL in our method and that of Calbiochem, were 10 and 40 ng (Sigma, USA), and standard deviations 0.45 and 0.96%, respectively. The SOD activity from 107 healthy donors (76 males and 31 females, 40 smokers and 67 non-smokers) has been determined and was found to be 15.1 +/- 0.4 ng SOD/1 x 10(9) erythrocytes.