Flow-injection chemiluminometric assay of superoxide dismutase

A rapid and sensitive FIA method for superoxide dismutase (SOD) activity was developed using the chemiluminescence of lucigenin, which is specific to superoxide anion (O2-). The immobilized xanthine oxidase in a reactor was used to generate O2- and hypoxanthine was selected as the substrate. In the absence of SOD, the maximum amount of lucigenin reduced with O2- is observed (control). The presence of SOD suppresses the reduction of lucigenin. Therefore, the suppression ratio against the control can be regarded as being the inhibition ratio of each sample. Under the optimum conditions, the concentration of the SOD preparation giving 50% inhibition (IC50) was <200ng/ml (about 3.6ng per injection) and the sampling frequency was about 60 samples/h. No tendency of the response to decrease was recognized by 200 repetitive injections. As another approach, a double-line flow system was constructed in which a carrier stream containing hypoxanthine and lucigenin was fed into the enzyme reactor after confluence with a sample stream. Optimized conditions were examined and the performance was compared with that of the above single line system. When the SOD activity in erythrocytes of rats was determined by the present FIA method, the values were linearly related to those obtained by the conventional nitroblue tetrazolium (NBT) assay (r= 0.980, n=19).