Development of a simultaneous multiple measurement method for superoxide generation from phagocytes using the cytochrome c reduction method.

It is important to measure superoxide generation in biological samples in research concerning reactive oxygen, because most reactive oxygen species biologically generated are derived from superoxides.This paper reports the establishment of a simultaneous multiple measurement method for detection of superoxides generated from phagocytes, human peripheral neutrophils and rat alveolar macrophages, using cvtochrome c reduction with stirring at 37°C. Using this method, the following results were obtained.1. For more objective presentation of obtained results, velocities of cytochrome c reduction were calculated by the linear regression method at each measurement point and new indicators of cytochrome c reduction, peak value (maximum reduction velocity) and peak time (time when peak value was observed), were introduced.2. Cytochrome c reduction was measured by dual wavelengths, 550nm and 492nm, under the conditions mentioned below.Final sample volume was 300 μl and phagocytes, 1.5 x 10(5) human neutrophils or 3.0 x 10(5) rat macrophages, were mixed with 2.0mg/ml of opsonized zymosan in the presence of 150 μM cytochrome c.3. In the case of human neutrophils, a small amount of cytochrome c reduction was induced by reactions with the microplate surface, whereas in the case of rat macrophages, which can phagocytize materials without opsonization, it was expected that substantial cytochrome c reduction would be observed. However, in comparison with the results of opsonized zymosan stimulation, peak value reached only 40% and peak time was significantly delayed.4. Peak value showed significant linear changes corresponding with changes of the opsonized zymosan concentration and phagocyte numbers when measurements were carried out 37°C.These results suggest that differences of phagocyte activity and serum opsonic activity, which are difficult to measure using previous methods, are clearly detected by the simultaneous measurement method and the indicators introduced in this paper.