Examination of the enzyme assay for NADPH oxidoreductase; application to polymorphonuclear leukocyte superoxide anion generation.

Superoxide anion production in intact polymorphonuclear leukocytes isolated from medically stable pediatric ICU patients was investigated by examining the initial velocity enzyme kinetics of NADPH oxidoreductase, under a variety of experimental conditions. Present findings confirm that enzyme activity requires stimulation of the respiratory burst, and that catalysis followed longitudinally reflects activation, activity, and termination phases. There was no superoxide anion production in unstimulated neutrophils, and only short-lived superoxide generation in neutrophils stimulated by phorbol myristate acetate. Moreover, the ability to activate resting neutrophils was a labile phenomenon, as reflected by an inducible enzyme half-life of 1.2 days. There were no apparent differences in enzyme activity from granulocytes isolated from venous vs. arterial blood. Enzyme activity in intact granulocytes peaked at pH 7 and 30 degrees C, where the ratio of enzyme activity to cell number was constant over the range 0.25 to 2.5 X 10(5) cells per assay. Utilizing the above strictly defined assay conditions, pediatric-derived polymorphonuclear leukocytes induced with phorbol myristate acetate were found to generate superoxide anion at rates equivalent to those in healthy adults.