Hydrogen peroxide release from human polymorphonuclear leukocytes measured with horseradish peroxidase and o-dianisidine. Effect of various stimulators and cytochalasin B.

A simple, rapid and inexpensive method (o-DD method) is described for the measurement of hydrogen peroxide released by human polymorphonuclear leukocytes (PMNL) stimulated with phorbol myristate acetate (PMA), n-formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A). The method is based on the horseradish peroxidase-catalysed oxidation of o-dianisidine by H2O2 which results in the formation of a compound exhibiting an increased absorbance at 470 nm. A linear relationship between the absorbance at 470 nm and the concentration of H2O2 was found in the 1-150 microM range. Using this assay the time course of H2O2 release by PMNL, the dependence of H2O2 release on cell number, the agonist concentration and the presence of cytochalasin B (4.8 micrograms/ml) were studied. The maximal PMNL H2O2 response was found for PMA at 10 ng/ml. Con A at 200 micrograms/ml, FMLP at 300 ng/ml and reached 39 +/- 1.5, 14 +/- 1.2, 2.2 +/- 0.7 nmol for 10(6) cells incubated for 60 min at 37 degrees C. respectively. FMLP under these conditions was a most potent stimulator of myeloperoxidase release (MPO). Cytochalasin B having a weak influence on FMLP- and Con A-mediated H2O2 production enhanced strongly their stimulatory effect on MPO release. It is suggested that measurement of the H2O2 release with the o-DD method could be useful for monitoring PMNL respiratory burst especially in the cases of low MPO release.