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.
Nowak
D
D
Department of Pneumonology and Allergology, Medical Academy Kopcińskiego, Lódź, Poland.
D003208:Concanavalin A / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology*; D003962:Dianisidine; D006735:Horseradish Peroxidase; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009195:Peroxidase; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003208:Concanavalin A; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D009195:Peroxidase; D003962:Dianisidine; D013755:Tetradecanoylphorbol Acetate