FAST AND SENSITIVE CHEMILUMINESCENCE DETERMINATION OF H2O2 CONCENTRATION IN STIMULATED HUMAN NEUTROPHILS

A fast and sensitive chemiluminescence assay for the determination of H2O2 in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide. Changes in H2O2 concentration in PMA-stimulated neutrophils were followed by injection of NaOCl to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H2O2 without influence of H2O2 decomposition by cellular enzymes or newly produced H2O2 due to dismutation of superoxide anion radicals. Concentrations of H2O2 were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H2O2 concentration upon stimulation could be observed at 3000 cells/mL.