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 develope d. The method is based on the oxidation of luminol by hypochlorous aci d. The chemiluminescence of this reaction is highly dependent on the c oncentration of hydrogen peroxide. Changes in H2O2 concentration in PM A-stimulated neutrophils were followed by injection of NaOCl to cell s uspension at different times after cell stimulation. The short integra tion time of 2 s permits calculation of actual concentrations of H2O2 without influence of H2O2 decomposition by cellular enzymes or newly p roduced H2O2 due to dismutation of superoxide anion radicals. Concentr ations of H2O2 were diminished by catalase and enhanced by sodium azid e owing to inhibition of cellular catalase and myeloperoxidase. Change s in H2O2 concentration upon stimulation could be observed at 3000 cel ls/mL.