7] Measurement of nitric oxide and hydrogen peroxide production from human neutrophils

Abstract

Publisher Summary This chapter discusses the measurement of nitric oxide (NO) and hydrogen peroxide production from human neutrophils. Human neutrophils, polymorphonucleocytes (PMNs) exposed to soluble or particulate stimuli largely increase cyanide-insensitive oxygen consumption with the production of reactive oxygen species in a process termed the respiratory burst, which seems responsible for the microbicidal activity of PMNs. Circulating human neutrophils release NO when they are exposed to appropriate stimuli by activation of a constitutive nitric oxide synthase (NOS), while inflammatory neutrophils appear to express an inducible NOS. The NO and superoxide anion (O 2 – ) are able to interact to form peroxynitrite 1° and other reactive species that apparently contribute to host defense against infections. NO production is measured in a double beam-double wavelength 356 Perkin–Elmer spectrophotometer. A direct measurement of released NO can be made with Clark-type nitric oxide selective electrodes. Neutrophils release NO after stimulation either with phorbol esters such as phorbol myristate acetate (PMA) or with formylmethionylleucylphenylalanine (fMLP); typical traces showing the spectrophotometric changes in performing the oxymyoglobin assay. The detection of nitrite (and nitrate) is useful for high cell concentrations such as those in culture.