title: Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a Ficoll-Hypaque gradient.
abstract: Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O(2).(-), H(2)O(2) and ONOO(-)) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H(2)O(2) reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results.
authors.affiliation: Departmento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo (USP), Brazil.
authors.forename: I M
authors.initials: IM
authors.lastname: Rebecchi
authors.forename: N
authors.initials: N
authors.lastname: Ferreira Novo
authors.forename: Y
authors.initials: Y
authors.lastname: Julian
authors.forename: A
authors.initials: A
authors.lastname: Campa
mesh_terms: D000166:Acridines / Q000378:metabolism; D001799:Blood Sedimentation; D002624:Chemistry, Clinical / Q000379:methods*; D003571:Cytochalasin B / Q000494:pharmacology; D003973:Diatrizoate / Q000737:chemistry*; D005362:Ficoll / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008163:Luminescent Measurements; D008165:Luminol / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology; D009195:Peroxidase / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015203:Reproducibility of Results; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
chemical_list: D000166:Acridines; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D003973:Diatrizoate; C033472:10,10'-dimethyl-9,9'-biacridinium; D005362:Ficoll; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008165:Luminol; D015054:Zymosan; D006861:Hydrogen Peroxide; D009195:Peroxidase; D013755:Tetradecanoylphorbol Acetate