Effect of chelating agents and superoxide on human neutrophil NAD(P)H oxidation

Abstract

NAD(P)H oxidation is frequently measured to assay the activity of the neutrophil O 2 −-generating oxidase. It was found that 10 −4 m ethylene glycol bis (β-aminoethyl ether)- N-N′-tetraacetic acid (EGTA) increased NAD(P)H oxidation by the 27,000g granule fraction of resting and stimulated human neutrophils without altering net O 2 − production. The commonly used chelating agents EDTA and diethylene triamine pentaacetic acid had similar effects. The addition of superoxide dismutase eliminated the effect of the chelating agents and thus demonstrated that the stimulated reaction was dependent upon O 2 −. KCN and bathophenanthroline disulfonate, an iron-chelating agent, prevented O 2 −-dependent NADPH oxidation by neutrophil granule fractions in the presence of EGTA. In contrast, bathocuproine disulfonate, a copper-chelating agent, mimicked the EGTA effect. The effects of both bathophenanthroline disulfonate and bathocuproine disulfonate were completely abolished when the agents were saturated with iron and copper, respectively. All the chelating agents studied, except bathophenonthroline disulfonate, also promoted O 2 −-dependent NADPH oxidation in a system wherein O 2 − was generated by xanthine oxidase. Thus, commonly used chelating agents, by interacting with available iron and copper, may after the apparent stoichiometry of the neutrophil O 2 −-generating oxidase and artifactually increase NADPH oxidation in other systems where O 2 − is present.