Evaluation and improvements of a rapid microassay for measuring superoxide anion production by phagocytes. 2. Biochemical aspects

Superoxide anion synthesis by stimulated human polymorphonuclear leukocytes (PMNL) may be measured as superoxide dismutase-inhibitable cytochrome C (Cytc) reduction in a rapid microplate assay. However, when determinations are made over a fixed time interval (15 min) a lack of proportionality is observed between the amount of Cytc reduced and the number of cells stimulated. Examination of the reduction kinetics indicated that depressed superoxide detection at higher cell densities might be a consequence of Cytc re-oxidation by later metabolites in the oxidative burst. This was confirmed using dithionite-reduced Cytc as substrate in the assay. A method of analysis which avoids this source of error in determining superoxide anion synthesis is proposed.