Reduction of ferricytochrome C may underestimate superoxide production by monocytes

Superoxide production by stimulated phagocytes is commonly measured by reduction of ferricytochrome C, with specificity of the assay assumed if the reaction is inhibited by superoxide dismutase (SOD). Most preparations of ferricytochrome C contain a small proportion in the reduced (ferro) form, and this is also formed by the reaction of ferricytochrome C with superoxide. The generation of other reactive oxygen intermediates, such as hydrogen peroxide or hydroxyl radical, could cause oxidation of ferrocytochrome C and consequent underestimation of superoxide production. In support of this, it has been demonstrated that exogenous catalase enhanced the reduction of ferricytochrome C by phorbol myristate acetate (PMA)-stimulated human monocytes. Control experiments confirmed that this was due to enhanced detection rather than increased production of superoxide. In addition, SOD was found to promote oxidation of ferrocytochrome C by PMA-stimulated human monocytes, but this was also inhibited by catalase. These effects of catalase and SOD on ferricytochrome C reduction/ferrocytochrome C oxidation were also demonstrated when superoxide was produced independently of monocytes by a xanthine and xanthine oxidase generating system. It is concluded that the assay of superoxide, using 'SOD inhibitable' reduction of ferricytochrome C, underestimates superoxide production.