Production of oxygen-centered radicals by neutrophils and macrophages as studied by electron spin resonance (ESR).

Neutrophils and macrophages undergo a respiratory burst and an increase in the activity of the hexose monophosphate pathway in response to particulate or soluble agents. The increase in oxygen consumption was found to be associated with the production of oxygen-centered radicals. The ESR technique of spin trapping showed that besides a superoxide spin adduct, a hydroxyl spin adduct is also produced. ESR is considered to be the least ambiguous technique for the detection of free radicals. The spin-trapping agents used for oxygen-centered radical detection are usually nitrones. The most commonly used nitrone is 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which reacts with O2-. to form 5,5-dimethyl-2-hydroperoxypyrroline-N-oxide (DMPO-OOH) and with OH. to form 5,5-dimethyl-2-hydroxypyrroline-N-oxide (DMPO-OH). Although spin-adduct formation is considered to be the most direct technique for the detection of free radicals, some disadvantages are encountered. There has been considerable interest in the isolation of the O2-. generating activity from phagocytic cells. The enzyme can be extracted with deoxycholate and gel filtration indicates that it is a high molecular weight complex. Maximum activity was between pH 7.0 and pH 7.5. The Km value was 15.8 microM for NADPH and 434 micron for NADH, indicating that NADPH is the preferred substrate.