Abstract
In view of the important role of superoxide in cellular injury, there has been a great need for methods suitable for quantitation of superoxide production from cells. Previous methods have had limited sensitivity or specificity as well as problems with side reactions in cellular systems. Recently, we have shown that the new spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide has ideal properties for quantitative superoxide measurement in chemical/biochemical systems; however, its suitability and potential for measurements in cellular systems has not been determined. Therefore, we evaluated the use of DEPMPO for quantitative measurement of superoxide formed by polymorphonuclear leukocytes. After activation of these cells with the phorbol ester (PMA, 200 ng/ml) or opsonized zymosan (1 mg/ml) at 24°C a strong signal of the superoxide adduct, DEPMPO–OOH, was observed. This technique was highly sensitive and enabled measurement of superoxide generation from as few as 2 × 103cells. The kinetics of adduct formation and decay were measured which enabled quantitation of superoxide formation. Spin label electron paramagneticresonance (EPR) oximetry was used to measure the oxygen consumption from these cells. With PMA activation rapid onset of superoxide generation occurred with a rate of 0.78 nmol/min/106cells while with zymosan a slower gradual onset of activation was seen to a peak rate of 0.061 nmol/min/106cells. With both stimulators the ratios of superoxide production to oxygen consumption were similar with values of approximately 50% obtained. Thus, EPR spin trapping with DEPMPO together with EPR oximetry methods can be used to provide sensitive and specific quantitation of cellular superoxide generation and oxygen consumption. These methods provide a promising new approach for the measurement of oxygen reductionand superoxide generation in cellular systems.
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