QUANTITATIVE MEASUREMENT OF SUPEROXIDE GENERATION AND OXYGEN-CONSUMPTION FROM LEUKOCYTES USING ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY

In view of the important role of superoxide in cellular injury, there has been a great need for methods suitable for quantitation of superox ide production from cells, Previous methods have had limited sensitivi ty or specificity as well as problems with side reactions in cellular systems, Recently, we have shown that the new spin trap -(diethoxyphos phoryI)-5-methyl-1-pyrroline-N-oxide has ideal properties for quantita tive 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 qu antitative measurement of superoxide formed by polymorphonuclear leuko cytes. After activation of these cells with the phorbol ester (PMA, 20 0 ng/ml) or opsonized zymosan (1 mg/ml) at 24 degrees C a strong signa l of the superoxide adduct, DEPMPO-OOH, was observed, This technique w as highly sensitive and enabled measurement of superoxide generation f rom as few as 2 x 10(3) cells, The kinetics of adduct formation and de cay were measured which enabled quantitation of superoxide formation, Spin label electron paramagnetic resonance (EPR) oximetry was used to measure the oxygen consumption from these cells, With PMA activation r apid onset of superoxide generation occurred with a rate of 0.78 nmol/ min/10(6) cells while with zymosan a slower gradual onset of activatio n was seen to a peak rate of 0.061 nmol/min/10(6) cells, With both sti mulators the ratios of superoxide production to oxygen consumption wer e similar with values of approximately 50% obtained, Thus, EPR spin tr apping with DEPMPO together with EPR oximetry methods can be used to p rovide sensitive and specific quantitation of cellular superoxide gene ration and oxygen consumption, These methods provide a promising new a pproach for the measurement of oxygen reduction and superoxide generat ion in cellular systems, (C) 1998 Academic Press.