Abstract
Electron spin resonance spin-trapping methods were used to investigate the free radical production kinetics of neutrophils stimulated with phorbol myristate acetate (PMA) and opsonized zymosan (OPZ). Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, the principle spin adduct observed is DMPO-OH (trapped hydroxyl radical). The DMPO-OH ESR signal amplitude was observed to decay exponentially. In such cases a simple method may be used to analyze the raw kinetics amplitude data to yield true production rate and net production data. The method, pitfalls, and self-consistency criteria are illustrated with PMA and OPZ-stimulated neutrophils at 25 and 37 degrees C under varying oxygen tensions, and with noise-free simulated data. The simulations demonstrate that rate results are relatively insensitive to the precise choice of decay time constant, tc, while net production results are very sensitive to the choice of tc used to analyze the raw data. OPZ (0.6-2.4 mg/ml) yields a strong, sharp neutrophil burst which peaks in 2 min or less while PMA yields a slower burst which peaks in 3.4-14 min for PMA concentrations of 500-50 ng/ml, respectively. Increased oxygen tension during the PMA experiments increased the spin adduct lifetime. The methods presented are applicable to other cell systems or spin adducts which exhibit first order decay.
Highlights
Spin trap5,5-dimethyl-1-pyrroline-N-oxide,the prin- havein the production of OH'.With ESR detection it is ciple spin adduct observedis DMPO-OH'.The DMPO-OH ESR signal amplitude was observed to decay exponentially
Itis applied ygen species including superoxide anions and hydrogen per- to severaltypical cases including neutrophils stimulated with oxideare generatedthat play an important role in thefunction opsonized zymosan(OPZ)and with phorbol myristate acetate of neutrophils
Hydroxyl radicals (OH')' may be pro- (PMA) at both 25 and 37 "Cto demonstrate the utility of the duced in this process, and there isa substantial interest and technique
Summary
Spin trap5,5-dimethyl-1-pyrroline-N-oxide,the prin- havein the production of OH'.With ESR detection it is ciple spin adduct observedis DMPO-OH' (trappedhy- argued that decaying superoxideadduct may be the principal droxyl radical).The DMPO-OH ESR signal amplitude was observed to decay exponentially In such cases a simple method mabye used to analyzethe raw kinetics amplitude data to yield true production rate and net production data. The source of the hydroxyl adduct (12, 18).None of the methods are ideal;the ESR spin trap method offerssome specific advantages despite the difficulties of interpretation Because it is a spectroscopictechnique capable of simultaneously detecting several radical species, e.g. OH ' , OX, and others ( E ) , with good time resolution (10, 11, 19) it has the potential to clarify some of the complex biochemical issues simulations demonstrate thraatte resultsarerelatively concerningthe existenceand role of OH' in activated neutroinsensitiveto the precise choice of decay time conspthailnst., t,, while net production results are very sensitive to Studies of neutrophil-generated free radicals have all utithe choice otf, used to analyzethe raw dataO. Several methods have been used to detect OH' direct comparison with other conventional assays of neutroincluding the formation of ethylene from methional (2-4) or phil activity such as net superoxide production via reduction 2-keto-4-thiomethylbutyricacid (5),the formation of I4CO2 of cytochrome e
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