TY - JOUR
T1 - Detection of phagocyte-derived free radicals with spin trapping techniques
T2 - Effect of temperature and cellular metabolism
AU - Rosen, Gerald M.
AU - Britigan, Bradley E.
AU - Cohen, Myron S.
AU - Ellington, Sharon P.
AU - Barber, Michael J.
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health, AM-3322, AI-15036, HL-33550 and AI-107001; the Knights Templer Eye Foundation and the American Cancer Society, BC-453. B.E. Britigan is a recipient of the Burroughs Welcome Fellowship of the Infectious Disease Society of America.
PY - 1988/5/13
Y1 - 1988/5/13
N2 - Human neutrophils activated with either particulate or soluble stimuli generate oxygen-centered free radicals which are detected by spin trapping in conjunction with electron spin resonance (ESR) spectroscopy. We investigated the effect of temperature on ESR spectra resulting from stimulation of human neutrophils with phorbol myristate acetate (PMA) or opsonized zymosan in the presence of the spin trap, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). At 20°C with either stimuli, neutrophil superoxide production was manifested predominantely as the superoxide spin-trapped adduct, 5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH). In contrast, at 37°C, the hydroxyl spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH) was dominant. No evidence of hydroxyl radical (defined as the methyl spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy, DMPO-CH3) was observed, suggesting that elevated temperatures increased the rate of DMPO-OOH conversion to DMPO-OH. In addition, the elevated temperature activated a neutrophil reductase which accelerated the rate of DMPO-OH reduction to its corresponding hydroxylamine, 2,2-dimethyl-5-hydroxy-1-hydroxypyrrolidine. This bioreduction was dependent upon the presence of both superoxide and a phagocyte-derived factor (possibly a thiol) released into the surrounding media.
AB - Human neutrophils activated with either particulate or soluble stimuli generate oxygen-centered free radicals which are detected by spin trapping in conjunction with electron spin resonance (ESR) spectroscopy. We investigated the effect of temperature on ESR spectra resulting from stimulation of human neutrophils with phorbol myristate acetate (PMA) or opsonized zymosan in the presence of the spin trap, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). At 20°C with either stimuli, neutrophil superoxide production was manifested predominantely as the superoxide spin-trapped adduct, 5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH). In contrast, at 37°C, the hydroxyl spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH) was dominant. No evidence of hydroxyl radical (defined as the methyl spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy, DMPO-CH3) was observed, suggesting that elevated temperatures increased the rate of DMPO-OOH conversion to DMPO-OH. In addition, the elevated temperature activated a neutrophil reductase which accelerated the rate of DMPO-OH reduction to its corresponding hydroxylamine, 2,2-dimethyl-5-hydroxy-1-hydroxypyrrolidine. This bioreduction was dependent upon the presence of both superoxide and a phagocyte-derived factor (possibly a thiol) released into the surrounding media.
KW - (Human)
KW - ESR
KW - Free radical
KW - Phagocyte stimulation
KW - Superoxide generation
KW - Temperature dependence
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U2 - 10.1016/0167-4889(88)90057-2
DO - 10.1016/0167-4889(88)90057-2
M3 - Article
C2 - 2835986
AN - SCOPUS:0023929881
SN - 0167-4889
VL - 969
SP - 236
EP - 241
JO - BBA - Molecular Cell Research
JF - BBA - Molecular Cell Research
IS - 3
ER -