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J. Biol. Chem., Vol. 265, Issue 5, 2650-2656, Feb, 1990
BE Britigan, TJ Coffman and GR Buettner
Failure to detect hydroxyl radical (.OH)-derived spin adducts of 5,5-
dimethyl-1-pyrroline N-oxide in electron spin resonance (ESR) spin trapping
experiments has been offered as evidence for the lack of the endogenous
capacity of stimulated human phagocytes (neutrophils, monocytes, and
monocyte-derived macrophages (MDM] to generate .OH. Recent reports that
5,5-dimethyl-1-pyrroline N-oxide spin adducts are unstable in the presence
of superoxide-generating systems such as stimulated neutrophils has raised
concerns regarding the sensitivity of spin trapping techniques for
assessment of phagocyte free radical formation. Consequently, we have
employed a new approach that uses the spin trap
N-t-butyl-alpha-phenyl-nitrone (PBN) and dimethyl sulfoxide. In the
presence of dimethyl sulfoxide and PBN, the formation of .OH via three
different mechanisms in air-saturated aqueous solutions all yielded a
single nitroxide species whose ESR peak amplitude remained stable in the
presence of superoxide (.O2-). This nitroxide, which we have assigned as
PBN/.OCH3, appears to be an oxygen-centered radical derived from the spin
trapping of the reaction product of O2 and methyl radical. When
neutrophils, monocytes, or MDM were stimulated with phorbol 12-myristate
13-acetate or opsonized zymosan in the presence of exogenous iron,
catalase-inhibitable PBN/.OCH3 was the sole nitroxide detected. In the
absence of exogenous iron, no nitroxide was observed, providing evidence
for the lack of the endogenous capacity of neutrophils, monocytes, and MDM
to generate .OH.
Spin trapping evidence for the lack of significant hydroxyl radical production during the respiration burst of human phagocytes using a spin adduct resistant to superoxide-mediated destruction
Department of Internal Medicine, Veterans Administration Medical Center, Iowa City, Iowa.
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