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

Bradley E Britigan, T. J. Coffman, G. R. Buettner

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Abstract

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 used the spin trap N-t-butyl-α-phenylnitrone (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.

Original languageEnglish (US)
Pages (from-to)2650-2656
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number5
StatePublished - Apr 2 1990

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Spin Trapping
Macrophages
Phagocytes
Superoxides
Hydroxyl Radical
Respiration
Neutrophils
Dimethyl Sulfoxide
Monocytes
Paramagnetic resonance
Electron Spin Resonance Spectroscopy
Iron
Zymosan
Reaction products
Catalase
Free Radicals
Reactive Oxygen Species
Acetates
Air
Experiments

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "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",
abstract = "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 used the spin trap N-t-butyl-α-phenylnitrone (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.",
author = "Britigan, {Bradley E} and Coffman, {T. J.} and Buettner, {G. R.}",
year = "1990",
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T1 - 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

AU - Britigan, Bradley E

AU - Coffman, T. J.

AU - Buettner, G. R.

PY - 1990/4/2

Y1 - 1990/4/2

N2 - 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 used the spin trap N-t-butyl-α-phenylnitrone (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.

AB - 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 used the spin trap N-t-butyl-α-phenylnitrone (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.

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