Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical

S. Pou, M. S. Cohen, B. E. Britigan, G. M. Rosen

Research output: Contribution to journalArticle

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Abstract

Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and an excess of dimethyl sulfoxide, we previously reported that in the absence of an exogenous iron catalyst, human neutrophils will not generate hydroxyl radical, manifested as the catalyse-inhibitable methyl radical spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy (DMPO-CH3) (Britigan, B.E., Rosen, G.M., Chai, Y., and Cohen, M.S. (1986) J. Biol. Chem. 261, 4426-4431). However, superoxide destroys the preformed hydroxyl radical spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH), and DMPO-CH3. The present study was undertaken to better resolve the limits of sensitivity of the spin-trapping method. Photolytically generated DMPO-CH3 and DMPO-OH slowly decomposed in the presence of a low flux (1 μM/min) of enzymatically (xanthine/xanthine oxidase)-generated superoxide, but more rapid decomposition of these adducts occurred with higher superoxide flux (5 μM/min). Inclusion of cysteine markedly increased the rate of DMPO-OH and DMPO-CH3 decomposition, masking the effects of superoxide alone. The addition of varying concentrations of superoxide dismutase did not lead to increased formation of DMPO-OH or DMPO-CH3, as should have occurred if these adducts were being destroyed by superoxide. As a positive control, we employed an iron-supplemented system with phorbol 12-myristate 13-acetate-stimulated neutrophils or xanthine/xanthine oxidase to generate DMPO-CH3. Addition of superoxide dismutase increased the magnitude of DMPO-CH3, primarily by increasing the rate of hydrogen peroxide formation, and to a lesser extent by prolonging the half-life of DMPO-CH3. Although spin-trapped adducts can be destroyed by a high concentration of superoxide, or by lower concentrations of superoxide in the presence of thiol-containing compounds, our results demonstrate that such decomposition does not interfere with the ability of the spin-trapping method to detect hydroxyl radical generated by human neutrophils. These data do not support the capacity of neutrophils to generate hydroxyl radical in the absence of an exogenous Haber-Weiss catalyst.

Original languageEnglish (US)
Pages (from-to)12299-12302
Number of pages4
JournalJournal of Biological Chemistry
Volume264
Issue number21
StatePublished - Jan 1 1989

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Spin Trapping
Hydroxyl Radical
Oxides
Limit of Detection
Neutrophils
Superoxides
Xanthine
Xanthine Oxidase
Decomposition
Superoxide Dismutase
pyrroline
Iron
Fluxes
Catalysts
Dimethyl Sulfoxide
Sulfhydryl Compounds
Hydrogen Peroxide
Cysteine
Half-Life
Acetates

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical. / Pou, S.; Cohen, M. S.; Britigan, B. E.; Rosen, G. M.

In: Journal of Biological Chemistry, Vol. 264, No. 21, 01.01.1989, p. 12299-12302.

Research output: Contribution to journalArticle

Pou, S. ; Cohen, M. S. ; Britigan, B. E. ; Rosen, G. M. / Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 21. pp. 12299-12302.
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