Measurement and characterization of postischemic free radical generation in the isolated perfused heart

J. L. Zweier, P. Kuppusamy, R. Williams, B. K. Rayburn, B Douglas Smith, Myron Weisfeldt, J. T. Flaherty

Research output: Contribution to journalArticle

Abstract

Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the post-ischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g(parallel) = 2.033 and g(perpendicular) = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and a(N) = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.

Original languageEnglish (US)
Pages (from-to)18890-18895
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number32
StatePublished - 1989

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Free Radicals
Deferoxamine
Ischemia
Oxides
Superoxide Dismutase
Reperfusion
Tissue
Spin Trapping
Electron Spin Resonance Spectroscopy
Mannitol
Paramagnetic resonance
Spectrum Analysis
Myocardium
Nitrogen
Spectroscopy
Rabbits
Temperature
pyrroline
5,5-dimethyl-1-pyrroline-1-oxide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zweier, J. L., Kuppusamy, P., Williams, R., Rayburn, B. K., Smith, B. D., Weisfeldt, M., & Flaherty, J. T. (1989). Measurement and characterization of postischemic free radical generation in the isolated perfused heart. Journal of Biological Chemistry, 264(32), 18890-18895.

Measurement and characterization of postischemic free radical generation in the isolated perfused heart. / Zweier, J. L.; Kuppusamy, P.; Williams, R.; Rayburn, B. K.; Smith, B Douglas; Weisfeldt, Myron; Flaherty, J. T.

In: Journal of Biological Chemistry, Vol. 264, No. 32, 1989, p. 18890-18895.

Research output: Contribution to journalArticle

Zweier, JL, Kuppusamy, P, Williams, R, Rayburn, BK, Smith, BD, Weisfeldt, M & Flaherty, JT 1989, 'Measurement and characterization of postischemic free radical generation in the isolated perfused heart', Journal of Biological Chemistry, vol. 264, no. 32, pp. 18890-18895.
Zweier, J. L. ; Kuppusamy, P. ; Williams, R. ; Rayburn, B. K. ; Smith, B Douglas ; Weisfeldt, Myron ; Flaherty, J. T. / Measurement and characterization of postischemic free radical generation in the isolated perfused heart. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 32. pp. 18890-18895.
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abstract = "Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the post-ischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g(parallel) = 2.033 and g(perpendicular) = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and a(N) = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.",
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AU - Weisfeldt, Myron

AU - Flaherty, J. T.

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AB - Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the post-ischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g(parallel) = 2.033 and g(perpendicular) = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and a(N) = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.

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