Myeloperoxidase produces nitrating oxidants in vivo

Joseph Gaut

Jaeman Byun

Hung Tran

Wendy Lauber

James Carroll

Richard Hotchkiss

Abderrazzaq Belaaouaj

Jay Heinecke

^{Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA}^{Pharmacia Corp., St. Louis, Missouri, USA}^{Department of Anesthesiology,}^{Department of Molecular Microbiology, and}^{Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri, USA}

Address correspondence to: Jay W. Heinecke, Division of Atherosclerosis, Nutrition and Lipid Research, Box 8046, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA. Fax: (314) 362-0811; E-mail: ude.ltsuw.mi@ekcenieh.

Received 2002 Jan 10; Accepted 2002 Apr 5.

Abstract

Despite intense interest in pathways that generate reactive nitrogen species, the physiologically relevant mechanisms for inflammatory tissue injury remain poorly understood. One possible mediator is myeloperoxidase, a major constituent of neutrophils, monocytes, and some populations of macrophages. The enzyme uses hydrogen peroxide and nitrite to generate 3-nitrotyrosine in vitro. To determine whether myeloperoxidase produces nitrating intermediates in vivo, we used isotope dilution gas chromatography/mass spectrometry to quantify 3-nitrotyrosine in two models of peritoneal inflammation: mice infected with Klebsiella pneumoniae and mice subjected to cecal ligation and puncture. Both models developed an intense neutrophil inflammatory response, and the inflammatory fluid contained markedly elevated levels of 3-chlorotyrosine, a marker of myeloperoxidase action. In striking contrast, 3-nitrotyrosine levels rose only in the mice infected with K. pneumoniae. Levels of total nitrite and nitrate were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to cecal ligation and puncture. Levels of 3-nitrotyrosine failed to increase in mice infected with K. pneumoniae that lacked functional myeloperoxidase. Our observations provide strong evidence that myeloperoxidase generates reactive nitrogen species in vivo and that it operates in this fashion only when nitrite and nitrate become available.

Abstract

Acknowledgments

We thank William Parks (Washington University) for assistance with immunohistochemical studies. Mass spectrometry experiments were performed at the Washington University School of Medicine Mass Spectrometry Resource. This work was supported by grants from the NIH (AG-121191, AG-19309, HL-64344, DK-56341, and RR-00954) and the Washington University–Pharmacia Research Program. J.P. Gaut was supported by a Cardiovascular Training Grant at Washington University School of Medicine and a grant from the Glenn/American Federation of Aging Research.

Acknowledgments

Footnotes

See the related Commentary beginning on page 1287.

Joseph P. Gaut and Jaeman Byun contributed equally to this work.

Footnotes

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