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. 2002 May;109(10):1311-9.
doi: 10.1172/JCI15021.

Myeloperoxidase produces nitrating oxidants in vivo

Joseph P Gaut  1 , Jaeman Byun, Hung D Tran, Wendy M Lauber, James A Carroll, Richard S Hotchkiss, Abderrazzaq Belaaouaj, Jay W Heinecke
Affiliations

Myeloperoxidase produces nitrating oxidants in vivo

Joseph P Gaut et al. J Clin Invest. 2002 May.

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. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

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Figures

Figure 1
Figure 1
Analysis of N-acetyl-L-tyrosine exposed to the myeloperoxidase-NO2–-Cl–-H2O2 system. (a) HPLC analysis. Inset: Absorbance spectra of authentic N-acetyl-L-3-nitrotyrosine (N-Ac-nitrotyrosine [N-Ac-NO2-Y]) and peak III. (b) Electrospray ionization tandem mass spectrometric analysis of peak III. (c) Aromatic region of a proton nuclear magnetic resonance spectrum of peak III. MPO, myeloperoxidase.
Figure 2
Figure 2
Reaction requirements for generation of N-Ac-nitrotyrosine by HOCl and myeloperoxidase. (a and b) Effect of pH. (c) Effect of taurine. Results represent means ± SEM of three independent experiments.
Figure 3
Figure 3
Western blot analysis and immunoprecipitation of K. pneumoniae proteins exposed to the myeloperoxidase-H2O2-NO2– system at neutral PH. (a) Western blot analysis of nitrated proteins isolated from a reaction mixture containing K. pneumoniae (KP), myeloperoxidase, H2O2, and NO2– (100 or 500 μM). Proteins were probed with a rabbit polyclonal antibody to 3-nitrotyrosine. (b) Solubilized proteins from K. pneumoniae exposed to myeloperoxidase, H2O2, and NO2– were immunoprecipitated with a mouse mAb to 3-nitrotyrosine. Only the protein of about 65 kDa was immunoreactive with a rabbit polyclonal antibody to 3-nitrotyrosine. (c) Tandem mass spectrometric analysis of a trypsin digest of the immunoreactive 65-kDa protein. (M+H)+, molecular ion.
Figure 4
Figure 4
Immunohistochemical staining of wild-type and myeloperoxidase-deficient neutrophils for 3-nitrotyrosine (red immunostaining; a–c) or inducible nitric oxide synthase (iNOS) (brown immunostaining; d–f). Cells were incubated with K. pneumoniae in the presence (a, c, d, and f) or absence (b and e) of NO2– and then subjected to immunostaining.
Figure 5
Figure 5
Detection of free 3-nitrotyrosine in peritoneal inflammatory exudate of a wild-type mouse infected with K. pneumoniae. Amino acids were isolated from peritoneal fluid by solid-phase chromatography, converted to their heptafluobutyryl t-butyl-dimethylsilyl derivatives, and subjected to GC/MS analysis. (a) Endogenous (m/z 518), (b) isotope-labeled (m/z 524), and (c) artifactual (m/z 528) 3-nitrotyrosine was monitored simultaneously using selected ion monitoring in the negative ion electron capture mode.
Figure 6
Figure 6
Quantification of (a) free 3-chlorotyrosine and (b) free 3-nitrotyrosine in peritoneal fluid isolated from wild-type and myeloperoxidase-deficient mice subjected to either CLP or infection with K. pneumoniae. 3-Chlorotyrosine (3-Cl-Tyr) and 3-nitrotyrosine (3-NO2-Tyr) were quantified by isotope dilution GC/MS with selected ion monitoring in negative ion electron capture mode. KP, infection with K. pneumoniae.
Figure 7
Figure 7
Quantification of NO2– and NO3– levels in peritoneal fluid isolated from wild-type and myeloperoxidase-deficient mice subjected to either CLP or infection with K. pneumoniae. Total NO2– and NO3– in peritoneal lavage fluid was quantified using the Griess reagent.
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Comment in

  • Whence nitrotyrosine?
    Hurst JK. Hurst JK. J Clin Invest. 2002 May;109(10):1287-9. doi: 10.1172/JCI15816. J Clin Invest. 2002. PMID: 12021243 Free PMC article. No abstract available.

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