Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD

Yoshiaki H. Hitomi, Junna Okuda, Hirohito Nishino, Yasuhiro Kambayashi, Yuri Hibino, Kei Takemoto, Tomoko Takigawa, Hideki Ohno, Naoyuki Taniguchi, Keiki Ogino

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10 Citations (Scopus)


3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 ± 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 ± 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 ± 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo.

Original languageEnglish
Pages (from-to)495-502
Number of pages8
JournalJournal of biochemistry
Issue number4
Publication statusPublished - Apr 1 2007



  • 3-nitrotyrosine
  • Electrochemical detection
  • HPLC
  • Lipopolysaccharide
  • Nitric oxide

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Hitomi, Y. H., Okuda, J., Nishino, H., Kambayashi, Y., Hibino, Y., Takemoto, K., Takigawa, T., Ohno, H., Taniguchi, N., & Ogino, K. (2007). Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD. Journal of biochemistry, 141(4), 495-502. https://doi.org/10.1093/jb/mvm050