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Journal of Biochemistry Advance Access first published online on January 29, 2007
This version published online on March 15, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm050
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© 2007 The Japanese Biochemical Society

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

Yoshiaki H. Hitomi1, Junna Okuda1, Hirohito Nishino2, Yasuhiro Kambayashi1, Yuri Hibino1, Kei Takemoto3, Tomoko Takigawa3, Hideki Ohno4, Naoyuki Taniguchi5 and Keiki Ogino3,*

1Department of Environmental and Preventive Medicine, Graduate School of Medical Science, Kanazawa University, 13-1, Takaramachi, Kanazawa, Ishikawa 920-8640; 2Eicom Corporation, 24-2, Enmenda, Shimotoba, Fushimi-ku, Kyoto 612-8474; 3Department of Public Health, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikada, Okayama 700-8558; 4Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo 181-8611, and
5Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita, Osaka 565-0871, Japan

* To whom correspondence should be addressed; Keiki Ogino, M.D., Ph.D. Department of Public Health, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikada, Okayama, 700-8558, Japan Tel: +81-86-235-7184. Fax: +81-86-226-0715. E-mail: kogino{at}md.okayama-u.ac.jp

Received November 30, 2006; Accepted January 24, 2007


  Abstract

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 one week. Our protocol is reproducible and suitable for analyzing multiple clinical samples to study RNS production in vivo.

Key Words: 3-nitrotyrosine, nitric oxide, HPLC, electrochemical detection, lipopolysaccharide


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