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Journal of Biochemistry Advance Access originally published online on January 29, 2007
Journal of Biochemistry 2007 141(4):495-502; doi:10.1093/jb/mvm050
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© 2007 The Japanese Biochemical Society.

Disposition of Protein-bound 3-nitrotyrosine in Rat Plasma Analysed 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. 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 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo.

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

Abbreviations: ECD, electrochemical detection; GS/MS, gas chromatography/mass spectrometry; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; LPS, lipopolysaccharide; NO, nitric oxide; NTyr, 3-nitrotyrosine; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; RNS, reactive nitrogen species; TBST, Tris-buffered saline with Tween 20


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