Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD.
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.
Hitomi
Yoshiaki H
YH
Department of Environmental and Preventive Medicine, Graduate School of Medical Science, Kanazawa University, 13-1, Takaramachi, Kanazawa, Ishikawa, Japan.
Okuda
Junna
J
Nishino
Hirohito
H
Kambayashi
Yasuhiro
Y
Hibino
Yuri
Y
Takemoto
Kei
K
Takigawa
Tomoko
T
Ohno
Hideki
H
Taniguchi
Naoyuki
N
Ogino
Keiki
K
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D004594:Electrophysiology; D007275:Injections, Intravenous; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D010949:Plasma / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D026361:Reactive Nitrogen Species / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism
D001798:Blood Proteins; D008070:Lipopolysaccharides; D026361:Reactive Nitrogen Species; C002744:3-nitrotyrosine; D014443:Tyrosine