Determination of nitrotyrosine by HPLC-ECD and its application.
3-nitrotyrosine, a product of tyrosine nitration, is a useful indicator of oxidative damage. We modified the previously reported HPLC-electrochemical detection (ECD) method: specifically, a through-type porous carbon electrode was used as a reducing electrode instead of the mercury-gold amalgam electrode, because the response of the latter changes over time. A combination of reverse-phase HPLC and electrochemical detector passed through -800 mV reduction potential and subsequently under +250 mV oxidation potential allows measurement of 3-nitrotyrosine. The detection limit of this assay was less than 10 fmol. In mice to which lipopolysaccharide (LPS) was administered intraperitoneally, plasma 3-nitrotyrosine levels were elevated, corresponding to LPS dosage. These findings suggest that the improved HPLC-ECD method can be used as a specific and sensitive assay of biological 3-nitrotyrosine and can be applied clinically.
Ishida
Nobushige
N
Equine Research Institute, Japan Racing Association, Utsunomiya, Japan.
Hasegawa
Telhisa
T
Mukai
Kazutaka
K
Watanabe
Masanobu
M
Nishino
Hirohito
H
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry / Q000379:methods*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000097:blood*
D008070:Lipopolysaccharides; C002744:3-nitrotyrosine; D014443:Tyrosine