title: Determination of nitrotyrosine and tyrosine by high-performance liquid chromatography with tandem mass spectrometry and immunohistochemical analysis in livers of mice administered acetaminophen.
abstract: Nitrotyrosine (NTYR) is used as a biomarker of nitrative pathology caused by peroxynitrite (ONOO-) formation. NTYR measurement in biological materials usually employs such methodologies as immunohistochemistry staining, high-performance liquid chromatography and gas chromatography. In this study, we developed a method for the determination of tyrosine (TYR) and NTYR, using liquid chromatography with tandem mass spectrometry (LC-MS/MS). In order to confirm the applicability of our method to an in vivo system, we measured protein-bound NTYR levels using by LC-MS/MS method and immunohistochemical analysis in liver of B6C3F1 mice at 2 h, 4 h and 8 h after administration of 300 mg/kg acetaminophen (APAP). A mass spectrometer equipped with an electrospray ionization source using a crossflow counter electrode and ran in the positive ion mode (ESI+) was set up for multiple reaction monitoring (MRM), which monitored the transitions 182.2>136.2, 227.1>181.2, 191.3>144.4 and 236.3>189.5, for TYR, NTYR, [13C9]-TYR and [13C9]-NTYR, respectively. The average recoveries from mice liver protein samples spiked with 25 microM TYR and 100 nM NTYR were 94.4% and 95.6%, respectively, with correction using the added surrogate standards. The limits of quantification were 100 nM for TYR and 0.5 nM for NTYR. NTYR was detected all liver samples of mice by the proposed LC-MS/MS method. The concentration range of NTYR per milligram protein in samples was 0.17-0.3 pmol/mg protein. And the level reached a maximum at 4 h. These data were well correlated with the result obtained by an immunohistochemical reaction with anti-NTYR antibody. The LC-MS/MS method was able to determine protein-bound NTYR in a small amount of tissue sample, and is therefore expected to be a very powerful tool for evaluating ONOO- generation in an in vivo system.
authors.affiliation: Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ishii
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Iijima
authors.forename: Takashi
authors.initials: T
authors.lastname: Umemura
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nishikawa
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Iwasaki
authors.forename: Rie
authors.initials: R
authors.lastname: Ito
authors.forename: Koichi
authors.initials: K
authors.lastname: Saito
authors.forename: Masao
authors.initials: M
authors.lastname: Hirose
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakazawa
mesh_terms: D000082:Acetaminophen / Q000633:toxicity*; D018712:Analgesics, Non-Narcotic / Q000633:toxicity*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis*
chemical_list: D018712:Analgesics, Non-Narcotic; C002744:3-nitrotyrosine; D000082:Acetaminophen; D014443:Tyrosine