Quantification of 3-nitrotyrosine in biological tissues and fluids: generating valid results by eliminating artifactual formation.
Reactive nitrogen species such as peroxynitrite can nitrate specific amino acids, whether free or protein bound, and 3-nitrotyrosine is believed to be one marker of this reaction. To examine the significance of this pathway in biological systems we have developed an accurate, sensitive, and specific assay for 3-nitrotyrosine based on combined liquid chromatography tandem mass spectrometry. Our approach allowed simultaneous analysis of both tyrosine and 3-nitrotyrosine and employs isotopomer standards (i.e., [15N1, 13C9]-tyrosine and [13C6]-3nitrotyrosine). Calibration curves were linear (r2 = 0.999) across the range 0.5-100 pg/microL (i.e., 2.2-442 fmol/microL), and the detection limit for standard samples was 0.5 pg/microL (2.2 fmol/microL, or 10 fmol on column; S/N = 5) or 1 pg/microL (4.4 fmol/microL) for extracted (biological) samples. As a component of this study we have undertaken an extensive investigation of artifactual formation of 3-nitrotyrosine under conditions that exist during sample extraction and derivatization. Our studies show that under appropriate conditions (low pH, elevated temperatures, and in the presence of a vast excess of the two substrates, tyrosine and the nitrate anion), 3-nitrotyrosine can readily be formed as an artifact.
Yi
D
D
Ray Williams Biomedical Mass Spectrometry Faculty, University of New South Wales, Sydney, Australia.
Ingelse
B A
BA
Duncan
M W
MW
Smythe
G A
GA
D000818:Animals; D016477:Artifacts; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D051381:Rats; D012015:Reference Standards; D013582:Synovial Fluid / Q000378:metabolism; D014018:Tissue Distribution; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000652:urine
C002744:3-nitrotyrosine; D014443:Tyrosine