title: A new pitfall in detecting biological end products of nitric oxide-nitration, nitros(yl)ation and nitrite/nitrate artefacts during freezing.
abstract: The present study shows that when freezing nitrite containing biological samples in the presence of sodium and phosphate, a process of tyrosine nitration and S-nitrosocysteine formation is observed. The underlying mechanism is obviously based on the already described pH decrease in sodium phosphate buffered solutions during the freezing process and probably involves nitrous acid as an intermediate. However, in pure potassium phosphate buffer freeze-artefacts were absent. The yield of 3-nitrotyrosine from albumin-bound or free tyrosine depends not only on the concentration of nitrite, tyrosine or protein, and sodium phosphate but also on the velocity of the freezing process. Nitrite and nitrate were quantified by the Griess/nitrate reductase assay. 3-nitrotyrosine formation was quantitatively measured by HPLC analysis with optical and electrochemical detection as well as qualitatively investigated by immunohistochemistry and slot blot analysis using 3-nitrotyrosine specific antibodies. The formation of S-nitrosocysteine was detected by S-nitrosothiol specific antibodies and quantified by a fluorometric assay. Irrespective of the mechanism and although the here presented results cannot be generalized, the data warrant caution for the analysis of nitration or nitros(yl)ation products following freezing of nitrite containing biological material.
authors.affiliation: Universitätsklinikum Hamburg-Eppendorf, Medizinische Klinik III, Angiologie und Kardiologie, Martinistr 51, Hamburg 20246, Germany. daiber@uke.uni-hamburg.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Daiber
authors.forename: Markus
authors.initials: M
authors.lastname: Bachschmid
authors.forename: Cengiz
authors.initials: C
authors.lastname: Kavaklí
authors.forename: Daniel
authors.initials: D
authors.lastname: Frein
authors.forename: Maria
authors.initials: M
authors.lastname: Wendt
authors.forename: Volker
authors.initials: V
authors.lastname: Ullrich
authors.forename: Thomas
authors.initials: T
authors.lastname: Munzel
mesh_terms: D000818:Animals; D001011:Aorta; D002021:Buffers; D015925:Cryopreservation; D003545:Cysteine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry; D005615:Freezing; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009566:Nitrates / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D009573:Nitrites / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D009596:Nitrophenols / Q000032:analysis; D009603:Nitroso Compounds / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D051381:Rats; D026403:S-Nitrosothiols / Q000097:blood / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry
chemical_list: D002021:Buffers; D009566:Nitrates; D009573:Nitrites; D009596:Nitrophenols; D009603:Nitroso Compounds; D010710:Phosphates; D017680:Potassium Compounds; D026403:S-Nitrosothiols; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; C021315:S-nitrosocysteine; C013216:potassium phosphate; D003545:Cysteine; C018279:sodium phosphate