Richards Douglas A, Silva Michael A, Devall Adam J
Department of Pharmacology, Division of Neuroscience, Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Anal Biochem. 2006 Apr 1;351(1):77-83. doi: 10.1016/j.ab.2006.01.001. Epub 2006 Jan 19.
3-Nitrotyrosine (3-NT) is formed by the reaction of peroxynitrite with either free or protein-bound tyrosine residues and has been proposed as a biomarker of oxidative stress caused by reactive nitrogen species. This study describes the development of an HPLC electrochemical detection assay for free 3-NT capable of measuring this metabolite at the very low (nanomolar) levels encountered physiologically. We employed a dual-cell coulometric approach in which 3-NT is first reduced at an upstream cell to 3-aminotyrosine, which itself is then oxidized at the downstream cell. The method was shown to be linear over the range of 1-500 nM (r = 0.999), with a detection limit (signal/noise ratio of 3) of 0.5 nM (25 fmol on column). Ten consecutive injections of 2 and 20 nM 3-NT standards produced coefficients of variation of 5.88 and 1.87%, respectively. Validation of the identity of the 3-NT peak was confirmed by coelution with authentic standards and by the in vitro production of 3-NT by incubation of 3-morpholinylsydnoneimine (SIN-1, 100 microM), a molecule releasing nitric oxide and superoxide in solution at a pH of 7.0 or higher with tyrosine (10 microM). Using this method, 3-NT was detected in human liver microdialysate (levels up to 2.6 nM), although levels in rat spinal cord dialysate were below the limit of detection.
3-硝基酪氨酸（3-NT）由过氧亚硝酸盐与游离或与蛋白质结合的酪氨酸残基反应形成，已被提议作为活性氮物种引起的氧化应激的生物标志物。本研究描述了一种用于游离3-NT的高效液相色谱电化学检测方法的开发，该方法能够在生理条件下遇到的极低（纳摩尔）水平下测量这种代谢物。我们采用了一种双池库仑法，其中3-NT首先在上游池中还原为3-氨基酪氨酸，然后3-氨基酪氨酸本身在下游池中被氧化。该方法在1-500 nM范围内呈线性（r = 0.999），检测限（信噪比为3）为0.5 nM（柱上25 fmol）。连续十次进样2 nM和20 nM的3-NT标准品，变异系数分别为5.88%和1.87%。通过与标准品共洗脱以及在pH值为7.0或更高的溶液中，将3-吗啉代亚胺（SIN-1，100 microM）与酪氨酸（10 microM）孵育体外产生3-NT，证实了3-NT峰的同一性。使用该方法，在人肝微透析液中检测到了3-NT（水平高达2.6 nM），尽管大鼠脊髓透析液中的水平低于检测限。
