Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatographyȁ3;tandem mass spectrometry and evaluation of the excretion after oral intake

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• 作者：Dimitrios Tsikas ; Anja Mitschke ; Maria-Theresia Suchy ; Frank-Mathias Gutzki and Dirk O. Stichtenoth
• 关键词：Reactive-nitrogen species ; 3-Nitrotyrosine ; Human ; Urine ; Biomarker
• 刊名：Journal of Chromatography B ; Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2005
• 期刊代码：124_15700232
• 类别：ch
• 出版时间：2005
• 卷：827
• 期：1
• 页码：146-156
• 文件大小：427 K

摘要

3-Nitrotyrosine (NO₂Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)ȁ3;tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[²H₃]tyrosine, and GCȁ3;tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5 ± 7.2) 3-nitrotyrosine levels were determined to be 8.4 ± 10.4 nM (range, 1.6ȁ3;33.2 nM) or 0.46 ± 0.49 nmol/mmol creatinine (range, 0.05ȁ3;1.30 nmol/mmol creatinine). The present GCȁ3;tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 μg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.