Quantification of Modified Tyrosines in Healthy and Diabetic Human Urine using Liquid Chromatography/Tandem Mass Spectrometry
Quantification of Modified Tyrosines in Healthy and Diabetic Human Urine using Liquid Chromatography/Tandem Mass Spectrometry
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DOI：
10.3164/jcbn.08-185
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发表时间：
2009-01-01
影响因子：
2.4
通讯作者：
Osawa, Toshihiko
中科院分区：
文献类型：
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作者：
Kato, Yoji;Dozaki, Natsuko;Osawa, Toshihiko
采用液相色谱-串联质谱法（LC/MS/MS）测定尿中氧化酪氨酸、二酪氨酸（DiY）、硝基酪氨酸（NY）、溴酪氨酸（BrY）和二溴酪氨酸（DiBrY）的含量。采用固相萃取法对样品进行部分纯化，然后采用多反应监测（MRM）方法进行LC/MS/MS分析。首先进行了MY定量分析。剩余样品用正丁醇/HCI进一步丁化，其他修饰的酪氨酸用同位素稀释法定量。测定了人尿中改性酪氨酸（DiY、NY、BrY和DiBrY）的MRM峰，洗脱时间符合真实标准和同位素标准。23例健康人尿液中酪氨酸修饰量分别为肌酐8.8 +/- 0.6 μ mol/mol （DiY）、1.4 +/- 0.4 μ mol/mol （NY）、3.8 +/- 0.3 μ mol/mol （BrY）和0.7 +/- 0.1 μ mol/mol （DiBrY）。还将修饰后的酪氨酸与尿8-氧脱氧鸟苷、戊苷和n- ε -（己醇）赖氨酸进行了比较。除NY外，几乎所有产品之间都表现出良好的相关性。糖尿病患者尿液中修饰型酪氨酸（NY、BrY和DiBrY）的含量高于健康人尿液。
The quantification of urinary oxidized tyrosines, dityrosine (DiY), nitrotyrosine (NY), bromotyrosine (BrY), and dibromotyrosine (DiBrY), was accomplished by quadruple liquid chromatography-tandem mass spectrometry (LC/MS/MS). The sample was partially purified by solid phase extraction, and was then applied to the LC/MS/MS using multiple-reaction monitoring (MRM) methods. The analysis for the MY quantification was done first. The residual samples were further butylated with n-butanolt/HCI, and the other modified tyrosines were then quantified with isotopic dilution methods. MRM peaks of the modified tyrosines (DiY, NY, BrY, and DiBrY) from human urine were measured and the elution times coincided with the authentic and isotopic standards. The amounts of modified tyrosines in healthy human urine (n = 23) were 8.8 +/- 0.6 (DiY), 1.4 +/- 0.4 (NY), 3.8 +/- 0.3 (BrY), and 0.7 +/- 0.1 (DiBrY) mu mol/mol of creatinine, respectively. A comparison of the modified tyrosines with urinary 8-oxo-deoxyguanosine, pentosidine, and N-epsilon-(hexanoyl)lysine was also performed. Almost all products, except for NY, showed good correlations with each other. The amounts of the modified tyrosines (NY, BrY, and DiBrY) in the diabetic urine were higher than those in the urine from healthy people.