Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry
Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry
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DOI：
10.1093/nar/30.2.e7
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发表时间：
2002-01-15
影响因子：
14.9
通讯作者：
Poulsen, HE
中科院分区：
文献类型：
--
作者：
Weimann, A;Belling, D;Poulsen, HE
氧化性DNA损伤与多种疾病（如癌症和衰老）有致病关系，可以通过测量尿液中特定的DNA修复产物来研究。在过去的十年中，由于确定这些产物会排泄到尿液中，在尿液中分析它们的进展有限。鸟嘌呤是最容易氧化的DNA碱基。我们提出了一种基于直接注射尿液的高效液相色谱-串联质谱系统测定尿中8-羟基鸟嘌呤的方法。分析包括8-羟基化碱基，核糖核苷和脱氧核苷，以及相应的非氧化物质。未经尿液预处理，核碱基的检出限近似于2 nM(50。0.5 nM （12.5 fmol注射）的核苷。以前，核碱基的液相色谱法一直存在问题，但通过低温反相C18色谱法可以实现，这种方法增加了柱上的保留。在核苷的情况下，保留几乎是全部的，并提供了一种手段，在柱上浓度较大的尿液样本和控制高峰梯度洗脱。8-羟化鸟嘌呤的总排泄量为212 nmol/24 h，氧化碱基占64%，核糖核苷占23%，脱氧核苷占13%，表明RNA在人体中大量氧化。在大鼠尿液中，氧化碱基的排泄更占优势，氧化碱基，核糖核苷和脱氧核苷的百分比。分别是89,8和3%这一发现与先前使用免疫亲和前纯化和hplc -电化学检测分析的报告不一致。开发的方法现在使得在流行病学和干预设置中测量RNA和DNA的氧化核酸应激成为可能，我们的研究结果表明除了DNA氧化外，还有大量的RNA氧化。该方法体积小，适用于小型实验动物。
Oxidative DNA damage, linked pathogenically to a variety of diseases such as cancer and ageing, can be investigated by measuring specific DNA repair products in urine. Within the last decade, since it was established that such products were excreted into urine, progress in their analysis in urine has been limited. Guanine is the DNA base most prone to oxidation. We present a method for determination of the urinary 8-hydroxylated species of guanine, based on direct injection of urine onto a high-performance liquid chromatography (HPLC)-tandem mass spectrometry system. The analysis covers the 8-hydroxylated base, ribonucleoside and deoxynucleoside, and the corresponding non-oxidised species. Without pretreatment of urine the detection limits for the nucleobases are similar to2 nM (50.fmol injected) and for the nucleosides similar to0.5 nM (12.5 fmol injected). Previously, liquid chromatography of the nucleobases has been problematic but is made possible by low-temperature reverse-phase C18 chromatography, a method that Increases retention on the column. In the case of the nucleosides, retention was almost total and provides a means for on-column concentration of larger urine samples and controlled high peak gradient elution. The total excretion of 8-hydroylated guanine species was 212 nmol/24 h. The oxidised base accounted for 64%, the ribonucleoside for 23% and the deoxynucleoside for 13%, indicating substantial oxidation of RNA In humans. In rat urine, excretion of the oxidised base was more dominant, the percentages of the oxidised base, ribonucleoside and deoxynucleosides. being 89, 8 and 3%. This finding is at odds with previous reports using immunoaffinity pre-purification and HPLC-electrochemical detection analysis. The developed method now makes it possible to measure oxidative nucleic acid stress to both RNA and DNA in epidemiological and intervention settings, and our findings indicate a substantial RNA oxidation in addition to DNA oxidation. The small volume needed also makes the method applicable to small experimental animals.