Analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine by ultra high pressure liquid chromatography-heat assisted electrospray ionization-tandem mass spectrometry
Analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine by ultra high pressure liquid chromatography-heat assisted electrospray ionization-tandem mass spectrometry
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DOI：
10.1016/j.jchromb.2009.12.004
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发表时间：
2010-02-01
影响因子：
3
通讯作者：
Swenberg, James A.
中科院分区：
文献类型：
--
作者：
Boysen, Gunnar;Collins, Leonard B.;Swenberg, James A.
活性氧（ROS）的增加，通常被称为氧化应激，经常被假设与许多疾病有因果关系。DNA和尿液中的8-氧-7,8-二氢-2'脱氧鸟苷（8-氧- dg）是被广泛应用于氧化应激的生物标志物。多年来，由于样品工作过程中的人工形成，分析DNA中的8-oxo-dG是具有挑战性的。本研究表明，如果采取适当的预防措施，8-氧-脱氧葡萄糖可以可靠和准确地测量。首先，在样品制备过程中，抗氧化剂、金属螯合剂或自由基捕获剂的存在提高了再现性。其次，采用高效液相色谱法富集样品，优化灵敏度。第三，热辅助电喷雾电离（HESI）消除了潜在的干扰，提高了分析性能和灵敏度。随后，采用UPLC-HESI-MS/MS方法研究8-oxo-dG在h2o2处理的HeLa细胞中的双相剂量响应。将该方法应用于人类淋巴细胞DNA (n = 156)，得到每10(6)dG平均+/- SD内源性加合物量为1.57 +/- 0.88，该值与欧洲氧化DNA损伤标准委员会（ESCODD）和其他机构先前估计的建议量一致。这些结果表明，该方法非常适合应用于氧化应激作用的分子毒理学和流行病学研究。Elsevier B.V.出版
Increased amounts of reactive oxygen species (ROS), generally termed oxidative stress, are frequently hypothesized to be Causally associated with many diseases. Analyses of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dG) in DNA and urine are widely used biomarkers for oxidative stress. Over the years it became clear that analysis of 8-oxo-dG in DNA is challenging due to artifactual formation during sample work up. The present study demonstrates that 8-oxo-dG can be measured reliably and accurately when appropriate precautions are taken. First, the presence of an antioxidant, metal chelator, or free radical trapping agent during sample preparation improves reproducibility. Second, sample enrichment by HPLC fraction collection was used to optimize sensitivity. Third, heat assisted electrospray ionization (HESI) eliminated potential interferences and improved assay performance and sensitivity. Subsequently, the UPLC-HESI-MS/MS method was applied to show the biphasic dose response of 8-oxo-dG in H2O2-treated HeLa cells. Application of this method to human lymphocyte DNA (n = 156) gave a mean +/- SD endogenous amount of 1.57 +/- 0.88 adducts per 10(6) dG, a value that is in agreement with the suggested amount previously estimated by European Standard Committee on Oxidative DNA Damage (ESCODD) and others. These results suggest that the present method is well suited For application to molecular toxicology and epidemiology studies investigating the role of oxidative stress. Published by Elsevier B.V.