Malayappan Bhaskar, Garrett Timothy J, Segal Mark, Leeuwenburgh Christiaan
Department of Aging and Geriatrics, College of Medicine, Institute on Aging, Division of Biology of Aging, University of Florida, Gainesville, FL 32611, USA.
J Chromatogr A. 2007 Oct 5;1167(1):54-62. doi: 10.1016/j.chroma.2007.08.024. Epub 2007 Aug 17.
A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2'-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and (13)C- and (15)N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.
一种旨在测量氧化核酸8-氧代鸟嘌呤（8-oxoGua）、2,6-二氨基-4-羟基-5-甲酰氨基嘧啶（Fapy-Gua）、8-氧代鸟苷（8-oxoGuo）、8-氧代-2'-脱氧鸟苷（8-oxodG）的灵敏且特异的检测方法，已通过将反相液相色谱（HPLC）与电喷雾串联质谱检测（MS/MS）及同位素稀释相结合而开发出来。采用多反应监测（MRM）的HPLC-MS/MS方法能够灵敏地测定人尿液样本中的8-oxoGua、Fapy-Gua、8-oxoGuo和8-oxodG。除了在将尿液注入HPLC-MS/MS系统之前向其中添加缓冲液以及（13）C和（15）N标记的内标外，无需进行样品制备。该方法在来自非吸烟者、吸烟者、患有慢性肾病（CKD）的非吸烟者以及患有CKD的吸烟者的尿液样本中进行了测试，以评估核酸的氧化损伤水平。与各自的对照受试者相比，患有和未患有CKD的吸烟者中RNA和DNA损伤的标志物均显著增加。这些发现表明，诸如同位素稀释HPLC-MS/MS这样高度特异且灵敏的分析方法可能是测量人体氧化应激的一种有价值的工具。
