Jaruga Pawel, Kirkali Güldal, Dizdaroglu Miral
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Free Radic Biol Med. 2008 Dec 15;45(12):1601-9. doi: 10.1016/j.freeradbiomed.2008.09.019. Epub 2008 Sep 27.
Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among major lesions in DNA generated by hydroxyl radical attack, UV radiation, or photosensitization in vitro and in vivo. FapyAde and FapyGua exist in living cells at detectable background levels and are formed by exposure of cells to DNA-damaging agents. Numerous prokaryotic and eukaryotic DNA glycosylases exist for the repair of formamidopyrimidines by base excision repair pathways in cells, indicating their biological significance. Moreover, they are premutagenic lesions, albeit to different extents, revealing a possible role in disease processes. Methodologies using gas chromatography/mass spectrometry (GC/MS) with capillary columns have been developed to accurately measure FapyAde and FapyGua in DNA in vitro and in vivo. Stable isotope-labeled analogues of these compounds have been synthesized and are commercially available to be used as internal standards for accurate quantification. GC/MS with isotope dilution provides excellent sensitivity and selectivity for positive identification and accurate quantification, and has widely been applied in the past to the measurement of formamidopyrimidines under numerous experimental conditions. This paper reports on the details of this GC/MS methodology.
甲酰胺嘧啶、4,6-二氨基-5-甲酰胺嘧啶（FapyAde）和2,6-二氨基-4-羟基-5-甲酰胺嘧啶（FapyGua）是体外和体内因羟基自由基攻击、紫外线辐射或光致敏作用而在DNA中产生的主要损伤产物。FapyAde和FapyGua以可检测的背景水平存在于活细胞中，是细胞暴露于DNA损伤剂后形成的。细胞中存在许多原核和真核DNA糖基化酶，可通过碱基切除修复途径修复甲酰胺嘧啶，这表明了它们的生物学意义。此外，它们虽然程度不同，但都是前诱变损伤，揭示了其在疾病过程中可能发挥的作用。已开发出使用带有毛细管柱的气相色谱/质谱联用仪（GC/MS）的方法，以准确测量体外和体内DNA中的FapyAde和FapyGua。这些化合物的稳定同位素标记类似物已合成并可商购，用作准确定量的内标。带有同位素稀释的GC/MS对阳性鉴定和准确定量具有出色的灵敏度和选择性，过去已广泛应用于在众多实验条件下对甲酰胺嘧啶的测量。本文报道了这种GC/MS方法的详细信息。
