Schneider Marc, Georgescu Adriana, Bidmon Clemens, Tutsch Martin, Fleischmann Erwin H, Popov Doina, Pischetsrieder Monika
Institute of Pharmacy and Food Chemistry, Friedrich-Alexander-University Erlangen-Nuremberg, Germany.
Mol Nutr Food Res. 2006 Apr;50(4-5):424-9. doi: 10.1002/mnfr.200500217.
Sugars and sugar degradation products are formed during food processing, but also endogenously in vivo. In vitro, nucleosides and DNA react readily with these carbonyl compounds during the formation of the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)), leading to a loss of DNA integrity. Only little is known about DNA glycation in vivo and about the influence of nutrition on CEdG formation. In this study, we developed a sensitive method to analyze DNA glycation by HPLC. For this purpose, immunoaffinity chromatography (IAC) using a polyclonal antibody against N(2)-carboxyethylguanine (CEguanine) was coupled to HPLC-DAD. In some samples, peak identity was confirmed by LC-MS/MS. The recovery of CEguanine from the IAC columns was 52.5% +/- 3.6 (n = 4). Thus, it was possible for the first time to detect CEdG(A,B), N(2)-carboxyethylguanosine (CEG(A,B)), and CEguanine in 11 human urine samples. However, due to imprecision of IAC, valid quantification of the adducts could not be achieved. Furthermore, CEdG was also detected in the DNA of cultured human smooth muscle cells (SMCs) and bovine aorta endothelium cells (BAECs). In BAECs, CEdG(A,B) were found by HPLC-DAD and LC-MS/MS after immunoaffinity purification, whereas in SMCs DNA-advanced glycation end-products were only detected with the more sensitive LC-MS/MS method.
糖和糖降解产物在食品加工过程中形成，也在体内内源性产生。在体外，核苷和DNA在形成N(2)-羧乙基-2'-脱氧鸟苷（CEdG(A,B)）的两种非对映异构体过程中很容易与这些羰基化合物发生反应，导致DNA完整性丧失。关于体内DNA糖基化以及营养对CEdG形成的影响，人们了解甚少。在本研究中，我们开发了一种通过高效液相色谱法（HPLC）分析DNA糖基化的灵敏方法。为此，使用针对N(2)-羧乙基鸟嘌呤（CE鸟嘌呤）的多克隆抗体的免疫亲和色谱法（IAC）与HPLC-二极管阵列检测器（DAD）联用。在一些样品中，通过液相色谱-串联质谱法（LC-MS/MS）确认了峰的同一性。CE鸟嘌呤从IAC柱上的回收率为52.5%±3.6（n = 4）。因此，首次在11份人类尿液样本中检测到了CEdG(A,B)、N(2)-羧乙基鸟苷（CEG(A,B)）和CE鸟嘌呤。然而，由于IAC的不精确性，无法实现对加合物的有效定量。此外，在培养的人平滑肌细胞（SMCs）和牛主动脉内皮细胞（BAECs）的DNA中也检测到了CEdG。在BAECs中，免疫亲和纯化后通过HPLC-DAD和LC-MS/MS发现了CEdG(A,B)，而在SMCs中，仅用更灵敏的LC-MS/MS方法检测到了DNA晚期糖基化终产物。
