Detection of DNA-bound advanced glycation end-products by immunoaffinity chromatography coupled to HPLC-diode array...

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.