Advanced Glycation End Products of DNA: Quantification of N2-(1-Carboxyethyl)-2′-deoxyguanosine in Biological Samples by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

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Abstract
Methylglyoxal (MG) and related α-oxoaldehydes react with proteins, lipids, and DNA to give rise to covalent adducts known as advanced glycation end products (AGEs). Elevated levels of AGEs have been implicated in the pathological complications of diabetes, uremia, Alzheimerʼs disease, and possibly cancer. There is therefore widespread interest in developing sensitive methods for the in vivo measurement of AGEs as prognostic biomarkers and for treatment monitoring. The two diastereomeric MG-DNA adducts of N2-(1-carboxyethyl)-2′-deoxyguanosine (CEdG) are the primary glycation products formed in DNA; however, accurate assessment of their distribution in vivo has not been possible since there is no readily available quantitative method for CEdG determination in biological samples. To address these issues, we have developed a sensitive and quantitative liquid chromatography electrospray ionization tandem mass spectrometry assay using the stable isotope dilution method with an 15N5-CEdG standard. Methods for CEdG determination in urine or tissue extracted DNA are described. Changes in urinary CEdG in diabetic rats in response to oral administration of the AGE inhibitor LR-90 are used to demonstrate the potential utility of the method for treatment monitoring. Both stereoisomeric CEdG adducts were detected in a human breast tumor and normal adjacent tissue at levels of 3−12 adducts/107 dG, suggesting that this lesion may be widely distributed in vivo. Strategies for dealing with artifactual adduct formation due to oxoaldehyde generation during DNA isolation and enzymatic workup procedures are described.