Determination of Glycated Nucleobases in Human Urine by a New Monoclonal Antibody Specific for N~2-Carboxyethyl-2'-deoxyguanosine

摘要

Sugars and sugar degradation products react in vivo readily with proteins(glycation)resulting in the formation of a heterogeneous group of reaction products,which are called advanced glycation end products(AGEs).AGEs notably change the structure and function of proteins so that extended protein-AGE formation is linked to complications such as nephropathy,atherosclerosis,and Cataract.DNA can be glycated in vitro in a similar way as proteins,and the two diastereomers of N~2-carboxyethyl-2'-deoxyguanosine(CEDG_(A,B))were identified as major DNA AGEs.It was postulated that DNA AGEs play an important role in aging,diabetes,and uremia.However,at the moment,sensitive methods to measure the extent and impact of DNA AGEs in vivo do not exist.In this study,we developed a monoclonal antibody,which recognized CEdG_(A,B)with high affinity and specificity(MAb M-5.1.6).The I_(50)value for CEdG_(A,B)was 2.1 ng/mL,whereas other modified nuclueobases and AGE proteins showed negligible cross-reactivity.Unmodified 2'-deoxyguanosine was only weakly recognized with an I_(50)value > 600 000 ng/mL,which is the limit of solubility.MAb M-5.1.6 was then used to measure the urinary excretion of AGE-modified nucleobases in a competitive enzyme-linked immunosorbent assay.The recovery of CEdG_(A,B)from human urine was between 87.4 and 99.7% with coefficients of variations between 8.0 and 22.2%.The detection limit was 0.06 ng/mL,and the determination limit was 0.15 ng/mL with a linear range between 0.3 and 100 ng/mL.CEdG equivalents were analyzed in urine samples from 121 healthy volunteers,and concentrations between 1.2 and 117 ng CEdG equiv/mg creatinine were detected.