Immunochemical quantification of crossline as a fluorescent advanced glycation endproduct in erythrocyte membrane proteins from diabetic patients with or without retinopathy

Abstract

Crossline is a novel advanced glycation endproduct (AGE) which has both a crosslink and fluorescence similar to AGE-protein in vivo. To assess the association of AGEs to the development of diabetic retinopathy we developed a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for crossline in blood samples and investigated the association of the development of retinopathy and erythrocyte membrane protein (EMP)- crossline concentrations in patients with Type 2 diabetes mellitus (Type 2 DM). Crossline formation in EMP exceeded that in haemoglobin and was detectable in normal EMP samples without pretreatment by this ELISA system. Mean (±SE) EMP crossline levels were elevated 1.6fold in diabetic patients without retinopathy (7.6 ± 0.5 pmol mg-1, p < 0.005), 2.2-fold in diabetic patients with non-proliferative retinopathy (10.5:1:0.6 pmol mg-1, p < 0.001) compared with healthy control subjects (4.7 ± 0.5 pmol mg-1). Type 2 DM patients with retinopathy had significantly higher EMP-crossline levels than those without retinopathy (p < 0.005). Our data suggest that elevated EMP-crossline concentrations are associated with the presence of retinopathy in patients with Type 2 DM and EMP-crossline measured by our ELISA may provide a useful marker for assessing the role of glycation in the development of diabetic retinopathy.