Laboratory measurements
Glycated haemoglobin HbA1c was measured in blood samples taken at time 0 and after 5 years, by high performance liquid chromatography (HPLC, Menarini Akray ADAM A1c HA-8180v), according to the standards of the International Federation of Clinical Chemistry (FICC) (AGEs were measured using an enzyme-linked immunosorbent assay (ELISA) according to Makita et al. method (s-RAGE were assayed by ELISA method (ELISA Kit, manufactured by Cusabio Biotech Co. Ltd.). Briefly, antibody specific for s-RAGE were pre-coated onto a microplate, where samples or standards were added, so any s-RAGE is bound by the immobilized antibody. Following removal of unbound conjugates, a biotin-conjugated antibody specific for s-RAGE was introduced into the wells, and after washing, avidin conjugated Horseradish Peroxidase (HRP) was added and developed on adequate substrate for reading absorbance at 450nm in a microplate reader. The linear range of the sRAGE measurements was from 78 to 5000 pg/mL, and the inter- and intra-assay coefficients of variation for sRAGE were <10% and 8.0%, respectively.