Autofluorescence characterization of advanced glycation end products of hemoglobin.

This article describes the analysis of autofluorescence of advanced glycation end products of hemoglobin (Hb-AGE). Formed as a result of slow, spontaneous and non-enzymatic glycation reactions, Hb-AGE possesses a characteristic autofluorescence at 308/345 nm (lambda(ex)/lambda(em)). Even in the presence of heme as a quenching molecule, the surface presence of the glycated adduct gave rise to autofluorescence with the quantum yield of 0.19. The specificity of monoclonal antibody developed against common AGE structure with Hb-AGE was demonstrated using reduction in fluorescence polarization value due to increased molecular volume while binding. The formation of fluorescent adduct in hemoglobin in the advanced stage of glycation and the non-fluorescent HbA(1c) will be of major use in distinguishing and to know the past status of diabetes mellitus. While autofluorescence correlated highly with HbA(1c) value under in vivo condition (r = 0.85), it was moderate in the clinical samples (r = 0.55). The results suggest a non-linear relation between glycemia and glycation, indicating the application of Hb-AGE as a measure of susceptibility to glycation rather than glycation itself.

Evaluation of autofluorescent property of hemoglobin-advanced glycation end product as a long-term glycemic index of diabetes.

Current methods for measuring long-term glycemia in patients with diabetes are HbA(1c) and advanced glycation end products (AGEs), which are estimated by phenyl boronate affinity chromatography and competitive enzyme-linked immunosorbent assay, respectively. In this study, we hypothesize that the intrinsic fluorescence property of hemoglobin-AGE (Hb-AGE) may be a simple, accurate, and therefore better index for long-term glycemic status due to its highly specific nature and longer half-life. To establish this contention, in vitro and in vivo experiments were carried out. The former was performed by incubating commercially available hemoglobin with 5 and 20 mmol/l glucose and the latter through experimentally induced (streptozotocin) diabetes in an animal model (male Wistar rats) to identify the new fluorophore formed due to the nonenzymatic glycosylation of hemoglobin. An adduct exhibiting fluorescence at 308/345 nm of excitation/emission wavelengths has been identified and its time-dependent formation established. Under in vitro conditions, the first appearance of the new fluorophore was noticed only after a period of 2 months, whereas under in vivo conditions, it increased significantly after 2 months of hyperglycemia. Consistent with the observations, studies on patients with type 2 diabetes demonstrated an elevated level of this new fluorescent adduct in patients with persisting high levels of plasma glucose for >2 months. Based on the results obtained, Hb-AGE appears to be an efficient fluorescence-based biosensing molecule for the long-term monitoring of glycemic control in diabetes.