Evaluation of Autofluorescent Property of Hemoglobin-Advanced Glycation End Product as a Long-Term Glycemic Index of Diabetes

Abstract

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 longterm 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.

Structural and Functional Characterization of Polyphenols Isolated from Acerola (Malpighia emarginataDC.) Fruit

“…Formation of AGE has been observed in vivo in several long-living proteins such as lens crystalline and collagen affected by diabetes mellitus. 9,11) Some radical species including oxygen are considered to play an important role in these processes. 10) Hence compounds that scavenge radical species are expected to prevent the formation of AGEs.…”

“…Recently, many reports have suggested that these complications are caused by the formation of advanced glycation end products (AGEs), which are generally formed through Amadori-type compounds from proteins and glucose. [7][8][9][10][11] AGEs are postulated to be physiological markers of the Maillard reaction in a biological system. Formation of AGE has been observed in vivo in several long-living proteins such as lens crystalline and collagen affected by diabetes mellitus.…”

Near-infrared absorbance measurements of hemoglobin solutions incubated with glucose

“…19,20 has investigated glycated hemoglobin fractions by fluorescence measurements. It was showed that in vitro glycated hemoglobin gave the maximum intensity of emitted light at 345 nm upon excitation at 308 nm.…”

“…3 Most methods quantify hemoglobin A1c, defined as hemoglobin A with glucose attached to the NH2-terminus valine of one or both beta chains. Over the last years various optical methods for monitoring of free glucose and glycated hemoglobin levels have been investigated, such as near-infrared spectroscopy 4-13 , optical coherence tomography 14 , polarimetry 15 , Raman spectroscopy [16][17][18] and fluorescence 19,20 .…”

Non-Enzymatic Glycation of Human Fibrillin-1

“…Examples include hemoglobin [2][3][4] , interferon-␤ 1b [5] , highand low-density lipoproteins [6,7] , antithrombin III [8] , enzymes [9] , insulin [10,11] , collagen [12][13][14][15][16] , ␣ -crystallines of lenses [17] , fibronectin [18,19] , laminin [20] , peripheral nerve myelin [6,21] , and IgG and human serum albumin [22] .…”