Comparison Of The Glycation Ability Of Ribose,Fructose And Glucose

Objective: The high glucose environment in diabetics promotes the nonenzymatic glycation of protein(NEG),which is an important factor inducing diabetic complications.NEG will not only lead to the alteration of protein structure and function,but also the formation of advanced glycation end-products(AGEs).The abnormal accumulation of AGEs in the body can eventually lead to abnormal metabolism and dysfunction in a variety of tissues and organs.Previous studies have found that under the same concentration conditions,the glycation rate of glucose is slower than that of ribose and fructose.However,under physiological conditions,the concentration of ribose and fructose is much lower than that of glucose.Clarifying the difference of their glycation rate and the glycation products has significant implications for understanding the occurrence and development of diabetic complications.Aiming at the above scientific problems,this study compared the glycation ability of ribose,fructose and glucose by simulating the concentration ratio under physiological conditions.Methods: Cells were cultured with three reducing sugars in different concentration separately.CCK-8 assay was performed to detect cell viability,while flow cytometry was performed to measure cell apoptosis,and the levels of AGEs in cells were detected by Western blotting(WB)and Enzyme-linked immunosorbent assay(ELISA)to explore the relationship among cell viability,apoptosis and the AGEs.According to the physiological concentration ratio of the three sugars,ribose(20m M),fructose(20m M)and glucose(1M)were incubated with bovine serum albumin(150μM,BSA)for 0-14 days.The alteration of kinetics of AGEs produced by the reaction system of three reducing sugars with BSA were monitored by fluorescence spectroscopy combined with Tsou’s kinetic method.The glycation modification sites of BSA were analyzed by mass spectrometry and the content of AGEs produced was analyzed by WB and ELISA.Fluorescence probe of ANS(8-Anilino-1-naphthalenesulfonic acid)and Th T(Thioflavin T),circular dichroism(CD),electrophoresis(denaturing /non-denaturing),transmission electron microscope(TEM)and dynamic light scattering(DLS)were used to monitor the changes of protein structure and properties in the three glycation reaction system.In addition,we also preliminarily explored the NEG of human hemoglobin(h Hb)by the fluorescence spectroscopy and Th T assay described above.For the further verification the difference in glycation among ribose,fructose and glucose,we collected the serum and urine of normal subjects and patients with type 2 diabetes mellitus(T2DM),then detected the levels of ribose and glucose with high performance liquid chromatography(HPLC),and the levels of fructose with fructose kit.We compared the differences of the three reducing sugars in normal subjects and T2 DM respectively,then investigated the correlation between sugars and glycated hemoglobin(Hb A1c)or glycated serum protein(GSP)which are important diagnostic indicators of diabetes.Results: Ribose induced more AGEs in cells,and declined cell viability,accelerated cell apoptosis.Fructose and glucose had no significant effect on cell cytotoxicity.Under the physiological concentration ratio,fluorescence kinetics of AGEs production indicated that the glycation rate of ribose was fastest,and then glucose,and fructose was the slowest when reacted with BSA.LC-MS/MS analysis revealed 48 ribose-glycated residues,44 glucose-glycated residues and19 fructose-glycated residues.Except for the shared Lys residues,ribose reacted selectively with 16 Lys and 1 Arg,and glucose reacted selectively with 7 Lys,while no selective Lys was found in fructose-glycated residues.Conformational analysis showed decrease of hydrophobicity and increase of β-sheet in glycated BSA,accompanied by the increase of inhomogeneity and hydration particle size.The changes occurred in BSA glycated by ribose were the most obvious,sequentially followed by those with glucose,but not with fructose.In the h Hb reaction system,the reaction rate of ribose was still the fastest,and the increasing of β-sheet was also found in BSA glycated by ribose.The levels of ribose and glucose in serum and urine in T2 DM patients were significantly higher than those in normal subjects,and positively correlated with Hb A1 c and GSP.The urine fructose in T2 DM was also consistent with the above,but the serum fructose was at the similar level with those in normal subjects,and had no correlation with Hb A1 c and GSP.Conclusion: In physiological concentration ratio,the glycation rates of the three reducing sugars were in the following order: ribose > glucose > fructose.Ribose plays an important role in the formation of Hb A1 c,GSP and AGEs.The above work will provide a new reference for the prediction,diagnosis and treatment of diabetic complications.