Non-enzymatic glycation of proteins: a cause for complications in diabetes

Diabetes mellitus is one of the most common non-communicable diseases, and is the fifth leading cause of death in most of the developed countries. It can affect nearly every organ and system in the body and may result in blindness, end stage renal disease, lower extremity amputation and increase risk of stroke, ischaemic heart diseases and peripheral vascular disease. Hyperglycemia in diabetes causes non-enzymatic glycation of free amino groups of proteins (of lysine residues) and leads to their structural and functional changes, resulting in complications of the diabetes. Glycation of proteins starts with formation of Shiff's base, followed by intermolecular rearrangement and conversion into Amadori products. When large amounts of Amadori products are formed, they undergo cross linkage to form a heterogeneous group of protein-bound moieties, termed as advanced glycated end products (AGEs). Rate of these reactions are quite slow and only proteins with large amounts of lysine residues undergo glycation with significant amounts of AGEs. The formation of AGEs is a irreversible process, causing structural and functional changes in protein leading to various complications in diabetes like nephropathy, retinopathy, neuropathy and angiopathy. The present review discusses about role of glycation in various complications of diabetes.     

Peptide and amino acid glycation: new insights into the Maillard reaction.

Nonenzymatic glycation of proteins, peptides and other macromolecules (the Maillard reaction) has been implicated in a number of pathologies, most clearly in diabetes mellitus. but also in the normal processes of aging and neurodegenerative amyloid diseases such as Alzheimer's. In the early stage, glycation results in the formation of Amadori-modified proteins. In the later stages, advanced glycation end products (AGE) are irreversibly formed from Amadori products leading to the formation of reactive intermediates, crosslinking of proteins, and the formation of brown and fluorescent polymeric materials. Although, the glycation of structural proteins has been attributed a key role in the complications of diabetes, recent attention has been devoted to the physiological significance of glycated peptide hormones. This review focuses on the physico-chemical properties of the Amadori compounds of bioactive peptides of endogenous and exogenous origin, such as Leu-enkephalin and morphiceptin, investigated under different conditions as well as on novel pathways in the Maillard reaction observed from investigating intramolecular events in ester-linked glycopeptides.     

Role of Early Glycation Amadori Products of Lysine-Rich Proteins in the Production of Autoantibodies in Diabetes Type 2 Patients

In diabetes, protein glycation mostly occurs at intrachain lysine residues resulting in the formation of early stage Amadori products which are finally converted to advance glycation end products (AGEs). Several studies have reported autoantibodies against AGEs in diabetes but not much data are found in respect of Amadori products. In this study, poly-l-lysine (PLL) was glycated with 50 mM glucose and the resultant Amadori products were estimated by fructosamine or nitroblue tetrazolium assay. We report high content of Amadori products in PLL upon glycation. Glycated PLL showed marked hyperchromicity in the UV spectrum, ellipticity changes in CD spectroscopy, and variations in ε-methylene protons shift in NMR. It was better recognized by autoantibodies in type 2 diabetics compared to the native PLL. Induced antibodies against glycated PLL were successfully used to probe early glycation in the IgG isolated from diabetes type 2 patients. Role of Amadori products of glycated proteins in the induction of autoantibodies in type 2 diabetes as well as in associated secondary complications has been discussed.     

Physicochemical analysis of structural changes in DNA modified with glucose

Reactions of reducing sugars with free amino groups of proteins can form advanced glycation end products (AGEs). While the formation of nucleoside AGEs has been studied in detail, no extensive work has been carried out to assess DNA Amadori and DNA advanced glycation end products. In this study, we report biophysical/chemical characterization of glucose-induced changes in DNA, as well as DNA Amadori and DNA advanced glycation end products. Glucose treated DNA exhibited hyperchromicity, decrease in melting temperature, and enhanced emission intensity in a time dependent manner. Formation of DNA Amadori product and DNA advanced glycation end products, mainly CEdG (N(2)-carboxyethyl-2'-deoxyguanosine), were the major outcome of the study.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

Structural and immunological characterization of Amadori-rich human serum albumin: role in diabetes mellitus

Proteins modifications in diabetes may lead to early glycation products (EGPs) as well as advanced glycation end products (AGEs). Whereas no extensive studies have been carried out to assess the role of EGPs in secondary complications of diabetes, numerous investigators have demonstrated the role of AGEs. Early glycation involves attachment of glucose on ε-NH2 of lysine residues of proteins leading to generation of the Amadori product (an early glycation species). This study reports the structural and immunological characterization of EGPs of HSA because we believe that during persistent hyperglycemia the HSA, one of the major blood proteins, can undergo fast glycation. Glucose mediated generation of EGPs of HSA was quantitated as Amadori products by NBT assay and authenticated by boronate affinity chromatography and LC/MS. Compared to native HSA changes in glycated-HSA were characterized by hyperchromicity, loss in fluorescence intensity and a new peak in the FTIR profile. Immunogenicity of native- and glycated-HSA was evaluated by inducing antibodies in rabbits. Results suggest generation of neo-epitopes on glycated-HSA rendering it highly immunogenic compared to native HSA. Quantization of EGPs of HSA by authentic antibodies against HSA-EGPs can be used as marker for early detection of the initiation/progression of secondary complications of diabetes.     

Gramicidin S: a peptide model for protein glycation and reversal of glycation using nucleophilic amines.

Nonenzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. Glycation of proteins proceeds through the stages of Schiff base formation, conversion to ketoamine product and advanced glycation end products. Gramicidin S, which has two ornithine residues, was used as a model system to study the various stages of glycation of proteins using electrospray ionization mass spectrometry. The proximity of two ornithine residues in the peptide favors the glycation reaction. Formation of advanced glycation end products and diglycation on ornithine residues in gramicidin S were observed. The formation of Schiff base adduct is reversible, whereas the Amadori rearrangement to the ketoamine product is irreversible. Nucleophilic amines and hydrazines can deglycate the Schiff base adduct of glucose with peptides and proteins. Hydroxylamine, isonicotinic acid hydrazide and aminoguanidine effectively removed glucose from the Schiff base adduct of gramicidin S. Hydroxylamine is more effective in deglycating the adduct compared with isonicotinic acid hydrazide and aminoguanidine. The observation that the hydrazines are effective in deglycating the Schiff base adduct even in the presence of high concentrations of glucose, may have a possible therapeutic application in preventing complications of diabetes mellitus. Hydrazines may be used to distinguish between the Schiff base and the ketoamine products formed at the initial stages of glycation.     

Inhibition of nonenzymatic protein glycation and lipid peroxidation by drugs with antioxidant activity

We studied the effects of aminoguanidine (AG), beta-resorcylidene aminoguanidine (RAG), DL-penicillamine (PNCA) and captopril on early and advanced glycation of human serum albumin (HSA). We also assessed inhibition of lipid peroxidation by AG and RAG in erythrocytes. Incubation of HSA with D-glucose (20 mM, 37 degrees C for 21 days) led to the formation of Amadori products and fluorescent advanced glycation end-products (AGE). Only PNCA markedly reduced the formation of Amadori products, while all tested compounds markedly reduced the formation of AGE. AG and RAG also inhibited malondialdehyde formation in erythrocytes incubated with hydrogen peroxide. Addition of AG at concentrations from 1 microM to 1 mM caused a 10-80% inhibition of lipid peroxidation. Thus, AG and RAG inhibit toxic oxidative processes and may have therapeutic potential in a number of human diseases.     

Production and characterization of antibodies to advanced glycation products on proteins

Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.     

Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. Evidence for the presence of a common structure

Reaction of protein amino groups with glucose (the Maillard reaction) leads from early stage products such as Schiff base and Amadori products to advanced glycation end products (AGE), structures implicated in diabetic complications and the aging process. We have prepared the polyclonal anti-AGE antibody and the monoclonal anti-AGE antibody against AGE-bovine serum albumin and made an immunochemical approach to characterize AGE structures. Both polyclonal and monoclonal antibodies reacted with AGE-proteins such as AGE-bovine serum albumin, AGE-human serum albumin, and AGE-hemoglobin but not with unmodified counterparts. Treatments of these AGE-proteins with borohydride had no effect on the immunoreactivity. Moreover, fructosyl-epsilon-caproic acid, a synthetic Amadori compound, did not serve as an antigen, indicating that these antibodies were specific for AGE products but not for early stage products of the Maillard reaction. In addition, these antibodies were also able to recognize AGE products prepared either from alpha-tosyl-1-lysine, alpha-tosyl-1-lysine methyl ester, monoaminocarboxylic acid such as epsilon-aminocaproic acid, gamma-amino-n-butyric acid, and beta-alanine. Thus, these results strongly suggest the presence of a common structure in AGE preparations, regardless of whether AGE products are generated from proteins, amino acids, or monoaminocarboxylic acids.     

Intracellular protein glycation in cultured retinal capillary pericytes and endothelial cells exposed to high-glucose concentration.

There is now increasing evidence suggesting that non-enzymatic glycation (NEG) of proteins is involved in the pathogenesis of chronic diabetic complication. In this study we demonstrate that chronic exposure to high-glucose concentration leads to intracellular protein glycation in cultured bovine retinal capillary pericytes and endothelial cells. The level of intracellular protein glycation, as measured using a competitive enzyme-linked immunoabsorbant assay (ELISA), was found to increase in both pericytes and endothelial cells as function of time. As expected products of NEG were only detected when the Schiff base and the Amadori products were chemically reduced to glucitollysine by sodium borohydride. Despite the accumulation of early glycation products on cellular proteins there was no further rearrangement reaction into advanced glycation endproducts (AGEs), even after 12 days of incubation in high-glucose medium. Immunofluorescence microscopy demonstrated that the monoclonal antibody reacting with glucitollysine stains the cytoplasm of both pericytes and endothelial cells in a finely punctate pattern. Further studies using Western blot analysis suggested that a number of cellular proteins, including smooth muscle actin in pericytes, become rapidly glycated. The results from this in vitro study suggest that excessive accumulation of early products of non-enzymatic glycation in pericytes and endothelial cells may play an important role in the pathogenesis of diabetic retinopathy.     

Hypochlorous acid generates N epsilon-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Comparative study of different glycating agents on human plasma and vascular cells

Molecular Biology Reports - Diabetic complications are associated with the glycation and formation of advanced glycation end products (AGEs) which leads to structural modifications of biomolecules...     

Acetoacetate enhancement of glucose mediated DNA glycation

Acetoacetate (AA) is a ketone body, which generates reactive oxygen species (ROS). ROS production is impacted by the formation of covalent bonds between amino groups of biomacromolecules and reducing sugars (glycation). Glycation can damage DNA by causing strand breaks, mutations, and changes in gene expression. DNA damage could contribute to the pathogenesis of various diseases, including neurological disorders, complications of diabetes, and aging. Here we studied the enhancement of glucose-mediated DNA glycation by AA for the first time. The effect of AA on the structural changes, Amadori and advanced glycation end products (AGEs) formation of DNA incubated with glucose for 4 weeks were investigated using various techniques. These included UV–Vis, circular dichroism (CD) and fluorescence spectroscopy, and agarose gel electrophoresis. The results of UV–Vis and fluorescence spectroscopy confirmed that AA increased the DNA-AGE formation. The NBT test showed that AA also increased Amadori product formation of glycated DNA. Based on the CD and agarose gel electrophoresis results, the structural changes of glycated DNA was increased in the presence of AA. The chemiluminescence results indicated that AA increased ROS formation. Thus AA has an activator role in DNA glycation, which could enhance the adverse effects of glycation under high glucose conditions.     

Chemical tests as alternatives to animal tests in research on late symptoms in diabetes

Glycation reactions, such as those seen in late diabetes, can be mimicked in purely chemical systems. The glycation is time-dependent, and in in vitro systems it can continue for days. Ascorbate seems to enhance the reactions. The reactions are associated with free-radical formation through transformation of an Amadori product to (deoxy-)glycoson, catalysed by heavy metals. Ascorbate enhances the reaction by a factor of 5-10 compared with in vitro systems without ascorbate. In vitro systems containing bovine serum albumin retard the formation of free-radicals, because of the formation of advanced glycation products.     

Inhibition of protein glycation and advanced glycation end products by ascorbic acid and other vitamins and nutrients

Nonenzymatic glycation, the reaction of glucose and other reducing sugars with protein, reversibly produces Amadori products and over a long period irreversible advanced glycation end products. In diabetes, these reactions are greatly accelerated and are important in the pathogenesis of diabetic complications.

In vitro glycation was studied with bovine albumin as the model protein. A mixture of 25 mM glucose/fructose was used as the glycating agent. The Amadori product was quantitated by thiobarbituric acid colorimetry after hydrolysis. Advanced glycation end products were measured by their intrinsic fluorescence. A number of vitamins and nutrients were found to be potent inhibitors of both the glycation reaction and the subsequent end products. The nutrients were effective at physiological concentrations and exhibited dose-response relationships. The inhibitors included ascorbic acid, tocopherol, pyridoxal, niacinamide, sodium selenite, selenium yeast, and carnosine. A significant correlation was found between the inhibition of glycation and the inhibition of AGE formation (P < 0.001). One of the nutrients, ascorbic acid, was used in a pilot study. Eighteen normal subjects, 7 college age and 10 middle age, were supplemented with 1,000 mg of ascorbic acid in the form of Re-Natured Vitamin C^® for a period of 4 weeks. Serum protein glycation was decreased an average of 46.8% (P < 0.01). These results underline the importance of nutrition in diabetes and indicate the possibility of therapeutic use of these nutrients for the prevention of diabetic complications.     

Hypochlorous acid generates Nε-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Glycation of H1 Histone by 3-Deoxyglucosone: Effects on Protein Structure and Generation of Different Advanced Glycation End Products

Advanced glycation end products (AGEs) culminate from the non-enzymatic reaction between a free carbonyl group of a reducing sugar and free amino group of proteins. 3-deoxyglucosone (3-DG) is one of the dicarbonyl species that rapidly forms several protein-AGE complexes that are believed to be involved in the pathogenesis of several diseases, particularly diabetic complications. In this study, the generation of AGEs (N^ε-carboxymethyl lysine and pentosidine) by 3-DG in H1 histone protein was characterized by evaluating extent of side chain modification (lysine and arginine) and formation of Amadori products as well as carbonyl contents using several physicochemical techniques. Results strongly suggested that 3-DG is a potent glycating agent that forms various intermediates and AGEs during glycation reactions and affects the secondary structure of the H1 protein. Structural changes and AGE formation may influence the function of H1 histone and compromise chromatin structures in cases of secondary diabetic complications.     

Screening for glucose‐triggered modifications of glutathione

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. These structures may undergo further Amadori rearrangement and free radical‐mediated oxidation to finally generate irreversible advanced glycation end products (AGEs). One of the factors known to modulate the glycation of proteins is glutathione, the most abundant nonprotein thiol tripeptide with the γ‐linkage, H‐Glu(Cys‐Gly‐OH)‐OH (GSH). Screening for products formed by GSH with D ‐glucose is an essential step in understanding the participation of GSH in glycation (the Maillard) reaction. Under the conditions used in these studies we observed N‐(1‐deoxy‐D ‐fructos‐1‐yl)‐pyroglutamic acid as the major glycation product formed in the mixtures of GSH and glucose in vitro. A RP HPLC/MS and tandem MS analyses of the GSH/glucose mixtures revealed that cleavage of the N‐terminal glutamic acid and the formation of pyroglutamic acid‐related Amadori product were accompanied by generation of Cys‐Gly‐derived Amadori and thiazolidine compounds. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Formation of the molten globule-like state during prolonged glycation of human serum albumin

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA (r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively. The formation of advanced glycation end products (AGE) was detected using the autofluorescence characteristic of samples. We identified three stages of Maillard reaction for HSA upon incubation with the physiological level of glucose (0-630 mg/dl): the early, intermediate and late stages, which occurred after 7-14, 21, and >28 days of incubation, respectively. Structural information, Stokes radius, and 1-anilinonaphthalene-8-sulfonate (ANS) binding data indicated the formation of a molten globule-like state of HSA after 21 days of incubation with 35 mM (630 mg/dl) glucose. Thus, the extent of the Maillard reaction was influenced by the concentration of glucose and incubation time, such that longer exposure of HSA to glucose may have a more deleterious effect on its structure and especially on its half-life and turnover in the circulation. Our results suggest that in acute diabetes mellitus patients, HSA, after 21 days of glycation, passes through a molten globule-like state and may contribute to the pathogenesis of diabetes, and perhaps other diseases.