Novel inhibitors of advanced glycation endproducts

A number of natural or synthetic compounds as AGE inhibitors have been proposed, discovered or currently being advanced by others and us. We have identified two new classes of aromatic compounds; aryl- (and heterocyclic) ureido and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids, and benzoic acid derivatives and related compounds, as potential inhibitors of glycation and AGE formation. Some of these novel compounds also showed "AGE-breaking" activities in vitro. Current evidence is that chelation of transition metals and/or trapping or indirect inhibition of formation of reactive carbonyl compounds are involved in the mechanisms of action of these novel AGE inhibitors and breakers. Here, we review the inhibitors of glycation and AGE-breakers published to date and present the results of our in vitro and in vivo investigations on a number of these novel AGE inhibitors. These AGE-inhibitors and AGE-breakers may find therapeutic use in the treatment of diseases that AGE formation and accumulation may be responsible for their pathogenesis such as diabetes, Alzheimer's, rheumatoid arthritis, and atherosclerosis.     

Pyridoxamine, an inhibitor of advanced glycation and lipoxidation reactions: a novel therapy for treatment of diabetic complications

Pyridoxamine (PM), originally described as a post-Amadori inhibitor of formation of advanced glycation end-products (AGEs), also inhibits the formation of advanced lipoxidation end-products (ALEs) on protein during lipid peroxidation reactions. In addition to inhibition of AGE/ALE formation, PM has a strong lipid-lowering effect in streptozotocin (STZ)-induced diabetic and Zucker obese rats, and protects against the development of nephropathy in both animal models. PM also inhibits the development of retinopathy and neuropathy in the STZ-diabetic rat. Several products of reaction of PM with intermediates in lipid autoxidation have been identified in model reactions in vitro and in the urine of diabetic and obese rats, confirming the action of PM as an AGE/ALE inhibitor. PM appears to act by a mechanism analogous to that of AGE-breakers, by reaction with dicarbonyl intermediates in AGE/ALE formation. This review summarizes current knowledge on the mechanism of formation of AGE/ALEs, proposes a mechanism of action of PM, and summarizes the results of animal model studies on the use of PM for inhibiting AGE/ALE formation and development of complications of diabetes and hyperlipidemia.     

Rutin metabolites: Novel inhibitors of nonoxidative advanced glycation end products

Glycation is a nonenzymatic condensation reaction between reducing sugars and amino groups of proteins that undergo rearrangements to stable ketoamines, leading to the formation of advanced glycation end products (AGEs) including fluorescent (argpyrimidine) and nonfluorescent (N^ε-carboxymethyllysine; CML) protein adducts and protein cross-links. AGEs are formed via protein glycation and correlate with processes resulting in aging and diabetes complications. Reactive carbonyl species such as glyoxal and methylglyoxal are ubiquitous by-products of cell metabolism that potently induce the formation of AGEs by nonenzymatic protein glycation and may achieve plasma concentrations of 0.3–1.5 μmol/L. In this in vitro study histone H1 glycation by glyoxal, methylglyoxal, or ADP-ribose was used to model nonoxidative protein glycation, permitting us to distinguish specific AGE inhibition from general antioxidant action. Rutin derivatives were tested as AGE inhibitors because rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, is metabolized by gut microflora to a range of phenolic compounds that are devoid of significant antioxidant activity and achieve blood concentrations in the μmol/L range. Our data show that in a 1:1 stoichiometry with glyoxal or methylglyoxal, 3,4-dihydroxyphenylacetic acid (DHPAA) and 3,4-dihydroxytoluene (DHT) are powerful inhibitors of CML and argpyrimidine histone H1 adduct formation, respectively. Furthermore, when DHPAA and DHT were tested as inhibitors of histone H1 glycation by the powerful glycating agent ADP-ribose, they inhibited glycation as effectively as aminoguanidine. These results suggest that dietary flavonoids may serve as effective AGE inhibitors and suggest mechanisms whereby fruit- and vegetable-rich diets contribute to the prevention of processes resulting in aging and diabetes complications.     

Alternative routes for the formation of immunochemically distinct advanced glycation end-products in vivo

The advanced stage of the glycation process (also called the "Maillard reaction") that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. AGEs elicit a wide range of cell-mediated responses that might contribute to diabetic complications, vascular disease, renal disease, and Alzheimer's disease. Recently, it has been proposed that AGE are not only created from glucose per se, but also from dicarbonyl compounds derived from glycation, sugar autoxidation, and sugar metabolism. However, this advanced stage of glycation is still only partially characterized and the structures of the different AGEs that are generated in vivo have not been completely determined. Because of their heterogeneity and the complexity of the chemical reactions involved, only some AGEs have been characterized in vivo, including N-carboxymethyllysine (CML), pentosidine, pyrraline, and crosslines. In this article, we provide a brief overview of the pathways of AGE formation and of the immunochemical methods for detection of AGEs, and we also provide direct immunological evidence for the existence of five distinct AGE classes (designated as AGE-1 to -5) within the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis. We also propose pathways for the in vivo formation of various AGEs by glycation, sugar autoxidation, and sugar metabolism.     

Inhibition of protein glycation by skins and seeds of the muscadine grape

The formation of advanced glycation end products (AGEs) leading to protein glycation and cross-linking is associated with the pathogenesis of diabetic complications. The inhibition of protein glycation by phenolic and flavonoid antioxidants demonstrates that the process is mediated, in part, by oxidative processes. In this study, the effects of seed and skin extracts of the muscadine grape on AGEs formation were examined. Seeds and skins were extracted (10% w/v) with 50% ethanol and incubated at 37 degrees C with a solution containing 250 mM fructose and 10 mg/ml albumin. After 72 h, fluorescence was measured at the wavelength pair of 370 and 440 nm as an index of the formation of AGEs. Both seed and skin extracts were found to be efficacious inhibitors of AGE formation. A 1:300 dilution of the seed extract decreased fluorescence by approximately 65%, whereas muscadine grape skin extract produced a 40% lowering. This difference correlates with the greater antioxidant activity found in muscadine seeds in comparison to skins, however, on a mass basis, the inhibitory activities of the seeds and skins were found to be nearly equivalent. Gallic acid, catechin and epicatechin, the three major polyphenols in the seeds, all significantly decreased the AGE product related fluorescence at a concentration of 50 microM. Neither muscadine seed extract nor skin extract inhibited the methylglyoxal-mediated glycation of albumin. These results suggest that consumption of the muscadine grape may have some benefit in altering the progression of diabetic complications.     

Novel inhibitors of glycation and AGE formation

Accelerated formation of advanced glycation/lipoxidation and endproducts (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several natural and synthetic compounds have been proposed and tested as inhibitors of AGE/ALE formation. We have previously reported the therapeutic effects of several new AGE/ALE inhibitors on the prevention of nephropathy and dyslipidemia in streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the effects of various concentrations of a compound, LR-90, on the progression of renal disease and its effects on AGE and receptor for AGE (RAGE) protein expression on the kidneys of diabetic STZ-rats. Diabetic male Sprague–Dawley rats were treated with or without LR-90 (0, 5, 20, 25, and 50 mg/l of drinking water). After 32 weeks, body weight, glycemic status, renal function, and plasma lipids were measured. Kidney histopathology and AGE/ALE accumulation and RAGE protein expression in tissues were also determined. In vitro studies were also performed to determine the possible mechanism of action of LR-90 in inhibiting AGE formation and AGE-protein cross-linking. LR-90 protected the diabetic kidneys by inhibiting the increase in urinary albumin-to-creatinine ratio and ameliorated hyperlipidemia in diabetic rats in a concentration-dependent fashion without any effects on hyperglycemia. LR-90 treatment also reduced kidney AGE/ALE accumulation and RAGE protein expression in a concentration-dependent manner. In vitro, LR-90 exhibited general antioxidant properties by inhibiting metal-catalyzed reactions and reactive oxygen species (^·OH radical) and reactive carbonyl species (methlyglyoxal, glyoxal) generations without any effect on pyridoxal 5′ phosphate. The compound also prevents AGE-protein cross-linking reactions. These findings demonstrate the bioefficacy of LR-90 in treating nephropathy and hyperlipidemia in diabetic animals by inhibiting AGE accumulation, RAGE protein expression, and protein oxidation in the diabetic kidney. Additionally, our study suggests that LR-90 may be useful also to delay the onset and progression of diabetic atherosclerosis as the compound can inhibit the expression of RAGE and inflammation-related pathology, as well as prevent lipid peroxidation reactions.     

Cyperus rotundus suppresses AGE formation and protein oxidation in a model of fructose-mediated protein glycoxidation

Non-enzymatic glycation, as the chain reaction between reducing sugars and the free amino groups of proteins, has been shown to correlate with severity of diabetes and its complications. Cyperus rotundus (Cyperaceae) is used both as a food to promote health and as a drug to treat certain diseases. In this study, considering the antioxidative effects of C. rotundus, we examined whether C. rotundus also protects against protein oxidation and glycoxidation. The protein glycation inhibitory activity of hydroalcoholic extract of C. rotundus was evaluated in vitro using a model of fructose-mediated protein glycoxidation. The C. rotundus extract with glycation inhibitory activity also demonstrated antioxidant activity when a ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays as well as metal chelating activity were applied. Fructose (100 mM) increased fluorescence intensity of glycated bovine serum albumin (BSA) in terms of total AGEs during 14 days of exposure. Moreover, fructose caused more protein carbonyl (PCO) formation and also oxidized thiol groups more in glycated than in native BSA. The extract of C. rotundus at different concentrations (25–250 μg/ml) has significantly decreased the formation of AGEs in term of the fluorescence intensity of glycated BSA. Furthermore, we demonstrated the significant effect of C. rotundus extract on preventing oxidative protein damages including effect on PCO formation and thiol oxidation which are believed to form under the glycoxidation process. Our results highlight the protein glycation inhibitory and antioxidant activity of C. rotundus. These results might lead to the possibility of using the plant extract or its purified active components for targeting diabetic complications.     

Advanced glycation end-products and the progress of diabetic vascular complications

Epidemiological studies have confirmed that hyperglycemia is the most important factor in the onset and progress of vascular complications, both in Type 1 and 2 diabetes mellitus. The formation of advanced glycation end-products (AGEs) correlates with glycemic control. The AGE hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications including nephropathy, retinopathy, neuropathy and atherosclerosis. Recent studies have shown that increased formation of serum AGEs exists in diabetic children and adolescents with or without vascular complications. Furthermore, the presence of diabetic complications in children correlates with elevated serum AGEs. The level of serum AGEs could be considered as a marker of later developments of vascular complications in children with Type 1 and 2 diabetes mellitus. The careful metabolic monitoring of young diabetics together with monitoring of serum AGEs can provide useful information about impending AGE-related diabetic complications. It is becoming clear that anti-AGE strategies may play an important role in the treatment of young and older diabetic patients. Several potential drug candidates such as AGE inhibitors have been reported recently.     

Ellagic acid, a new antiglycating agent: its inhibition of Nϵ-(carboxymethyl)lysine

Non-enzymatic glycation is a complex series of reactions between reducing sugars and amino groups of proteins. Accumulation of AGEs (advanced glycation end-products) due to non-enzymatic glycation has been related to several diseases associated with aging and diabetes. The formation of AGEs is accelerated in hyperglycaemic conditions, which alters the structure and function of long-lived proteins, thereby contributing to long-term diabetic complications. The present study describes AGE inhibition and the mechanism of action of a new antiglycating agent, EA (ellagic acid), a flavonoid present in many dietary sources. Inhibition of AGE formation by EA was demonstrated with different proteins, namely eye lens TSP (total soluble protein), Hb (haemoglobin), lysozyme and BSA, using different glycating agents such as fructose, ribose and methylglyoxal by a set of complementary methods. These results suggest that the antiglycating action of EA seems to involve, apart from inhibition of a few fluorescent AGEs, predominantly inhibition of CEL [N?-(carboxyethyl)lysine] through scavenging of the dicarbonyl compounds. Furthermore, MALDI-TOF-MS (matrix-assisted laser-desorption ionisation-time-of-flight MS) analysis confirms inhibition of the formation of CEL on lysozyme on in vitro glycation by EA. Prevention of glycation-mediated β-sheet formation in Hb and lysozyme by EA confirm its antiglycating ability. Inhibition of glycosylated Hb formation in human blood under ex vivo high-glucose conditions signifies the physiological antiglycating potential of EA. We have also determined the effectiveness of EA against loss of eye lens transparency through inhibition of AGEs in the lens organ culture system. These findings establish the antiglycating potential of EA and its in vivo utility in controlling AGE-mediated diabetic pathologies.     

Amadorins: novel post-Amadori inhibitors of advanced glycation reactions

The present review focuses on the background and progress that led to discovery of specific inhibition of post-Amadori formation of advanced glycation end products, or AGEs. The "classic" or Hodge pathway begins with glucose condensation with amino groups to form a Schiff base aldimine adduct that undergoes rearrangement to a ketoamine Amadori product. This pathway is considered an important route to AGE formation that has been implicated in glucose-mediated damage in vivo (3-5). We recently described a facile procedure for isolation of proteins rich in Amadori adducts but free of AGEs, thus permitting study of pathways of conversion of Amadori compounds to AGEs. This in turn led to a unique and rapid post-Amadori screening assay for putative "Amadorins," which we define here as inhibitors of the conversion of Amadori intermediates to AGEs in the absence of excess free or reversibly bound (Schiff base) sugar. Our screening assay then led to the identification of pyridoxamine (Pyridorin) as the first member of this class of Amadorin compounds. Rather unexpectedly, the assay also led to the clear demonstration that the well-known AGE inhibitor aminoguanidine, currently in Phase 3 clinical trials for treatment of diabetic nephropathy, has negligible Amadorin activity. In view of the importance of Amadori compounds as intermediates in AGE formation in vivo, the therapeutic potential of Pyridorin is currently being investigated and is now showing highly promising results in different animal models.     

Pyridoxamine traps intermediates in lipid peroxidation reactions in vivo: evidence on the role of lipids in chemical modification of protein and development of diabetic complications

Maillard or browning reactions between reducing sugars and protein lead to formation of advanced glycation end products (AGEs) and are thought to contribute to the pathogenesis of diabetic complications. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) inhibit both the formation of AGEs and development of complications in animal models of diabetes. PM also inhibits the chemical modification of protein by advanced lipoxidation end products (ALEs) during lipid peroxidation reactions in vitro. We show here that several PM adducts, formed in incubations of PM with linoleate and arachidonate in vitro, are also excreted in the urine of PM-treated animals. The PM adducts N-nonanedioyl-PM (derived from linoleate), N-pentanedioyl-PM, N-pyrrolo-PM, and N-(2-formyl)-pyrrolo-PM (derived from arachidonate), and N-formyl-PM and N-hexanoyl-PM (derived from both fatty acids) were quantified by liquid chromatography-mass spectrometry analysis of rat urine. Levels of these adducts were increased 5-10-fold in the urine of PM-treated diabetic and hyperlipidemic rats, compared with control animals. We conclude that the PM functions, at least in part, by trapping intermediates in AGE/ALE formation and propose a mechanism for PM inhibition of AGE/ALE formation involving cleavage of alpha-dicarbonyl intermediates in glycoxidation and lipoxidation reactions. We also conclude that ALEs derived from polyunsaturated fatty acids are increased in diabetes and hyperlipidemia and may contribute to development of long term renal and vascular pathology in these diseases.     

Inhibition of advanced glycation end product formation on collagen by rutin and its metabolites

Several lines of evidence suggest that rutin, flavonoid in fruits and vegetables, or one of its metabolites may effectively modulate advanced glycation end product (AGE) formation. Following ingestion, rutin forms metabolites that include 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3,4-dihydroxytoluene (3,4-DHT), m-hydroxyphenylacetic acid (m-HPAA), 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA) and 3,5,7,3',5'-pentahydroxyflavonol (quercetin). We studied the effects of rutin and its metabolites on the formation of AGE biomarkers such as pentosidine, collagen-linked fluorescence, N(epsilon)-carboxymethyllysine (CML) adducts, glucose autoxidation and collagen glycation, using an in vitro model where collagen I was incubated with glucose. Rutin metabolites containing vicinyl dihydroxyl groups, i.e., 3,4-DHT, 3,4-DHPAA and quercetin, inhibited the formation of pentosidine and fluorescent adducts, glucose autoxidation and glycation of collagen I in a dose-dependent manner, whereas non-vicinyl dihydroxyl group-containing metabolites, i.e., HVA and m-HPAA, were much less effective. All five metabolites of rutin effectively inhibited CML formation. In contrast, during the initial stages of glycation and fluorescent AGE product accumulation, only vicinyl hydroxyl group-containing rutin metabolites were effective. These studies demonstrate that rutin and circulating metabolites of rutin can inhibit early glycation product formation, including both fluorescent and nonfluorescent AGEs induced by glucose glycation of collagen I in vitro. These effects likely contribute to the beneficial health effects associated with rutin consumption.     

Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: characterization of a major product from the reaction of pyridoxamine and methylglyoxal

Advanced glycation end products (AGEs) from the Maillard reaction contribute to protein aging and the pathogenesis of age- and diabetes-associated complications. The alpha-dicarbonyl compound methylglyoxal (MG) is an important intermediate in AGE synthesis. Recent studies suggest that pyridoxamine inhibits formation of advanced glycation and lipoxidation products. We wanted to determine if pyridoxamine could inhibit MG-mediated Maillard reactions and thereby prevent AGE formation. When lens proteins were incubated with MG at 37 degrees C, pH 7.4, we found that pyridoxamine inhibits formation of methylglyoxal-derived AGEs concentration dependently. Pyridoxamine reduces MG levels in red blood cells and plasma and blocks formation of methylglyoxal-lysine dimer in plasma proteins from diabetic rats and it prevents pentosidine (an AGE derived from sugars) from forming in plasma proteins. Pyridoxamine also decreases formation of protein carbonyls and thiobarbituric-acid-reactive substances in plasma proteins from diabetic rats. Pyridoxamine treatment did not restore erythrocyte glutathione (which was reduced by almost half) in diabetic animals, but it enhanced erythrocyte glyoxalase I activity. We isolated a major product of the reaction between MG and pyridoxamine and identified it as methylglyoxal-pyridoxamine dimer. Our studies show that pyridoxamine reduces oxidative stress and AGE formation. We suspect that a direct interaction of pyridoxamine with MG partly accounts for AGE inhibition.     

AGE-breakers cleave model compounds,but do not break Maillard crosslinks in skin and tail collagen from diabetic rats

Advanced glycation end products (AGE), formed by nonenzymatic Maillard reactions between carbohydrate and protein, contribute to the increase in chemical modification and crosslinking of tissue proteins with age. Acceleration of AGE formation in collagen during hyperglycemia, with resultant effects on vascular elasticity and basement membrane permeability, is implicated in the pathogenesis of diabetic complications. AGE-breakers, such as N-phenacylthiazolium (PTB) and N-phenacyl-4,5-dimethylthiazolium (PMT) halides, have been proposed as therapeutic agents for reversing the increase in protein crosslinking in aging and diabetes. We have confirmed that these compounds, as well as the AGE-inhibitor pyridoxamine (PM), cleave the model AGE crosslink, phenylpropanedione, and have studied the effects of these compounds in reversing the increased crosslinking of skin and tail collagen isolated from diabetic rats. Crosslinking of skin collagen, measured as the half-time for solubilization of collagen by pepsin in 0.5M acetic acid, was increased approximately 5-fold in diabetic, compared to nondiabetic rats. Crosslinking of tail tendon collagen, measured as insolubility in 0.05 N acetic acid, was increased approximately 10-fold. Collagen preparations were incubated in the presence or absence of AGE-breakers or PM in phosphate buffer, pH 7.4, for 24h at 37 degrees C. These treatments did not decrease the half-time for solubilization of diabetic skin collagen by pepsin or increase the acid solubility of diabetic tail tendon collagen. We conclude that, although AGE-breakers and PM cleave model crosslinks, they do not significantly cleave AGE crosslinks formed in vivo in skin collagen of diabetic rats.     

Genistein prevents the glucose autoxidation mediated atherogenic modification of low density lipoprotein

Hyperglycemia has been assumed to be responsible for oxidative stress in diabetes. In this respect, glucose autoxidation and advanced glycation end products (AGE) may play a causal role in the etiology of diabetic complications as e.g. atherosclerosis. There is now growing evidence that the oxidative modification of LDL plays a potential role in atherogenesis. Glucose derived oxidants have been shown to peroxidise LDL. In the present study, genistein, a compound derived from soy with a flavonoid chemical structure (4′, 5, 7-trihydroxyisoflavone) has been evaluated for its ability to act as an antioxidant against the atherogenic modification of LDL by glucose autoxidation radical products. Daidzein, (4′, 7-dihydroxyisoflavone) an other phytoestrogen of soy, was tested in parallel. Genistein — in contrast to daidzein — effectively prevented the glucose mediated LDL oxidation as measured by thiobarbituric acid-reactive substance formation (TBARS), alteration in electrophoretic mobility, lipid hydroperoxides and fluorescence quenching of tryptophan residues of the lipoprotein. In addition the potential of glucose-oxidized LDL to increase tissue factor (TF) synthesis in human endothelial cells (HUVEC) was completely inhibited when genistein was present during LDL oxidative modification by glucose. Both phytoestrogens did not influence the nonenzymatic protein glycation reaction as measured by the in vitro formation of glycated LDL. As the protective effect of genistein on LDL atherogenic modification was found at glucose/genistein molar ratios which may occur in vivo, our findings support the suggested beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.     

Overexpression of glyoxalase-I reduces hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ～50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.     

A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation.

Reactive carbonyl compounds are formed during autoxidation of carbohydrates and peroxidation of lipids. These compounds are intermediates in the formation of advanced glycation end products (AGE) and advanced lipoxidation end products (ALE) in tissue proteins during aging and in chronic disease. We studied the reaction of carbonyl compounds glyoxal (GO) and glycolaldehyde (GLA) with pyridoxamine (PM), a potent post-Amadori inhibitor of AGE formation in vitro and of development of renal and retinal pathology in diabetic animals. PM reacted rapidly with GO and GLA in neutral, aqueous buffer, forming a Schiff base intermediate that cyclized to a hemiaminal adduct by intramolecular reaction with the phenolic hydroxyl group of PM. This bicyclic intermediate dimerized to form a five-ring compound with a central piperazine ring, which was characterized by electrospray ionization-liquid chromatography/mass spectrometry, NMR, and x-ray crystallography. PM also inhibited the modification of lysine residues and loss of enzymatic activity of RNase in the presence of GO and GLA and inhibited formation of the AGE/ALE N(epsilon)-(carboxymethyl)lysine during reaction of GO and GLA with bovine serum albumin. Our data suggest that the AGE/ALE inhibitory activity and the therapeutic effects of PM observed in diabetic animal models depend, at least in part, on its ability to trap reactive carbonyl intermediates in AGE/ALE formation, thereby inhibiting the chemical modification of tissue proteins.     

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.     

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.     

The pathogenic role of Maillard reaction in the aging eye

The proteins of the human eye are highly susceptible to the formation of advanced glycation end products (AGEs) from the reaction of sugars and carbonyl compounds. AGEs progressively accumulate in the aging lens and retina and accumulate at a higher rate in diseases that adversely affect vision such as, cataract, diabetic retinopathy and age-related macular degeneration. In the lens AGEs induce irreversible changes in structural proteins, which lead to lens protein aggregation and formation of high-molecular-weight aggregates that scatter light and impede vision. In the retina AGEs modify intra- and extracellular proteins that lead to an increase in oxidative stress and formation of pro-inflammatory cytokines, which promote vascular dysfunction. This review outlines recent advances in AGE research focusing on the mechanisms of their formation and their role in cataract and pathologies of the retina. The therapeutic action and pharmacological strategies of anti-AGE agents that can inhibit or prevent AGE formation in the eye are also discussed.