Characterization of advanced glycation end products for biochemical studies: side chain modifications and fluorescence characteristics

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases and therefore effects of AGEs on cells are the objective of numerous investigations. Since AGEs used in biochemical studies are usually not chemically characterized, comparison of data is difficult if not impossible. To find a suitable characterization protocol, human serum albumin was reacted with different concentrations of glucose, methyl glyoxal, and glyoxylic acid. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine), the carboxymethyl lysine and carbonyl content, and the fibrillar state. Additionally, their fluorescence and absorbance characteristics were extensively studied. Although we found significant differences in the degree of modification and in AGE-specific fluorescence when using different modifiers, the results provide important information and allow comparing AGEs derived from different modifier concentrations. The results also suggest strong conformational changes within the modified proteins. In the present paper we propose a set of parameters that is sufficient to partially characterize AGEs used for biochemical studies.     

Studies on advanced glycation end products by recent mass spectrometric techniques

Summary The results obtained by different mass spectrometric approaches in the field of advanced glycation of proteins are reported and discussed in detail in comparison with those obtained by other analytical methodologies (fluorescence and absorbance spectroscopies, radioimmunoassay, enzyme-linked immunosorbent assay). They have been subdivided in three main groups: analysis on degraded glycated proteins, direct analysis of glycated proteins and studies on the reaction between protected lysine and glucose. The general overview so achieved indicate mass spectrometry as a particularly valid analytical method in this field of research.     

Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes

In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18–36 years) and eighteen individuals without diabetes (17–35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (μg/l) and CEC (μg/l) were significantly higher in DM (55.73 ± 29.43, 521.47 ± 239.13, respectively) compared to controls (24.25 ± 10.26, 262.85 ± 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.     

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.     

Receptor for advanced glycation end products (RAGE)-mediated cytotoxicity of 3-hydroxypyridinium derivatives

Advanced glycation end products (AGEs) formed from glyceraldehyde (Gcer) and glycolaldehyde (Gcol) are involved in the pathogenesis of diabetic complications, via interactions with a receptor for AGEs (RAGE). In this study, we aimed to elucidate the RAGE-binding structure in Gcer and Gcol-derived AGEs and identify the minimal moiety recognized by RAGE. Among Gcer and Gcol-derived AGEs, GLAP (glyceraldehyde-derived pyridinium) and GA-pyridine elicited toxicity in PC12 neuronal cells. The toxic effects of GLAP and GA-pyridine were suppressed in the presence of anti-RAGE antibody or the soluble form of RAGE protein. Furthermore, the cytotoxicity test using GLAP analog compounds indicated that the 3-hydroxypyridinium (3-HP) structure is sufficient for RAGE-dependent toxicity. Surface plasmon resonance analysis showed that 3-HP derivatives directly interact with RAGE. These results indicate that GLAP and GA-pyridine are RAGE-binding epitopes, and that 3-HP, a common moiety of GLAP and GA-pyridine, is essential for the interaction with RAGE.     

Reduced cell replication and induction of apoptosis by advanced glycation end products in rat Schwann cells

We investigated the effects of advanced glycation end products (AGEs) derived from glucose, glyceraldehyde, and glycolaldehyde (designated as AGE-1, -2, and -3, respectively) on the viability, replication rate, and cytokine production of cultured Schwann cells. AGE-2 and -3, but not AGE-1, induced apoptosis, and significantly decreased the viability measured by MTT assay. The decrease was prevented completely by antioxidant alpha-lipoic acid and was prevented partially by p38 mitogen-activated protein kinase inhibitor SB202190. The decrease in mitochondrial membrane potential by AGE-2 and -3 was also observed. In addition, AGE-2 and -3 significantly suppressed the replication rate as shown by reduced bromodeoxyuridine uptake, whereas they enhanced the release of TNF-alpha and IL-1beta into the medium and activated nuclear factor-kappaB. The effects of AGE-1 on these measures were equivocal. The series of events elicited by AGE-2 and -3 may be responsible for some of the aspects of pathogenetic mechanisms in patients with diabetic neuropathy.     

Atorvastatin exerts its anti-atherosclerotic effects by targeting the receptor for advanced glycation end products

Recent studies demonstrated the beneficial role of atorvastatin in reducing the risk of cardiovascular morbidity and mortality in patients with diabetes mellitus and/or metabolic syndrome. To investigate the mechanisms underlying the anti-atheroscleroic action of atorvastatin, we examined the expression of the receptor for advanced glycation end products (RAGE) and its downstream target gene, monocyte chemoattractant protein-1 (MCP-1) using real-time PCR. In in vitro studies, exposure to high glucose or AGE induced oxidative stress and activation of the AGE/RAGE system in human umbilical vein endothelial cells. Treatment of the cells with atorvastatin significantly released the oxidative stress by restoring the levels of glutathione and inhibited the RAGE upregulation. In diabetic Goto Kakisaki (GK) rats fed with a high-fat diet for 12 weeks, RAGE and MCP-1 were upregulated in the aortas, and there was a significant correlation between RAGE and MCP-1 mRNA abundance (r = 0.482, P = 0.031). Treatment with atorvastatin (20 mg/kg qd) significantly downregulated the expression of RAGE and MCP-1. These data thus demonstrate a novel “pleiotropic” activity of atorvastatin in reducing the risk of cardiovascular diseases by targeting RAGE expression.     

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.     

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolase-derived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K_M value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V_max and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.     

Identification of a novel advanced glycation end product derived from lactaldehyde

Advanced glycation end products (AGEs) are implicated in the development of diabetic complications via the receptor for AGEs (RAGE). We have reported that the 3-hydroxypyridinium (3HP)-containing AGEs derived from α-hydroxyaldehydes physically interact with RAGE and show cytotoxicity. Lactaldehyde (LA) is formed from a reaction between threonine and myeloperoxidase, but no LA-derived AGEs have been characterized. Here, we identify the structure and physiological effects of an AGE derived from LA. We isolated a novel 3HP derivative, 2-acetamido-6-(3-hydroxy-5-methyl-pyridin-1-ium-1-yl)hexanoate, named as N-acetyl-LAPL (lactaldehyde-derived pyridinium-type lysine adduct), from a mixture of LA with N^α-acetyl-L-lysine. LAPL was also detected in the LA-modified protein. LAPL elicited toxicity in PC12 neuronal cells, but the effect was suppressed by the soluble form of RAGE as a decoy receptor. Moreover, surface plasmon resonance-based analysis revealed that LAPL specifically binds to recombinant RAGE. These results indicate that LA generates an AGE containing the 3HP moiety and contributes to RAGE-dependent cytotoxicity.

Abbreviations: AGEs: advanced glycation end products; RAGE: receptor for advanced glycation end products; 3HP: 3-hydroxypyridinium; LA: lactaldehyde; LAPL: lactaldehyde-derived pyridinium-type lysine adduct; BSA: bovine serum albumin; GLAP: glyceraldehyde-derived pyridinium; MPO: myeloperoxidase; HFBA: heptafluorobutyric acid; TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; LC-ESI-QTOF-MS: liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry; NMR: nuclear magnetic resonance; LA-BSA: lactaldehyde-modified bovine serum albumin; PBS: phosphate buffered saline, GST, glutathione S-transferase; SPR: surface plasmon resonance; OP-lysine: 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate; GLO1: glyoxalase 1; MG, methylglyoxal     

晚期糖化终产物诱导内皮细胞通透性增高

本文探讨了晚期糖化终产物(advanrced glycation end products,AGEs)修饰蛋白对内皮细胞通透性及细胞骨架肌动蛋白的形态学影响,以及特异的AGEs受体(receptors for AGEs,RAGE)、氧化应激和p38 MAPK通路在此病理过程中的作用。用不同浓度的AGEs修饰人血清白蛋白(AGE-HSA)与人脐静脉内皮细胞株ECV304在体外共同培养不同时间,并设立对照组进行比较,采用TRITC荧光标记白蛋白漏出法测定单层内皮细胞的通透系数Pa值,荧光染色法示细胞骨架的形态学改变。与对照组相比,AGE-HSA以时间和剂量依赖的方式引起单层内皮细胞通透性的升高及细胞骨架肌动蛋白F-actin形态的改变;可溶性RAGE的抗体(anti-RAGE IgG)、NADPH氧化酶抑制剂(apocynin)及p38抑制剂SB203580均可减轻AGEs对内皮细胞屏障功能和形态的影响。结果提示,AGEs修饰蛋白对单层内皮细胞通透性及骨架重排的作用可能通过与内皮细胞上的RAGE结合,引起细胞内的氧化应激,并激活p38 MAPK通路所介导。     

Therapeutic potential of breakers of advanced glycation end product-protein crosslinks

Long-lived structural proteins, collagen and elastin, undergo continual non-enzymatic crosslinking during aging and in diabetic individuals. This abnormal protein crosslinking is mediated by advanced glycation end products (AGEs) generated by non-enzymatic glycosylation of proteins by glucose. The AGE-derived protein crosslinking of structural proteins contributes to the complications of long-term diabetes such as nephropathy, retinopathy, and neuropathy. AGE-crosslinks have also been implicated in age-related cardiovascular diseases. Potential treatment strategies for these AGE-derived complications include prevention of AGE-formation and breaking of the existing AGE-crosslinks. The therapeutic potential of the AGE-inhibitor, pimagedine (aminoguanidine), has been extensively investigated in animal models and in Phase 3 clinical trials. This review presents the pre-clinical and clinical studies using ALT-711, a highly potent AGE-crosslink breaker that has the ability to reverse already-formed AGE-crosslinks. Oral administration of ALT-711 has resulted in a rapid improvement in the elasticity of stiffened myocardium in experimental animals. Topical administration of ALT-711 was effective in improving the skin hydration of aged rats. The therapeutic potential of crosslink breakers for cardiovascular complications and dermatological alterations associated with aging and diabetes is discussed.     

渐进性糖化终产物与糖尿病肾病

高级糖化终末产物(advanced glycation end product,AGE)参与了糖尿病、动脉粥样硬化、癌症等多种疾病的发生和发展,尤其是其导致的糖尿病肾病(diabetic nephropathy,DN)是终末期肾衰竭的主要病因,因此探索以AGEs为靶点的DN治疗手段成为了国内外研究的热点。本文概述了国内外关于AGE参与DN的发病机制,靶向AGE的DN治疗策略,以及天然中药基于AGE为靶点干预DN的研究进展,初步探讨了靶向AGE的DN天然药物的筛选模型。     

Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoptosis

Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-kappaB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-kappaB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.     

Prevention and reversible solubilization of advanced glycation and products (AGE) by organic germanium compounds as derivatives of amino acids

Summary The amino-carbonyl reaction (The Maillard reaction) of bovine lens crystallin, serum albumin or skin collagen with glucose was investigated to find effective means to prevent the formation of Advanced Glycation End Products (AGE) and induce the reversible solubilization of polymerized glycated proteins. The organic germanium compounds (Ge-132, 373, 385), derivatives of amino acids containing germanium as the linker of framework, were combined by the box titration method to determine the dose that would be most effective, compared with Aminoguanidine-HCl (AMG),-tocopherol (VE), and pirenoxine (Catalin-K, CK). Although AMG suppressed the formation of AGE, effective concentrations were higher than 20 mM. Ge-385, when administered by itself at a low dose, induced the reversible solubilization of AGE made from crystallin, and albumin. The addition of any two reagents such as AMG, VE, CK and Ge-132 or 385 together to proteins lessened the effective range, and the peaks of smaller molecules in the profiles of HPLC and PAGE were quite remarkable. Examination was made of the effects of Ge-132 on the eyes of SAM mice, which show senescence accelerated cataracts at a relatively young age. The prevention of cataract-genesis and induction of reversible transparency of turbid lenses became evident following the administration of Ge-132 to the eyes 4 times a day. The mode of action of organic germanium compounds was demonstrated quite capable of disconnecting the sugar-parts from AGE by decarbonylation, resulting in the formation of glucosone and amino residues, and further leading subsequently to fewer AGE.Abbreviations used in this paper: BLC bovine lens crystallin; BSA bovine serum albumin; AsCol acid soluble bovine skin collagen type III; AGE advanced glycation end products; Ge-132 2-Carboxyethylgermanium sesquioxide, Ge-373 2-Carboxy-2-amino-6-phenyl germanium sesquioxide; Ge-385 2-Carboxy-ethyl-2-aminogermanium sesquioxide; AMG or AG aminoguanidine-HCl; V. E. vitamin E or-tocopherol; CK 1-Hydroxy-5-oxo-5H-pyrido [3, 2-a] phenoxazine-3-carboxylic acid or catalin-K or pirenoxine; PACE polyacrylamide gel electrophoresis; SAM senescence accelerated mouse; HPLC high pressure liquid chromatography; SDS sodium laurylsulfate; FT fructose-p-toluidine.     

Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.     

COMP-Ang1 inhibits apoptosis as well as improves the attenuated osteogenic differentiation of mesenchymal stem cells induced by advanced glycation end products

Background

In the present study, we have investigated the possibility that cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), important factor in angiogenesis, osteogenesis and the survival of mesenchymal stem cells (MSCs) through the Ang1/Tie2 pathway has beneficial effects on osteogenic differentiated cells (ODCs) from MSCs treated by advanced glycation end products (AGE), which are pathological factors of diabetes.

Methods

Primary culture of MSCs was used. For comparison analysis of AGE and COMP-Ang1 effects, we performed cell viability assay with each treated variety concentration for 24 h. Apoptosis rate and Caspase-3 activity were measured by each ELISA assay. To make sure with Ang1/Tie2 pathway, we performed small interfering RNA transfected to MSCs. Real-time RT-PCR was performed to identify ODCs marker genes. Immunoblotting was used to evaluate the expression of Tie2, AKT, p38 and ERK.

Results

Our results clearly demonstrate that COMP-Ang1 upregulates the phosphorylation of AKT and p38 by activating the Ang1/Tie2 signaling pathway, indicating that COMP-Ang1 affects both AGE-induced apoptosis and the attenuated osteogenic differentiation of MSCs through the p38/MAPK and PI3K/AKT pathways.

Conclusions

COMP-Ang1 improves cell viability and differentiation function of ODCs against AGE via Ang/Tie2 signaling pathway.

General significance

Our results suggest the potential importance of COMP-Ang1 as a new therapy for impaired bone formation that is associated with diabetes and advanced age.     

缬沙坦对人肾小球系膜细胞糖基化终产物受体表达的影响

目的：本实验探讨缬沙坦对糖基化终产物诱导的人肾小球系膜细胞氧化应激水平及糖基化终产物受体（RAGE）表达的影响。方法：体外常规培养人肾小球系膜细胞,运用糖基化修饰的牛血清白蛋白（AGE-BSA）和缬沙坦进行干预,流式细胞术检测细胞内活性氧（ROS）,RT-PCR法检测NADPH氧化酶的亚基p47^phox的mRNA表达,RT-PCR和细胞免疫化学法检测RAGE的表达量。结果：缬沙坦干预组人肾小球系膜细胞的ROS产生量、NADPH氧化酶的亚基p47^phox mRNA表达量、RAGE表达量均低于AGE-BSA组（P〈0．05）,且缬沙坦的抑制作用呈浓度和时间依赖性。结论：缬沙坦可能通过降低氧化应激水平来抑制RAGE的表达。     

Accumulation of advanced glycation end products decreases collagen turnover by bovine chondrocytes

The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that chondrocytes maintain a functional collagen network. We investigated the effects of advanced glycation end products (AGEs) on the turnover of collagen by articular cartilage chondrocytes. Increased AGE levels (by culturing in the presence of ribose) resulted in decreased collagen synthesis (P < 0.05) and decreased MMP-mediated collagen degradation (P < 0.02). The latter could be attributed to increased resistance of the collagen network to MMPs (P < 0.05) as well as the decreased production of MMPs by chondrocytes (P < 0.02). Turnover of a protein is determined by its synthesis and degradation rates and therefore these data indicate that collagen turnover is decreased at enhanced AGE levels. Since AGE levels in human cartilage increase approximately 50 fold between age 20 and 80, cartilage collagen turnover likely decreases with increasing age. Impaired collagen turnover adversely affects the capacity of chondrocytes to remodel and/or repair its extracellular matrix. Consequently, age-related accumulation of AGE (via decreased collagen turnover) may contribute to the development of cartilage damage in osteoarthritis.     

Aspartic acid functions as carbonyl trapper to inhibit the formation of advanced glycation end products by chemical chaperone activity

Advanced glycation end products (AGEs) were implicated in pathology of numerous diseases. In this study, we present the bioactivity of aspartic acid (Asp) to inhibit the AGEs. Hemoglobin and bovine serum albumin (BSA) were glycated with glucose, fructose, and ribose in the presence and absence of Asp (100–200 μM). HbA₁c inhibition was investigated using human blood and characterized by micro-column ion exchange chromatography. The effect of methyl glyoxal (MG) on hemoglobin and BSA was evaluated by fluorescence spectroscopy and gel electrophoresis. The effect of MG on red blood cells morphology was characterized by scanning electron micrographs. Molecular docking was performed on BSA with Asp. Asp is capable of inhibiting the formation of fluorescent AGEs by reacting with the reducing sugars. The presence of Asp as supplement in whole blood reduced the HbA₁c% from 8.8 to 6.1. The presence of MG showed an increase in fluorescence and the presence of Asp inhibited the glycation thereby the fluorescence was quenched. MG also affected the electrophoretic mobility of hemoglobin and BSA by forming high molecular weight aggregates. Normal RBCs showed typical biconcave shape. MG modified RBCs showed twisted and elongated shape whereas the presence of ASP tends to protect RBC from twisting. Asp interacted with arginine residues of bovine serum albumin particularly ARG 194, ARG 198, and ARG 217 thereby stabilized the protein complex. We conclude that Asp has dual functions as a chemical chaperone to stabilize protein and as a dicarbonyl trapper, and thereby it can prevent the complications caused by glycation.