Glycation Adducts Research Articles

Analysis of Chemically Labile Glycation Adducts in Seed Proteins: Case Study of Methylglyoxal-Derived Hydroimidazolone 1 (MG-H1).

Seeds represent the major source of food protein, impacting on both human nutrition and animal feeding. Therefore, seed quality needs to be appropriately addressed in the context of viability and food safety. Indeed, long-term and inappropriate storage of seeds might result in enhancement of protein glycation, which might affect their quality and longevity. Glycation of seed proteins can be probed by exhaustive acid hydrolysis and quantification of the glycation adduct Nɛ-(carboxymethyl)lysine (CML) by liquid chromatography-mass spectrometry (LC-MS). This approach, however, does not allow analysis of thermally and chemically labile glycation adducts, like glyoxal-, methylglyoxal- and 3-deoxyglucosone-derived hydroimidazolones. Although enzymatic hydrolysis might be a good solution in this context, it requires aqueous conditions, which cannot ensure reconstitution of seed protein isolates. Because of this, the complete profiles of seed advanced glycation end products (AGEs) are not characterized so far. Therefore, here we propose the approach, giving access to quantitative solubilization of seed proteins in presence of sodium dodecyl sulfate (SDS) and their quantitative enzymatic hydrolysis prior to removal of SDS by reversed phase solid phase extraction (RP-SPE). Using methylglyoxal-derived hydroimidazolone 1 (MG-H1) as a case example, we demonstrate the applicability of this method for reliable and sensitive LC-MS-based quantification of chemically labile AGEs and its compatibility with bioassays.

Suppression in advanced glycation adducts of human serum albumin by bio-enzymatically synthesized gold and silver nanoformulations: A potential tool to counteract hyperglycemic condition

Advanced glycation end-products (AGEs) from non-enzymatic glycation are implicated in several degenerative diseases, including being a crucial contributor in secondary complications of diabetes. This has garnered significant scientific interest in inhibiting agents of AGEs to prevent and remediate disorders arising due to glycation. In the current study, inhibitory effects on AGEs formation were investigated using bio-enzymatically synthesized nanoformulations of gold (AuNPs) and silver (AgNPs) by physiologically important enzyme, β galactosidase. Human serum albumin, most abundant protein in human blood plasma, was glycated by incubating with glucose leading to AGEs formation. The AGEs formation was significantly minimized with both AuNPs and AgNPs, as confirmed by various biophysical and biochemical techniques. Circular dichroism and Fourier transform infrared spectroscopy further affirmed antiglycation potential of AuNPs and AgNPs. The results were corroborated with thiol group, free lysine and carbonyl content estimation for native, glycated and nanoparticles (NPs) treated samples. Confocal microscopic imaging was performed to exhibit glycation inhibiting potential of the NPs. The inhibition of AGEs was observed to be slightly stronger in case of AgNPs than AuNPs with both exhibiting promising results as potential anti-glycating agents. The study sheds light on potential of non-toxic NPs being utilized as controlling agents against hyperglycemic conditions and diabetes management.

Comparison of imaged capillary isoelectric focusing and cation exchange chromatography for monitoring dextrose-mediated glycation of monoclonal antibodies in infusion solutions

Intravenous (IV) infusion of therapeutic proteins typically involves dilution of the formulated product into infusion media such as normal saline or dextrose, 5% m/v in water. We report results from a rigorous evaluation of imaged capillary isoelectric focusing (iCIEF) for monitoring dextrose-mediated glycation of proteins in IV infusion solutions. In addition to detecting stable Amadori glycation products, iCIEF was able to detect the labile Schiff base (SB) glycation adducts since the equilibrium with free dextrose is maintained on capillary. Method parameters such as sample dilution factor and ampholyte composition (but not urea) were found to influence the observed level of SB glycation adducts. The impacts of dextrose and urea on the apparent pI values are also reported. iCIEF results were compared with results from cation exchange chromatography, which was found to preferentially detect the more stable Amadori glycation products due to the on-column decomposition of the SB adducts resulting from the separation of the protein from free dextrose which in turn altered the SB adduct- free dextrose equilibrium. These results demonstrate the need for careful consideration when selecting the analytical methodology to investigate protein sensitivity to dextrose and to monitor protein stability in dextrose-containing infusion solutions.

Methylglyoxal modified IgG generates autoimmune response in rheumatoid arthritis

The detection of autoantibodies generated against modified proteins that stimulate cellular and humoral immune response has developed a lot of interest in the recent years and a search for biomarkers for the early detection of diseases has increased. IgG protein has earned attention for its possible modifications under hyperglycaemic conditions in rheumatoid arthritis, wherein dicarbonyl stress has been reported to alter the structural integrity of the protein. This report suggests that the interaction of the methylglyoxal with the IgG has consequences in the autoimmunopathology of rheumatoid arthritis. Our molecular docking analysis of methylglyoxal and IgG revealed a close interaction between the two molecules. TNBS studies confirmed the interaction by showing a decline in free lysine–arginine content post-MG modifications in IgG. The modified IgG was thermally more stable and showed the generation of glycation adducts N-epsilon-carboxyethyllysine. Rheumatoid arthritis patients showed enhanced carbonyl stress which was expected to induce structural changes in the epitope makeup of IgG. The ELISA studies and gel retardation assay confirmed auto-antibodies against MG modified IgG (MG-IgG) pointing towards the generation of neoepitopes upon IgG after interaction with MG. This study establishes the IgG modification in RA patients under alter carbonyl concentrations.

Modification of histone by glyoxal: recognition of glycated histone containing advanced glycation adducts by serum antibodies of type 1 diabetes patients.

Dicarbonyl compounds react more rapidly, than glucose, with arginine and lysine in proteins to form advanced glycation end products (AGEs) and further produce free radicals which cause DNA damage. AGEs are reliable diagnostic biomarkers for most of the age-related diseases. In the present study histone was modified with glyoxal and it was characterized by various spectral techniques. Binding characteristics of the modified histone towards serum antibodies from type 1 diabetes patients was evaluated by solid phase enzyme immunoassay and the results were compared with normal human subjects. Fluorescence and Fourier transformed infrared analysis of the nuclear protein clearly indicated changes in their respective intensities upon modification with glyoxal. Liquid chromatography together with mass spectrometry showed new peaks and m/z values related to AGE adducts of dihydroimidazolidines/hydroimidazolones. This glyoxal modified protein was recognized by serum antibodies of the diabetes patients while it showed negligible binding with that of normal human subjects. Glyoxal modification of histone causes structural turbulence and formation of advanced glycation adducts in histone. These adducts might be the main antigenic epitope of the modified histone, leading to its recognition by circulating type 1 diabetes antibodies.

Probing Protein Glycation by Chromatography and Mass Spectrometry: Analysis of Glycation Adducts.

Glycation is a non-enzymatic post-translational modification of proteins, formed by the reaction of reducing sugars and α-dicarbonyl products of their degradation with amino and guanidino groups of proteins. Resulted early glycation products are readily involved in further transformation, yielding a heterogeneous group of advanced glycation end products (AGEs). Their formation is associated with ageing, metabolic diseases, and thermal processing of foods. Therefore, individual glycation adducts are often considered as the markers of related pathologies and food quality. In this context, their quantification in biological and food matrices is required for diagnostics and establishment of food preparation technologies. For this, exhaustive protein hydrolysis with subsequent amino acid analysis is the strategy of choice. Thereby, multi-step enzymatic digestion procedures ensure good recoveries for the most of AGEs, whereas tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode with stable isotope dilution or standard addition represents “a gold standard” for their quantification. Although the spectrum of quantitatively assessed AGE structures is continuously increases, application of untargeted profiling techniques for identification of new products is desired, especially for in vivo characterization of anti-glycative systems. Thereby, due to a high glycative potential of plant metabolites, more attention needs to be paid on plant-derived AGEs.

Identification and characterization of glycation adducts on osteocalcin

Osteocalcin is an important extracellular matrix bone protein that contributes to the structural properties of bone through its interactions with hydroxyapatite mineral and with collagen I. This role may be affected by glycation, a labile modification the levels of which has been shown to correlate with bone fragility. Glycation starts with the spontaneous addition of a sugar onto a free amine group on a protein, forming an Amadori product, and then proceeds through several environment-dependent stages resulting in the formation of an advanced glycation end product. Here, we induce the first step of this modification on synthetic osteocalcin, and then use multiple mass spectrometry fragmentation techniques to determine the location of this modification. Collision-induced dissociation resulted in spectra dominated by neutral loss, and was unable to identify Amadori products. Electron-transfer dissociation showed that the Amadori product formed solely on osteocalcin's N-terminus. This suggests that the glycation of osteocalcin is unlikely to interfere with osteocalcin's interaction with hydroxyapatite. Instead, glycation may interfere with its interaction with collagen I or another bone protein, osteopontin. Potentially, the levels of glycated osteocalcin fragments released from bone during bone resorption could be used to assess bone quality, should the N-terminal fragments be targeted.

Free and protein-bound Nε-carboxymethyllysine and Nε-carboxyethyllysine in fish muscle: Biological variation and effects of heat treatment

Nε-Carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) are typical advanced glycation end-products (AGEs) found in foods, which have been linked to various health risks. Little is known about AGEs formation in fish muscle and the variability in AGEs formation from one animal to another. In this study, free CML and CEL (glycated amino acids) and their protein-bound forms (protein glycation adducts) in fresh grass carp (Ctenopharyngodon idellus) and catfish (Clarias leather) muscle before and after heating (100°C, 5, 10, 30min) were determined by an HPLC-MS/MS method. High biological variation in CML and CEL levels was found between individual fish, particularly for CEL in catfish muscle [n=21, free CEL 0.18–30.1(6.50±7.19)mg/kg; protein-bound CEL 0.48–8.63 (3.08±2.70)mg/kg]. Heating resulted in great increase of protein-bound CML (2.1–10.8 fold increase) and CEL (27%–242% increase) in fish muscle, but had little or no effect on free CML and CEL contents. Simple kinetic functions did not fit well for the formation rate of protein-bound AGEs during heating, although zero-order reaction fitted very well for some individual fish, which further indicated the complexity of AGEs formation and the strong impact of biological variation of individual fish on AGEs; formation.

Boiling-induced nanoparticles and their constitutive proteins from Isatis indigotica Fort. root decoction: Purification and identification

Colloidal particles are essential components of sun-dried Isatis indigotica Fort. roots (Ban-Lan-Gen in Chinese, BLG) decoction. Nanoparticles (NPs) were isolated from BLG decoction with size exclusion chromatography and characterized. Their average diameter is ∼120 nm, reversibly responding to pH and temperature changes. They promoted the growth of normal cells but suppressed that of cancerogenic cells and macrophages. Two constitutive glycated proteins were identified from the NPs, namely BLGP1 and BLGP2. Their N-terminal amino acid sequences were V-X-R-E-V-V-K-D-I and V-V-R-E-V-V-K-D-I-A-G-A-V-Q-T-N-E-Q-Y. Their full-length cDNA sequences were cloned to obtain the highly homological amino acid sequences of non-glycated proteins, whose theoretical molecular weights are 21831.64 Da and 21841.67 Da. Using pepsin hydrolysis and mass spectrometry, four possible glycation adducts were identified in BLGP1, whereas one in BLGP2. To conclude, bioactive nanoparticles isolated from the herbal decoction are intelligent nanoassemblies composed of a new boiling-stable protein. Glycation plays a critical role in heat-induced formation of these nanoassemblies. The novel, intelligent, safe and stable nano-carriers for drug delivery may be developed using BLG NPs as prototype.

Relationship between plasma glycation with membrane modification, oxidative stress and expression of glucose trasporter-1 in type 2 diabetes patients with vascular complications

Background of studyEnhanced protein glycation in diabetes causes irreversible cellular damage through membrane modifications. Erythrocytes are persistently exposed to plasma glycated proteins; however, little are known about its consequences on membrane. Aim of this study was to examine the relationship between plasma protein glycation with erythrocyte membrane modifications in type 2 diabetes patients with and without vascular complications. MethodWe recruited 60 healthy controls, 85 type 2 diabetic mellitus (DM) and 75 type 2 diabetic patients with complications (DMC). Levels of plasma glycation adduct with antioxidants (fructosamine, protein carbonyl, β-amyloids, thiol groups, total antioxidant status), erythrocyte membrane modifications (protein carbonyls, β-amyloids, free amino groups, erythrocyte fragility), antioxidant profile (GSH, catalase, lipid peroxidation) and Glut-1 expression were quantified. ResultCompared with controls, DM and DMC patients had significantly higher level of glycation adducts, erythrocyte fragility, lipid peroxidation and Glut-1 expression whereas declined levels of plasma and cellular antioxidants. Correlation studies revealed positive association of membrane modifications with erythrocyte sedimentation rate, fragility, peroxidation whereas negative association with free amino groups, glutathione and catalase. ConclusionOur data suggest that plasma glycation is associated with oxidative stress, Glut-1 expression and erythrocyte fragility in DM patients. This may further contribute to progression of vascular complications.

Mass spectrometric determination of early and advanced glycation in biology.

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and Nε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.

Plasma Proteins Modified by Advanced Glycation End Products (AGEs) Reveal Site-specific Susceptibilities to Glycemic Control in Patients with Type 2 Diabetes

Protein glycation refers to the reversible reaction between aldoses (or ketoses) and amino groups yielding relatively stable Amadori (or Heyns) products. Consecutive oxidative cleavage reactions of these products or the reaction of amino groups with other reactive substances (e.g. α-dicarbonyls) yield advanced glycation end products (AGEs) that can alter the structures and functions of proteins. AGEs have been identified in all organisms, and their contents appear to rise with some diseases, such as diabetes and obesity. Here, we report a pilot study using highly sensitive and specific proteomics approach to identify and quantify AGE modification sites in plasma proteins by reversed phase HPLC mass spectrometry in tryptic plasma digests. In total, 19 AGE modification sites corresponding to 11 proteins were identified in patients with type 2 diabetes mellitus under poor glycemic control. The modification degrees of 15 modification sites did not differ among cohorts of normoglycemic lean or obese and type 2 diabetes mellitus patients under good and poor glycemic control. The contents of two amide-AGEs in human serum albumin and apolipoprotein A-II were significantly higher in patients with poor glycemic control, although the plasma levels of both proteins were similar among all plasma samples. These two modification sites might be useful to predict long term, AGE-related complications in diabetic patients, such as impaired vision, increased arterial stiffness, or decreased kidney function.

Antiglycation and cell protective actions of metformin and glipizide in erythrocytes and monocytes

Chronic hyperglycaemia causes glycation which subsequently results in the long-term complications of diabetes. Albumin, the major plasma protein is more sensitive to glycation resulting in structural, biological and physiological modifications. The long-term benefits of commonly used anti-diabetic drugs such as metformin and glipizide in diabetic patients are well understood. However, no extensive study has been performed to assess their role in the glycation induced albumin modifications and cellular protection. We carried out the glycation of bovine serum albumin using methylglyoxal as a glycating agent in absence or presence of metformin and glipizide to establish their anti-glycation action. Different glycation markers (fructosamine, carbonyl groups, free thiol groups and β-amyloid aggregation) and protein structural markers (absorption spectroscopy and native-polyacrylamide gel electrophoresis) were examined. Further THP-1 cells (monocytes) and erythrocytes were treated with drugs that were exposed to glycated albumin samples for 24 h, respectively at 37 °C to investigate the cytoprotective actions of drugs against glycation. After the treatment different anti-oxidant indices (catalase, glutathione, superoxide dismutase and nitric oxide), cell viability, lipid peroxidation and erythrocyte hemolysis were determined. Treatment with metformin and glipizide during in vitro albumin glycation significantly reduced the formation of glycation adducts and inhibited structural modifications. They restored the level of antioxidants in THP-1 and erythrocytes cells treated with glycated albumin thus protecting cells. Our results suggested protection mode of albumin glycation through inhibition by metformin and glipizide. Additionally, they exerted inhibitory actions on glycation-induced cellular damage by restoring cellular antioxidant defense.

Increased DNA dicarbonyl glycation and oxidation markers in patients with type 2 diabetes and link to diabetic nephropathy.

Aim. The aim of this study was to assess the changes of markers of DNA damage by glycation and oxidation in patients with type 2 diabetes and the association with diabetic nephropathy. Methodology. DNA oxidation and glycation adducts were analysed in plasma and urine by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. DNA markers analysed were as follows: the oxidation adduct 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-OxodG) and glycation adducts of glyoxal and methylglyoxal—imidazopurinones GdG, MGdG, and N2-(1,R/S-carboxyethyl)deoxyguanosine (CEdG). Results. Plasma 8-OxodG and GdG were increased 2-fold and 6-fold, respectively, in patients with type 2 diabetes, with respect to healthy volunteers. Median urinary excretion rates of 8-OxodG, GdG, MGdG, and CEdG were increased 28-fold, 10-fold, 2-fold, and 2-fold, respectively, in patients with type 2 diabetes with respect to healthy controls. In patients with type 2 diabetes, nephropathy was associated with increased plasma 8-OxodG and increased urinary GdG and CEdG. In a multiple logistic regression model for diabetic nephropathy, diabetic nephropathy was linked to systolic blood pressure and urinary CEdG. Conclusion. DNA oxidative and glycation damage-derived nucleoside adducts are increased in plasma and urine of patients with type 2 diabetes and further increased in patients with diabetic nephropathy.

Differential effects of glyoxalase 1 overexpression on diabetic atherosclerosis and renal dysfunction in streptozotocin-treated, apolipoprotein E-deficient mice.

The reactive dicarbonyls, glyoxal and methylglyoxal (MG), increase in diabetes and may participate in the development of diabetic complications. Glyoxal and MG are detoxified by the sequential activities of glyoxalase 1 (GLO1) and glyoxalase 2. To determine the contribution of these dicarbonyls to the etiology of complications, we have genetically manipulated GLO1 levels in apolipoprotein E‐null (Apoe−/−) mice. Male Apoe−/− mice, hemizygous for a human GLO1 transgene (GLO1TGApoe−/− mice) or male nontransgenic Apoe−/− litter mates were injected with streptozotocin or vehicle and 6 or 20 weeks later, aortic atherosclerosis was quantified. The GLO1 transgene lessened streptozotocin (STZ)‐induced increases in immunoreactive hydroimidazolone (MG‐H1). Compared to nondiabetic mice, STZ‐treated GLO1TGApoe−/− and Apoe−/− mice had increased serum cholesterol and triglycerides and increased atherosclerosis at both times after diabetes induction. While the increased GLO1 activity in the GLO1TGApoe−/− mice failed to protect against diabetic atherosclerosis, it lessened glomerular mesangial expansion, prevented albuminuria and lowered renal levels of dicarbonyls and protein glycation adducts. Aortic atherosclerosis was also quantified in 22‐week‐old, male normoglycemic Glo1 knockdown mice on an Apoe−/− background (Glo1KDApoe−/− mice), an age at which Glo1KD mice exhibit albuminuria and renal pathology similar to that of diabetic mice. In spite of ~75% decrease in GLO1 activity and increased aortic MG‐H1, the Glo1KDApoe−/− mice did not show increased atherosclerosis compared to age‐matched Apoe−/− mice. Thus, manipulation of GLO1 activity does not affect the development of early aortic atherosclerosis in Apoe−/− mice but can dictate the onset of kidney disease independently of blood glucose levels.

Assay of methylglyoxal-derived protein and nucleotide AGEs

Glyoxalase- and methylglyoxal-related research has required the development of quantitative and reliable techniques for the measurement of methylglyoxal-derived glycation adducts of protein and DNA. There are also other glycation adducts, oxidation adducts and nitration adducts of proteins and oxidation adducts of DNA. Proteolysis of protein releases glycation, oxidation and nitration free adducts (glycated, oxidized and nitrated amino acids) in plasma and nuclease digestion of DNA releases glycated and oxidized nucleosides into plasma and other body fluids for excretion in urine. The gold standard method for quantifying these adducts is stable isotopic dilution analysis LC-MS/MS. Protein and DNA adduct residues are determined by assay of enzymatic hydrolysates of protein and DNA extracts prepared using cocktails of proteases and nucleases respectively. Free adducts are determined by analysis of ultrafiltrates of plasma, urine and other physiological fluids. Protein damage markers (13 glycation adducts, five oxidation adducts and 3-nitrotyrosine) and DNA damage markers (three glycation adducts and one oxidation adduct) are quantified using 25 μg of protein, 10 μg of DNA or 5 μl of physiological fluid. Protein and nucleotide AGE (advanced glycation end-product) assay protocols resistant to interferences is described.

A new glycation product 'norpronyl-lysine,' and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen.

NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-13C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, ‘norpronyl-lysine’, as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) 13C–13C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of 13C5-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products.

Special edition of Amino Acids of selected papers from the eleventh international symposium on the Maillard reaction, September 16–20, 2012

We introduce this special edition of Amino Acids with selected papers from the eleventh international symposium on the Maillard reaction held in Nancy, France, September 16–20, 2012. Glycation of proteins occurs by a complex series of sequential and parallel reactions called collectively the Maillard reaction—named after the leading pioneer of glycation research, Louis Camille Maillard (1978–1936). Glycation researchers gathered at the symposium in 2012 organised by the International Maillard Reaction Society to commemorate the centennial of Maillard reaction research and review achievements and current standing of glycation research. In this special edition there are selected papers from this meeting. Subjects of study include: the involvement of advanced glycation endproducts (AGEs) in complications of diabetes, association of AGEs with insulin secretion, enzymatic metabolism of early glycation adducts, absorption into the body of early and advanced glycation endproducts (AGEs) of dietary origin, association of AGEs in dietary protein with chronic disease, the study of the effect of AGEs on bacterial growth and inhibition of AGE formation by natural products. The Centennial Maillard Symposium began with a presentation by Nobel laureate Jean-Marie Lehn (University of Strasbourg, France) on ‘‘Towards a chemistry of complex matter’’. The Maillard reaction exemplifies how a relatively simple chemical reaction of a reducing sugar or sugar derivative with an amino group becomes highly complex through multiple parallel and sequential reactions. In recent decades there has also been increasing awareness of dicarbonyl compounds and metabolites reacting with guanidino groups in the Maillard reaction. This expands the locus of glycation adduct formation from N-terminal and lysine side chain amino groups in amino acids and proteins to guanidino groups of arginine and arginine residues in proteins. Indeed, arginine-derived AGEs are now considered to be quantitatively major AGEs in physiological systems. In 100 years the Maillard reaction has seen applications in chemistry, food, nutrition, health and disease, microbiology and clinical diagnostics and repeated, on-going development of applications in pharmacology and therapeutics. We celebrate the achievements of Louis Camille Maillard in glycation research and suggest that, if he was here today, he would marvel at the advances and development that led from his earlier studies.

Glycoxidative damage to human DNA: Neo-antigenic epitopes on DNA molecule could be a possible reason for autoimmune response in type 1 diabetes

Advanced glycation end-products (AGEs) are known to be mutagenic, diabetogenic and vascular disease risk factors. Methylglyoxal (MG) is a dicarbonyl species that reacts with biological macromolecule (proteins, DNA and lipids) to give AGEs. Nonenzymatic glycation of MG with lysine (Lys) in the presence of copper (Cu(2+)) is reported to generate reactive oxygen species (ROS) capable of causing DNA damage. We show that DNA modification in MG-Lys-Cu(2+) system results in the generation of strand breaks, base modification, hyperchromicity and increased fluorescence intensity. Superoxide generation in the MG-Lys system was found to be significantly higher when compared with that in the MG and Lys alone. Moreover, d-penicillamine and pyridoxal phosphate significantly inhibited the formation of glycation products. The presence of a major DNA glycation adduct, N(2)-carboxyethyl-2'-deoxyguanosine (CEdG), was detected by high performance liquid chromatography (HPLC) and confirmed by nuclear magnetic resonance (NMR). As reported earlier, modified DNA (MG-Lys-Cu(2+)-DNA) was highly immunogenic in experimental animals. Furthermore, induced anti-MG-Lys-Cu(2+)-DNA antibodies were effective probe for detecting glycoxidative lesions in human genomic DNA of type I diabetes patients. Our results clearly imply that interaction of MG-Lys and Cu(2+) leads to the formation of AGEs and also the production of potent ROS, capable of causing DNA damage, thereby playing an important role in diabetes mellitus.

Can We Target Tubular Damage to Prevent Renal Function Decline in Diabetes?

The glomerulus has been at the center of attention as the primary site of injury in diabetic nephropathy (DN). Although there is no question that there are changes seen in the glomerulus, it is also well known that tubulointerstitial changes are a prominent component of the disease, especially in patients with type 2 diabetes. The level of albuminuria and DN disease progression best correlate with tubular degeneration and interstitial fibrosis. Nephrotoxicity studies in animals reveal that albuminuria is a highly sensitive marker of early tubular toxicity even in the absence of glomerular pathology. Urinary biomarker data in human beings support the view that proximal tubule injury contributes in a primary way, rather than in a secondary manner, to the development of early DN. I present a model in which very specific injury to the proximal tubule in vivo in the mouse results in severe inflammation, loss of blood vessels, interstitial fibrosis, and glomerulosclerosis. Increased glucose levels, free glycation adducts, reactive oxygen species, and oxidized lipids result in toxicity to tubule epithelia. This results in loss of cells with a stimulus to repair the epithelium. However, because of sublethal injury there is cell-cycle arrest in epithelial cells attempting to replace damaged cells. This leads to epithelial secretion of both profibrogenic growth factors, collagens, and factors that cause pericytes to proliferate and differentiate into myofibroblasts, leading to endothelial destabilization and capillary rarefaction. Local ischemia ensues with further injury to the tubules, more profibrogenic mediators, matrix protein deposition, fibrosis, and glomerulosclerosis.