Non-enzymatic Glycation Products Research Articles

Boronate crosslinking-based ratiometric electrochemical assay of glycated albumin

As a product of nonenzymatic glycation, glycated albumin (GA) is a promising serum marker for the short-term glycemic monitoring in patients with diabetes. On the basis of the boronate crosslinking (BCL)-enabled direct labeling of ferrocene (Fc) tags to the nonenzymatically glycated (NEG) sites, we report herein a novel aptamer-based ratiometric electrochemical (apt-REC) platform for the point-of-care (POC) assay of GA. This apt-REC platform is based on the recognition of GA proteins by the methylene blue (MB)-modified aptamer receptors and the labeling of the Fc tags to the NEG sites via the BCL. Using MB as the reference tag and Fc as the quantification tag, the ratio of the oxidation currents (i.e., IFc/IMB) can serve as the yardstick for the ratiometric assay of GA. Due to the presence of tens of the NEG sites, each GA protein can be labeled with tens of quantification tags, permitting the amplified assay in a simple, time-saving, and low-cost manner. The ratiometric signal exhibited a good linear response over the range from 0.1 to 100 μg/mL, with a detection limit of 45.5 ng/mL. In addition to the superior reproducibility and robustness, this apt-REC platform is highly selective (capable of discriminating GA against human serum albumin (HSA)) and applicable to GA assay in serum samples. Due to its low cost, high reproducibility and robustness, simple operation, and high sensitivity and selectivity, this apt-REC platform holds great promise in the POC assay of GA for diabetes management.

Antiglycation and antioxidant potential of coumaric acid isomers: a comparative in-vitro study

Advanced glycation end products (AGEs) are the product of non-enzymatic glycation of serum proteins. AGEs increase reactive oxygen species (ROS) formation, which leads to diabetic complications. Phytochemicals exhibit lesser side effects as compared to conventional therapy. In this study, three isomers of coumaric acid (ortho, meta, para) were used to deduce the better one in terms of reducing diabetic complications. For this purpose, human serum albumin (HSA) was incubated with glucose in the absence and presence of isomers for 28 days. To avoid any growth, NaN3 was added and temperature was kept constant throughout the incubation period. Studies like fluorescence, circular dichroism spectroscopy, fructosamine analysis, free lysine estimation, free thiol group estimation were done. To investigate the ROS production, fluorescence microscopy of isolated lymphocytes using DAPI and dichloro-dihydro-fluorescein diacetate were performed. Molecular docking and molecular dynamic simulations (root-mean-square deviation, root-mean-square fluctuations, radius of gyration and solvent-accessible surface area) of HSA and peroxisome proliferator activated receptor (PPAR) alpha and gamma were also done. It was observed that in glycated protein samples, the level of absorbance, fluorescence, fructosamine and carbonyl group increased along with the loss of secondary structure, free lysine and thiol group. These parameters were found gradually recovered in treated samples. ROS production and apoptosis were found to be reduced in lymphocytes treated with p-Coumaric acid (pCA)-treated protein samples as compared to lymphocyte treated with glycated protein. Computational modelling suggested a stable complex formation of HSA and PPARs with pCA. Results with pCA at 200 µM were consistently better than other two isomers. Our next step is to evaluate this study in rats. Communicated by Ramaswamy H. Sarma

A-027 The Clinical Indications of Low or Non-reportable HbA1c Results

Abstract Background Hemoglobin A1c (HbA1c) is the major non-enzymatic glycation product of hemoglobin A (HbA) and usually measured as a percentage of total hemoglobin. It is widely used in the screening, diagnosis, and monitoring of diabetes because HbA1c correlates with the average blood glucose level over the past 8–12 weeks in patients with a normal RBC lifespan. However, many factors can cause inaccurate HbA1c measurements or invalidate this correlation. While a high HbA1c raises the concern of inadequate glycemic control, a low or non-reportable HbA1c may also indicate diverse clinical conditions or technical issues. This study analyzes the pathophysiological conditions and technical limits leading to a low or non-reportable HbA1c value and discusses the use of other lab values in HbA1c result interpretation. Methods HbA1c was measured by cation exchange HPLC (Tosoh G8, v5.24F). 37 cases with a low or non-reportable HbA1c (<4.0%) from January 1, 2022 to November 30, 2022 were reviewed. The relevant diagnoses, medical history, medications, and recent (±1 week from the HbA1c test) lab results (CBC, blood glucose, fructosamine, haptoglobin, LDH) were examined to identify the causes of low HbA1c values. The causes were grouped by pathological mechanisms or technical issues to calculate the frequency, and the association with other lab values was also explored. Results Low or non-reportable HbA1c cases accounted for 0.1% of total HbA1c tests (37/42 771), and about 30% (11/37) had been diagnosed with diabetes by the end of the review period. From the chart, 41% (15/37) cases had elevated blood glucose (>109 mg/dL), 73% (27/37) were anemic (Hb < 13.0 or 11.7 g/dL for male or female), and 46% (17/37) showed a high MCV (>96 fL). Among those cases, a minor portion was caused by method-specific technical issues (8%, 3/37) or unknown reasons (5%, 2/37). The majority (86%, 32/37) was attributed to the following mechanisms: intravascular hemolysis (51%, 19/37), hepatosplenic RBC destruction (27%, 10/37), and increased erythropoiesis (8%, 3/37). For those cases with intravascular hemolysis (51%), the causes were categorized as inherited RBC disorders (19%, 7/37, including hemoglobinopathy and G6PD deficiency), acquired hemolytic diseases (19%, 7/37, including autoimmune hemolytic anemia), and adverse effects of medications (13%, 5/37, including dapsone and hydroxychloroquine). For those cases with hepatosplenic RBC destruction (27%), most of them were caused by cirrhosis (19%, 7/37). In addition, increased erythropoiesis (8%) was observed in patients with recent hemorrhage or EPO treatment. Some hemoglobin variants like homozygous HbE resulted in non-reportable HbA1c (8%), which represented a technical limit of the HPLC method. Conclusion Although low or non-reportable HbA1c cases are rare (0.1%), a significant portion (30%) of them are diabetic patients, and their HbA1c results can be misleading. The majority of low HbA1c cases (86%) are associated with clinical conditions causing a shortened RBC lifespan, and the most common causes include inherited RBC diseases (19%), cirrhosis (19%), medication adverse effects (14%), and autoimmune diseases (11%). Because increased RBC destruction and compensatory erythropoiesis are the underlying mechanisms, hemoglobin, MCV, and markers assessing hemolytic anemia (haptoglobin, LDH, reticulocyte%) can aid in HbA1c interpretation.

The alteration of advanced glycation end products and its potential role on bone loss under microgravity

Bone loss occurs in astronauts during long-term space flight, and hardly recover to the normal level after back to the earth. This is because the mechanism of microgravity-induced osteoporosis is still unclear. Advanced glycation end products (AGEs) are formed between reducing sugars and long-lived proteins and are verified to be the pathogenic mechanism for diabetic and aging osteoporosis. These non-enzymatic glycation products are considered to have adverse effects on bone metabolism and bone quality which consist of bone microstructure and bone organic matrix. However, whether AGEs accumulation happened under microgravity is not clear yet. AGEs accumulation can be induced by hyperglycemia in diabetic patients and senior people. Hyperglycemia was also found to occur in astronauts during spaceflight or under simulated microgravity, however, whether it also can cause bone matrix AGEs accumulation remains unrevealed, and whether AGEs involve in the disuse osteoporosis is as well not clear yet. Therefore, in this study, we explored the AGEs changes and genesis, together with its potential relationship with disuse osteoporosis under simulated microgravity by using a rat tail-suspended model. Sixteen 8-week-old female Sprague-Dawley rats were randomly distributed into control (CON) and tail-suspension (TS) groups. After 21-days of an experiment, fluorescent total AGEs and pentosidine in bone matrix were separately detected by fluorescence microscope and High Performance Liquid Chromatography (HPLC); soluble AGEs and glucose-related biomarkers in serum were detected by ELISA; bone microstructure was evaluated by Micro-CT, and mRNA expression of bone metabolism biomarkers (OCN, ALP, RANKL/OPG) in bone was determined by QPCR. Results showed that after tail-suspension AGEs fluorescent intensity and PEN content increased in trabecular bone while no significant changes in cortical bone; serum glucose increased and was positively correlated with bone matrix AGEs; trabecular bone microstructure was deteriorated, ALP and OCN mRNA expression decreased while RANKL/OPG mRNA expression increased, and all of them showed correlations with bone matrix AGEs. The results suggested under simulated microgravity, bone matrix AGEs accumulation may be attributed to the elevated serum glucose, and preferred to occur in trabecular bone first. AGEs accumulation in bone matrix accompanied negative effects on trabecular bone microstructure and bone metabolism activities under simulated microgravity, suggesting AGEs accumulation may be a culprit in disused bone loss.

Role of Nitric Oxide-Derived Metabolites in Reactions of Methylglyoxal with Lysine and Lysine-Rich Protein Leghemoglobin.

Carbonyl stress occurs when reactive carbonyl compounds (RCC), such as reducing sugars, dicarbonyls etc., accumulate in the organism. The interaction of RCC carbonyl groups with amino groups of molecules is called the Maillard reaction. One of the most active RCCs is α-dicarbonyl methylglyoxal (MG) that modifies biomolecules forming non-enzymatic glycation products. Organic free radicals are formed in the reaction between MG and lysine or Nα-acetyllysine. S-nitrosothiols and nitric oxide (•NO) donor PAPA NONOate increased the yield of organic free radical intermediates, while other •NO-derived metabolites, namely, nitroxyl anion and dinitrosyl iron complexes (DNICs) decreased it. At the late stages of the Maillard reaction, S-nitrosoglutathione (GSNO) also inhibited the formation of glycation end products (AGEs). The formation of a new type of DNICs, bound with Maillard reaction products, was found. The results obtained were used to explain the glycation features of legume hemoglobin-leghemoglobin (Lb), which is a lysine-rich protein. In Lb, lysine residues can form fluorescent cross-linked AGEs, and •NO-derived metabolites slow down their formation. The knowledge of these processes can be used to increase the stability of Lb. It can help in better understanding the impact of stress factors on legume plants and contribute to the production of recombinant Lb for biotechnology.

Omeprazole inhibits α-glucosidase activity and the formation of nonenzymatic glycation products: Activity and mechanism

In this study, the inhibitory effect and mechanism of omeprazole on α-glucosidase and nonenzymatic glycation were investigated invitro by using multi-spectroscopic methods and molecular docking. Enzyme kinetic results showed that omeprazole inhibited α-glucosidase in a reversible and noncompetitive manner (IC50= 0.595 ± 0.003mM). The results from fluorescence quenching and thermomechanical analyses signified that omeprazole reduced the fluorescence intensity of α-glucosidase by forming an omeprazole-α-glucosidase complex primarily driven by hydrogen bonds. Molecular docking further confirmed that hydrogen bonds and hydrophobic forces were the major driving forces for omeprazole binding to α-glucosidase. The nonenzymatic glycation assays revealed that omeprazole had a moderate inhibition against the formation of fructosamine, dicarbonyl compounds, and advanced glycation end products (AGEs). This study provides a new inhibitor of both α-glucosidase and nonenzymatic glycation and provides a practicable candidate for treating diabetes and its complications.

Glycated albumin as a biomarker for diagnosis of diabetes mellitus: A systematic review and meta-analysis.

BACKGROUNDGlycated albumin (GA), the non-enzymatic glycation product of albumin in plasma, became a glycemic marker in the beginning of the 21st century. The assay is not affected by hemoglobin levels and reflects the glycemic status over a shorter period as compared to HbA1c measurements. Thus, GA may contributes as an intermediate glucose index in the current diabetes mellitus (DM) diagnostic system.AIMTo search and summarize the available data on glycated albumin measurements required for the diagnosis of diabetes mellitus.METHODSDatabases, including PubMed, Embase, Web of Science, and Cochrane Central Register of Controlled Trials (CENTRAL), among others, were systematically searched. The Quality Assessment of Diagnostic Accuracy Studies-2 tool was applied for the assessment of quality, and the bivariate model was used to pool the sensitivity and specificity. The hierarchical summary receiver operator characteristic curves (HSROC) model was utilized to estimate the summary receiver operating characteristics curve (SROC). Sensitivity analysis was performed to investigate the association of the study design and patient characteristics with the test accuracy and meta-regression to find the source of heterogeneity.RESULTSThree studies regarding gestational diabetes mellitus (GDM) and a meta-analysis of 16 non-GDM studies, comprising a total sample size of 12876, were included in the work. Results reveal that the average cut-off values of GA reported for the diagnosis of GDM diagnosis was much lower than those for non-GDM. For non-GDM cases, diagnosing DM with a circulating GA cut-off of 14.0% had a sensitivity of 0.766 (95%CI: 0.539, 0.901), specificity of 0.687 (95%CI: 0.364, 0.894), and area under the curve of 0.80 (95%CI: 0.76, 0.83) for the SROC. The estimated SROC at different GA cut-off values for non-GDM exhibited that the average location parameter lambda of 16 non-GDM studies was 2.354 (95%CI: 2.002, 2.707), and the scale parameter beta was -0.163 (95%CI: -0.614, 0.288). These non-GDM studies with various thresholds had substantial heterogeneity, which may be attributed to the type of DM, age, and body mass index as possible sources.CONCLUSIONGlycated albumin in non-DM exhibits a moderate diagnostic accuracy. Further research on the diagnostic accuracy of GA for GDM and combinational measurements of GA and other assays is suggested.

Skin advanced glycation end products as a diagnostic and monitoring tool among psoriatic patients: how the therapy helps reduce cardiovascular disease risk.

Psoriasis is a disturbing and burdensome inflammatory skin disorder, with a global prevalence of 2-3%. An increased risk of cardiometabolic disease between psoriatic patients has been recently demonstrated. This is probably due to the psoriasis systemic inflammation and the increased levels of inflammatory cytokines, such as IL-17, IL-23, and TNF-α. Advanced glycation end products (AGEs) are the products of nonenzymatic glycation and oxidation of proteins and lipids which modify their structure and function. They have a significant role in the pathogenesis of diabetic nephropathy, atherosclerosis, and cardiovascular diseases of diabetic adults and children. The accumulation of AGEs can be measured by skin autofluorescence (SAF). Adalimumab (Humira ®) is a fully human monoclonal antibody, administered via subcutaneous injection, which binds the tumor necrosis factor (TNF) and is used to treat moderate-to-severe chronic plaque psoriasis. We performed an observational prospective study of 24 weeks to assess the reduction of AGEs through SAF measurement during treatment with adalimumab. SAF measurements in patients were performed at T0 and after 24 weeks of therapy. Adalimumab efficacy was assessed using Psoriasis Area and Severity Index (PASI), Visual Analogue Scale (VAS) for pain, and erythrocyte sedimentation rate (ESR). ESR, AGEs, PASI, and VAS for pain decreased throughout the study period. Adalimumab reduced AGEs in psoriatic patients. Biologic therapies may also prevent cardiovascular disease, suggesting a new approach of combined therapy for psoriasis and cardiovascular diseases.

A UPLC-MRM-MS method for comprehensive profiling of Amadori compound-modified phosphatidylethanolamines in human plasma.

Phosphatidylethanolamines (PEs) are targets of non-enzymatic glycation, a chemical process that occurs between glucose and primary amine-containing biomolecules. As the early-stage non-enzymatic glycation products of PE, Amadori-PEs are implicated in the pathogenesis of various diseases. However, only a few Amadori-PE molecular species have been identified so far; a comprehensive profiling of these glycated PE species is needed to establish their roles in disease pathology. Herein, based on our previous work using liquid chromatography-coupled neutral loss scanning and product ion scanning tandem mass spectrometry (LC-NLS-MS and LC-PIS-MS) in tandem, we extend identification of Amadori-PE to the low-abundance species, which is facilitated by using plasma lipids glycated in vitro. The confidence of identification is improved by high-resolution tandem mass spectrometry and chromatographic retention time regression. A LC-coupled multiple reaction monitoring mass spectrometry (LC-MRM-MS) assay is further developed for more sensitive quantitation of the Amadori compound-modified lipids. Using synthesized stable isotope-labeled Amadori lipids as internal standards, levels of 142 Amadori-PEs and 33 Amadori-LysoPEs are determined in the NIST human plasma standard reference material. These values may serve as an important reference for future investigations of Amadori-modified lipids in human diseases.

Polyphenols Attenuate Highly-Glycosylated Haemoglobin-Induced Damage in Human Peritoneal Mesothelial Cells.

We investigated the cytoprotective role of the dietary polyphenols on putative damage induced by Amadori adducts in Human Peritoneal Mesothelial Cells (HPMCs). Increased accumulation of early products of non-enzymatic protein glycation—Amadori adducts—in the peritoneal dialysis fluid due to their high glucose, induces severe damage in mesothelial cells during peritoneal dialysis. Dietary polyphenols reportedly have numerous health benefits in various diseases and have been used as an efficient antioxidant in the context of several oxidative stress-related pathologies. HPMCs isolated from different patients were exposed to Amadori adducts (highly glycated haemoglobin, at physiological concentrations), and subsequently treated with several polyphenols, mostly presented in our Mediterranean diet. We studied several Amadori-induced effects in pro-apoptotic and oxidative stress markers, as well as the expression of several pro-inflammatory genes (nuclear factor-kappaB, NF-kB; inducible Nitric Oxide synthetase, iNOS), different caspase-activities, level of P53 protein or production of different reactive oxygen species in the presence of different polyphenols. In fact, cytoprotective agents such as dietary polyphenols may represent an alternate approach to protect mesothelial cells from the cytotoxicity of Amadori adducts. The interference with the Amadori adducts-triggered mechanisms could represent a therapeutic tool to reduce complications associated with peritoneal dialysis in the peritoneum, helping to maintain peritoneal membrane function longer in patients undergoing peritoneal dialysis.

Receptor for Advanced Glycation End Products (RAGE) and Mechanisms and Therapeutic Opportunities in Diabetes and Cardiovascular Disease: Insights From Human Subjects and Animal Models

Obesity and diabetes are leading causes of cardiovascular morbidity and mortality. Although extensive strides have been made in the treatments for non-diabetic atherosclerosis and its complications, for patients with diabetes, these therapies provide less benefit for protection from cardiovascular disease (CVD). These considerations spur the concept that diabetes-specific, disease-modifying therapies are essential to identify, especially as the epidemics of obesity and diabetes continue to expand. Hence, as hyperglycemia is a defining feature of diabetes, it is logical to probe the impact of the specific consequences of hyperglycemia on the vessel wall, immune cell perturbation, and endothelial dysfunction—all harbingers to the development of CVD. In this context, high levels of blood glucose stimulate the formation of the irreversible advanced glycation end products, the products of non-enzymatic glycation and oxidation of proteins and lipids. AGEs accumulate in diabetic circulation and tissues and the interaction of AGEs with their chief cellular receptor, receptor for AGE or RAGE, contributes to vascular and immune cell perturbation. The cytoplasmic domain of RAGE lacks endogenous kinase activity; the discovery that this intracellular domain of RAGE binds to the formin, DIAPH1, and that DIAPH1 is essential for RAGE ligand-mediated signal transduction, identifies the specific cellular means by which RAGE functions and highlights a new target for therapeutic interruption of RAGE signaling. In human subjects, prominent signals for RAGE activity include the presence and levels of two forms of soluble RAGE, sRAGE, and endogenous secretory (es) RAGE. Further, genetic studies have revealed single nucleotide polymorphisms (SNPs) of the AGER gene (AGER is the gene encoding RAGE) and DIAPH1, which display associations with CVD. This Review presents current knowledge regarding the roles for RAGE and DIAPH1 in the causes and consequences of diabetes, from obesity to CVD. Studies both from human subjects and animal models are presented to highlight the breadth of evidence linking RAGE and DIAPH1 to the cardiovascular consequences of these metabolic disorders.

Differences in non-enzymatic glycation products in human dentine and clavicle: changes with aging.

In recent decades, several methods based on biochemical and molecular changes caused by aging have been proposed to improve the accuracy of forensic age estimation. The present study aimed to measure changes in furosine and pentosidine, two markers of non-enzymatic glycation of proteins (NEGs), in human dentine and clavicle with aging, and to identify possible differences between turnover rates in different mineralized tissues. Furosine and pentosidine were quantified in 32 dentine samples from living donors between 14 and 80years of age, and in a second group of samples consisting of a tooth and a piece of clavicle collected from the same cadaver (15 individuals aged 18 to 85years). Furosine concentration was much higher than pentosidine concentration in the same tissue, although they were strongly correlated in both dentine and bone. A close relationship between furosine and/or pentosidine content and chronological age was found in both tissues (r > 0.93). Moreover, age estimation was more accurate when furosine or pentosidine content was determined in dentine, with specificity values for the tests higher than 82% in all age groups. In clavicle, furosine concentration and pentosidine concentration were much lower (2.6-fold and 3.1-fold, respectively) than in dentine from the same individuals. In conclusion, although the results show strong correlations between chronological age and furosine or pentosidine concentrations determined in mineralized tissues, there is still a need for further research with larger data sets, including patients with diabetes.

Galangin inhibits α-glucosidase activity and formation of non-enzymatic glycation products

Inhibition of α-glucosidase and non-enzymatic glycation is considered as an effective approach to treat type 2 diabetes. Herein, multispectroscopic techniques and molecular docking analysis were used to investigate the inhibition of galangin on α-glucosidase and non-enzymatic glycation. Galangin showed a reversible inhibition on α-glucosidase activity in a mixed-type manner through a monophasic kinetic process, and induced the fluorescence quenching and conformational changes of α-glucosidase by forming α-glucosidase-galgangin complex. Molecular docking revealed that galangin primarily interacted with the amino acid residues within the active site of α-glucosidase, which may prevent the entrance of substrate resulting in a decrease in catalytic efficiency of α-glucosidase. Moreover, galangin moderately inhibited the formation of intermediates of non-enzymatic glycation, fructosamine and α-dicarbonyl compounds and strongly inhibited the formation of advanced glycation end products.

About the HbA1c in the elderly

HbA1c product of non enzymatic glycation of HbA increases in relation with the mean blood glucose level during the former 2-3 months. HbA1c levels are correlated with the development of diabetic complications and HbA1c assessment is now the gold standard for evaluation of diabetes control. HbA1c level should not be higher than 7% to avoid these complications. However, in aged peoples, the objectives of diabetes control vary according to their health status. It must be good with HbA1c lower than 7-7.5% in healthy subjects and more relax in subjects with symptoms of frailty and risks of non perceived and self corrected hypoglycemia. Under these conditions, HbA1c values lower than 8 to 9% are advised. Nevertheless, hypoglycemia episodes may occur in patients with high HbA1c and capillary glucose follow-up is necessary for detection of such complications.

The non-aggregated aggrecan in the human intervertebral disc can arise by a non-proteolytic mechanism.

Analysis of both the aggregated and non-aggregated fractions of aggrecan isolated from adult human intervertebral disc using immunoblotting with antibodies specific for the different domains constituting the aggrecan core protein or atomic force microscopy revealed that many components contained the G1 domain. However, little of the disc aggrecan was able to reform aggregates with hyaluronan, as determined by gel filtration chromatography, suggesting that the G1 domains had been rendered non-functional. Since previous studies have shown that disc aggrecan undergoes non-enzymatic glycation with age, the functional effect of such modification was investigated in vitro using bovine aggrecan isolated from young animals. Incubation of monomeric aggrecan with ribose to induce glycation rendered it unable to form complexes with hyaluronan stable to agarose gel electrophoresis or gel filtration chromatography. Similarly, extended treatment of intact proteoglycan aggregate with ribose resulted in destabilisation of the complex with separation of the aggrecan from the hyaluronan. Although it is clear that proteolysis occurs in the intervertebral disc and gives rise to some non-aggregating molecules, a different mechanism is required to explain the presence of many non-aggregating molecules bearing the G1 domain. The products of non-enzymatic glycation of the globular domains of aggrecan would account for this phenomenon and explain why some of the non-aggregating molecules are still large proteoglycans. While such molecules may be retained in the nucleus pulposus, they may be able to diffuse within it, reducing the ability of the tissue to resist compression under asymmetric loading such as bending and ultimately contributing to disc degeneration.

Glycoxidation of biological macromolecules: a critical approach to halt the menace of glycation.

Glycation is the result of covalent bonding of a free amino group of biological macromolecules with a reducing sugar, which results in the formation of a Schiff base that undergoes rearrangement, dehydration and cyclization to form a more stable Amadori product. The final products of nonenzymatic glycation of biomacromolecules like DNA, proteins and lipids are known as advanced glycation end products (AGEs). AGEs may be generated rapidly or over long times stimulated by distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. Both Schiff base and Amadori glycation products generate free radicals resulting in decline of antioxidant defense mechanisms and can damage cellular organelles and enzymes. This critical review primarily focuses on the mechanistic insight of glycation and the most probable route for the formation of glycation products and their therapeutic interventions. Furthermore, the prevention of glycation reaction using therapeutic drugs such as metformin, pyridoxamine and aminoguanidine (AG) are discussed with special emphasis on the novel concept of the bioconjugation of these drugs like, AG with gold nanoparticles (GNPs). At or above 10 mM concentration, AG is found to be toxic and therefore has serious health concerns, and the study warrants doing this novel bioconjugation of AG with GNPs. This approach might increase the efficacy of the AG at a reduced concentration with low or no toxicity. Using the concept of synthesis of GNPs with abovementioned drugs, it is assumed that toxicity of various drugs which are used at high doses can be minimized more effectively.

Skin Autofluorescence, 5-Year Mortality, and Cardiovascular Events in Peripheral Arterial Disease

The hunt for predictive biomarkers in diseases of the cardio-, cerebro-, and peripheral vascular systems is an ongoing effort and one whose importance cannot be overstated. The identification of subjects most at risk for death or major adverse cardiovascular events may provide a means to stratify diagnostic and therapeutic interventions based on validated predictive algorithms. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , de Vos et al1 report that in 252 eligible subjects with documented peripheral arterial disease (PAD) studied at a single center, the measurement of skin autofluorescence (SAF) was independently associated with all-cause mortality and fatal or nonfatal major adverse cardiovascular events after a follow-up period of 5 years. Importantly, the authors excluded subjects with hemodialysis, kidney transplantation, recent myocardial infarction, or recent stroke and report that even after adjustment for cardiovascular risk factors and the use of lipid-lowering drugs, increased SAF remained associated with increased risk for death and major adverse cardiovascular events. A previous report from these authors compared subjects with PAD versus control PAD-free subjects and showed that increased SAF was significantly associated with PAD in a manner independent of traditional cardiovascular risk factors, although such risk factors were associated with further increases in SAF.2 See accompanying article on page 933 The AGE reader was used as a biological marker of advanced glycation endproducts (AGEs) in the skin and is usually tested in the forearm. AGEs are the products of nonenzymatic glycation and oxidation of proteins and lipids. AGEs are increased not only in diabetes mellitus but also in natural aging, oxidative stress, and inflammatory conditions, and in renal failure.3 AGEs form on lysine and arginine residues and are heterogeneous; some AGEs are associated with crosslinking (such as pentosidine), and some AGEs do not fluoresce, such as the carboxy methyl …

Metabolism of AGEs – Bacterial AGEs Are Degraded by Metallo-Proteases

Advanced Glycation End Products (AGEs) are the final products of non-enzymatic protein glycation that results in loss of protein structure and function. We have previously shown that in E. coli AGEs are continually formed as high-molecular weight protein complexes. Moreover, we showed that AGEs are removed from the cells by an active, ATP-dependent secretion and that these secreted molecules have low molecular weight. Taken together, these results indicate that E. coli contains a fraction of low molecular weight AGEs, in addition to the high-molecular weight AGEs. Here we show that the low-molecular weight AGEs originate from high-molecular weight AGEs by proteolytic degradation. Results of in-vitro and in vivo experiments indicated that this degradation is carried out not by the major ATP-dependent proteases that are responsible for the main part of bacterial protein quality control but by an alternative metal-dependent proteolysis. This proteolytic reaction is essential for the further secretion of AGEs from the cells. As the biochemical reactions involving AGEs are not yet understood, the implication of a metalloprotease in breakdown of high molecular weight AGEs and their secretion constitutes an important step in the understanding of AGEs metabolism.

Anti-glycation and Antioxidant Activities of Australian Centella species

The products of nonenzymatic glycation and oxidation of extracellular matrix contribute to the delayed healing of diabetic ulcers. Centella plant species have been used medicinally for various dermatological disorders including wound healing. The aim of this study was to evaluate the anti-glycation and antioxidant activities of the methanolic extract of two species of Australian Centella; Centella asiatica (L.) Urb. (Apiaceae/Umbelliferae) and Centella erecta (L.f.) Fern. The anti-glycation activity of the extracts was assessed by measuring the fluorescence intensity of advanced glycation end products (AGEs) formed after incubating collagen-type I and bovine serum albumin with methylglyoxal (MGO) for two and five days, respectively. The antioxidant activity was evaluated by interrogating the ability of each extract to scavenge DPPH free radicals. The phytochemical profile of both species was also examined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis. The methanolic extracts (100 µg/mL – 1200 µg/mL) of both species decreased significantly the formation of AGEs induced by MGO in a dose dependent manner. Free radical scavenging inhibition was 80.2 ± 4.4% for C. asiatica and 77.8 ± 3.9% for C. erecta. HPLC and TLC showed the presence of the major Centella saponins (asiaticoside, madecassoside) and their aglycones (asiatic and madecassic acids), phenolic acids (chlorogenic acid) and flavanoids (quercetin, isoquercetin and kaempferol) in both extracts. The results suggest that Centella species are not only capable of inhibiting the glycation of collagen but also exhibit substantial antioxidant activity, which may be clinically effective in impaired wound healing in diabetes.

Receptor for AGE (RAGE): signaling mechanisms in the pathogenesis of diabetes and its complications

The receptor for advanced glycation endproducts (RAGE) was first described as a signal transduction receptor for advanced glycation endproducts (AGEs), the products of nonenzymatic glycation and oxidation of proteins and lipids that accumulate in diabetes and in inflammatory foci. The discovery that RAGE was a receptor for inflammatory S100/calgranulins and high mobility group box 1 (HMGB1) set the stage for linking RAGE to both the consequences and causes of types 1 and 2 diabetes. Recent discoveries regarding the structure of RAGE as well as novel intracellular binding partner interactions advance our understanding of the mechanisms by which RAGE evokes pathological consequences and underscore strategies by which antagonism of RAGE in the clinic may be realized. Finally, recent data tracking RAGE in the clinic suggest that levels of soluble RAGEs and polymorphisms in the gene encoding RAGE may hold promise for the identification of patients who are vulnerable to the complications of diabetes and/or are receptive to therapeutic interventions designed to prevent and reverse the damage inflicted by chronic hyperglycemia, irrespective of its etiology.