Non-enzymatic Glycation Reaction Research Articles

Development of simultaneous quantitation method for 20 free advanced glycation end products using UPLC–MS/MS and clinical application in kidney injury

Advanced glycation end products (AGEs), derived from the non-enzymatic glycation reaction, are defined as glycotoxins in various diseases including aging, diabetes and kidney injury. Exploring AGEs as potential biomarkers for these diseases holds paramount significance. Nevertheless, the high chemical structural similarity and great heterogeneity among AGEs present a formidable challenge when it comes to the comprehensive, simultaneous, and accurate detection of multiple AGEs in biological samples. In this study, an UPLC/MS/MS method for simultaneous quantification of 20 free AGEs in human serum was firstly established and applied to quantification of clinical samples from individuals with kidney injury. Simple sample preparation method through protein precipitation without derivatization was used. Method performances including imprecision, accuracy, sensitivity, linearity, and carryover were systematically validated. Intra- and inter- imprecision of 20 free AGEs were 1.93–5.94 % and 2.30–8.55 %, respectively. The method accuracy was confirmed with good recoveries ranging from 96.40 % to 103.25 %. The LOD and LOQ were 0.1–3.13 ng/mL and 0.5–6.25 ng/mL, respectively. Additionally, the 20 free AGEs displayed excellent linearity (R2 >0.9974) across a wide linear range (1.56–400 ng/mL). Finally, through simultaneous quantitation of 20 Free AGEs in 100 participants including kidney injury patient and healthy controls, we identified six free AGEs, including N6-carboxyethyl-L-arginine (CEA), N6-carboxymethyl-L-lysine (CML), methylglyoxal-derived hydroimidazolones (MG-H), N6-formyl-lysine, N6-carboxymethyl-L-arginine (CMA), and glyoxal-derived hydroimidazolone (G-H), could well distinguish kidney injury patients and healthy individuals. Among them, the levels of four free AGEs including CML, CEA, MG-H, and G-H strongly correlate with traditionally clinical markers of kidney disease. The high area under the curve (AUC) values (AUC=0.965) in receiver operating characteristic (ROC) curve indicated that these four free AGEs can be served as combined diagnostic biomarkers for the diagnosis of kidney disease.

Effect of Total Hemoglobin on Glycated Hemoglobin (HbA1c) in Type 2 Diabetes Mellitus

Background: Glycated hemoglobin (HbA1c) is a pivotal marker in the diagnosis, prognosis, and therapeutic monitoring of diabetes mellitus. Given the involvement of hemoglobin in non-enzymatic glycation reactions, it is hypothesized that total hemoglobin concentration might impact HbA1c levels alongside blood glucose concentrations. Objectives: This study aimed to estimate and compare plasma fasting glucose level and total hemoglobin level in type 2 diabetes mellitus (T2DM) patients with and without anemia. The study also aimed to estimate HbA1C and compare with calculated HbA1C using formula in T2DM patients with and without anemia and to correlate between total hemoglobin levels with directly measured HbA1c in T2DM patients with and without anemia. Methodology: This cross-sectional study, employing purposive sampling, investigated 30 anemic and 30 non-anemic Type 2 Diabetes Mellitus (T2DM) patients at the Central Diagnostic Laboratory. Total Hemoglobin, HbA1C, and Fasting Plasma Glucose were measured for the patients and the data was analyzed. Results: Independent 't' tests revealed significant differences (p < 0.0001) in estimated HbA1c, fasting blood sugar, calculated HbA1c, and Hb levels between the two groups. Subgroup analyses exhibited significant differences in estimated HbA1c for both groups individually (p = 0.003 for T2DM without anemia and p<0.0001 for T2DM with anemia). Pearson's correlation analysis indicated no significant correlation between HbA1c and hemoglobin in either subgroup. Similarly linear regression analysis treating HbA1c as the dependent variable yielded a non-significant p-value, suggesting that hemoglobin levels do not significantly influence HbA1c. Conclusion: In conclusion, regardless of anemia status, our findings suggest that hemoglobin is not a significant predictor of HbA1c levels in T2DM patients, offering valuable insights into factors influencing HbA1c variability in diabetic populations.

AGEs accumulation with vascular complications, glycemic control and metabolic syndrome: A narrative review.

Multiple pathogenetic mechanisms are involved in the genesis of various microvascular and macrovascular complications of diabetes mellitus. Of all these, advanced glycation end products (AGEs) have been strongly implicated. The present narrative review aims to summarize the available literature on the genesis of AGEs and their potential role in the causation of both micro- and macrovascular complications of diabetes mellitus. Uncontrolled hyperglycemia triggers the formation of AGEs through non-enzymatic glycation reactions between reducing sugars and proteins, lipids, or nucleic acids. AGEs accumulate in bloodstream and bodily tissues under chronic hyperglycemia. AGEs create irreversible cross-linkages of various intra- and extracellular molecules and activate the receptor for advanced glycation end products (RAGE), which stimulates downstream signaling pathways that generate reactive oxygen species (ROS) and contribute to oxidative stress. Additionally, intracellular glycation of mitochondrial respiratory chain proteins by AGEs contributes to the further generation of ROS, which, in turn, sets a vicious cycle that further promotes the production of endogenous AGEs. Through these pathways, AGEs play a principal role in the pathogenesis of various diabetic complications, including diabetic retinopathy, nephropathy, neuropathy, bone disease, atherosclerosis and non-alcoholic fatty liver disease. Multiple clinical studies and meta-analyses have revealed a positive association between tissue or circulating levels of AGEs and development of various diabetic complications. Besides, exogenous AGEs, primarily those derived from diets, promote insulin resistance, obesity, and metabolic syndrome. AGEs, triggered by chronic hyperglycemia, play a pivotal role in the pathogenesis of various complications of diabetes mellitus.

Glycolaldehyde-derived advanced glycation end products promote macrophage proliferation via the JAK-STAT signaling pathway.

Advanced glycation end products (AGEs) are heterogeneous proinflammatory molecules produced by a non-enzymatic glycation reaction between reducing sugars (and their metabolites) and biomolecules with amino groups, such as proteins. Although increases in and the accumulation of AGEs have been implicated in the onset and exacerbation of lifestyle- or age-related diseases, including diabetes, their physiological functions have not yet been elucidated in detail. The present study investigated the cellular responses of the macrophage cell line RAW264.7 stimulated by glycolaldehyde-derived AGEs (Glycol-AGEs) known as representative toxic AGEs. The results obtained showed that Glycol-AGEs significantly promoted the proliferation of RAW264.7 cells at a low concentration range (1-10µg/mL) in a concentration-dependent manner. On the other hand, neither TNF-α production nor cytotoxicity were induced by the same concentrations of Glycol-AGEs. The increases observed in cell proliferation by low concentrations of Glycol-AGEs were also detected in receptor triple knockout (RAGE-TLR4-TLR2 KO) cells as well as in wild-type cells. Increases in cell proliferation were not affected by various kinase inhibitors, including MAP kinase inhibitors, but were significantly suppressed by JAK2 and STAT5 inhibitors. In addition, the expression of some cell cycle-related genes was up-regulated by the stimulation with Glycol-AGEs. These results suggest a novel physiological role for AGEs in the promotion of cell proliferation via the JAK-STAT pathway.

Advanced Glycation End Products and Diabetes Mellitus: Mechanisms and Perspectives.

Persistent hyperglycemic state in type 2 diabetes mellitus leads to the initiation and progression of non-enzymatic glycation reaction with proteins and lipids and nucleic acids. Glycation reaction leads to the generation of a heterogeneous group of chemical moieties known as advanced glycated end products (AGEs), which play a central role in the pathophysiology of diabetic complications. The engagement of AGEs with its chief cellular receptor, RAGE, activates a myriad of signaling pathways such as MAPK/ERK, TGF-β, JNK, and NF-κB, leading to enhanced oxidative stress and inflammation. The downstream consequences of the AGEs/RAGE axis involve compromised insulin signaling, perturbation of metabolic homeostasis, RAGE-induced pancreatic beta cell toxicity, and epigenetic modifications. The AGEs/RAGE signaling instigated modulation of gene transcription is profoundly associated with the progression of type 2 diabetes mellitus and pathogenesis of diabetic complications. In this review, we will summarize the exogenous and endogenous sources of AGEs, their role in metabolic dysfunction, and current understandings of AGEs/RAGE signaling cascade. The focus of this review is to recapitulate the role of the AGEs/RAGE axis in the pathogenesis of type 2 diabetes mellitus and its associated complications. Furthermore, we present an overview of future perspectives to offer new therapeutic interventions to intervene with the AGEs/RAGE signaling pathway and to slow down the progression of diabetes-related complications.

Association of Glycative Stress With Motor and Muscle Function.

Glycative stress is a type of biological stress caused by non-enzymatic glycation reactions, which include advanced glycation end product (AGE) formation, AGE accumulation, glycation-driven dysfunction of proteins and cellular signaling, inflammation, oxidation, and tissue damage. Increased glycative stress derived from hyperglycemia and lifestyle disorders is a risk factor in metabolic and age-related diseases, such as type 2 diabetes, cardiovascular disease, cancer, Alzheimer’s disease, osteoporosis, and dementia. Studies have shown that AGE accumulation is correlated with the age-related loss of muscle mass and power output, also called sarcopenia. Mechanistically, dysfunctions of contractile proteins, myogenic capacity, and protein turnover can cause glycative stress-induced skeletal muscle dysfunction. Because the skeletal muscle is the largest metabolic organ in the body, maintaining skeletal muscle health is essential for whole-body health. Increasing awareness and understanding of glycative stress in the skeletal muscle in this review will contribute to the maintenance of better skeletal muscle function.

Physicochemical Characterization of In Vitro LDL Glycation and Its Inhibition by Ellagic Acid (EA): An In Vivo Approach to Inhibit Diabetes in Experimental Animals.

Hundreds of millions of people around the globe are afflicted by diabetes mellitus. The alteration in glucose fixation process might result into hyperglycaemia and could affect the circulating plasma proteins to undergo nonenzymatic glycation reaction. If it is unchecked, it may lead to diabetes with increase in advanced glycation end products (AGEs). Therefore, the present study was designed to inhibit the diabetes and glycation by using natural antioxidant “ellagic acid” (EA). In this study, we explored the antidiabetes and antiglycation potential of EA in both in vitro (EA at micromolar concentration) and in vivo systems. The EA concentrations of 10 and 20 mg kg−1B.W./day were administered orally for 25 days to alloxan-induced diabetic rats, a week after confirmation of stable diabetes in animals. Intriguingly, EA supplementation in diabetic rats reversed the increase in fasting blood sugar (FBS) and hemoglobin A1c (HbA1c) level. EA also showed an inhibitory role against glycation intermediates including dicarbonyls, as well as AGEs, investigated in a glycation mixture with in vitro and in vivo animal plasma samples. Additionally, EA treatment resulted in inhibition of lipid peroxidation-mediated malondialdehyde (MDA) and conjugated dienes (CD). Furthermore, EA exhibited an antioxidant property, increased the level of plasma glutathione (GSH), and also helped to decrease histological changes evaluated by histoimmunostaining of animal kidney tissues. The results from our investigation clearly indicates the antiglycative property of EA, suggesting EA as an adequate inhibitor of glycation and diabetes, which can be investigated further in preclinical settings for the treatment and management of diabetes-associated complications.

In vitro Antidiabetic, Antioxidant and Antiglycation Activity of Ethanolic Leaf Extract of Gomphrena globosa (Linn.)

G. globosa L. is an everlasting tropical annual plant reported to possess several biological activities for curing ailments such as arthritis, diabetes, asthma, bronchitis and also known to possess anti tussive, anti-oxidant, anti-asthmatic and anti-cancer activities. The amino group of protein and carbonyl group of reducing sugar undergoes a non-enzymatic protein glycation reaction that leads to major complications in Diabetic patients. Several antiglycation agents of natural or synthetic origin are there to inhibit protein glycation reaction. But due to its toxicity, antiglycation agents of plant origin are gaining importance as an effective strategy to minimize diabetic complications. The aim of this study was to evaluate the antidiabetic, antioxidant and antiglycation activity of ethanolic leaf extract of G. globosa. The ethanolic leaf extract exerted its antidiabetic effect by inhibiting the activity of α-amylase (p<0.05) resulting in the delayed digestion of the dietary carbohydrates and lowering the amount of glucose liberated. The highest scavenging was observed at 500 μg concentrations and the percent inhibition was found to be 50.80% for DPPH assay and 57.24% (p<0.05) for hydroxyl free radical scavenging assay. 25mM of glucose concentration showed the highest uptake of glucose. The glucose adsorption capacities of G. globosa ethanolic leaf extract was directly proportional to the glucose concentration in the medium resulting in significantly higher glucose adsorption. The effect of antiglycation activity of G. globosa ethanolic leaf extract was found to be increasing with increase in time. The inhibition of ethanolic leaf extract was found to be maximum (27.66%) at 72 hr, followed by 23.7% at 48 hr and 13.7% at 24 hr for fructation with ROS modification/UV/guanosine. This result indicates that ethanolic leaf extract of G. globosa possess significant activity at desirable concentration.

Dietary advanced glycation end-products: Perspectives linking food processing with health implications.

Dietary advanced glycation end products (dAGEs) are complex and heterogeneous compounds derived from nonenzymatic glycation reactions during industrial processing and home cooking. There is mounting evidence showing that dAGEs are closely associated with various chronic diseases, where the absorbed dAGEs fuel the biological AGEs pool to exhibit noxious effects on human health. Currently, due to the uncertain bioavailability and rapid renal clearance of dAGEs, the relationship between dAGEs and biological AGEs remains debatable. In this review, we provide the most updated information on dAGEs including their generation in processed foods, analytical and characterization techniques, metabolic fates, interaction with AGE receptors, implications on human health and reducing strategies. Available evidence demonstrating a relevance between dAGEs and food allergy is also included. AGEs are ubiquitous in foods and their contents largely depend on the reactivity of carbonyl and amino groups, along with surrounding condition mainly pH and heating procedures. Once being digested and absorbed into the circulation, two separate pathways can be involved in the deleterious effects of dAGEs: an AGE receptor-dependent way to stimulate cell signals, and an AGE receptor-independent way to dysregulate proteins via forming complexes. Inhibition of AGEs formation during food processing and reduction in the diet are two potent approaches to restrict health-hazardous dAGEs. To elucidate the biological role of dAGEs toward human health, the following significant perspectives are raised: molecular size and complexity of dAGEs; interactions between unabsorbed dAGEs and gut microbiota; and roles played by concomitant compounds in the heat-processed foods.

Les réactions d’oxydation des protéines et leurs biomarqueurs

The oxidation reaction corresponds to the action of reactive oxygen species and is responsible for the formation of advanced oxidation protein products, the main ones are methionine sulfoxide, dityrosine and 3-nitrotyrosine. These products are potential markers in Alzheimer’s disease or systemic lupus erythematosus. The oxidation reaction is also at the origin of the formation of reactive metabolites which are substrates for other reactions: carbonylation and non-enzymatic glycation. The main products formed during the carbonylation reaction, called advanced lipoxidation end products, are hydroxynonenal-lysine, malonic dialdehyde-lysine and carboxy-ethyl-lysine. During the non-enzymatic glycation reaction, the main products studied, called advanced glycation end products, are carboxymethyl-lysine and pentosidine, which constitute markers for the occurrence of adverse events in patients suffering from chronic diseases, like diabetes or kidney failure.

Bioconjugation of gold nanoparticles with aminoguanidine as a potential inhibitor of non-enzymatic glycation reaction

The unavoidable glycation reaction is the major cause of diabetes and metabolic disorders. The glycation reaction is enhanced in case when the glycating agent is reactive carbonyl species (RCS) like, methylglyoxal (MG). The impact of RCS may result into the diabetes mellitus and its secondary complications along-with its role in cancers too. This reaction can be discontinued by using natural product inhibitors or by chemically synthesized drugs, like aminoguanidine (AG). However, AG is reported to be nephrotoxic (toxic to kidneys) at a concentration of 10 mM or more and has therefore serious health concerns. In the present study, bioconjugation of AG was done with the gold nanoparticles (Gnps) to mitigate its toxic effect and upsurge the efficacy of AG on RCS induced glycation. The AG-Gnps formed waas characterized by UV-Vis. spectroscopy and it reveals a peak at 529 nm corresponding to AG-gold nanoparticles. The particle size of the AG-Gnp was found to be 12 nm in TEM while in DLS it was found to be 54.07 nm. The fluorescence studies in combination with GK-peptide and δ-Glu assay support the inhibition of AGEs by AG-Gnps. Based on the idea of gold nano-particle synthesis, it is anticipated, the toxicity of numerous drugs used at high doses can be diminished with additional efficiency. Communicated by Ramaswamy H. Sarma

Glycation With Fructose: The Bitter Side of Nature's Own Sweetener.

Fructose is a ketohexose and sweetest among all the natural sugars. Like other reducing sugars, it reacts readily with the amino- and nucleophilic groups of proteins, nucleic acids and other biomolecules resulting in glycation reactions. The non-enzymatic glycation reactions comprise Schiff base formation, their Amadori rearrangement followed by complex and partly incompletely understood reactions culminating in the formation of Advance Glycation End products (AGEs). The AGEs are implicated in complications associated with diabetes, cardiovascular disorders, Parkinson's disease, etc. Fructose is highly reactive and forms glycation products that differ both in structure and reactivity as compared to those formed from glucose. Nearly all tissues of higher organisms utilize fructose but only a few like the ocular lens, peripheral nerves erythrocytes and testis have polyol pathway active for the synthesis of fructose. Fructose levels rarely exceed those of glucose but, in tissues that operate the polyol pathway, its concentration may rise remarkably during diabetes and related disorders. Diet contributes significantly to the body fructose levels however, availability of technologies for the large scale and inexpensive production of fructose, popularity of high fructose syrups as well as the promotion of vegetarianism have resulted in a remarkable increase in the consumption of fructose. In vivo glycation reactions by fructose, therefore, assume remarkable significance. The review, therefore, aims to highlight the uniqueness of glycation reactions with fructose and its role in some pathophysiological situations.

Caffeic Acid Inhibits the Formation of Advanced Glycation End Products (AGEs) and Mitigates the AGEs-Induced Oxidative Stress and Inflammation Reaction in Human Umbilical Vein Endothelial Cells (HUVECs).

The advanced glycation end products (AGEs) are the compounds produced by non-enzymatic glycation reaction of proteins and sugars, which can induce the generation of free radicals and the expression of inflammatory factors, thereby playing an important role in vascular dysfunction in diabetes. To investigate the effects of caffeic acid (CA) on glycation formed by glucose and protein, various spectroscopic techniques and molecular docking methods were carried out. Furthermore, the protective effects of CA on human umbilical vein endothelial cells (HUVECs) damaged by AGEs were detected. The results indicated that CA inhibited AGEs formation in vitro, decreased the expression of IL-1β, IL-18, ICAM-1, VCAM-1, NLRP3, Caspase-1 and CRP (C-reactive protein) and reduced the ROS in HUVECs exposed to AGEs. Our findings suggested that the supplementation with dietary CA could prevent and delay the AGEs-induced vascular dysfunction in diabetes.

Screening for active components of Sophorae Flos on inhibiting AGEs formation based on non-enzymatic glycation reaction

Sugar-poison caused blood-heat is the pathological basis of many complications of diabetes. Advanced glycation end products( AGEs) are considered as the potential glycotoxic factor that can cause blood-heat. Sophorae Flos hold the effect of removing pathogenic heat from blood. In this study,chromatographic non-enzymatic glycation reaction system of bovine serum albumin( BSA)/methylglyoxal( MGO) and Sophorae Flos was established to identify active components in Sophorae Flos inhibiting AGEs formation. The HPLC was used to analyze chromatograms before and after the incubation of Sophorae Flos and methylglyoxal. Changes of chromatographic peaks of eight compounds was found. It is speculated that this change may be due to new substance produced by the reaction of active components in Sophorae Flos and methylglyoxal,and these active components may be flavonoid component rutin. Further investigation for the effects of rutin and MGO reaction( 1 ∶ 1,1 ∶ 3,3 ∶ 1) for 6 days on the formation of AGEs was performed. The results showed that the inhibition activity of rutin on AGEs production was most obvious when the reaction ratio was 1 ∶3,and the most inhibition was in 24 h and stabilized after 3 d. The product of the reaction of rutin with MGO was identified by LC-ESI-MS/MS,which indicated that the newly formed seven substances were the mono-and di-MGO adducts of rutin. This study showed that rutin is the active component on Sophorae Flos for removing pathogenic heat from blood by forming new compounds to inhibit the formation of sugar poison products,which provides reference for rational application of Sophorae Flos.

Fructose-induced AGEs-RAGE signaling in skeletal muscle contributes to impairment of glucose homeostasis

Increased fructose intake has been linked to the development of dyslipidemia, obesity and impaired glucose tolerance. Due to its specific metabolic fate, fructose impairs normal lipid and carbohydrate metabolism and facilitates the non-enzymatic glycation reaction leading to enhanced accumulation of advanced glycation end products (AGEs). However, the formation of fructose-AGEs under in vivo setup and its tissue specific accumulation is less explored. Here, we investigated the impact of high fructose on AGEs accumulation in skeletal muscle and its causal role in impaired glucose homeostasis. In L6 rat skeletal muscle cells, chronic exposure to fructose induced AGEs accumulation and the cellular level of the receptor for AGEs (RAGE) and the effect was prevented by pharmacological inhibition of glycation. Under in vivo settings, Sprague Dawley rats exposed to 20% fructose in drinking water for 16 weeks, displayed increased fasting glycemia, impaired glucose tolerance, decreased skeletal muscle Akt (Ser-473) phosphorylation, and enhanced triglyceride levels in serum, liver and gastrocnemius muscle. We also observed a high level of AGEs in serum and gastrocnemius muscle of fructose-supplemented animals, associated with methylglyoxal accumulation and up regulated expression of RAGE in gastrocnemius muscle. Treatment with aminoguanidine inhibited fructose-induced AGEs accumulation and normalized the expression of RAGE and Dolichyl-Diphosphooligosaccharide-Protein Glycosyltransferase (DDOST) in gastrocnemius muscle. Inhibition of AGEs-RAGE axis counteracted fructose-mediated glucose intolerance without affecting energy metabolism. These data reveal diet-derived AGEs accumulation in skeletal muscle and the implication of tissue specific AGEs in metabolic derangement, that may open new perspectives in pathogenic mechanisms and management of metabolic diseases.

Correlation of Glycated Albumin and Glycated Hemoglobin with Glycemic Control in Patients with Diabetic Chronic Kidney Disease on Hemodialysis

Background and aims. Diabetes mellitus (DM) is the most common cause of end-stage renal disease (ESRD) worldwide and is the primary etiology of ESRD in Indonesia. It is estimated that there are 10 million patients with DM in Indonesia and about 25-40% of diabetics develop diabetic nephropathy (DN) within 25 years. Poor glycemic control is associated with increased mortality in a large observational study among diabetics on hemodialysis. Glycated hemoglobin, or known as HbA1c, was the currently recommended biomarker to monitor long-term glycemic control in diabetes mellitus guideline. However, it becomes underestimated in patients with DN on hemodialysis (DN-HD), because of 20- 50% reduction of erythrocyte lifespan, clinical use of iron therapy, and effects of recombinant human erythropoietin. Glycated albumin (GA), ketoamine formed via a non-enzymatic glycation reaction of serum albumin, is free from interference by erythrocyte lifespan or erythropoietin therapy and subsequently can be used as an alternative biomarker to monitor glycemic control in DN-HD. As albumin has a half-life of only 14-21 days* compared to Hb 60-100 days in well ESKD patients, thus GA may provide a better reflection of recent diabetic control than HbA1c. The aim of this study was to analyze the correlation between GA and HbA1c with glycemic control in DN-HD. Methods The study was an analytical observational study with a cross-sectional design. The subjects were consecutive patients with DN-HD who visited Hemodialysis Unit at Mohammad Hoesin General Hospital Palembang during August-November 2014. The glycemic control index was determined by the average value of 2 times a week pre-HD random blood glucose for 4 weeks (aRBG). Results. The subjects were 25 patients with an average age of 56.16±7.49 years old. The average value of GA was 26.94±7.74%. GA was strongly correlated with aRBG with r=0.776; p=0.000. After correcting for age, sex, and BMI, the correlations became significantly very strong (r=0.809, p <0.001). The simple linear regression for the relationship between GA and aRBG was aRBG=4,62×GA+42.74 (R2=0.602, P<0.001), estimating that a 1% increase of GA was associated with 4.62mg/dL increase of aRBG. After correcting for age, sex, & BMI, the correlations between HbA1c and aRBG were significant (r=0.852, p<0.001). Conclusion. GA was strongly correlated with glycemic control in patients with DN-HD and HbA1c was correlated better.

The Relevance of Toxic AGEs (TAGE) Cytotoxicity to NASH Pathogenesis: A Mini-Review.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common feature of chronic liver disease. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD, and one of its risk factors is hyperglycemia. The chronic ingestion of excessive amounts of high-fructose corn syrup is associated with an increased prevalence of fatty liver. Under hyperglycemic conditions, advanced glycation end-products (AGEs) are generated through a non-enzymatic glycation reaction between the ketone or aldehyde groups of sugars and amino groups of proteins. Glyceraldehyde (GA) is a metabolic intermediate of sugars, and GA-derived AGEs (known as toxic AGEs (TAGE)) have been implicated in the development of NASH. TAGE accumulates more in serum or liver tissue in NASH patients than in healthy controls or patients with simple steatosis. Furthermore, the TAGE precursor, GA, causes cell damage through protein dysfunctions by TAGE modifications and induces necrotic-type hepatocyte death. Intracellular TAGE may leak outside of necrotic-type cells. Extracellular TAGE then induce inflammatory or fibrotic responses related to the pathology of NASH in surrounding cells, including hepatocytes and hepatic stellate cells. This review focuses on the contribution of TAGE to the pathology of NASH, particularly hepatic cell death related to NASH.

Development of quantitation method for glycated aminophospholipids at the molecular species level in powdered milk and powdered buttermilk

The Maillard reaction is a nonenzymatic glycation reaction between a reducing sugar and a free amino group, known to naturally occur during heat processing of food. In this study, we especially focused on phosphatidylethanolamine (PE)-linked Amadori products (Amadori-PE) in powdered milk, since the analysis of these products at the molecular species level has not yet been evaluated. Analysis of Amadori-PE was conducted by using liquid chromatography-tandem mass spectrometry in three different modes. The main Amadori-PE species in a powdered milk sample were first identified as 34:1, 36:1, 36:2 and 36:3 in the total ion current mode. Additionally, by using the characteristic product ions observed in the presence of sodium, we quantified the main Amadori-PE species in the multiple reaction monitoring mode, and evaluated their total concentrations in the precursor ion scan (PIS) mode for the first time. Powdered milk contained much Amadori-PE with concentrations ranging from 4.3 to 8239 mg/100 g, quantified by the PIS mode. The newly developed methods represent powerful tools for detailed analysis of glycated lipids including Amadori-PE in powdered milk, which may further be applied to research relating to infant food and nutrition.

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated.ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion.Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.

The Effects of Rosella Extract (Hibiscus sabdariffa) against the n-carboxymethyl-lysine, NF-κβ, TNF-α in the Rats Heating Food Diets

The food processing by heating can increase the formation of AGEs (Advanced Glycation End Products). AGEs are compounds that formed from a non-enzymatic continuous glycation reaction between proteins and sugar residues. The CML ( N-carboxymethyl-lysine ) is used as a marker for AGEs cause most commonly found in vivo. The bond of AGEs and RAGE (receptor for AGEs) induce various signaling pathways that trigger inflammation and increase oxidative stress. The AGE and RAGE interaction activate the transcription factor of NF-κβ. NF-κβ activates gene transcription to release proinflammatory cytokines such as TNF-α. Anthocyanins are compounds that can prevent the formation of AGEs and muffle the adverse effects of AGEs. Rosella contains anthocyanin such as: delphinidin-3-O-glucoside, delphinidin-3-O-sambubioside, and cyanidin-3-O-sambubioside. This study is to determine whether the daily intake of Rosella extract can reduce the levels of n-carboxymethyl-lysine in serum, expression of NF-κβ and TNF α in the rats fed with heated food. This study applied experimental post test control using Rattus norvegicus Wistar strain. The samples were divided into 5 groups: KN (negative control/fed without heating), KP (positive control/fed heated food but not treated by Rosella extract), R 1 (fed heated food and treated by 200 mg.kg -1 BW Rosella extract), R 2 (fed heated food and treated by 300 mg.kg -1 BW Rosella extract), and R 3 (fed heated food and treated by 400 mg.kg -1 BW Rosella extract). The n -carboxymethyl-lysine levels were measured by using the ELISA, the expression of NF-κβ is analyzed by using immunofluorescence, and expression of TNF - α is observed by immunohistochemistry. There was significantly decreased the levels of n -carboxymethyl-lysine in all groups which were treated by Rosella extracts (R 1 ,R 2 ,R 3 ); p= 0.000, α = 0.05 (p< α). Decreased activation of NF-κβ in all groups which were treated by Rosella extract is significant (p = 0.000), and decreased expression of TNF α in all groups which were treated by Rosella extract is also significant (p = 0.000). Rosella extract can reduce the levels of n-carboxymethyl-lysine , expression of NF-κβ, and TNF α. Keywords: AGEs, anthocyanin, Rosella flower extract, n -carboxymethyl-lysine , NF-κβ, TNF α.