HILIC UPLC/ QTof MS Method Development for the Quantification of AGEs Inhibitors - Trouble Shooting Protocol.

OBJECTIVE: The paper reports an attempt to develop and validate a HILIC UPLC/ QTof MS method for quantifying N-ε-carboxymethyl-L-lysine (CML) in vitro, testing N-ε-carboxy[D2]methyl-L-lysine (d2-CML), and N-ε-carboxy[4,4,5,5-D4]methyl-L-lysine (d4-CML) as internal standards. METHOD: During the method development, several challenging questions occurred that hindered the successful completion of the method. The study emphasizes the impact of issues, generally overlooked in the development of similar analytical protocols. For instance, the use of glassware and plasticware was critical for the accurate quantification of CML. Moreover, the origin of atypical variation in the response of the deuterated internal standards, though widely used in other experimental procedures, was investigated. RESULT: A narrative description of the systematic approach used to address the various drawbacks during the analytical method development and validation is presented. CONCLUSION: Reporting those findings can be considered beneficial while bringing an insightful notion about critical factors and potential interferences. Therefore, some conclusion and ideas can be drawn from these trouble-shooting questions, which might help other researchers to develop more reliable bioanalytical methods, or to raise their awareness of stumbling blocks along the way.

Ezetimibe attenuates experimental diabetes and renal pathologies via targeting the advanced glycation, oxidative stress and AGE-RAGE signalling in rats.

The current in-vivo study was premeditated to uncover the protective role of ezetimibe (EZ) against advanced glycation endproducts (AGEs)-related pathologies in experimental diabetes. Our results showed that EZ markedly improved the altered biochemical markers of diabetes mellitus (DM) (FBG, HbA1c, insulin, microalbumin, and creatinine) and cardiovascular disease (in-vivo lipid/lipoprotein level and hepatic HMG-CoA reductase activity) along with diminished plasma carboxymethyl-lysine (CML) and renal fluorescent AGEs level. Gene expression study revealed that EZ significantly down-regulated the renal AGEs-receptor (RAGE), nuclear factor-κB (NFκB-2), transforming growth factor-ß (TGF-ß1), and matrix metalloproteinase-2 (MMP-2) mRNA expression, however, the neuropilin-1 (NRP-1) mRNA expression was up-regulated. In addition, EZ also maintained the redox status via decreasing the lipid peroxidation and protein-bound carbonyl content (CC) and increasing the activity of high-density lipoprotein (HDL)-associated-paraoxonase-1 (PON-1) and renal antioxidant enzymes as well as also protected renal histopathological features. We conclude that EZ exhibits antidiabetic and reno-protective properties in diabetic rats.

Studies about the Dietary Impact on "Free" Glycation Compounds in Human Saliva.

Glycation reactions play a key role in post-translational modifications of amino acids in food proteins. Questions have arisen about a possible pathophysiological role of dietary glycation compounds. Several studies assessed the metabolic fate of dietary glycation compounds into blood and urine, but studies about saliva are rare. We investigated here the dietary impact on salivary concentrations of the individual Maillard reaction products (MRPs) N-ε-fructosyllysine, N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine (CEL), pyrraline (Pyr), and methylglyoxal-derived hydroimidazolone 1 (MG-H1). Quantitation was performed using stable isotope dilution analysis (LC-MS/MS). We describe here, that a low MRP diet causes a significant lowering of salivary levels of Pyr from 1.9 ± 0.4 ng/mL to below the LOD and MG-H1 from 2.5 ± 1.5 ng/mL to 0.7 ± 1.8 ng/mL. An impact on the salivary protein fraction was not observed. Furthermore, salivary Pyr and MG-H1 levels are modified in a time-dependent manner after a dietary intervention containing 1.2 mg Pyr and 4.7 mg MG-H1. An increase in mean salivary concentrations to 1.4 ng/mL Pyr and 4.2 ng/mL MG-H1 was observed within 30-210 min. In conclusion, saliva may be a useful tool for monitoring glycation compound levels by using Pyr and MG-H1 as biomarkers for intake of heated food.

Diet-induced weight loss reduces postprandial dicarbonyl stress in abdominally obese men: Secondary analysis of a randomized controlled trial.

AIMS: Dicarbonyl compounds contribute to the formation of advanced glycation endproducts (AGEs) and the development of insulin resistance and vascular complications. Dicarbonyl stress may already be detrimental in obesity. We evaluated whether diet-induced weight loss can effectively reverse dicarbonyl stress in abdominally obese men. MATERIALS AND METHODS: Plasma samples were collected from lean (n = 25) and abdominally obese men (n = 52) in the fasting state, and during a mixed meal test (MMT). Abdominally obese men were randomized to 8 weeks of dietary weight loss or habitual diet, followed by a second MMT. The α-dicarbonyls methylglyoxal (MGO), glyoxal (GO) and 3-deoxyglucosone (3-DG) and AGEs were measured by UPLC-MS/MS. Skin autofluorescence (SAF) was measured using the AGE reader. T-tests were used for the cross-sectional analysis and ANCOVA to assess the treatment effect. RESULTS: Postprandial glucose, MGO and 3-DG concentrations were higher in obese men as compared to lean men (p < 0.05 for all). Fasting dicarbonyls, AGEs, and SAF were not different between lean and obese men. After the weight loss intervention, fasting MGO levels tended to decrease by 25 nmol/L (95%-CI: -51-0.5; p = 0.054). Postprandial dicarbonyls were decreased after weight loss as compared to the control group: iAUC of MGO decreased by 57% (5280 nmol/L∙min; 95%-CI: 33-10526; p = 0.049), of GO by 66% (11,329 nmol/L∙min; 95%-CI: 495-22162; p = 0.041), and of 3-DG by 45% (20,175 nmol/L∙min; 95%-CI: 5351-35000; p = 0.009). AGEs and SAF did not change significantly after weight loss. CONCLUSION: Abdominal obesity is characterized by increased postprandial dicarbonyl stress, which can be reduced by a weight loss intervention. Registered under ClinicalTrials.gov Identifier no. NCT01675401.

Advances in Glycation: from food to human health and disease.

This Special Issue on "Advances in Glycation: from food to human health and disease" was planned after the XXV International Symposium on Glycoconjugates (Glyco25) in Milan in order to ask special attention of importance of glycation to glycoscience community. In addition, we also celebrate the 30th anniversary of JMARS (Japan Maillard Reaction Society), and dedicated to one of the pioneers of this field, Professor Vincent Monnier, MD. He contributed enormously to studies on glycation related to aging and diseases to date and also he contributed to establish IMARS (International Maillard Reaction Society) as well as JMARS.

Role of Advanced Glycation Endproduct (AGE)-Receptor for Advanced Glycation Endproduct (RAGE) Axis in Cardiovascular Disease and Its Therapeutic Intervention.

Despite the early loss of glycemic differences between the original intensive therapy group and conventional treatment in the DCCT/EDIC and UKPDS 80 trials, a continued reduction in microvascular risk and risk reductions for emergency myocardial infarction and all-cause death were observed 10-30 years after the end of these trials. These observations demonstrated that so-called "metabolic memory" could cause chronic abnormalities in diabetic vessels that are not easily reversed, even by subsequent improvement in blood glucose levels, thus suggesting a long-term beneficial influence of early metabolic control; that is, legacy effects on the risk of vascular complications and death in patients with both type 1 and type 2 diabetes. Formation and accumulation of advanced glycation endproducts (AGEs) are known to progress at an accelerated rate under diabetes. Furthermore, AGEs are hardly degraded and remain for a long time in diabetic vessels even after glycemic control is improved. Therefore, AGEs could explain why former cumulative diabetic exposure could contribute to current progression of vascular complications in diabetes. Here, the clinical utility of measurement of serum and tissue accumulation levels of AGEs for evaluating the prevalence and severity of numerous types of cardiovascular disease is reviewed and novel therapeutic strategies that could target the AGE-RAGE axis in CVD are discussed.

Advanced glycation endproducts, dityrosine and arginine transporter dysfunction in autism - a source of biomarkers for clinical diagnosis.

Background: Clinical chemistry tests for autism spectrum disorder (ASD) are currently unavailable. The aim of this study was to explore the diagnostic utility of proteotoxic biomarkers in plasma and urine, plasma protein glycation, oxidation, and nitration adducts, and related glycated, oxidized, and nitrated amino acids (free adducts), for the clinical diagnosis of ASD. Methods: Thirty-eight children with ASD (29 male, 9 female; age 7.6 ± 2.0 years) and 31 age-matched healthy controls (23 males, 8 females; 8.6 ± 2.0 years) were recruited for this study. Plasma protein glycation, oxidation, and nitration adducts and amino acid metabolome in plasma and urine were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Machine learning methods were then employed to explore and optimize combinations of analyte data for ASD diagnosis. Results: We found that children with ASD had increased advanced glycation endproducts (AGEs), Nε-carboxymethyl-lysine (CML) and Nω-carboxymethylarginine (CMA), and increased oxidation damage marker, dityrosine (DT), in plasma protein, with respect to healthy controls. We also found that children with ASD had increased CMA free adduct in plasma ultrafiltrate and increased urinary excretion of oxidation free adducts, alpha-aminoadipic semialdehyde and glutamic semialdehyde. From study of renal handling of amino acids, we found that children with ASD had decreased renal clearance of arginine and CMA with respect to healthy controls. Algorithms to discriminate between ASD and healthy controls gave strong diagnostic performance with features: plasma protein AGEs-CML, CMA-and 3-deoxyglucosone-derived hydroimidazolone, and oxidative damage marker, DT. The sensitivity, specificity, and receiver operating characteristic area-under-the-curve were 92%, 84%, and 0.94, respectively. Conclusions: Changes in plasma AGEs were likely indicative of dysfunctional metabolism of dicarbonyl metabolite precursors of AGEs, glyoxal and 3-deoxyglucosone. DT is formed enzymatically by dual oxidase (DUOX); selective increase of DT as an oxidative damage marker implicates increased DUOX activity in ASD possibly linked to impaired gut mucosal immunity. Decreased renal clearance of arginine and CMA in ASD is indicative of increased arginine transporter activity which may be a surrogate marker of disturbance of neuronal availability of amino acids. Data driven combination of these biomarkers perturbed by proteotoxic stress, plasma protein AGEs and DT, gave diagnostic algorithms of high sensitivity and specificity for ASD.

Association between habitual dietary and lifestyle behaviours and skin autofluorescence (SAF), a marker of tissue accumulation of advanced glycation endproducts (AGEs), in healthy adults.

PURPOSE: Advanced glycation endproducts (AGEs) are produced endogenously and also enter the body during the consumption of AGEs present in heat-processed food. It is unknown whether AGEs of dietary origin accumulate within the body of healthy individuals. AGEs can deposit within skin tissue long-term by crosslinking extracellular matrix proteins. The fluorescent nature of many AGEs enables their detection within the skin by non-invasively measuring skin autofluorescence (SAF). This study aimed to identify habitual dietary and lifestyle behaviours cross-sectionally associated with SAF in an adult population sample. METHODS: 251 Healthy adult volunteers completed validated food frequency and physical activity questionnaires. Waist circumference, BMI, blood pressure and blood glucose was also measured. SAF was measured using an AGE Reader. RESULTS: Significant positive correlations were found between SAF and chronological age (r = 0.63, P < 0.001), waist circumference (r = 0.28, P < 0.01), body weight (r = 0.24, P < 0.05), BMI (r = 0.23, P < 0.05) and consumption of meat and meat products (r = 0.22, P < 0.05). A negative correlation was found between SAF and cereal consumption (r = -0.21, P < 0.05). Cigarette smokers also had a significantly higher SAF than non-smokers (2.4 vs 2.0 U, P < 0.05). Regression analysis identified age, cigarette smoking, waist circumference and intake of meat products as significant predictors of SAF. The regression model explained 48% of the variation in SAF. CONCLUSIONS: Age, cigarette smoking, waist circumference and dietary consumption of meat/meat products were positively associated with SAF in this sample. Further research is required to determine whether frequent consumption of foods containing large quantities of dietary AGEs contribute to pathological disease processes in healthy individuals.

The AGE-RAGE axis in an Arab population: The United Arab Emirates Healthy Futures (UAEHFS) pilot study.

AIMS: The transformation of the United Arab Emirates (UAE) from a semi-nomadic to a high income society has been accompanied by increasing rates of obesity and Type 2 diabetes mellitus. We examined if the AGE-RAGE (receptor for advanced glycation endproducts) axis is associated with obesity and diabetes mellitus in the pilot phase of the UAE Healthy Futures Study (UAEHFS). METHODS: 517 Emirati subjects were enrolled and plasma/serum levels of AGE, carboxy methyl lysine (CML)-AGE, soluble (s)RAGE and endogenous secretory (es)RAGE were measured along with weight, height, waist and hip circumference (WC/HC), blood pressure, HbA1c, Vitamin D levels and routine chemistries. The relationship between the AGE-RAGE axis and obesity and diabetes mellitus was tested using proportional odds models and linear regression. RESULTS: After covariate adjustment, AGE levels were significantly associated with diabetes status. Levels of sRAGE and esRAGE were associated with BMI and levels of sRAGE were associated with WC/HC. CONCLUSIONS: The AGE-RAGE axis is associated with diabetes status and obesity in this Arab population. Prospective serial analysis of this axis may identify predictive biomarkers of obesity and cardiometabolic dysfunction in the UAEHFS.

Novel insights in the dysfunction of human blood-brain barrier after glycation.

The blood-brain barrier (BBB) provides a dynamic and complex interface consisting of endothelial cells, pericytes and astrocytes, which are embedded in a collagen and fibronectin-rich basement membrane. This complex structure restricts the diffusion of small hydrophilic solutes and macromolecules as well as the transmigration of leukocytes into the brain. It has been shown that carbonyl stress followed by the formation of advanced glycation endproducts (AGE=glycation) interfere with the BBB integrity and function. Here, we present data that carbonyl stress induced by methylglyoxal leads to glycation of endothelial cells and the basement membrane, which interferes with the barrier-function and with the expression of RAGE, occludin and ZO-1. Furthermore, methylglyoxal induced carbonyl stress promotes the expression of the pro-inflammatory interleukins IL-6 and IL-8. In summary, this study provides new insights into the relationship between AGE formation by carbonyl stress and brain microvascular endothelial barrier dysfunction.

Analysis of advanced glycation endproducts in selected food items by ultra-performance liquid chromatography tandem mass spectrometry: Presentation of a dietary AGE database.

The aim of this study was to validate an ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the determination of advanced glycation endproducts (AGEs) in food items and to analyze AGEs in a selection of food items commonly consumed in a Western diet. N(ε)-(carboxymethyl)lysine (CML), N(ε)-(1-carboxyethyl)lysine (CEL) and N(δ)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were quantified in the protein fractions of 190 food items using UPLC-MS/MS. Intra- and inter-day accuracy and precision were 2-29%. The calibration curves showed perfect linearity in water and food matrices. We found the highest AGE levels in high-heat processed nut or grain products, and canned meats. Fruits, vegetables, butter and coffee had the lowest AGE content. The described method proved to be suitable for the quantification of three major AGEs in food items. The presented dietary AGE database opens the possibility to further quantify actual dietary exposure to AGEs and to explore its physiological impact on human health.

Glycation of the high affinity NGF-receptor and RAGE leads to reduced ligand affinity.

AGEs are posttranslational modifications generated by irreversible non-enzymatic crosslinking reactions between sugars and proteins - a reaction referred to as glycation. Glycation, a feature of ageing, can lead to non-degradable and less functional proteins and enzymes and can additionally induce inflammation and further pathophysiological processes such as neurodegeneration. In this study we investigated the influence of glycation on the high affinity NGF-receptor TrkA and the AGE-receptor RAGE. We quantified the binding affinity of the TrkA-receptor and RAGE to their ligands by surface plasmon resonance (SPR) and compared these to the binding affinity after glycation. At the same time, we established a glycation procedure using SPR. We found that glycation of TrkA reduced the affinity to NGF by a factor of three, which could be shown to lead to a reduction of NGF-dependent neurite outgrowth in PC12 cells. Glycation of RAGE reduced binding affinity of AGEs by 10-fold.

Antioxidation and antiglycation of Fagopyrum tataricum ethanol extract.

Fagopyrum tataricum is used for the treatment of type 2 diabetes mellitus in Taiwan. The aim of this study was to evaluate the inhibitory effects of 75 % ethanol extract of buckwheat (EEB) and rutin on carbohydrate-metabolized enzymes, including α-amylase and α-glucosidase, which are related to hyperglycemia. The rutin dosage (40 µg/mL) was equivalent to that of EEB (200 µg/mL). In addition, the antioxidant and antiglycation activities of EEB and rutin were investigated. Results showed that both EEB and rutin exerted free radical (DPPH and ABTS) scavenging activity. They also attenuated protein glycation to lower the generation of advanced glycation end-products (AGEs) through the suppression of fructosamine and α-dicarbonyl compounds. Moreover, EEB and rutin also inhibited α-amylase and α-glucosidase activity. Taken together, these findings suggest that EEB and rutin may reduce oxidative stress, AGEs formation, and carbohydrate-metabolized enzymes hence EEB may use as protection agent in diabetic patients.