Methylglyoxal Adducts Research Articles

Trapping mechanism by di-d-psicose anhydride with methylglyoxal for prevention of diabetic nephropathy

Di-d-psicose anhydride (DPA), derived from functional rare saccharide as d-psicose, is investigated for its strong chelating ability. Methylglyoxal (MGO), an important precursor of advanced glycation end-products (AGEs), promotes obesity, and causes complications such as diabetic nephropathy. On mesangial cells, DPA can substantially reduce the negative effects of MGO. DPA effectively trapping MGO in mesangial cells. The bonding properties of the DPA–MGO adduct were discussed by mass spectrometry and nuclear magnetic resonance (NMR). The NMR spectra of the DPA–MGO adduct provide evidence for chelation bonding. The inhibition of AGE formation and the mass spectrometry results of the DPA–MGO adduct indicate that DPA can scavenge MGO at a molar ratio of 1:1. DPA suppressed 330 % of the up-regulated receptor for an AGEs protein expression to a normal level and restored the suppressed glyoxalase 1 level to 86 % of the normal group. This research provides important evidence and theoretical basis for the development of AGE inhibitors derived from rare saccharide.

Scavenging Glyoxal and Methylglyoxal by Synephrine Alone or in Combination with Neohesperidin at High Temperatures.

α-Dicarbonyl compounds, such as glyoxal (GO) and methylglyoxal (MGO), are a series of chemical hazards that exist in vivo and in vitro, posing a threat to human health. We aimed to explore the scavenging effects on GO/MGO by synephrine (SYN) alone or in combination with neohesperidin (NEO). First, through LC-MS/MS, we confirmed that both SYN and NEO could effectively remove GO and form GO adducts, while NEO could also clear MGO by forming MGO adducts, and its ability to clear MGO was stronger than that of GO. Second, a synergistic inhibitory effect on GO was found when SYN and NEO were used in combination by using the Chou-Talalay method; on the other hand, SYN could promote NEO to clear more MGO, although SYN could not capture MGO. Third, after synthesizing four GO/MGO-adducts (SYN-GO-1, SYN-GO-3, NEO-GO-7, and NEO-MGO-2) and identifying their structure through NMR, strict correlations between the GO/MGO-adducts and the GO/MGO-clearance rate were found when using SYN and NEO alone or in combination. Furthermore, it was inferred that the synergistic effect between SYN and NEO stems from their mutual promotion in capturing more GO by the quantitative analysis of the adducts in the combined model. Finally, a study was conducted on flowers of Citrus aurantium L. var. amara Engl. (FCAVA, an edible tea) rich in SYN and NEO, which could serve as an effective GO and MGO scavenger in the presence of both GO and MGO. Therefore, our study provided well-defined evidence that SYN and NEO, alone or in combination, could efficiently scavenge GO/MGO at high temperatures, whether in the pure form or located in FCAVA.

Methylglyoxal-Derived Nucleoside Adducts Drive Vascular Dysfunction in a RAGE-Dependent Manner.

Diabetic kidney disease (DKD) is a leading cause of death in patients with diabetes. An early precursor to DKD is endothelial cell dysfunction (ECD), which often precedes and exacerbates vascular disease progression. We previously discovered that covalent adducts formed on DNA, RNA, and proteins by the reactive metabolic by-product methylglyoxal (MG) predict DKD risk in patients with type 1 diabetes up to 16 years pre-diagnosis. However, the mechanisms by which MG adducts contribute to vascular disease onset and progression remain unclear. Here, we report that the most predominant MG-induced nucleoside adducts, N2-(1-carboxyethyl)-deoxyguanosine (CEdG) and N2-(1-carboxyethyl)-guanosine (CEG), drive endothelial dysfunction. Following CEdG or CEG exposure, primary human umbilical vein endothelial cells (HUVECs) undergo endothelial dysfunction, resulting in enhanced monocyte adhesion, increased reactive oxygen species production, endothelial permeability, impaired endothelial homeostasis, and exhibit a dysfunctional transcriptomic signature. These effects were discovered to be mediated through the receptor for advanced glycation end products (RAGE), as an inhibitor for intracellular RAGE signaling diminished these dysfunctional phenotypes. Therefore, we found that not only are MG adducts biomarkers for DKD, but that they may also have a role as potential drivers of vascular disease onset and progression and a new therapeutic modality.

Methylglyoxal Adducts Are Prognostic Biomarkers for Diabetic Kidney Disease in Patients With Type 1 Diabetes.

Over 30% of patients with type 1 diabetes develop diabetic kidney disease (DKD), significantly increasing mortality risk. The Diabetes Control and Complications Trial (DCCT) and follow-up study Epidemiology of Diabetes Interventions and Complications (EDIC) established that glycemic control measured by HbA1c predicts DKD risk. However, the continued high incidence of DKD reinforces the urgent need for additional biomarkers to supplement HbA1c. Here, we assessed biomarkers induced by methylglyoxal (MG), a metabolic by-product that forms covalent adducts on DNA, RNA, and protein called MG-adducts. Urinary MG-adducts were measured from patients with type 1 diabetes enrolled in DCCT/EDIC who did (cases, n=90) or did not (controls, n=117) develop DKD. Univariate and multivariable analysis revealed that measurements of MG-adducts independently predict DKD, even before established DKD biomarkers such as glomerular filtration rate and albumin excretion rate. Elevated levels of MG-adducts bestowed the greatest risk of developing DKD in a multivariable model that included HbA1c and other clinical covariates. Our work establishes a novel class of biomarkers to predict DKD risk and suggests that inclusion of MG-adducts may be a valuable tool to improve existing predictors of complications like DKD prior to overt disease to aid in identifying at-risk individuals and personalized risk management.

Mechanism of Pyrazine Formation Intervened by Oxidized Methionines during Thermal Degradation of the Methionine-Glucose Amadori Compound.

Methionine (Met) oxidation was observed during thermal degradation of methionine/glucose-derived Amadori rearrangement product (MG-ARP). The effects of oxidized methionine products, methionine sulfoxide (MetSO) and methionine sulfone (MetSO2), on pyrazine yields of the MG-ARP model were investigated. The pyrazine contents in the MG-ARP/Met and MG-ARP/MetSO models were found lower compared to those in the MG-ARP/MetSO2 model, and the inefficiency of pyrazine formation in the MG-ARP/Met model was proposed due to the fact that Met oxidation competitively inhibited the oxidation of dihydropyrazines for pyrazine formation in spite of relatively high methylglyoxal (MGO) content. The models of MGO mixed with Met, MetSO, or MetSO2 were established for further investigation of the mechanism for the involvement of Met oxidation in pyrazine formation. It was observed that the aldolization or carbonyl-amine reaction of MetSO with MGO was another important reason for the inhibition of pyrazine formation, except for the competitive inhibition of oxidative formation of MetSO on dihydropyrazine oxidation, and the adduct of MGO-MetSO was identified by MS/MS. These results also accounted for the phenomenon of low pyrazine yields but high yields of long-chain substituted pyrazines, which were converted from dihydropyrazines with the aldehyde involvement.

Major anthocyanins in elderberry effectively trap methylglyoxal and reduce cytotoxicity of methylglyoxal in HepG2 cell line.

The accumulation of advanced glycation end-products (AGEs) in the body is implicated in numerous diseases, being methylglyoxal (MGO) one of the main precursors. One of the strategies to reduce AGEs accumulation might be acting in an early stage of glycation by trapping MGO. Thus, this work aimed to evaluate, for the first time, the potential of elderberries polyphenols to trap MGO, access the formation of MGO adducts, and evaluate the cytoprotection effect in HepG2 and Caco-2 cells. The results demonstrated that monoglycosylated anthocyanins (cyanidin-3-glucoside and cyanidin-3-sambubioside) are very efficient in trapping MGO, forming mono- and di-adducts. Quercetin-3-glucoside and quercetin-3-rutinoside reacted slowly, while diglycosylated anthocyanins did not react. The trapping of MGO by elderberry monoglycosylated anthocyanins significantly decreased the MGO cytotoxicity in HepG2 cells (∼70% of cell viability), while the effect in Caco-2 cells was lower (∼50%). Thus, elderberry phenolics present antiglycation potential by trapping MGO.

Recombinant Human Collagen Hydrogel Rapidly Reduces Methylglyoxal Adducts within Cardiomyocytes and Improves Borderzone Contractility after Myocardial Infarction in Mice

AbstractMethylglyoxal (MG) production after myocardial infarction (MI) leads to advanced glycation end‐product formation, adverse remodeling, and loss of cardiac function. The extracellular matrix (ECM) is a main target for MG glycation. This suggests that ECM‐mimicking biomaterial therapies may protect the post‐MI environment by removing MG. In this study, mechanisms by which a recombinant human collagen type I hydrogel therapy confers cardioprotection are investigated. One‐week post‐MI, mice receive intramyocardial injection of hydrogel or PBS. The hydrogel improves border zone contractility after 2 days, which is maintained for 28 days. RNA sequencing shows that hydrogel treatment decreases the expression of erythroid differentiation regulator 1, a factor associated with apoptosis. Hydrogel treatment reduces cardiomyocyte apoptosis and oxidative stress at 2 days with greater myocardial salvage seen at 28 days. The hydrogel located at the epicardial surface is modified by MG, and less MG‐modified proteins are observed in the underlying myocardium of hydrogel‐treated mice. Biomaterials that can be a target for MG glycation may act as a sponge to remove MG from the myocardium post‐MI. This leads to less oxidative stress, greater survival and contractility of cardiomyocytes, which altogether suggests a novel mechanism by which biomaterials improve function of the infarcted heart.

Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress.

Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all samples. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts.

Methylglyoxal induces ambience for cancer promotion in HepG2 cells via Warburg effect and promotes glycation.

Methylglyoxal (MGO) is a toxic, highly reactive metabolite derived mainly from glucose and amino acids degradation. MGO is also one of the prime precursors for advanced glycation end products formation. The present research was performed to check whether MGO has any role in the promotion of cancer in HepG2 cells. For this, cells were incubated with MGO (50 µM) for 24 h and subjected to various analyses. Aminoguanidine (200 µM)was positive control. The various biochemical and protein expression studies, relevant to the MGO detoxification system, oxidative stress, and glycolysis were performed. MGO caused the reduction of expression of GLO 1 (27%) and GLO 2 (11%) causing weakening of the innate detoxification system. This is followed by an increase of RAGE (95%), AGEs or methylglyoxal adducts. We also observed hypoxia via estimation of oxygen consumption rate and surplus reactive oxygen species (ROS) (24%). To investigate the off-target effect of MGO we checked its effect on glucose transport, and its associated proteins. Glucose uptake was found to increase (15%) significantly with overexpression of GLUT 1 (35%). We also found a significant increase of glycolytic enzymes such as hexokinase II, phosphofructokinase 1, and lactate dehydrogenase along with lactate production. Observation of surplus ROS and enhanced glycolysis led us to check the expression of HIF 1α which is their downstream signaling pathway. Interestingly HIF 1α was found to increase significantly (35%). It is known that enhanced glycolysis and oxidative stress are catalysts for the overexpression of HIF 1α which in turn creates an ambience for the promotion of cancer. Aminoguanidine was able to prevent the adverse effect of MGO partially. This is the first study to show the potential of MGO for the promotion of cancer in the non-tumorigenic HepG2 cells via the Warburg effect and glycation.

Identification and cytotoxic evaluation of the novel rutin–methylglyoxal adducts with dione structures in vivo and in foods

Flavonoids with meta-hydroxyl groups have been proven to react with methylglyoxal (MGO) and form mono- and di-MGO adducts via nucleophilic addition reactions. Rutin, a rutinoside of quercetin with typical meta-phenol structure, is widely distributed in plant-sourced materials. Interestingly, different from the adducts reported between flavonoids and MGO, new rutin–MGO adducts with dione structures on the moiety of MGO were identified and proven to occur in various foods (0.66–6.58 mg/kg in total) and in vivo (up to 5.01 μg/L in plasma of rats administered with 100 mg/kg bodyweight of rutin). The three adducts discovered were assigned as 6-(1,2-propanedione)-8-(1-acetol)-rutin, 6-(1-acetol)-8-(1,2-propanedione)-rutin, and 6-(1,2-propanedione)-8-(1,2-propanedione)-rutin. Cytotoxicity evaluation in different cell lines indicated that the formation of these rutin–MGO adducts remarkably reduced the toxicity of MGO, which provide further promise for the application of rutin as a scavenger of dicarbonyl compounds by dietary supplement and addition in foods.

Methylglyoxal Adducts Levels in Blood Measured on Dried Spot by Portable Near-Infrared Spectroscopy.

The altered glucose metabolism characterising cancer cells determines an increased amount of methylglyoxal in their secretome. Previous studies have demonstrated that the methylglyoxal, in turn, modifies the protonation state (PS) of soluble proteins contained in the secretomes of cultivated circulating tumour cells (CTCs). In this study, we describe a method to assess the content of methylglyoxal adducts (MAs) in the secretome by near-infrared (NIR) portable handheld spectroscopy and the extreme learning machine (ELM) algorithm. By measuring the vibration absorption functional groups containing hydrogen, such as C-H, O-H and N-H, NIR generates specific spectra. These spectra reflect alterations of the energy frequency of a sample bringing information about its MAs concentration levels. The algorithm deciphers the information encoded in the spectra and yields a quantitative estimate of the concentration of MAs in the sample. This procedure was used for the comparative analysis of different biological fluids extracted from patients suspected of having cancer (secretome, plasma, serum, interstitial fluid and whole blood) measured directly on the solute left on a surface upon a sample-drop cast and evaporation, without any sample pretreatment. Qualitative and quantitative regression models were built and tested to characterise the different levels of MAs by ELM. The final model we selected was able to automatically segregate tumour from non-tumour patients. The method is simple, rapid and repeatable; moreover, it can be integrated in portable electronic devices for point-of-care and remote testing of patients.

Genistein Inhibits Protein Glycation in Healthy and Pre-Obese Mice Treated With High-Fat Diet via Trapping and Activating the Detoxification Pathways of Methylglyoxal

Abstract Objectives High levels of methylglyoxal (MGO) and advanced glycation end products (AGEs) play important roles in the pathogenesis of diabetes and other chronic diseases. This study aimed to investigate the underlying mechanisms that dietary genistein inhibits the accumulations of MGO and AGEs in healthy and pre-obese mice treated with high-fat diet. Methods In Study 1, male C57BL/6J mice (6-wk old, n = 15) were fed a low-fat (LF) diet (10% fat energy) or a very-high-fat diet (VHF, 60% fat energy) alone or including 0.25% genistein (VHF-G) for 16 weeks. In study 2, the mice were fed the LF diet (LF) or HF diet alone (HF, 45% fat energy) or in combination with up to 0.2% MGO in water (HFM), and 0.067% (HFM-GL) or 0.2% (HFM-GH) dietary genistein for 18 weeks. In study 3, the mice were induced with obesity after being fed HF diet (45% fat energy) for 9 weeks before being administrated with genistein at two doses of 0.1% (G 0.1) and 0.2% (G 0.2) in the HF diet for additional 19 weeks. The concentrations of MGO and AGEs in mouse samples and other metabolic data of the mice were measured. Moreover, the potential mechanisms of genistein on lowering MGO and AGEs were investigated. Results The plasma MGO concentration in VHF-G mice was 52% lower than that in VHF mice. Also, the AGE levels in plasma, liver, and kidney of VHF-G mice were 73%, 52%, and 49% lower than in the VHF group (Study 1). Similarly, the concentrations of plasma MGO and AGEs in plasma, liver, and kidney of HFM-GH mice were 33.5%, 49%, 69%, and 54% lower than in HFM mice (Study 2). Moreover, dietary genistein at 0.2% (G 0.2) significantly decreased the MGO levels in plasma and liver by 43.9% and 30.4% compared with HF mice and decreased the AGE level in the kidney by 48.3% in pre-obese mice (Study 3). Mechanistically, genistein lowered MGO concentrations and inhibited AGE formation through direct trapping both endogenous and exogenous MGO to form the MGO adducts and activating the MGO detoxification systems of glyoxalase and aldose reductase (AR) to lower MGO levels indirectly in vivo. Conclusions The present studies provide novel insights into the anti-obesity and anti-glycation roles of dietary genistein in mice. However, whether genistein could exert similar beneficial effects in humans needs to be further investigated. Funding Sources USDA/NIFA.

Methylglyoxal-Induced Dysfunction in Brain Endothelial Cells via the Suppression of Akt/HIF-1α Pathway and Activation of Mitophagy Associated with Increased Reactive Oxygen Species.

Methylglyoxal (MG) is a dicarbonyl compound, the level of which is increased in the blood of diabetes patients. MG is reported to be involved in the development of cerebrovascular complications in diabetes, but the exact mechanisms need to be elucidated. Here, we investigated the possible roles of oxidative stress and mitophagy in MG-induced functional damage in brain endothelial cells (ECs). Treatment of MG significantly altered metabolic stress as observed by the oxygen-consumption rate and barrier-integrity as found in impaired trans-endothelial electrical resistance in brain ECs. The accumulation of MG adducts and the disturbance of the glyoxalase system, which are major detoxification enzymes of MG, occurred concurrently. Reactive oxygen species (ROS)-triggered oxidative damage was observed with increased mitochondrial ROS production and the suppressed Akt/hypoxia-inducible factor 1 alpha (HIF-1α) pathway. Along with the disturbance of mitochondrial bioenergetic function, parkin-1-mediated mitophagy was increased by MG. Treatment of N-acetyl cysteine significantly reversed mitochondrial damage and mitophagy. Notably, MG induced dysregulation of tight junction proteins including occludin, claudin-5, and zonula occluden-1 in brain ECs. Here, we propose that diabetic metabolite MG-associated oxidative stress may contribute to mitochondrial damage and autophagy in brain ECs, resulting in the dysregulation of tight junction proteins and the impairment of permeability.

Classically activated mouse macrophages produce methylglyoxal that induces a TLR4- and RAGE-independent proinflammatory response.

The highly reactive compound methylglyoxal (MG) can cause direct damage to cells and tissues by reacting with cellular macromolecules. MG has been identified as a biomarker associated with increased sepsis-induced mortality. Patients undergoing septic shock have significantly elevated circulating MG levels compared to postoperative patients and healthy controls. Furthermore, MG has been implicated in the development of type II diabetes mellitus and Alzheimer's disease. Because MG is generated during glycolysis, we hypothesized that MG may be produced by classically activated (M1) macrophages, possibly contributing to the inflammatory response. LPS and IFN-γ-treated macrophages acquired an M1 phenotype (as evidenced by M1 markers and enhanced glycolysis) and formed MG adducts, MG-H1, MG-H2, and MG-H3, which were detected using antibodies specific for MG-modified proteins (methylglyoxal 5-hydro-5-methylimidazolones). MG adducts were also increased in the lungs of LPS-treated mice. Macrophages treated with LPS and IFN-γ also exhibited decreased expression of glyoxalase 1 (Glo1), an enzyme that metabolizes MG. Concentrations of exogenous, purified MG>0.5mM were toxic to macrophages; however, a nontoxic dose of 0.3mM induced TNF-α and IL-1β, albeit to a lesser extent than LPS stimulation. Despite prior evidence that MG adducts may signal through "receptor for advanced glycation endproducts" (RAGE), MG-mediated cell death and cytokine induction by exogenous MG was RAGE-independent in primary macrophages. Finally, RAGE-deficient mice did not exhibit a significant survival advantage following lethal LPS injection. Overall, our evidence suggests that MG may be produced by M1 macrophages during sepsis, following IFN-γ-dependent down-regulation of Glo1, contributing to over-exuberant inflammation.

The inhibition of oleanolic acid on protein non-enzymatic glycation

Non-enzymatic glycation can produce advanced glycation end products (AGEs), inducing a series of diabetic complications. In this study, oleanolic acid (OA) was found to inhibit the formation of fructosamine and α-dicarbonyl compounds, while AGEs was almost suppressed by OA at the concentration of 500 μg/mL. Moreover, OA exhibited strong anti-fibrillation and antioxidant abilities. It decreased the production of dityrosine, N′- formylkynurenine and kynurenine with strong ·OH and O2·− scavenging capacities. The binding of OA with lysine and arginine of bovine serum albumin (BSA) prevented BSA from the attack of sugars. The C−12 in the C ring of OA was predicted to trap methylglyoxal (MGO), forming the OA−MGO adduct. The antioxidant and MGO trapping abilities together with the secondary structure protection may be the main mechanism of anti-glycation ability of OA. These findings suggest that foods rich in OA are promising sources of anti-glycation agents. The study also provides a theoretical basis for the further functional research of OA in the prevention and treatment of diabetic complications.

Impact of Phenolic Compounds in Strecker Aldehyde Formation in Wine Model Systems: Target and Untargeted Analysis.

The Strecker degradation of phenylalanine has been studied in a phenolic compound/phenylalanine wine model system. Six phenolic compounds (3,4-dihydroxybenzoic acid, gallic acid, caffeic acid, ferulic acid, catechin, and epicatechin) were compared in the formation of phenylacetaldehyde when in the presence of glucose or methylglyoxal (MG). The addition of glucose reduced the formation of Strecker aldehyde, independently of the phenolic compound. The addition of MG, on the other hand, increased phenylacetaldehyde formation for hydroxybenzoic acids and decreased phenylacetaldehyde formation for flavan-3-ols, confirming their capacity to trap the dicarbonyl compound. As a target phenolic compound, catechin was chosen to perform kinetic studies to further understand the reaction intermediates involved in the mechanism of phenylacetaldehyde formation, in particular, catechin o-quinone and catechin-MG adduct. The addition of glucose and MG increased the consumption of catechin, while a reduction in the respective o-quinone was observed, suggesting that these substrates have an impact in other reactions involving catechin. In that regard, for the first time, it was demonstrated that the catechin-MG adduct was capable of oxidizing and forming a new o-quinone, contributing to wine instability promoted by oxidation reactions.

Methylglyoxal Acts as a Tumor-Promoting Factor in Anaplastic Thyroid Cancer.

Methylglyoxal (MG) is a potent inducer of advanced glycation end products (AGEs). MG, long considered a highly cytotoxic molecule with potential anticancer value, is now being re-evaluated to a protumorigenic agent in some malignancies. Anaplastic thyroid cancer (ATC) is an extremely aggressive and highly lethal cancer for which conventional therapies have proved ineffective. Successful therapeutic intervention in ATC is undermined by our poor understanding of its molecular etiology. In the attempt to understand the role of MG in ATC aggressiveness, we used immunohistochemistry to examine the level of MG protein adducts in ATC and slow-growing papillary thyroid cancer (PTC). We detected a high level of MG adducts in ATC compared to PTC ones, suggesting a protumor role for MG-mediated dicarbonyl stress in ATC. Accordingly, MG adduct accumulation in ATC cells in vitro was associated with a marked mesenchymal phenotype and increased migration/invasion, which were both reversed by aminoguanidine (AG)—a scavenger of MG—and resveratrol—an activator of Glyoxalase 1 (Glo1), the key metabolizing enzyme of MG. Our study represents the first demonstration that MG, via AGEs, acts as a tumor-promoting factor in ATC and suggests that MG scavengers and/or Glo1 activators merit investigations as potential therapeutic strategies for this malignancy.

Phloretin and its methylglyoxal adduct: Implications against advanced glycation end products-induced inflammation in endothelial cells

Methylglyoxal (MGO), a cytotoxic factor, reacts irreversibly with the side chains of lysine, cysteine, and arginine residues in proteins to form advanced glycation end products (AGEs) which might be a major pathological factor associated with diabetic complications. Thus, it is necessary to prevent or alleviate such diseases through inhibiting the formation of AGEs or lowering these AGEs-induced cellular damages. Based on our previous work, it was known that phloretin, an apple polyphenol, can inhibit the formation of AGEs under simulated physiological conditions. In this study, we found that phloretin prevented the formation of AGEs through trapping MGO in human umbilical endothelial cells (HUVECs). The phloretin-MGO adducts were analyzed in PBS and HUVECs. Surprisingly, only 1 MGO-phloretin adduct was detected in HUVECs, which was formed within 0.5 h and metabolized eventually within 24 h. The specific phloretin-MGO adduct was synthesized and identified by MS and NMR analysis. Its anti-inflammatory effect against AGEs was further investigated together with the parent compound, phloretin, which was proved to be through RAGE/p38 MAPK/NF-κB signaling pathway. Taken together, our data indicated the positive role of phloretin-MGO adduct on phloretin's protective effects, which might offer a new insight into the action mechanism of polyphenols against AGEs-induced damages.

Dual effects of propyl gallate and its methylglyoxal adduct on carbonyl stress and oxidative stress

In the present study, we investigated the trapping of methylglyoxal (MGO) by propyl gallate (PG), a known food grade antioxidant, and the anti-carbonyl and anti-oxidative properties of the mono-MGO adduct of PG (MM-PG). Our result indicated that more than 77.5% MGO was suppressed by PG after a 30 min incubation of PG with MGO, which was much more effective than gallic acid (15.2%). For the first time, MM-PG was purified, and its structure was elucidated based on the analysis of its 1H, 13C, and 2D-NMR data. We also demonstrated that MM-PG had strong anti-oxidative and anti-carbonyl activities. Furthermore, PG could trap the MGO generated during the preparation of roasted pork, and both mono- and di- MGO adducts of PG were detected in the roasted pork system using LC/MS technique. Thus, PG could be widely applied in the food system for inhibiting the formation of both carbonyl species and oxidative species.

Changes in MHC-II/peptide binding affinity following protein glycation, as observed in Diabetes

Abstract Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in several metabolic diseases including diabetes. These highly reactive moieties bind to the cellular proteome to form advanced glycation end-products (AGEs), which can alter the structure and function of targeted proteins. To address whether the AGE-mediated modification of immunologically relevant proteins could have functional consequences we incubated soluble recombinant HLA- DR1 (HLA-DRA*01:01/DRB1*01:01) in presence of glucose at concentration commonly observed in diabetic subjects (15–20 mM). Analysis of the AGE adducts was achieved by using bottom-up proteomics and tandem mass spectrometry analysis. Major observed modifications were glyoxal-derived hydroimiadazolone, argpyrimidine, dihydroxy and methylglyoxal adducts on arginine residues, pyralline and glyceraldehyde-derived pyridinium compound adducts of lysine and carboxy ethylation and methylation of both arginine and lysine residues. These AGE-related modifications were comparable to what observed on MHCII, immunoprecipitated from the PBMC of Diabetic patients. AGE PTMs mapped to both the binding groove and HLA-DM binding domains. AGE-modified and unmodified HLA-DR1 were then assessed for their binding affinity of several relevant peptides in a fluorescence polarization assay in the presence or absence of HLA-DM. Changes in binding affinity for the selected peptides was observed in AGE-HLA-DR1 as compared to the unmodified HLA-DR1. Altogether our data indicate that protein PTM, as commonly observed in conditions of metabolic and oxidative stress can change HLA-DR1/peptide binding affinity thus compromising MHC-II restricted immune responses.