Advanced glycation end-product crosslinking activates a type VI secretion system phospholipase effector protein.

Advanced glycation end-products (AGE) are a pervasive form of protein damage implicated in the pathogenesis of neurodegenerative disease, atherosclerosis and diabetes mellitus. Glycation is typically mediated by reactive dicarbonyl compounds that accumulate in all cells as toxic byproducts of glucose metabolism. Here, we show that AGE crosslinking is harnessed to activate an antibacterial phospholipase effector protein deployed by the type VI secretion system of Enterobacter cloacae. Endogenous methylglyoxal reacts with a specific arginine-lysine pair to tether the N- and C-terminal α-helices of the phospholipase domain. Substitutions at these positions abrogate both crosslinking and toxic phospholipase activity, but in vitro enzyme function can be restored with an engineered disulfide that covalently links the N- and C-termini. Thus, AGE crosslinking serves as a bona fide post-translation modification to stabilize phospholipase structure. Given the ubiquity of methylglyoxal in prokaryotic and eukaryotic cells, these findings suggest that glycation may be exploited more generally to stabilize other proteins. This alternative strategy to fortify tertiary structure could be particularly advantageous in the cytoplasm, where redox potentials preclude disulfide bond formation.

Balancing Maillard reaction products formation and antioxidant activities for improved sensory quality and health benefit properties of pan baked buns.

This study investigated the impact of processing temperatures (190 °C, 210 °C, and 230 °C) and durations (7 min, 10 min, and 14 min) on the formation of Maillard reaction products (MRPs) and antioxidant activities in pan baked buns. Key Maillard reaction indicators, including glyoxal (GO), methylglyoxal (MGO), 5-hydroxymethylfurfural (5-HMF), melanoidins, and fluorescent advanced glycation end products (AGEs) were quantified. The results demonstrated significant increases in GO, MGO, 5-HMF contents (p < 0.05), and antioxidant activities (p < 0.05) when the buns were baked at 210 °C for 14 min, 230 °C for 10 min and 14 min. However, the interior MRPs of baked buns were minimally affected by the baking temperature and duration. Prolonged heating temperatures and durations exacerbated MRPs production (43.8 %-1038 %) in the bottom crust. Nonetheless, this process promoted the release of bound phenolic compounds and enhanced the antioxidant activity. Heating induces the thermal degradation of macromolecules in food, such as proteins and polysaccharides, which releases bound phenolic compounds by disrupting their chemical bonds within the food matrix. Appropriate selections of baking parameters can effectively reduce the formation of MRPs while simultaneously improve sensory quality and health benefit of the pan baked buns. Considering the balance between higher antioxidant properties and lower MRPs, the optimal thermal parameters for pan baked buns were 210 °C for 10 min. Furthermore, a normalized analysis revealed a consistent trend for GO, MGO, 5-HMF, fluorescent AGEs, and melanoidins. Moreover, MRPs were positively correlated with total contents of phenolic compounds, ferric-reducing antioxidant power (FRAP), and color, but negatively correlated with moisture contents and reducing sugars. Additionally, the interaction between baking conditions and Maillard reactions probably contributed to enhanced primary flavors in the final product. This study highlights the importance of optimizing baking parameters to achieve desirable MRPs levels, higher antioxidant activity, and optimal sensory attributes in baked buns.

Quantitative NMR Analysis of Marine Macroalgae for AGE Inhibition by Methylglyoxal Scavenging.

Reactive carbonyl species (RCS) induce a fundamental form of biological stress that has driven the evolution of diverse mechanisms for minimizing its impact on organismal health. The complications that accompany uncontrolled hyperglycemia exemplify the health implications when RCS stress exceeds the body's capacity to prevent the excessive formation of advanced glycation end-products. Presented here is a novel quantitative NMR (qNMR) technique for evaluating scavengers of the prominent sugar-derived carbonyl methylglyoxal (MGO). This tool was employed to screen the chemical diversity of marine macroalgae extracts, with a focus on species that have a history of consumption by the World's healthiest populations and are subject to global scale aquacultural production. Fucus vesiculosus demonstrated the highest capacity for inhibiting glycation and scavenging MGO. Additionally, the Chondrus cripsus, Gracilaria vermiculophyla, and Gracilaria tikvahiae extracts had a high capacity for scavenging MGO, representing the first report of this activity. This new qNMR methodology presented is highly applicable for screening extracts and compounds from diverse sources, and the results highlight the potential of macroalgae extracts to be employed as RCS and AGE targeting therapeutics and food additives.

Solvent-Dependent Chemoselectivity Switch to Arg-Lys Imidazole Cross-Links.

Herein, we report a trifluoroethanol-mediated, chemoselective method for the formation of Arg-Lys imidazole cross-links with methylglyoxal and its application in the selective macrocyclization of peptides between Lys and Arg and the late-stage diversification of Lys-containing peptides with guanidine. Our findings highlight the critical role of solvent choice in controlling chemoselectivity, providing valuable insights into solvent-dependent peptide modification.

Formation of methylglyoxal (CH3C(O)CHO) in interstellar analog ices - a key intermediate in cellular metabolism.

Ketoaldehydes are key intermediates in biochemical processes including carbohydrate, lipid, and amino acid metabolism. Despite their crucial role in the interstellar synthesis of essential biomolecules necessary for the Origins of Life, their formation mechanisms have largely remained elusive. Here, we report the first bottom-up formation of methylglyoxal (CH3C(O)CHO)-the simplest ketoaldehyde-through the barrierless recombination of the formyl (HCO) radical with the acetyl (CH3CO) radical in low-temperature interstellar ice analogs upon exposure to energetic irradiation as proxies of galactic cosmic rays. Utilizing vacuum ultraviolet photoionization reflectron time-of-flight mass spectrometry and isotopic substitution studies, methylglyoxal and its enol tautomer 2-hydroxypropenone (CH3C(OH)CO) were identified in the gas phase during the temperature-programmed desorption of irradiated carbon monoxide-acetaldehyde (CO-CH3CHO) ices, suggesting their potential as promising candidates for future astronomical searches. Once synthesized in cold molecular clouds, methylglyoxal can serve as a key precursor to sugars, sugar acids, and amino acids. Furthermore, this work provides the first experimental evidence for tautomerization of a ketoaldehyde in interstellar ice analogs, advancing our fundamental knowledge of how ketoaldehydes and their enol tautomers can be synthesized in deep space.

Scavenging of Methylglyoxal by the Total Flavonoids of Apocyni Veneti Folium in Mice.

Scavenging MGO has been considered as an effective strategy for preventing atherosclerosis. A previous study showed that the total flavonoids of Apocyni Veneti Folium (TFAVF) had a significant antiatherosclerotic effect. However, there are no studies that have investigated the MGO scavenging capacities of TFAVF in mice. We found that TFAVF consisted mainly of quercetin glycosides and kaempferol glycosides using ultrahigh performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). TFAVF was first demonstrated to effectively scavenge MGO in mice based on the formation of mono-MGO-quercetin, mono-MGO-dehydroquercetin, mono-MGO-isorhamnetin, mono-MGO-dehydroisorhamnetin, mono-MGO-kaempferol, and mono-MGO-dehydrokaempferol. In addition, one mono-MGO-quercetin was separated and purified, and its structure was elucidated as 8-MGO-quercetin based on UHPLC-QTOF-MS/MS and NMR data. Quantification studies have demonstrated that kaempferol, dehydrokaempferol, quercetin, dehydroquercetin, isorhamnetin, and dehydroisorhamnetin can dose dependently scavenge MGO in mice. Taken together, these results indicated that TFAVF showed a significant antiatherosclerotic effect, which might be based on MGO detoxification.

Capture of single or multiple reactive carbonyl species by mangiferin under high temperatures.

Reactive carbonyl species (RCS), including acrolein (ACR), methylglyoxal (MGO), and glyoxal (GO), are typically generated in food processing and accumulate in the body for ages, triggering various chronic diseases. Here, we investigated the capture capability and reaction pathways of mangiferin one-to-one and one-to-many on RCS in high temperatures using UPLC-MS/MS. We found that mangiferin can capture ACR/MGO/GO to form their adducts, yet, the ability to capture RCS is arranged in different orders, with ACR > MGO > GO for a single RCS and MGO > ACR > GO for multiple RCS. After synthesizing and identifying the structures of the ACR- and MGO-adducts of MGF, our results indicated that MGF-ACR-MGO produced in the multiple-RCS-MGF system was formed by capturing MGO through MGF-ACR rather than through MGF-MGO capturing ACR, which resulting in higher inhibitory activity of MGF against MGO than against ACR. Then, the capture ability and path of MGF on RCS were verified in the coffee-leaves tea and cake.

Impact of Extrusion Parameters on the Formation of Nε-(Carboxymethyl)lysine, Nε-(Carboxyethyl)lysine and Acrylamide in Plant-Based Meat Analogues.

To investigate the impact of extrusion parameters on the formation of Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL) and acrylamide in plant-based meat analogues (PBMAs), the content changes and the correlations of compounds related to their formation were studied. The extrusion promoted CML, CEL and acrylamide formation, with more CEL being formed than CML. Variations in the moisture level and barrel temperature exerted a greater influence on the CML, CEL, acrylamide and α-dicarbonyl compounds than the screw speed and the feed rate. An increase in the moisture content led to a decrease in the CEL content, whereas it enhanced CML formation. The impact of moisture on acrylamide formation varied depending on whether low- or high-moisture extrusion was applied. Elevated temperatures promoted the accumulation of CEL, methylglyoxal and 2,3-butanedione while diminishing the accumulation of CML, acrylamide, glyoxal and 3-deoxyglucosone. CML and CEL were positively correlated with glyoxal and methylglyoxal, respectively. CEL and methylglyoxal were negatively correlated with protein and water content, whereas CML, glyoxal and 3-deoxyglucosone displayed positive correlations. In summary, higher moisture levels and feed rates and lower screw speeds and barrel temperatures are advantageous for producing PBMAs with lower CEL and total advanced glycation end-products contents, while lower or higher moisture contents, a lower feed rate and a higher barrel temperature are beneficial to reducing the acrylamide content.

Catalytic elevation effect of methylglyoxal on invertase and characterization of MGO modification products.

Reactive carbonyl species can modify digestive enzymes upon intake due to their electrophilic nature. This study evaluated the effects of methylglyoxal (MGO), glyoxal, acrolein, and formaldehyde on invertase, an enzyme presents in digestive tract. Unexpectedly, MGO enhanced, rather than inhibited, invertase activity. Moreover, MGO counteracted the inhibitory effects of the other three carbonyls on invertase activity. Kinetic analyses revealed that 150 mmolLexp.-1 MGO resulted in a 2-fold increase in the Km and a 3.3-fold increase in Vmax, indicating that MGO increased the turnover rate of sucrose while reducing the substrate binding affinity of invertase. Additionally, MGO induced dynamic quenching of fluorescence, reduced free amino groups, increased hydrophobicity, the content of Amadori products, fluorescent and nonfluorescent AGEs, and amyloid fibrils of invertase. The specific modifications responsible for the elevated activity of MGO on invertase require further investigation.

The kinetic study of 2-acetyl-1-pyrroline accumulation in the model system: An insight into enhancing rice flavor through the Maillard reaction.

Controlling the Maillard reaction may affect the generation of 2-acetyl-1-pyrroline, the key aroma compound in rice. In this study, the kinetics of 2-acetyl-1-pyrroline accumulation in the glucose/proline model system was comprehensively investigated and extra methylglyoxal or glyoxal was added to enhance 2-acetyl-1-pyrroline concentrations during rice cooking. Using the multi-response kinetic modeling to derive kinetic parameters, the formation of glyoxal, as the reactive intermediate, was rate-determining for the overall generation rate of 2-acetyl-1-pyrroline. Besides, although 2-acetyl-1-pyrroline generation was easier to occur with lower activation energy, much higher depletion rates of 2-acetyl-1-pyrrroline at 120 °C and 140 °C led to maximal 2-acetyl-1-pyrroline accumulation at the lower temperature of 100 °C. Furthermore, the inclusion of 0.05 µmol/kg additional methylglyoxal in cooked rice significantly enhanced 2-acetyl-1-pyrroline generation. The work suggested that the development of rice products with superior flavor quality may be achieved by the slight accumulation of intermediates prior to thermal processing.

Label-free visualization of unfolding and crosslinking mediated protein aggregation in nonenzymatically glycated proteins.

Nonenzymatic glycation (NEG) unfolds and crosslinks proteins, resulting in aggregation. Label-free evaluation of such structural changes, without disturbing molecular integrity, would be beneficial for understanding the fundamental mechanisms of protein aggregation. The current study demonstrates the assessment of NEG-induced protein aggregation by combining autofluorescence (AF) spectroscopy and imaging. The methylglyoxal (MG) induced protein unfolding and the formation of cross-linking advanced glycation end-products (AGEs) leading to aggregation were evaluated using deep-UV-induced-autofluorescence (dUV-AF) spectroscopy in proteins with distinct structural characteristics. Since the AGEs formed on proteins are fluorescent, the study demonstrated the possibility of autofluorescence imaging of NEG-induced protein aggregates. Autofluorescence spectroscopy can potentially reveal molecular alterations such as protein unfolding and cross-linking. In contrast, AGE-based autofluorescence imaging offers a means to visually explore the structural arrangement of aggregates, regardless of whether they are amyloid or non-amyloid in nature.

Fenfuro®-mediated arrest in the formation of protein-methyl glyoxal adducts: a new dimension in the anti-hyperglycemic potential of a novel fenugreek seed extract.

The fenugreek plant (Trigonella foenum-graecum) is traditionally known for its anti-diabetic properties owing to its high content of furostanolic saponins, which can synergistically treat many human ailments. Non-enzymatic protein glycation leading to the formation of Advanced Glycation End products (AGE) is a common pathophysiology observed in diabetic or prediabetic individuals, which can initiate the development of neurodegenerative disorders. A potent cellular source of glycation is Methyl Glyoxal, a highly reactive dicarbonyl formed as a glycolytic byproduct. We demonstrate the in vitro glycation arresting potential of Fenfuro®, a novel patented formulation of Fenugreek seed extract with clinically proven anti-diabetic properties, in Methyl-Glyoxal (MGO) adducts of three abundant amyloidogenic cellular proteins, alpha-synuclein, Serum albumin, and Lysozyme. A 0.25% w/v Fenfuro® was able to effectively arrest glycation by more than 50% in all three proteins, as evidenced by AGE fluorescence. Glycation-induced amyloid formation was also arrested by more than 36%, 14% and 15% for BSA, Alpha-synuclein and Lysozyme respectively. An increase in MW by attachment of MGO was also partially prevented by Fenfuro® as confirmed by SDS-PAGE analysis. Glycation resulted in enhanced aggregation of the three proteins as revealed by Native PAGE and Dynamic Light Scattering. However, in the presence of Fenfuro®, aggregation was arrested substantially, and the normal size distribution was restored. The results cumulatively indicated the lesser explored potential of direct inhibition of glycation by fenugreek seed in addition to its proven role in alleviating insulin resistance. Fenfuro® boosts its therapeutic potential as an effective phytotherapeutic to arrest Type 2 diabetes.

Fenfuro^® is a novel patented formulation of Fenugreek seed extract with more than 45% furostanolic saponins and anti-diabetic property free from any side effect as established through clinical study.In the present study, the role of Fenfuro® in arresting in vitro AGE formation and glycation-induced amyloid formation has been demonstrated with the help of three amyloidogenic proteins, namely Human Lysozyme, Human alpha-synuclein and Bovine Serum Albumin using Methyl Glyoxal as the glycating agent.A 0.25% (w/v) ethanolic solution of Fenfuro® resulted in more than 50% arrest in glycation with simultaneous prevention of aggregation as demonstrated by native PAGE, DLS and inhibition of development of Thio-T positive amyloid like entities.The studies collectively aim toward the development of a safe therapeutic method for arresting protein glycation through direct physical intervention.

Theanine Capture of Reactive Carbonyl Species in Humans after Consuming Theanine Capsules or Green Tea.

Acrolein (ACR), methylglyoxal (MGO), and glyoxal (GO) are a class of reactive carbonyl species (RCS), which play a crucial role in the pathogenesis of chronic and age-related diseases. Here, we explored a new RCS inhibitor (theanine, THE) and investigated its capture capacity on RCS in vivo by human experiments. After proving that theanine could efficiently capture ACR instead of MGO/GO by forming adducts under simulated physiological conditions, we further detected the ACR/MGO/GO adducts of theanine in the human urine samples after consumption of theanine capsules (200 and 400 mg) or green tea (4 cups, containing 200 mg of theanine) by using ultraperformance liquid chromatography-time-of-flight-high-resolution mass spectrometry. Quantitative assays revealed that THE-ACR, THE-2ACR-1, THE-MGO, and THE-GO were formed in a dose-dependent manner in the theanine capsule groups; the maximum value of the adducts of theanine was also tested. Furthermore, besides the RCS adducts of theanine, the RCS adducts of catechins could also be detected in the drinking tea group. Whereas, metabolite profile analysis showed that theanine could better capture RCS produced in the renal metabolic pathway than catechins. Our findings indicated that theanine could reduce RCS in the body in two ways: as a pure component or contained in tea leaves.

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.

Insight into the Effect of Methylglyoxal on the Conformation, Function, and Aggregation Propensity of α-Synuclein.

It is well-known that people suffering from hyperglycemia have a higher propensity to develop Parkinson's disease (PD). One of the most plausible mechanisms linking these two pathologies is the glycation of neuronal proteins and the pathological consequences of it. α-Synuclein, a key component in PD, can be glycated at its fifteen lysine. In fact, the end products of this process have been detected on aggregated α-synuclein isolated from in vivo. However, the consequences of glycation are not entirely clear, which are of crucial importance to understand the mechanism underlying the connection between diabetes and PD. To better clarify this, we have here examined how methylglyoxal (the most important carbonyl compound found in the cytoplasm) affects the conformation and aggregation propensity of α-synuclein, as well as its ability to cluster and fuse synaptic-like vesicles. The obtained data prove that methylglyoxal induces the Lys-Lys crosslinking through the formation of MOLD. However, this does not have a remarkable effect on the averaged conformational ensemble of α-synuclein, although it completely depletes its native propensity to form soluble oligomers and insoluble amyloid fibrils. Moreover, methylglyoxal has a disrupting effect on the ability of α-synuclein to bind, cluster and fusion synaptic-like vesicles.

Formation of Hesperetin-Methylglyoxal Adducts in Food and In Vivo, and Their Metabolism In Vivo and Potential Health Impacts.

Polyphenols with a typical meta-phenol structure have been intensively investigated for scavenging of methylglyoxal (MGO) to reduce harmful substances in food. However, less attention has been paid to the formation level of polyphenol-MGO adducts in foods and in vivo and their absorption, metabolism, and health impacts. In this study, hesperitin (HPT) was found to scavenge MGO by forming two adducts, namely, 8-(1-hydroxyacetone)-hesperetin (HPT-mono-MGO) and 6-(1-hydroxyacetone)-8-(1-hydroxyacetone)-hesperetin (HPT-di-MGO). These two adducts were detected (1.6-15.9 mg/kg in total) in cookies incorporated with 0.01%-0.5% HPT. HPT-di-MGO was the main adduct detected in rat plasma after HPT consumption. The adducts were absorbed 8-30 times faster than HPT, and they underwent glucuronidation and sulfation in vivo. HPT-mono-MGO would continue to react with endogenous MGO in vivo to produce HPT-di-MGO, which effectively reduced the cytotoxicity of HPT and HPT-mono-MGO. This study provided data on the safety of employing HPT as a dietary supplement to scavenge MGO in foods.

The Interaction between Human Microbes and Advanced Glycation End Products: The Role of Klebsiella X15 on Advanced Glycation End Products' Degradation.

Previous studies have shown that advanced glycation end products (AGEs) are implicated in the occurrence and progression of numerous diseases, with dietary AGEs being particularly associated with intestinal disorders. In this study, methylglyoxal-beta-lactoglobulin AGEs (MGO-ß-LG AGEs) were utilized as the exclusive nitrogen source to investigate the interaction between protein-bound AGEs and human gut microbiota. The high-resolution mass spectrometry analysis of alterations in peptides containing AGEs within metabolites before and after fermentation elucidated the capacity of intestinal microorganisms to enzymatically hydrolyze long-chain AGEs into short-chain counterparts. The 16S rRNA sequencing revealed Klebsiella, Lactobacillus, Escherichia-Shigella, and other genera as dominant microbiota at different fermentation times. A total of 187 potential strains of AGE-metabolizing bacteria were isolated from the fermentation broth at various time points. Notably, one strain of Klebsiella exhibited the most robust growth capacity when AGEs served as the sole nitrogen source. Subsequently, proteomics was employed to compare the changes in protein levels of Klebsiella X15 following cultivation in unmodified proteins and proteins modified with AGEs. This analysis unveiled a remodeled amino acid and energy metabolism pathway in Klebsiella in response to AGEs, indicating that Klebsiella may possess a metabolic pathway specifically tailored to AGEs. This study found that fermenting AGEs in healthy human intestinal microbiota altered the bacterial microbiota structure, especially by increasing Klebsiella proliferation, which could be a key factor in AGEs' role in causing diseases, particularly intestinal inflammation.

Inhibitory effects and mechanisms of phenolic compounds in rapeseed oil on advanced glycation end product formation in chemical and cellular models in vitro.

Sinapic acid (SA), canolol (CAO) and canolol dimer (CAO dimer) are the main phenolic compounds in rapeseed oil. However, their possible efficacy against glycation remains unclear. This study aims to explore the impacts of these substances on the formation of advanced glycation end products (AGEs) based on chemical and cellular models in vitro. Based on fluorescence spectroscopy results, three chemical models of BSA-fructose, BSA-methylglyoxal (MGO), and arginine (Arg)-MGO showed that SA/CAO/CAO dimer could effectively reduce AGE formation but with different abilities. After SA/CAO/CAO dimer incubation, effective protection against BSA protein glycation was observed and three different MGO adducts were formed. In MGO-induced HUVEC cell models, only CAO and CAO dimer significantly inhibited oxidative stress and cell apoptosis, accompanied by the regulation of the Nrf2-HO-1 pathway. During the inhibition, 20 and 12 lipid mediators were reversed in the CAO and CAO dimer groups compared to the MGO group.

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.

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.