Quantifying carboxymethyl lysine and carboxyethyl lysine in human plasma: clinical insights into aging research using liquid chromatography-tandem mass spectrometry.

OBJECTIVE: The objective of this study was to establish a methodology for determining carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) concentrations in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test results were also used for clinical aging research. METHODS: Human plasma samples were incubated with aqueous perfluorovaleric acid (NFPA), succeeded by precipitation utilizing trichloroacetic acid, hydrolysis facilitated by hydrochloric acid, nitrogen drying, and ultimate re-dissolution utilizing NFPA, followed by filtration. Cotinine-D3 was added as an internal standard. The separation was performed on an Agela Venusil ASB C18 column (50 mm × 4.6 mm, 5 µm) with a 5 mmol/L NFPA and acetonitrile/water of 60:40 (v/v) containing 0.15% formic acid. The multiple reaction monitoring mode was used for detecting CML, CEL, and cotinine-D3, with ion pairs m/z 205.2 > 84.1 (for quantitative) and m/z 205.2 > m/z 130.0 for CML, m/z 219.1 > 84.1 (for quantitative) and m/z 219.1 > m/z 130.1 for CEL, and m/z 180.1 > 80.1 for cotinine-D3, respectively. RESULTS: The separation of CML and CEL was accomplished within a total analysis time of 6 minutes. The retention times of CML, CEL, and cotinine-D3 were 3.43 minutes, 3.46 minutes, and 4.50 minutes, respectively. The assay exhibited linearity in the concentration range of 0.025-1.500 µmol/L, with a lower limit of quantification of 0.025 µmol/L for both compounds. The relative standard deviations of intra-day and inter-day were both below 9%, and the relative errors were both within the range of ±4%. The average recoveries were 94.24% for CML and 97.89% for CEL. CONCLUSION: The results indicate that the developed methodology is fast, highly sensitive, highly specific, reproducible, and suitable for the rapid detection of CML and CEL in clinical human plasma samples. The outcomes of the clinical research project on aging underscored the important indicative significance of these two indicators for research on human aging.

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.

Chemical synthesis of site-selective advanced glycation end products in α-synuclein and its fragments.

Advanced glycation end products (AGEs) arise from the Maillard reaction between dicarbonyls and proteins, nucleic acids, or specific lipids. Notably, AGEs are linked to aging and implicated in various disorders, spanning from cancer to neurodegenerative diseases. While dicarbonyls like methylglyoxal preferentially target arginine residues, lysine-derived AGEs, such as N(6)-(1-carboxymethyl)lysine (CML) and N(6)-(1-carboxyethyl)lysine (CEL), are also abundant. Predicting protein glycation in vivo proves challenging due to the intricate nature of glycation reactions. In vitro, glycation is difficult to control, especially in proteins that harbor multiple glycation-prone amino acids. α-Synuclein (aSyn), pivotal in Parkinson's disease and synucleinopathies, has 15 lysine residues and is known to become glycated at multiple lysine sites. To understand the influence of glycation in specific regions of aSyn on its behavior, a strategy for site-specific glycated protein production is imperative. To fulfill this demand, we devised a synthetic route integrating solid-phase peptide synthesis, orthogonal protection of amino acid side-chain functionalities, and reductive amination strategies. This methodology yielded two disease-related N-terminal peptide fragments, each featuring five and six CML and CEL modifications, alongside a full-length aSyn protein containing a site-selective E46CEL modification. Our synthetic approach facilitates the broad introduction of glycation motifs at specific sites, providing a foundation for generating glycated forms of synucleinopathy-related and other disease-relevant proteins.

Comprehensive characterization of differential glycation in hepatocellular carcinoma using tissue proteomics with stable isotopic labeling.

Glycation is a non-enzymatic posttranslational modification coming from the reaction between reducing sugars and free amino groups in proteins, where early glycation products (fructosyl-lysine, FL) and advanced glycation end products (AGEs) are formed. The occurrence of glycation and accumulation of AGEs have been closely associated with hepatocellular carcinoma (HCC). Here, we reported the characterization of differential glycation in HCC using tissue proteomics with stable isotopic labeling; early glycation-modified peptides were enriched with boronate affinity chromatography (BAC), and AGEs-modified peptides were fractionated with basic reversed-phase separation. By this integrated approach, 3717 and 1137 early and advanced glycated peptides corresponding to 4007 sites on 1484 proteins were identified with a false discovery rate (FDR) of no more than 1%. One hundred fifty-five sites were modified with both early and advanced end glycation products. Five early and 7 advanced glycated peptides were quantified to be differentially expressed in HCC tissues relative to paired adjacent tissues. Most (8 out of 10) of the proteins corresponding to the differential glycated peptides have previously been reported with dysregulation in HCC. The results together may deepen our knowledge of glycation as well as provide insights for therapeutics.

Mango peel extracts and mangiferin chromatographic Fourier-transform infrared correlation with antioxidant, antidiabetic, and advanced glycation end product inhibitory potentials using in silico modeling and in vitro assays.

Mangifera indica peels are a rich source of diverse flavonoids and xanthonoids; however, generally these are discarded. Computational studies revealed that mangiferin significantly interacts with amino acid residues of transcriptional regulators 1IK3, 3TOP, and 4f5S. The methanolic extract of Langra variety of mangoes contained the least phenol concentrations (22.6 ± 0.32 mg/gGAE [gallic acid equivalent]) compared to the chloroform (214.8 ± 0.12 mg/gGAE) and ethyl acetate fractions (195.6 ± 0.14 mg/gGAE). Similarly, the methanolic extract of Sindhri variety contained lower phenol concentrations (42.3 ± 0.13 mg/gRUE [relative utilization efficiency]) compared with the chloroform (85.6 ± 0.15 mg/gGAE) and ethyl acetate (76.1 ± 0.32 mg/gGAE) fractions. Langra extract exhibited significant α-glucosidase inhibition (IC50 0.06 mg/mL), whereas the ethyl acetate fraction was highly active (IC50 0.12 mg/mL) in Sindhri variety. Mangiferin exhibited significant inhibition (IC50 0.026 mg/mL). A moderate inhibition of 15-LOX was observed in all samples, whereas mangiferin was least active. In advanced glycation end product inhibition assay, the chloroform fraction of Langra variety exhibited significant inhibition in nonoxidative (IC50 64.4 µg/mL) and oxidative modes (IC50 54.7 µg/mL). It was concluded that both Langra and Sindhri peel extracts and fractions possess significant antidiabetic activities. The results suggest the potential use of peel waste in the management and complications of diabetes.

In Vitro Spectroscopic Investigation of Losartan and Glipizide Competitive Binding to Glycated Albumin: A Comparative Study.

Understanding the interaction between pharmaceuticals and serum proteins is crucial for optimizing therapeutic strategies, especially in patients with coexisting chronic diseases. The primary goal of this study was to assess the potential changes in binding affinity and competition between glipizide (GLP, a second-generation sulfonylurea hypoglycemic drug) and losartan (LOS, a medication commonly prescribed for hypertension, particularly for patients with concurrent diabetes) with non-glycated (HSA) and glycated (gHSAGLC, gHSAFRC) human serum albumin using multiple spectroscopic techniques (fluorescence, UV-visible absorption, and circular dichroism spectroscopy). The results indicated that FRC is a more effective glycation agent for HSA than GLC, significantly altering the albumin structure and affecting the microenvironment around critical amino acid residues, Trp-214 and Tyr. These modifications reduce the binding affinity of LOS and GLP to gHSAGLC and gHSAFRC, compared to HSA, resulting in less stable drug-protein complexes. The study revealed that LOS and GLP interact nonspecifically with the hydrophobic regions of the albumin surface in both binary (ligand-albumin) and ternary systems (ligand-albumin-ligandconst) and specifically saturate the binding sites within the protein molecule. Furthermore, the presence of an additional drug (GLP in the LOS-albumin complex or LOS in the GLP-albumin complex) complicates the interactions, likely leading to competitive binding or displacement of the initially bound drug in both non-glycated and glycated albumins. Analysis of the CD spectra suggests mutual interactions between GLP and LOS, underscoring the importance of closely monitoring patients co-administered these drugs, to ensure optimal therapeutic efficacy and safety.

Comprehensive research on the properties of advanced glycation end products in food and biological samples and their harmful role in inducing metabolic diseases.

Advanced glycation end products (AGEs) are formed by the Maillard reaction, a nonenzymatic process that occurs widely in cooking, food processing, and within the human body. Primarily, AGEs are formed by the glycation of reducing sugars with amino groups, and this process is heat-dependent. With changes in lifestyle, there has been an increase in the diversity of dietary habits, including those patterns associated with Western diets, which include the consumption of processed foods that are rich in AGEs. Excessive intake and exposure to AGEs are known to cause abnormalities in body function such as obesity, diabetes, and fatty liver, and the beneficial effects of AGEs in food processing in improving food flavor and quality. To obtain meaningful data regarding AGEs in a variety of food and human samples, it is necessary to more precisely characterize and analyze the AGEs extracted from samples to obtain accurate results. This review explores the recent analytical research and characterization of AGEs in foods, including casein, ß-lactoglobulin, soy protein, and meat protein, and in human samples, such as glycated-albumin, hemoglobin, and plasma. Additionally, it explores the metabolic fate of AGEs in the body and the mechanisms of disease associated with metabolic abnormalities that may be caused by the consumption of foods containing AGEs. This review aims to provide an overview of the perspectives of relevant recent and future research on metabolic abnormalities caused by foods containing AGEs or by AGEs produced in the body.

The Inhibition Mechanisms of Three Structurally Different Salvianolic Acids on the Non-Enzymatic Glycation of Bovine Serum Albumin.

The antiglycation mechanisms of three structurally different salvianolic acids (Sals) including salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and salvianolic acid C (Sal-C) were investigated using the bovine serum albumin (BSA)-fructose model. The results showed that the three compounds could inhibit the formation of glycation products, maintain protein structural stability, mitigate the development of amyloid fibrils and scavenge radicals. Notably, Sal-A possessed the highest anti-glycated activity compared with Sal-B and Sal-C. This may be related to the fact that Sal-A contained the most molecules of caffeic acid (Sal-A, Sal-B, and Sal-C possessing two, one, and zero caffeic acid units, respectively), and caffeic acid played a leading role in the antiglycation properties relative to Danshensu. Moreover, these compounds quenched the intrinsic fluorescence intensity of BSA in a static mode, with the binding constants in the order of Sal-A > Sal-B > Sal-C. Obviously, Sal-A possessed the strongest binding affinity among these compounds, which may be one of the reasons why it exhibited the optimal antiglycation capability. Furthermore, molecular docking demonstrated that the three Sals exerted protective effects on BSA by preventing glycation modification of lysine and arginine residues. These findings would provide valuable insights into the potential application of Sals for alleviating non-enzymatic glycation of protein.

Unveiling the molecular basis of inflamm-aging induced by advanced glycation end products (AGEs)-modified human serum albumin (AGE-HSA) in patients with different immune-mediated diseases.

Increased serum advanced glycation end products (AGEs) are commonly found in the patients with Diabetes mellitus (DM), aging-related diseases, and immune-mediated diseases. These diseases are notorious for vasculopathy, immune dysfunctions, and low-grade inflammation mimicking inflamm-aging. However, the molecular basis of inflamm-aging related to AGEs remains elucidation. In this study, we incubated human serum albumin (HSA) and glucose at 37 °C in 5% CO2 incubator for 0-180 days to generate AGE-HSA. We found the mixture gradually changing the color from transparancy to brown color and increased molecular weight during incubation. The pH value also gradually decreased from 7.2 to 5.4 irrelevant to ionic charge or [Ca2+] concentration, but dependent on gradual glycation of the alkaline amino acids, lysine and arginine. Functionally, 40 µg/mL of AGE-HSA decreased IL-2 production from human Jurkat T cell line via suppressing p-STAT3, p-STAT4, and p-STAT6 with an increased tendency of senescence-associated ß-galactosidase (SA-ßgal) expression but irrelevant to change of Th1/Th2/Treg subpopulations. In contrast, AGE-HSA enhanced CC motif chemokine ligand 5 (CCL-5), IL-8, macrophage migration inhibitor factor (MIF), and interleukin 1 receptor antagonist (IL-1Ra) but suppressed SA-ßgal expression by human macrophage-like THP-1 cells. Interestingly, AGE-HSA abrogated the HSA-induced soluble intercellular adhesion molecules 1 (sICAM-1), sE-selectin and endothelin release from human coronary artery endothelial cells (HCAEC) and enhanced SA-ßgal expression. The accelerated and increased HSA glycations by individual inflammation-related cytokine such as IL-2, IL-6, IL-17, TGF-ß, or TNF-α in the in vitro study reflect increased serum AGE levels in patients with immune-mediated diseases. In conclusion, AGE-HSA can exert immunosuppresive, inflammatory and vasculopathic effects mimicking inflamm-aging in these patients.

Preparation and antimicrobial mechanism of Maillard reaction products derived from ε-polylysine and chitooligosaccharides.

Chitooligosaccharides can be combined with amino acids or polypeptide to form Maillard reaction products (MRPs) with the antibacterial characteristics through Maillard reaction. This research aims to clarify the structure, antimicrobial effect and mechanism against Shewanella putrefaciens (S. putrefaciens) of ε-polylysine and chitooligosaccharides Maillard reaction products (LC-MRPs). The results of intrinsic fluorescence (IF) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction, proton nuclear magnetic resonance (1H NMR) spectra and scanning electron microscope (SEM) indicated Maillard reaction occurred between ε-polylysine and chitooligosaccharides. The observation of confocal laser scanning microscopy (CLSM), SEM and growth curves of S. putrefaciens evidenced that LC-MRPs have the strongest antibacterial effects. The leakage of alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) implied that LC-MRPs sabotaged bacterial barrier (cell wall and cell membrane). The changes in content of nucleic acids, reactive oxygen species (ROS) level, lipid peroxidation content (LPO), succinate dehydrogenase (SDH) activity and adenosine triphosphate (ATP) content showed LC-MRPs will affect bacterial genetic gene transcription, material and energy metabolism. Therefore, the LC-MRPs were effective antibacterial agents to inhibit S. putrefaciens, which will help to preserve food with S. putrefaciens as the main spoilage bacteria.

Methods to investigate advanced glycation end-product and their application in clinical practice.

Advanced glycation end-products (AGEs) are heterogeneous compounds of irreversible adducts principally derived from nonenzymatic glycation and glycoxidation of proteins. An increase in AGEs may be involved in the pathogenesis of metabolic and cardiovascular diseases, chronic degenerative diseases, neurological diseases and cancer, and it has been suggested as a biomarker of oxidative stress. AGEs have been evaluated in different biological fluids, as well as in tissues. The most utilized techniques for AGE measurement can be divided into immunochemical methods, such as ELISA, and bioanalytical methods, including fluorescence spectroscopy, high-performance liquid chromatography, liquid chromatography-mass spectroscopy, gas chromatography-mass spectroscopy. However, the lack of reference values, well-established standard molecules, and standardized methods to measure these compounds, could limit the application of AGE evaluation for clinical purpose. Aim of this review is to provide an overview on the state of the art of the most employed techniques for detection and measurement of AGEs and their application in clinical practice.

In Vitro Antiglycation and Methylglyoxal Trapping Effect of Peppermint Leaf (Mentha × piperita L.) and Its Polyphenols.

The most significant reactive α-dicarbonyl RCS involved in the pathomechanism of glycation and related diseases is methylglyoxal (MGO). Hyperglycemia promotes the generation of MGO and leads to the formation of advanced glycation end products (AGEs). Therefore, MGO trapping and glycation inhibition appear to be important therapeutic targets in prediabetes, diabetes, and in the early prevention of hyperglycemic complications. Peppermint leaf is commonly used as herbal tea, rich in polyphenols. Eriocitrin, its predominant component, in a double-blind, randomized controlled study reversed the prediabetic condition in patients. However, the antiglycation activity of this plant material and its polyphenols has not been characterized to date. Therefore, the aim of this study was to evaluate the ability of a peppermint leaf dry extract and its polyphenols to inhibit non-enzymatic protein glycation in a model with bovine serum albumin (BSA) and MGO as a glycation agent. Peppermint polyphenols were also evaluated for their potential to trap MGO in vitro, and the resulting adducts were analyzed by UHPLC-ESI-MS. To relate chemical composition to glycation inhibitory activity, the obtained peppermint extract was subjected to qualitative and quantitative analysis. The capability of peppermint leaf polyphenols to inhibit glycation (27.3-77.2%) and form adducts with MGO was confirmed. In the case of flavone aglycones, mono- and di-adducts with MGO were observed, while eriodictyol and eriocitrin effectively produced only mono-adducts. Rosmarinic acid and luteolin-7-O-glycosides did not reveal this action. IC50 of the peppermint leaf dry extract was calculated at 2 mg/mL, equivalent to a concentration of 1.8 µM/mL of polyphenols, including ~1.4 µM/mL of flavonoids and ~0.4 µM/mL of phenolic acids. The contribution of the four major components to the anti-AGE activity of the extract was estimated at 86%, including eriocitrin 35.4%, rosmarinic acid 25.6%, luteolin-7-O-rutinoside 16.9%, luteolin-7-O-ß-glucuronoside 8.1%, and others 14%. The effect of peppermint dry extract and polyphenols in inhibiting MGO-induced glycation in vitro was comparable to that of metformin used as a positive control.

Differentiation and Quantitation of Coeluting Isomeric Amadori and Heyns Peptides Using Sugar-Specific Fragment Ion Ratios.

d-glucose and d-fructose present in blood, tissues, and organs of all mammals can react with amino groups, leading to glucated (Amadori) and fructated (Heyns) products, i.e., proteins glycated at lysine residues. While typically present at low concentration in humans, metabolic diseases including diabetes elevate sugar levels, favoring glycation and consecutive reactions leading to advanced glycation end products (AGEs) linked to diabetic complications and cardiovascular diseases. Analytical methods able to differentiate and to individually quantify Amadori- and Heyns-modified proteins in complex sample mixtures, e.g., serum, are still very limited. Here, we show that the reported and supposedly specific neutral losses displayed in tandem mass spectra of Heyns peptides cannot be used for a reliable differentiation as they were also observed for Amadori peptides. However, the combination of several neutral loss signals in fragment ion ratios at both precursor and fragment ion signals allowed the differentiation and relative quantitation of coeluting isomeric Amadori and Heyns peptides at different concentrations and peptide ratios. This was also true for digested human plasma. Thus, the presented strategy allows the quantitation of Amadori and Heyns peptides in complex samples, especially by spiking isotope-labeled peptides. This will allow searching for glucated and fructated biomarkers in clinical samples.

On the Prediction of In Vitro Arginine Glycation of Short Peptides Using Artificial Neural Networks.

One of the hallmarks of diabetes is an increased modification of cellular proteins. The most prominent type of modification stems from the reaction of methylglyoxal with arginine and lysine residues, leading to structural and functional impairments of target proteins. For lysine glycation, several algorithms allow a prediction of occurrence; thus, making it possible to pinpoint likely targets. However, according to our knowledge, no approaches have been published for predicting the likelihood of arginine glycation. There are indications that arginine and not lysine is the most prominent target for the toxic dialdehyde. One of the reasons why there is no arginine glycation predictor is the limited availability of quantitative data. Here, we used a recently published high-quality dataset of arginine modification probabilities to employ an artificial neural network strategy. Despite the limited data availability, our results achieve an accuracy of about 75% of correctly predicting the exact value of the glycation probability of an arginine-containing peptide without setting thresholds upon whether it is decided if a given arginine is modified or not. This contribution suggests a solution for predicting arginine glycation of short peptides.

AGEomics Biomarkers and Machine Learning-Realizing the Potential of Protein Glycation in Clinical Diagnostics.

Protein damage by glycation, oxidation and nitration is a continuous process in the physiological system caused by reactive metabolites associated with dicarbonyl stress, oxidative stress and nitrative stress, respectively. The term AGEomics is defined as multiplexed quantitation of spontaneous modification of proteins damage and other usually low-level modifications associated with a change of structure and function-for example, citrullination and transglutamination. The method of quantitation is stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS). This provides robust quantitation of normal and damaged or modified amino acids concurrently. AGEomics biomarkers have been used in diagnostic algorithms using machine learning methods. In this review, I describe the utility of AGEomics biomarkers and provide evidence why these are close to the phenotype of a condition or disease compared to other metabolites and metabolomic approaches and how to train and test algorithms for clinical diagnostic and screening applications with high accuracy, sensitivity and specificity using machine learning approaches.

Free L-Lysine and Its Methyl Ester React with Glyoxal and Methylglyoxal in Phosphate Buffer (100 mM, pH 7.4) to Form Nε-Carboxymethyl-Lysine, Nε-Carboxyethyl-Lysine and Nε-Hydroxymethyl-Lysine.

Glyoxal (GO) and methylglyoxal (MGO) are highly reactive species formed in carbohydrate metabolism. Nε-Carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) are considered to be the advanced glycation end-products (AGEs) of L-lysine (Lys) with GO and MGO, respectively. Here, we investigated the reaction of free L-lysine (Lys) with GO and MGO in phosphate buffer (pH 7.4) at 37 °C and 80 °C in detail in the absence of any other chemicals which are widely used to reduce Schiff bases. The concentrations of Lys, GO and MGO used in the experiments were 0.5, 2.5, 5.0, 7.5 and 10 mM. The reaction time ranged between 0 and 240 min. Experiments were performed in triplicate. The concentrations of remaining Lys and of CML and CEL formed in the reaction mixtures were measured by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS). Our experiments showed that CML and CEL were formed at higher concentrations at 80 °C compared to 37 °C. CML was found to be the major reaction product. In mixtures of GO and MGO, MGO inhibited the formation of CML from Lys (5 mM) in a concentration-dependent manner. The highest CML concentration was about 300 µM corresponding to a reaction yield of 6% with respect to Lys. An addition of Lys to GO, MGO and their mixtures resulted in strong reversible decreases in the Lys concentration up to 50%. It is assumed that free Lys reacts rapidly with GO and MGO to form many not yet identified reaction products. Reaction mixtures of Lys and MGO were stronger colored than those of Lys and GO, notably at 80 °C, indicating higher reactivity of MGO towards Lys that leads to polymeric colored MGO species. We have a strong indication of the formation of Nε-(hydroxymethyl)-lysine (HML) as a novel reaction product of Lys methyl ester with MGO. A mechanism is proposed for the formation of HML from Lys and MGO. This mechanism may explain why Lys and GO do not react to form a related product. Preliminary analyses show that HML is formed at higher concentrations than CEL from Lys methyl ester and MGO. No Schiff bases or their hydroxylic precursors were identified as reaction products. In their reactions with Lys, GO and MGO are likely to act both as chemical oxidants on the terminal aldehyde group to a carboxylic group (i.e., R-CHO to R-COOH) and as chemical reductors on labile Schiff bases (R-CH=N-R to R-CH2-NH-R) presumably via disproportionation and hydride transfer. Our study shows that free non-proteinic Lys reacts with GO and MGO to form CML, CEL and HML in very low yield. Whether proteinic Lys also reacts with MGO to form HML residues in proteins remains to be investigated. The physiological occurrence and concentration of HML in biological fluids and tissues and its relation to CML and CEL are elusive and warrant further investigations in health and disease. Chemical synthesis and structural characterization of HML are expected to advance and accelerate the scientific research in this topic.

Ultrasound-Assisted Preparation of Maillard Reaction Products Derived from Hydrolyzed Soybean Meal with Meaty Flavor in an Oil-In-Water System.

In the present work, we prepared Maillard reaction products (MRPs) derived from enzyme hydrolyzed soybean meal with ultrasound assistance in an oil-(oxidized lard)-in-water system (UEL-MRPs) or oil-free system (UN-MRPs), and the effect of ultrasound on the properties of the obtained MRPs was evaluated. The analysis of fatty acids in lard with different treatments showed that ultrasound can generate more unsaturated fatty acids in the aqueous phase. The UV-Vis absorbances of UEL-MRPs, UN-MRPs, and MRPs obtained in an oil-in-water system (EL-MRPs) and MRPs obtained in an oil-free system (N-MRPs) at 294 and 420 nm indicated that ultrasound could increase the amount of Maillard reaction intermediates and melanoids in the final products of the Maillard reaction. This was in line with the result obtained from color change determination-that ultrasound can darken the resultant MRPs. Volatile analysis showed ultrasound can not only increase the number of volatile substances, but also greatly increase the composition of volatile substances in UEL-MRPs and UN-MRPs, especially the composition of those contributing to the flavor of the MRPs, such as oxygen-containing heterocycles, sulfur-containing compounds, and nitrogen-containing heterocycles. Descriptive sensory evaluation revealed that UN-MRPs and UEL-MRPs had the highest scores in total acceptance, ranking in the top two, and UEL-MRPs had the strongest meaty flavor among these four kinds of MRPs. Furthermore, the measurements of antioxidant activities, including DPPH radical-scavenging activity, hydroxyl radical scavenging ability, and ferric ion reducing antioxidant power, were conducted, showing that UN-MRPs exhibited the highest antioxidant activity among all the MRPs.

Polyphenols and Maillard Reaction Products in Dried Prunus spinosa Fruits: Quality Aspects and Contribution to Anti-Inflammatory and Antioxidant Activity in Human Immune Cells Ex Vivo.

Dried Prunus spinosa fruits (sloes) are folk phytotherapeutics applied to treat chronic inflammatory disorders. However, their pharmacological potential, activity vectors, and drying-related changes in bioactive components remain unexplored. Therefore, the present research aimed to evaluate the anti-inflammatory and antioxidant effects of dried sloes in ex vivo models of human neutrophils and peripheral blood mononuclear cells (PMBCs) and establish their main active components. It was revealed that the fruit extracts significantly and dose-dependently inhibited the respiratory burst, downregulated the production of elastase (ELA-2) and TNF-α, and upregulated the IL-10 secretion by immune cells under pro-inflammatory and pro-oxidant stimulation. The slightly reduced IL-6 and IL-8 secretion was also observed. The structural identification of active compounds, including 45 phenolics and three Maillard reaction products (MRPs) which were formed during drying, was performed by an integrated approach combining LC-MS/MS, preparative HPLC isolation, and NMR studies. The cellular tests of four isolated model compounds (chlorogenic acid, quercetin, procyanidin B2, and 5-hydroxymethylfurfural), supported by statistical correlation studies, revealed a significant polyphenolic contribution and a slight impact of MRPs on the extracts' effects. Moreover, a substantial synergy was observed for phenolic acids, flavonoids, condensed proanthocyanidins, and MPRs. These results might support the phytotherapeutic use of dried P. spinosa fruits to relieve inflammation and establish the quality control procedure for the extracts prepared thereof.

Digestive properties of half-fin anchovy hydrolysates/glucose Maillard reaction products and modulation effects on intestinal microbiota.

BACKGROUND: The consumption of dietary Maillard reaction products (MRPs) might lead to positive or negative effects on health. The digestibility of half-fin anchovy hydrolysates/glucose MRPs (HAHp(9.0)-G MRPs) was therefore determined. The intestinal microbiota modulation of HAHp(9.0)-G MRPs in mice was also evaluated after administration for 14 days (1 g kg-1 •bodyweight). RESULTS: Different levels of digestibility of MRPs of fructosamine and advanced glycation products of Nε -carboxymethyllysine were detected in HAHp(9.0)-G MRPs during simulated gastrointestinal digestion. An increased relative proportion of soluble fluorescent melanoidins (SFMs) was observed during gastric digestion as compared to that in the original HAHp(9.0)-G MRPs, followed by decreases in SFMs in intestinal digestion. After feeding with HAHp(9.0)-G MRPs for 14 days, increased goblet cells were observed in the ileum regions of female and male mice. High-throughput 16S ribosomal RNA gene sequencing of fecal samples revealed that HAHp(9.0)-G MRPs administration increased the density of the phylum Bacteriodetes and reduced the density of the phylum Firmicutes in male mice. By comparison, a relatively higher density of members of the phylum Saccharibacteria was observed in female mice. A consistent increase in the abundance of Bacteroidales_S24-7_group_norank was found in female and male groups fed with HAHp(9.0)-G MRPs. Female and male mice treated with HAHp(9.0)-G MRPs also showed higher levels of propionic and butyric acids in feces than their corresponding controls. CONCLUSION: Half-fin anchovy hydrolysates/glucose MRPs can be partly hydrolyzed in the simulated gastrointestinal digestion system. Treatment with HAHp(9.0)-G MRPs induced sex-related differences in bacterial abundance and diversity in mice; however, the up-regulation of anti-inflammatory activity was predicted in both female and male mice. © 2021 Society of Chemical Industry.

Lysine-arginine advanced glycation end-product cross-links and the effect on collagen structure: A molecular dynamics study.

The accumulation of advanced glycation end-products is a fundamental process that is central to age-related decline in musculoskeletal tissues and locomotor system function and other collagen-rich tissues. However, although computational studies of advanced glycation end-product cross-links could be immensely valuable, this area remains largely unexplored given the limited availability of structural parameters for the derivation of force fields for Molecular Dynamics simulations. In this article, we present the bonded force constants, atomic partial charges and geometry of the arginine-lysine cross-links DOGDIC, GODIC, and MODIC. We have performed in vacuo Molecular Dynamics simulations to validate their implementation against quantum mechanical frequency calculations. A DOGDIC advanced glycation end-product cross-link was then inserted into a model collagen fibril to explore structural changes of collagen and dynamics in interstitial water. Unlike our previous studies of glucosepane, our findings suggest that intra-collagen DOGDIC cross-links furthers intra-collagen peptide hydrogen-bonding and does not promote the diffusion of water through the collagen triple helices.