RESUMO
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.
Assuntos
Lisina , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Lisina/análise , Lisina/química , Cotinina , Gerociência , Produtos Finais de Glicação Avançada/análise , Produtos Finais de Glicação Avançada/química , Cromatografia Líquida de Alta PressãoRESUMO
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.
Assuntos
Produtos Finais de Glicação Avançada , Agregados Proteicos , Desdobramento de Proteína , Espectrometria de Fluorescência , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Espectrometria de Fluorescência/métodos , Glicosilação , Aldeído Pirúvico/química , Humanos , Animais , Proteínas/química , Proteínas/metabolismo , Reagentes de Ligações Cruzadas/química , Proteínas GlicadasRESUMO
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.
Assuntos
Produtos Finais de Glicação Avançada , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Produtos Finais de Glicação Avançada/química , Lisina/química , Aldeído Pirúvico/metabolismo , AminoácidosRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Produtos Finais de Glicação Avançada , Marcação por Isótopo , Neoplasias Hepáticas , Proteômica , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/química , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/química , Humanos , Proteômica/métodos , Glicosilação , Marcação por Isótopo/métodos , Produtos Finais de Glicação Avançada/análise , Produtos Finais de Glicação Avançada/metabolismo , Produtos Finais de Glicação Avançada/química , Espectrometria de Massas em Tandem/métodos , Masculino , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Antioxidantes , Produtos Finais de Glicação Avançada , Hipoglicemiantes , Mangifera , Extratos Vegetais , Xantonas , Xantonas/química , Xantonas/farmacologia , Xantonas/análise , Mangifera/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Antioxidantes/análise , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipoglicemiantes/análise , Produtos Finais de Glicação Avançada/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Simulação de Acoplamento Molecular , Frutas/química , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/análise , Simulação por ComputadorRESUMO
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.
Assuntos
Produtos Finais de Glicação Avançada , Doenças Metabólicas , Produtos Finais de Glicação Avançada/efeitos adversos , Produtos Finais de Glicação Avançada/metabolismo , Produtos Finais de Glicação Avançada/química , Humanos , Doenças Metabólicas/etiologia , Análise de Alimentos , Reação de Maillard , Animais , Manipulação de Alimentos/métodosRESUMO
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.
Assuntos
Benzofuranos , Ácidos Cafeicos , Lactatos , Polifenóis , Soroalbumina Bovina , Soroalbumina Bovina/química , Ácidos Cafeicos/farmacologia , Ácidos Cafeicos/química , Glicosilação/efeitos dos fármacos , Polifenóis/farmacologia , Polifenóis/química , Benzofuranos/farmacologia , Benzofuranos/química , Lactatos/farmacologia , Lactatos/química , Alcenos/farmacologia , Alcenos/química , Animais , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Bovinos , Simulação de Acoplamento Molecular , DepsídeosRESUMO
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.
Assuntos
Produtos Finais de Glicação Avançada , Óxido de Magnésio , Compostos de Vinila , Produtos Finais de Glicação Avançada/química , Óleo de Brassica napus , Fenóis/química , Aldeído Pirúvico/químicaRESUMO
Plant-based proteins, represented by amaranth in our study, embrace a potential as an ingredient for the functional-food formulation. However, their efficacy is hindered by inherent limitations in solubility, emulsification, and antioxidant traits. The Maillard reaction, a complex chemical-process resulting in a diverse array of products, including Maillard conjugates and Maillard reaction products (MRPs), can employ variable effects on these specific attributes. To elucidate the influence of this reaction and the MRPs on the aforementioned properties, we used a complex blend of dehydrated seaweed Gracilaria and amaranth protein to create a conjugate-MRP blend. Our investigations revealed that the resultant incorporation enhanced solubility, emulsification, and antioxidant properties, while the intermediates formed did not progress to advanced glycation stages. This change is likely attributed to the dual effect of conjugates that altered the secondary protein structure, while the generation and/or preservation of MRPs post ultrasonication and spray drying enhanced its antioxidant potential.
Assuntos
Antioxidantes , Reação de Maillard , Antioxidantes/química , Proteínas de Plantas , Produtos Finais de Glicação Avançada/químicaRESUMO
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.
Assuntos
Nefropatias , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Produtos Finais de Glicação Avançada/química , Espectrometria de Massa com Cromatografia Líquida , Aldeído Pirúvico/química , Rim/química , Arginina , BiomarcadoresRESUMO
The century old Maillard reactions continue to draw the interest of researchers in the fields of Food Science and Technology, and Health and Medical Sciences. This chapter seeks to simplify and update this highly complicated, multifaceted topic. The simple nucleophilic attack of an amine onto a carbonyl group gives rise to a series of parallel and subsequent reactions, occurring simultaneously, resulting into a vast array of low and high mass compounds. Recent research has focused on: (1) the formation and transformation of α-dicarbonyl compounds, highly reactive intermediates which are essential in the development of the desired color and flavor of foods, but also lead to the production of the detrimental advanced glycation end products (AGEs); (2) elucidation of the structures of melanoidins in different foods and their beneficial effects on human health; and (3) harmful effects of AGEs on human health. Considering that MRs have both positive and negative consequences, their control to accentuate the former and to mitigate the latter, is also being conscientiously investigated with the use of modern techniques and technology.
Assuntos
Produtos Finais de Glicação Avançada , Reação de Maillard , Humanos , Produtos Finais de Glicação Avançada/metabolismo , Produtos Finais de Glicação Avançada/química , Polímeros/química , AnimaisRESUMO
The study investigated and compared physicochemical, functional, antioxidant properties, phenolic profile and Maillard reaction products (MRP) of flaxseed flour (FF) and flaxseed cake flour (FCF) upon dry-air roasting (DaR) of flaxseeds at 140, 160 and 180 °C for 5 and 10 min. This information on FF and FCF is limited and has considerable gaps. The raw FF exhibited higher fat, ash, antioxidant and functional properties while lower protein than the FCF. Upon increasing DaR conditions, the ash and protein increased in FCF and decreased in FF. DaR at 180 °C for 10 min augmented water solubility index, ΔE, MRP, free rutin and syringic acid, bound epicatechin, gallic acid and syringic acid while lowered moisture, L*, b*, hue, chroma, potassium, iron, selenium, emulsion indexes, caffeic acid, flavonoids and free resveratrol in FF and FCF. In conclusion, DaR improves phenolic profile, antioxidant properties, MRP, water solubility and oil absorption capacity of FF and FCF.
Assuntos
Antioxidantes , Linho , Ácido Gálico/análogos & derivados , Antioxidantes/química , Linho/química , Farinha/análise , Fenóis/análise , Produtos Finais de Glicação Avançada/química , ÁguaRESUMO
This study aimed to better understand whether and how the reactive 1,2-dicarbonyl precursors of advanced glycation end products (AGEs), glyoxal (GO) and methylglyoxal (MGO), cross the intestinal barrier by studying their transport in the in vitro Caco-2 transwell system. The results reveal that GO, MGO and Nε-(carboxymethyl)lysine (CML), the latter studied for comparison, are transported across the intestinal cell layer via both active and passive transport and accumulate in the cells, albeit all to a limited extent. Besides, the transport of the dicarbonyl compounds was only partially affected by the presence of amino acids and protein, suggesting that scavenging by a food matrix will not fully prevent their intestinal absorption. Our study provides new insights into the absorption of the two major food-borne dicarbonyl AGE precursors and provides evidence of their potential systemic bioavailability but also of factors limiting their contribution to the overall exposome.
Assuntos
Produtos Finais de Glicação Avançada , Glioxal , Aldeído Pirúvico , Humanos , Células CACO-2 , Produtos Finais de Glicação Avançada/metabolismo , Produtos Finais de Glicação Avançada/química , Aldeído Pirúvico/metabolismo , Glioxal/metabolismo , Glioxal/química , Modelos Biológicos , Transporte Biológico , Absorção IntestinalRESUMO
Myricetin and its derivatives, myricitrin and dihydromyricetin, are flavonoids widely presented in foods and phytomedicine that possess tremendous health potential. In this study, we compared the antiglycation activity of myricetin and its derivatives, then investigated the underlying mechanism using proteomic modification and fluorescence spectroscopy analysis. All three compounds exhibited thorough inhibition on nonenzymatic glycation process, with the inhibitory effects on AGEs reaching 85% at 40 µmol/L. They effectively protected bovine serum albumin (BSA) structure by inhibiting protein oxidation, preventing the conversion from α-helix to ß-sheet, and reducing amyloid-like cross-ß structure formation. Among the three compounds, myricetin showed a predominant antiglycation activity. Proteomic analysis identified the early glycated sites that were protected by myricetin, including lysine K235, 256, 336, 421, 420, 489, etc. Additionally, fluorescence spectroscopy revealed spontaneous interactions between BSA and myricetin. Overall, myricetin holds promise as an antiglycation agent in both the food and drug industries.
Assuntos
Flavonoides , Proteômica , Soroalbumina Bovina , Espectrometria de Fluorescência , Flavonoides/química , Flavonoides/farmacologia , Glicosilação/efeitos dos fármacos , Soroalbumina Bovina/química , Bovinos , Animais , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismoRESUMO
As a product of nonenzymatic glycation, glycated albumin (GA) is a promising serum marker for the short-term glycemic monitoring in patients with diabetes. On the basis of the boronate crosslinking (BCL)-enabled direct labeling of ferrocene (Fc) tags to the nonenzymatically glycated (NEG) sites, we report herein a novel aptamer-based ratiometric electrochemical (apt-REC) platform for the point-of-care (POC) assay of GA. This apt-REC platform is based on the recognition of GA proteins by the methylene blue (MB)-modified aptamer receptors and the labeling of the Fc tags to the NEG sites via the BCL. Using MB as the reference tag and Fc as the quantification tag, the ratio of the oxidation currents (i.e., IFc/IMB) can serve as the yardstick for the ratiometric assay of GA. Due to the presence of tens of the NEG sites, each GA protein can be labeled with tens of quantification tags, permitting the amplified assay in a simple, time-saving, and low-cost manner. The ratiometric signal exhibited a good linear response over the range from 0.1 to 100 µg/mL, with a detection limit of 45.5 ng/mL. In addition to the superior reproducibility and robustness, this apt-REC platform is highly selective (capable of discriminating GA against human serum albumin (HSA)) and applicable to GA assay in serum samples. Due to its low cost, high reproducibility and robustness, simple operation, and high sensitivity and selectivity, this apt-REC platform holds great promise in the POC assay of GA for diabetes management.
Assuntos
Ácidos Borônicos , Técnicas Eletroquímicas , Albumina Sérica Glicada , Humanos , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Ácidos Borônicos/química , Reagentes de Ligações Cruzadas/química , Técnicas Eletroquímicas/métodos , Produtos Finais de Glicação Avançada/química , Limite de Detecção , Albumina Sérica/química , Albumina Sérica/análise , Albumina Sérica Humana/química , Albumina Sérica Humana/análiseRESUMO
A novel analytical strategy was proposed to simultaneously quantify two advanced glycation end products (AGEs) including Nε-(Carboxymethyl)lysine (CML), Nε-(Carboxyethyl)lysine (CEL) and eight heterocyclic amines (HAs) including IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, PhIP, Harman, and Norharman. The procedure was based on a two-step extraction, solid phase extraction (SPE) purification followed by ultra performance liquid chromatography tandem mass spectrometry. The established method showed a good linearity (R2 ≥ 0.9950), rapid processing time (8 min per sample), satisfactory recoveries (matrix spiked recoveries range from 72.2% to 119.6%) and precision (intra-day and inter-day RSDs were <19.3%). The limit of quantification (LOQ) and limit of detection (LOD) resulted to be between 0.05-15 ng/g and 0.2-50 ng/g, respectively. The validated technique was further applied to determine HAs and AGEs in eight stewed meat product samples consumed in Shanghai, with the amount of HAs and AGEs ranging from 2.851 to 18.289 ng/g and 118.158-543.493 ng/g, respectively.
Assuntos
Aminas , Produtos Finais de Glicação Avançada , Compostos Heterocíclicos , Produtos da Carne , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/análise , Aminas/análise , Aminas/química , Cromatografia Líquida de Alta Pressão/métodos , Produtos da Carne/análise , Animais , Compostos Heterocíclicos/análise , Compostos Heterocíclicos/química , Extração em Fase Sólida/métodos , Limite de Detecção , Suínos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
The baking process has the potential to generate health-risk compounds, including products from lipid oxidation and Maillard reaction. Pre- and post-digestion levels of hydroxymethylfurfural (HMF), malondialdehyde (MDA), glyoxal (GO), and methylglyoxal (MGO) were studied in cakes formulated with hazelnut and sunflower oil, along with their oleogels as margarine substitutes. The concentration of HMF in oil and oleogel-formulated cakes increased after digestion compared to cakes formulated with margarine. The MDA values were between 82 and 120 µg/100 g in oil and oleogel formulated cakes before digestion and a decrease was observed after digestion. The substitution of margarine with oil and oleogels resulted in the production of high amounts of GO and MGO in cakes. However, the highest bioaccessibility as 318.2% was found in cakes formulated by margarine for GO. Oleogels may not pose a potential health benefit compared to margarines due to the formation of HMF, MDA, GO, and MGO.
Assuntos
Digestão , Furaldeído , Malondialdeído , Compostos Orgânicos , Furaldeído/análogos & derivados , Furaldeído/química , Furaldeído/análise , Malondialdeído/metabolismo , Malondialdeído/química , Compostos Orgânicos/química , Humanos , Trato Gastrointestinal/metabolismo , Corylus/química , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Modelos Biológicos , Óleo de Girassol/química , Margarina/análiseRESUMO
Glycation and the subsequent formation of advanced glycation end products (AGEs) disrupt and impair the physiological functions of proteins. This study presents a comprehensive glycation site mapping of human serum albumin (HSA) utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both in vitro glycation experiments and patient samples were investigated, exploring various enzymes, processing techniques, and their impacts on glycation site detection. A pilot study was conducted, analyzing sixteen serum samples, which spanned from healthy individuals to severe diabetic patients (with HbA1c values ranging from 5.7% to 18.1%). The aim was to comprehend the progression of glycation on various sites of HSA with increasing levels of glycation. Their glycated albumin levels (GA) spanned from 19.7% to 62.3%. Trypsin-mediated proteolytic digestion unveiled 12 glycation sites through direct in-solution digestion of whole serum. However, isolating albumin from serum enabled the identification of a higher number of glycation sites in each sample compared to direct serum digestion. Boronate affinity chromatography facilitated the segregation of less glycated albumin (LGA) from the more glycated albumin (MGA) fraction. Subsequent proteolytic digestion of both LGA and MGA samples revealed similar glycation sites. The MGA fraction exhibited a greater number of identified glycation sites, thereby elucidating which sites are particularly prone to glycation in highly glycated albumin samples. Changes in relative glycation levels were noted in the tryptic digests of albumin samples following the sample enrichment steps, as opposed to direct in-solution digestion of whole serum. Two enzymes, trypsin and Glu-C, were evaluated for efficacy in sequence coverage and glycation site analysis of HSA, with trypsin demonstrating superior efficiency over Glu-C.
Assuntos
Albumina Sérica Glicada , Produtos Finais de Glicação Avançada , Albumina Sérica Humana , Espectrometria de Massas em Tandem , Humanos , Produtos Finais de Glicação Avançada/química , Espectrometria de Massas em Tandem/métodos , Albumina Sérica Humana/química , Glicosilação , Cromatografia Líquida/métodos , Albumina Sérica/química , Albumina Sérica/análise , Albumina Sérica/metabolismo , Projetos Piloto , Diabetes Mellitus/sangue , Tripsina/química , Tripsina/metabolismoRESUMO
Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.
Assuntos
Antocianinas , Glucosídeos , Soroalbumina Bovina , Glicosilação , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Antocianinas/química , Antocianinas/metabolismo , Glucosídeos/metabolismo , Glucosídeos/química , Animais , Sítios de Ligação , Bovinos , Estrutura Secundária de Proteína , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Ligação Proteica , Espectrometria de Massas em Tandem , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Protein glycation closely intertwines with the pathogenesis of various diseases, sparking a growing interest in exploring natural antiglycation agents. Herein, high-purity betacyanins (betanin and phyllocactin) derived from Hylocereus polyrhizus peel were studied for their antiglycation potential using an in vitro bovine serum albumin (BSA)-glucose model. Notably, betacyanins outperformed aminoguanidine, a recognized antiglycation agent, in inhibiting glycation product formation across different stages, especially advanced glycation end-products (AGEs). Interestingly, phyllocactin displayed stronger antiglycation activity than betanin. Subsequent mechanistic studies employing molecular docking analysis and fluorescence quenching assay unveiled that betacyanins interact with BSA endothermically and spontaneously, with hydrophobic forces playing a dominant role. Remarkably, phyllocactin demonstrated higher binding affinity and stability to BSA than betanin. Furthermore, the incorporation of betacyanins into bread dose-dependently suppressed AGEs formation during baking and shows promise for inhibiting in vivo glycation process post-consumption. Overall, this study highlights the substantial potential of betacyanins as natural antiglycation agents.