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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.

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Glycated albumin (GA), defined as the percentage of serum albumin glycation, is a mid-term glycemic control marker for diabetes. The concentrations of both glycated human serum albumin (GHSA) and total human serum albumin (HSA) are required to calculate GA. Here, we report the development of a GA sensor employing two albumin aptamers: anti-GHSA aptamer which is specific to GHSA and anti-HSA aptamer which recognizes both glycated and non-glycated HSA. We combine these aptamers with extended gate field effect transistors (EGFETs) to realize GA monitoring without the need to pretreat serum samples, and therefore suitable for point of care and home-testing applications. Using anti-GHSA aptamer-immobilized electrodes and EGFETs, we measured GHSA concentrations between 0.1-10 µM within 20 min. The sensor was able to measure GHSA concentration in the presence of BSA for a range of known GA levels (5-29%). With anti-HSA aptamer-immobilized electrodes and EGFETs, we measured total HSA concentrations from 1-17 µM. Furthermore, GHSA and total HSA concentrations of both healthy and diabetic-level samples were determined with GHSA and HSA sensors. The measured GHSA and total HSA concentrations in three samples were used to determine respective GA percentages, and our calculations agreed with GA levels determined by reference methods. Thus, we developed simple and rapid dual aptamer-based EGFET sensors to monitor GA through measuring GHSA and total HSA concentration, without the need for sample pretreatment, a mandatory step in the current standard of enzymatic GA monitoring.

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BACKGROUND: Serum fructosamine (sFA) is used to assess glycaemic control in dogs with diabetes mellitus (DM). Nevertheless, its interpretation is hindered by several limitations. METHODS: This retrospective study evaluates the long-term diagnostic performance of sFA for monitoring clinical control of DM. sFA, bodyweight, appetite, presence of polyuria/polydipsia and clinical scores (CS; well-controlled DM, CS-0; uncontrolled DM, CS-1) were recorded. RESULTS: The study included 75 dogs (321 visits; median 3 visits/dog; range 1-19). Mean sFA was higher (p < 0.001) on visits with CS-1 (584 µmol/L; 95% confidence interval [95% CI] 561-608) than on visits with CS-0 (506 µmol/L; 95% CI 484-528). Increases in sFA increased   the odds ratio for CS-1 (1.37; 95% CI 1.24-1.52, p < 0.001). sFA was moderately predictive of the CS (area under receiver operating characteristic curve = 0.75; 95% CI 0.70-0.80; p < 0.0001), with a 486 µmol/L cutoff yielding 80% sensitivity and 59% specificity. Mean sFA was lower (p = 0.005) when hypoglycaemic episodes were suspected (496 µmol/L; 95% CI 450-541) than in their absence (572 µmol/L; 95% CI 548-596). sFA is moderately accurate for classifying CS in diabetic dogs. Decreasing sFA over follow-ups indicates improved CS but might suggest occurrence of hypoglycaemic episodes. LIMITATIONS: Retrospective design, variable treatments and comorbidities are limitations of this study. CONCLUSION: sFA has a moderate clinical utility in the long-term monitoring of diabetic dogs, but may serve as a first-line, accessible diagnostic tool. Discordant CS and sFA evaluation, or decreased sFA, warrants additional monitoring (i.e., continuous glucose monitoring).

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Haemoglobin A1c (hemoglobin A1c, HbA1c) is an important long-term glycemic control marker for diabetes. The aim of this study was to develop an enzyme flow injection analysis (FIA) system using engineered fructosyl peptide oxidase (FPOx) based on 2.5th generation principle for an HbA1c automated analytical system. FPOx from Phaeosphaeria nodorum (PnFPOx) was engineered by introducing a Lys residue at the R414 position, to be modified with amine reactive phenazine ethosulfate (arPES) in proximity of FAD. The engineered PnFPOx mutant with minimized oxidase activity, N56A/R414K, showed quasi-direct electron transfer (quasi-DET) ability after PES-modification. The FIA system was constructed by employing a PES-modified PnFPOx N56A/R414K and operated at 0 V against Ag/AgCl. The system showed reproducible responses with a linear range of 20-500 µM for both fructosyl valine (FV) and fructosyl valylhistidine (FVH), with sensitivities of 0.49 nA µM-1 and 0.13 nA µM-1, and the detection limits of 1.3 µM and 2.0 µM for FV and FVH, respectively. These results indicate that the enzyme electrochemical FIA system covers the clinical range of HbA1c detection for more 200 consecutive measurements. Protease digested three different levels of HbA1c samples including healthy and diabetic range subjects were also measured with the FIA system. Thus, it will be possible to develop an integrated system consisting of sample pretreatment and sample electrochemical measurement based on an FIA system possessing quasi-DET type PnFPOx.

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Non-enzymatic glycation of proteins is believed to be the root cause of high dietary sugar associated pathophysiological maladies. We investigated the structural changes in protein during progression of glycation using ribosylated Bovine Serum Albumin (BSA). Non enzymatic attachment of about 45 ribose molecules to BSA resulted in gradual reduction of hydrophobicity and aggregation as indicated by red-shifted tryptophan fluorescence, reduced ANS binding and lower anisotropy of FITC-conjugated protein. Parallely, there was a significant decrease of alpha helicity as revealed by Circular Dichroism (CD) and Fourier transformed-Infra Red (FT-IR) spectra. The glycated proteins assumed compact globular structures with enhanced Thioflavin-T binding resembling amyloids. The gross structural transition affected by ribosylation led to enhanced thermostability as indicated by melting temperature and Transmission Electron Microscopy. At a later stage of glycation, the glycated proteins developed non-specific aggregates with increase in size and loss of amyloidogenic behaviour. A parallel non-glycated control incubated under similar conditions indicated that amyloid formation and associated changes were specific for ribosylation and not driven by thermal denaturation due to incubation at 37 °C. Functionality of the glycated protein was significantly altered as probed by Isothermal Titration Calorimetry using polyphenols as substrates. The studies demonstrated that glycation driven globular amyloids form and persist as transient intermediates during formation of misfolded glycated adducts. To the best of our knowledge, the present study is the first systematic attempt to understand glycation associated changes in a protein and provides important insights towards designing therapeutics for arresting dietary sugar induced amyloid formation.

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Extracellular fungal cellobiases develop large stable aggregates by reversible concentration driven interaction. In-vitro addition of trehalose resulted in bigger cellobiase assemblies with increased stability against heat and dilution induced dissociation. In presence of 0.1 M trehalose, the size of aggregates increased from 344 nm to 494 nm. The increase in size was also observed in zymography of cellobiase. Activation energy of the trehalose stabilised enzyme (Ea = 220.9 kJ/mol) as compared to control (Ea = 257.734 kJ/mol), suggested enhanced thermostability and also showed increased resistance to chaotropes. Purified cellobiase was found to contain 196.27 µg of sugar/µg of protein. It was proposed that presence of glycan on protein's surface impedes and delays trehalose docking. Consequently, self-association of cellobiase preceded coating by trehalose leading to stabilisation of bigger cellobiase aggregates. In unison with the hypothesis, ribosylated BSA failed to get compacted by trehalose and developed into bigger aggregates with average size increasing from 210 nm to 328 nm. Wheat Germ Lectin, in presence of trehalose, showed higher molecular weight assemblies in DLS, native-PAGE and fluorescence anisotropy. This is the first report of cross-linking independent stabilisation of purified fungal glycosidases providing important insights towards understanding the aggregation and stability of glycated proteins.

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BACKGROUND: Plasma fructosamine concentration ([FRA]) is a widely used long term hyperglycemic biomarker in humans and dogs, but its clinical usefulness as a hypoglycemic biomarker in dairy cattle is uncertain. OBJECTIVES: To evaluate the relationship between plasma [FRA] and glucose concentration ([gluc]) as well as indices of energy balance during early lactation in dairy cattle, and to characterize the influence of plasma total protein concentration ([TP]) and albumin concentration ([albumin]) on [FRA]. ANIMALS: Convenience sample comprising 103 periparturient Holstein-Friesian cattle. METHODS: Plasma [gluc], [TP], [albumin], and other clinicopathologic indices of energy status were determined periodically from Day 4 postpartum. Body condition score (BCS) was assessed, and backfat thickness (BFT) and longissimus dorsi muscle thickness (LDT) were measured ultrasonographically. Plasma [FRA] was measured at approximately 28 days postpartum. Associations between plasma [FRA] and study variables were evaluated using Spearman's rho and stepwise forward linear regression. Statistical significance was declared at P < 0.05. RESULTS: A positive association was detected between plasma [FRA] and mean plasma [gluc] from Days 4-28 postpartum (rs = +0.36, P < 0.001), and between plasma [FRA] and LDT (rs = +0.28, P = 0.007), BCS (rs = +0.23, P = 0.029), and BFT (rs = +0.21, P = 0.043). Multivariable regression identified a positive association between plasma [FRA] and mean plasma [gluc] and [albumin] from Days 4-28 postpartum. Correcting plasma [FRA] for [albumin] improved the association (rs = +0.46, P < 0.001) between plasma [FRA] and mean plasma [gluc]. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma [FRA] does not provide a clinically useful method for quantifying the magnitude of hypoglycemia or negative energy balance in dairy cows during early lactation.

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Colloidal particles are essential components of sun-dried Isatis indigotica Fort. roots (Ban-Lan-Gen in Chinese, BLG) decoction. Nanoparticles (NPs) were isolated from BLG decoction with size exclusion chromatography and characterized. Their average diameter is ∼120 nm, reversibly responding to pH and temperature changes. They promoted the growth of normal cells but suppressed that of cancerogenic cells and macrophages. Two constitutive glycated proteins were identified from the NPs, namely BLGP1 and BLGP2. Their N-terminal amino acid sequences were V-X-R-E-V-V-K-D-I and V-V-R-E-V-V-K-D-I-A-G-A-V-Q-T-N-E-Q-Y. Their full-length cDNA sequences were cloned to obtain the highly homological amino acid sequences of non-glycated proteins, whose theoretical molecular weights are 21831.64 Da and 21841.67 Da. Using pepsin hydrolysis and mass spectrometry, four possible glycation adducts were identified in BLGP1, whereas one in BLGP2. To conclude, bioactive nanoparticles isolated from the herbal decoction are intelligent nanoassemblies composed of a new boiling-stable protein. Glycation plays a critical role in heat-induced formation of these nanoassemblies. The novel, intelligent, safe and stable nano-carriers for drug delivery may be developed using BLG NPs as prototype.

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Currently available enzymatic methods for the measurement of glycated proteins utilize fructosyl amino acid/peptide oxidases (FAOXs/FPOXs) as sensing elements. FAOXs/FPOXs oxidize glycated amino acids or glycated dipeptides but they are not able to accept longer glycated peptides or intact glycated proteins as substrates. Therefore, pretreatment via proteolytic digestion is unavoidable with the current enzymatic methods, and there remains a need for simpler measurement methods for glycated proteins. In this study, in order to develop a novel sensing system for glycated albumin (GA), a marker for diabetes, with no requirement for proteolytic digestion, we created an electrochemical sensor based on fructosamine 6-kinase (FN6K) from Escherichia coli. Uniquely, FN6K can react directly with intact GA unlike FAOXs/FPOXs. The concentration of GA in samples was measured using a carbon-printed disposable electrode upon which FN6K as well as two additional enzymes, pyruvate kinase and pyruvate dehydrogenase were overlaid. A clear correlation between the response current and the concentration of GA was observed in the range of 20-100 µM GA, which is suitable for measurement of GA in diluted blood samples from both healthy individuals and patients with diabetes. The sensing system reported here could be applied to point-of-care-testing devices for measurement of glycated proteins.

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Research advances in biochemical molecules have led to the development of convenient and reproducible biosensing molecules for glycated proteins, such as those based on the enzymes fructosyl amino acid oxidase (FAOX) or fructosyl peptide oxidase (FPOX). Recently, more attractive biosensing molecules with potential applications in next-generation biosensing of glycated proteins have been aggressively reported. We review 2 such molecules, fructosamine 6-kinase (FN6K) and fructosyl amino acid-binding protein, as well as their recent applications in the development of glycated protein biosensing systems. Research on FN6K and fructosyl amino acid-binding protein has been opening up new possibilities for the development of highly sensitive and proteolytic-digestion-free biosensing systems for glycated proteins.

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Hemoglobin A1c (HbA1c) is considered the gold standard for assessment of glycemic control in diabetic patients. HbA1c is inadequate in individuals homozygous or compound heterozygous for hemoglobin variants or in conditions with an altered red blood cell turnover. In these cases glycated albumin (GA) is proposed as an alternative assay. We aimed to evaluate the analytical performance of the Diazyme glycated serum protein (GSP) assay on an automated analyzer, to establish a reference interval (RI), and to compare from a clinical perspective, GSP/GA with glycated Hb (glyHb) results. Validation studies followed the CLSI guidelines and included precision, linearity, interferences, concordance of results with glyHb, and RI calculation. GSP was analyzed on representative samples with previously ordered HbA1c and albumin from the Dyna LIFE : DX laboratory. Samples from patients with bisalbuminemia, hemoglobinopathies, and multiple myeloma were also included. Within-run and total imprecision was <3.0% at both levels of control, analytical sensitivity was 5.31 µmol/L, and linearity was verified from 10 to 1150 µmol/L (total allowable error of 5%). Clinical concordance between %GA and glyHb was substantial (n = 175, R2 = .91, kappa = .78, P = .167). GSP RI was 160 to 340 µmol/L or if expressed as %GA 10.5 to 17.5%. Analytical performance of the Diazyme GSP assay on the Siemens ADVIA 1800 is acceptable for clinical use. The RI obtained was higher than that suggested by the manufacturer.

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OBJECTIVES: Blood and saliva samples were obtained to examine if there is a correlation between saliva glycated protein and blood glycated protein. METHODS: Blood and saliva samples of 51 male workers were collected. The fructosamine and hydrazine methods were used to measure saliva glycated protein. HbA1c, fructosamine and blood glucose were measured as indices of blood glycated protein, and the correlation between blood glycated protein and saliva glycated protein was examined. RESULTS: Saliva fructosamine glycated protein showed a significant correlation with HbA1c and blood glucose (r=0.449; p=0.001 and r=0.445; p=0.001, respectively). No correlation was identified between saliva hydrazine glycated protein and the index of blood glycated protein. CONCLUSIONS: Blood glycated protein and blood glucose could be estimated by measuring saliva glycated protein.

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Objectives: Blood and saliva samples were obtained to examine if there is a correlation between saliva glycated protein and blood glycated protein. Methods: Blood and saliva samples of 51 male workers were collected. The fructosamine and hydrazine methods were used to measure saliva glycated protein. HbA1c, fructosamine and blood glucose were measured as indices of blood glycated protein, and the correlation between blood glycated protein and saliva glycated protein was examined. Results: Saliva fructosamine glycated protein showed a significant correlation with HbA1c and blood glucose (r=0.449; p=0.001 and r=0.445; p=0.001, respectively). No correlation was identified between saliva hydrazine glycated protein and the index of blood glycated protein. Conclusions: Blood glycated protein and blood glucose could be estimated by measuring saliva glycated protein.

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<p><b>OBJECTIVES</b>Blood and saliva samples were obtained to examine if there is a correlation between saliva glycated protein and blood glycated protein.</p><p><b>METHODS</b>Blood and saliva samples of 51 male workers were collected. The fructosamine and hydrazine methods were used to measure saliva glycated protein. HbA1c, fructosamine and blood glucose were measured as indices of blood glycated protein, and the correlation between blood glycated protein and saliva glycated protein was examined.</p><p><b>RESULTS</b>Saliva fructosamine glycated protein showed a significant correlation with HbA1c and blood glucose (r=0.449; p=0.001 and r=0.445; p=0.001, respectively). No correlation was identified between saliva hydrazine glycated protein and the index of blood glycated protein.</p><p><b>CONCLUSIONS</b>Blood glycated protein and blood glucose could be estimated by measuring saliva glycated protein.</p>