Amadori Products Research Articles

Open Search of Peptide Glycation Products from Tandem Mass Spectra

Identification of chemically modified peptides in mass spectrometry (MS)-based glycation studies is a crucial yet challenging task. There is a need to establish a mode for matching tandem mass spectrometry (MS/MS) data, allowing for both known and unknown peptide glycation modifications. We present an open search approach that uses classic and modified peptide fragment ions. The latter are shifted by the mass delta of the modification. Both provide key structural information that can be used to assess the peptide core structure of the glycation product. We also leverage redundant neutral losses from the modification side chain, introducing a third ion class for matching referred to as characteristic fragment ions. We demonstrate that peptide glycation product MS/MS spectra contain multidimensional information and that most often, more than half of the spectral information is ignored if no attempt is made to use a multi-step matching algorithm. Compared to regular and/or modified peptide ion matching, our triple-ion strategy significantly increased the median interpretable fraction of the glycation product MS/MS spectra. For reference, we apply our approach for Amadori product characterization and identify all established diagnostic ions automatically. We further show how this method effectively applies the open search concept and allows for optimized elucidation of unknown structures by presenting two hitherto undescribed peptide glycation modifications with a delta mass of 102.0311 and 268.1768 Da. We characterize their fragmentation signature by integration with isotopically labeled glycation products, which provides high validity for non-targeted structure identification.

Non-inhibitory effects of the potent antioxidant C-phycocyanin from Plectonema sp. on the in vitro glycation reaction

Abstract When glucose and Amadori products are auto-oxidized, glycation occurs, resulting in the formation of early (Amadori) and late advanced glycation end products (AGEs), as well as free radicals. Glycation and an increase in free radical activity induce diabetic complications. Antioxidant and antiglycation compounds may aid in the prevention of oxidation and glycation. The goal of this study was to assess the antiglycation and antioxidant capacity of C-phycocyanin (C-PC) derived from Plectonema sp. The DPPH (1, 1-diphenyl-2-picrylhydrazyl), nitric oxide, hydroxyl radical scavenging activities and ferric ions reducing antioxidant power (FRAP) assays were used to assess antioxidant activity, while an in vitro bovine serum albumin-methyl glyoxal glycation (BSA-MG) model was used to assess glycation inhibitory potential. Glycation inhibition was measured using a variety of spectroscopic and biochemical parameters, including UV-visible & fluorescence spectroscopy, ketoamine, carbonyl and hydroxymethyl furfural content, as well as free lysine & free arginine estimations. In vitro, C-PC exhibited dose-dependent potent antioxidant activity, but lacked significant antiglycation potential. As a result, it is recommended that further studies be conducted to evaluate the antiglycation potential of C-PC.

Antidiabetic and antiglycating potential of chrysin is enhanced after nano formulation: An in vitro approach

Chrysin is a well-known flavonoid possessing variety of therapeutic benefits and showing antioxidant, anti-inflammatory, anticancer, anti-diabetic and other such properties. However, its proper application is hindered because of its poor solubility and poor bioavailability. To overcome these limiting factors, nanotization appears to be a promising method improving the therapeutic efficacy of the compound. Here, we have synthesized the nano formulation of chrysin by solvent-evaporation method and characterized it. The antioxidant potential was determined and in vitro anti-diabetic activity was assessed by ɑ-amylase and ɑ-glycosidase inhibition assays and the results showed a dose-dependent increase in percent inhibition of the enzyme. Glycation was reduced to a high extent in presence of chrysin nanoparticles as compared to its bulk form and this was estimated by decrease in synthesis of Amadori products as well as advanced glycation end products (AGEs). The results were further confirmed by spectroscopic techniques showing structural changes in HSA glycated in the absence and presence of chrysin or its nanoparticle. The antiglycating effect was also evident by estimating free lysine residues, and protein oxidation. All these results suggest that the nano formulation of chrysin possess significantly greater antioxidant, antidiabetic and antiglycating activities than the bulk form. The experimental outcomes were supported by molecular docking studies.

Insight into Isomeric Diversity of Glycated Amino Acids in Maillard Reaction Mixtures.

Maillard reactions generate a wide array of amino acid- and sugar-derived intermediates; the isomeric mixtures of glycated amino acids are of particular interest. Excluding stereoisomers, regioisomers, and various anomers, most amino acids can form two monoglycated and three N,N-diglycated isomers when reacted with sugars during the Maillard reaction. Using synthetic Schiff bases and Amadori compounds as standards, we have demonstrated that diagnostic ions obtained from MS/MS fragmentations in negative ionization mode can be used effectively for the discrimination between glucose-derived Schiff bases and their corresponding Amadori compounds in both mono- and diglycated forms. The utilization of these diagnostic ions and isotopic labeling in the glycine/glucose model system revealed that milling glucose/glycine mixtures for 30 min/30 Hz at ambient temperature produced monoglycated glycine in equal proportions of Amadori and Schiff base forms, whereas diglycated glycine was a mixture of the three isomers: Schiff-Schiff, Schiff-Amadori, or Amadori-Amadori in approximately equal molar proportions. The above results were further corroborated using a synthetic histidine Amadori product, N,N-difructosyl-β-alanine, dipeptides, and ribose. Using mechanochemistry as a convenient synthetic tool in combination with MS/MS diagnostic ions, the isomeric diversity of the early stages of the Maillard reaction can be revealed.

Attenuation of hyperglycemia and amadori products by aminoguanidine in alloxan-diabetic rabbits occurs via enhancement in antioxidant defenses and control of stress.

The micro- and macro-complications in diabetes mellitus (DM) mainly arise from the damage induced by Amadori and advanced glycation end products, as well as the released free radicals. The primary goal of DM treatment is to reduce the risk of micro- and macro-complications. In this study, we looked at the efficacy of aminoguanidine (AG) to prevent the production of early glycation products in alloxan-diabetic rabbits. Type1 DM was induced in rabbits by a single intravenous injection of alloxan (90 mg/kg body weight). Another group of rabbits was pre-treated with AG (100 mg/kg body weight) prior to alloxan injection; this was followed by weekly treatment with 100 mg/kg of AG for eight weeks. Glucose, insulin, and early glycation products (HbA1C and fructosamine) were measured in control, diabetic and AG treated diabetic rabbits. The effects of hyperglycemia on superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), reduced glutathione (rGSH), nitric oxide, lipid peroxides, and protein carbonyl were investigated. Alloxan-diabetic rabbits had lower levels of SOD, CAT, Gpx, and rGSH than control rabbits. Nitric oxide levels were considerably greater. AG administration restored the activities of SOD, CAT, Gpx enzymes up to 70–80% and ameliorated the nitric oxide production. HbA1c and fructosamine levels were considerably lower in AG-treated diabetic rabbits. The observed control of hyperglycemia and amadori adducts in alloxan-diabetic rabbits by AG may be attributed to decrease of stress and restoration of antioxidant defenses.

Biophysical Characterisation of Amadori Modified Human Serum Albumin: A Prognostic Biomarker for Diabetic Complications

Introduction: Glycation of biological macromolecules particularly protein leads to the generation of early and Advanced Glycation End (AGE) products. The interest in early glycation of protein is driven due to the fact of Amadori modified proteins having role in diabetic complications. Aim: To analyse the biophysical characterisation of Amadori modified human serum albumin as a prognostic biomarker for diabetic complications. Materials and Methods: This in-vitro experimental study was conducted at Department of Biochemistry, J.N. Medical College, Aligarh Muslim University, Uttar Pradesh, India, from May 2010 to December 2012. The structural characterisation of EGPs was generated by incubating Human Serum Albumin (HSA) with glucose for about a week. The generation of EGPs of HSA was quantitated as Hydroxy Methyl Furfural (HMF) by ThioBarbituric Acid (TBA) assay and authenticated by boronate affinity chromatography. Moreover, High Performance Liquid Chromatography (HPLC) and Electro Spray Ionisation/Mass Spectrometry (ESI/MS) was carried out to validate the presence of Amadori product formed. Additionally, Circular Dichroism (CD) and thermal denaturation studies were used to investigate the structural changes in Amadori albumin. Results: Glycated HSA was obtained as detected by the presence of HMF and chromatography peaks. On stratification, the structural perturbation was observed in Amadori HSA. Furthermore, the generation of furosine was also confirmed by obtaining a new peak in the HPLC profile of glycated HSA. The ESI/MS result also substantiated the presence of Amadori products. Conclusion: The therapeutic strategies that negate the Amadori modification of albumin might be a logical approach in the prevention of diabetic complications.

Bis(alaninato)iron(II) complexes as molecular scaffolds for the generation of N,N-di-glycated alanine derivatives in the presence of glucose

Metal ions are known to influence the course of the Maillard reaction through formation of various complexes such as bis(amino acids), these complexes are known to undergo more facile reaction with sugars. Due to this enhanced reactivity, the possible formation of di-glycated amino acids in glucose/alanine model systems with and without FeCl2 was investigated using isotope labelling technique in conjunction with ESI/qTOF/MS/MS. Forty-eight derivatives of bis[N,N'-di-glycated alanine] iron(II) complexes were tentatively identified. These complexes incorporated one iron atom, two [13C-3] atoms from alanine and up-to twenty-four carbon atoms from glucose [13C-U], were incorporated as triose (C3), tetrose (C4), pentose (C5) or hexose (C6) moieties. Furthermore, the dissociation of the above complexes released variously substituted N,N-di-glycated alanine derivatives incorporating one alanine [13C-3] atom and up to twelve carbon atoms from glucose [13C-U]. The MS/MS analysis of diglycated alanines indicated that they follow similar fragmentation pathways as Amadori product of alanine.

Improved heat stability of recombined filled evaporated milk emulsions by wet heat pre-treatment of skim milk powder dispersions at different pH values

In this study, skim milk powder (SMP) dispersions were subjected to wet heat pre-treatment at 80 °C at pH 6.6, 7.1 and 7.6 to improve the heat stability of recombined filled evaporated milk (RFEM) emulsions. The results of free amino group content, Amadori products formation and color measurement revealed that the Maillard conjugation was generally faster at higher pH. SDS-PAGE clearly implied the conjugation of whey proteins upon wet heating at different pH values. Non-sedimentable protein content and composition analysis showed that extensive casein dissociation from micelles was found upon wet heat incubation at pH 7.1 and 7.6. The obtained SMP dispersions were subsequently used to prepare RFEM emulsions. The heat stability of RFEM emulsions derived from SMP dispersions incubated at pH 6.6 and 7.6 was improved greatly upon selecting an appropriate incubation time, while the heat stability improvement was less distinct upon incubation at pH 7.1. Furthermore, the incubation time needed to obtain a heat-stable RFEM emulsion against heating at 120 °C for 30 min was shorter at pH 7.6 as compared to the original pH 6.6 (0.5 h versus 2 h). On the other hand, prolonged incubation had a detrimental effect on the heat stability of RFEM.

Characterization of Catecholaldehyde Adducts with Carnosine and l-Cysteine Reveals Their Potential as Biomarkers of Catecholaminergic Stress.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of dopamine and norepinephrine to produce 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), respectively. Both of these aldehydes are potently cytotoxic and have been implicated in pathogenesis of neurodegenerative and cardiometabolic disorders. Previous work has demonstrated that both the catechol and aldehyde moieties of DOPAL are reactive and cytotoxic via their propensity to cause macromolecular cross-linking. With certain amines, DOPAL likely reacts via a Schiff base before oxidative activation of the catechol and rearrangement to a stable indole product. Our current work expands on this reactivity and includes the less-studied DOPEGAL. Although we confirmed that antioxidants mediated DOPAL’s reactivity with carnosine and N-acetyl-l-lysine, antioxidants had no effect on reactivity with l-cysteine. Therefore, we propose a non-oxidative mechanism where, following Schiff base formation, the thiol of l-cysteine reacts to form a thiazolidine. Similarly, we demonstrate that DOPEGAL forms a putative thiazolidine conjugate with l-cysteine. We identified and characterized both l-cysteine conjugates via HPLC-MS and additionally identified a DOPEGAL adduct with carnosine, which is likely an Amadori product. Furthermore, we were able to demonstrate that these conjugates are produced in biological systems via MAO after treatment of the cell lysate with norepinephrine or dopamine along with the corresponding nucleophiles (i.e., l-cysteine and carnosine). As it has been established that metabolic and oxidative stress leads to increased MAO activity and accumulation of DOPAL and DOPEGAL, it is conceivable that conjugation of these aldehydes to carnosine or l-cysteine is a newly identified detoxification pathway. Furthermore, the ability to characterize these adducts via analytical techniques reveals their potential for use as biomarkers of dopamine or norepinephrine metabolic disruption.

Effects of age and gender on the redox homeostasis of morbidly obese people

Obesity is a chronic disease of complex etiology. Recent evidence suggests that obesity is caused by inflammation of adipose tissue leading to metabolic disorders, cardiovascular disease and cancer. This is the first study to evaluated the effects of age and gender on redox homeostasis, glutathione metabolism, and oxidative damage to plasma/serum lipids and proteins in morbidly obese patients. The study included 120 (60 men and 60 women) morbidly obese patients with class 3 obesity (BMI > 40 kg/m2), classified into three groups depending on age: 20–39 years (n = 20), 40–59 years (n = 20) and 60 years or older (n = 20). The number of patients was calculated a priori based on our previous experiment. We observed a reduction in serum activity of antioxidant enzymes (↓SOD) and plasma concentration of non-enzymatic antioxidants (↓GSH) in obese patients compared to the lean controls, which further decreased with age. Redox status (↑TAC, ↑TOS and ↓OSI) in morbidly obese men and women was shifted towards oxidation. Moreover, lipid (↑MDA and ↑LOOH) and protein (↑AOPP, ↑AGE and ↑Amadori products) damage products of oxidation and nitrosylation/nitration (↑total NO, ↑S-nitrosothiols, ↑peroxynitrite and ↑nitrotyrosine) were elevated in both genders of morbidly obese patients and were higher in the elderly. Interestingly, the concentrations of oxidative and nitrosative stress markers were generally higher in obese men compared to obese women at the same age. Summarizing, we showed that the total antioxidant/oxidant potential of obese patients is significantly increased and shifted towards oxidation. Obese patients have increased lipid and protein oxidation, glycation and nitration as compared to the lean controls. Disturbances in redox homeostasis increase with age in obese patients. Oxidative and nitrosative stress are more intense in men than in women at the same age.

Molecular characterization of sequence-driven peptide glycation

Peptide glycation is an important, yet poorly understood reaction not only found in food but also in biological systems. The enormous heterogeneity of peptides and the complexity of glycation reactions impeded large-scale analysis of peptide derived glycation products and to understand both the contributing factors and how this affects the biological activity of peptides. Analyzing time-resolved Amadori product formation, we here explored site-specific glycation for 264 peptides. Intensity profiling together with in-depth computational sequence deconvolution resolved differences in peptide glycation based on microheterogeneity and revealed particularly reactive peptide collectives. These peptides feature potentially important sequence patterns that appear in several established bio- and sensory-active peptides from independent sources, which suggests that our approach serves system-wide applicability. We generated a pattern peptide map and propose that in peptide glycation the herein identified molecular checkpoints can be used as indication of sequence reactivity.

Sulfated Modification of Polysaccharides from Sweet Corncob and Its Antiglycation Activity in Streptozotocin Induced Diabetic Rats

Polysaccharides extracted from sweet corncob (SCP) were modified by sulfuric acid to sulfated sweet corncob (SSCP) with a molecular weight of 13.412 kDa, and their antiglycation activity was studied. SSCP had high inhibitory effects on glycation and showed antiglycation activity stronger than that of SCPin vitro.The maximum inhibition rates of the Amadori products, dicarbonyl compounds, caboxymethyl-lysine (CML), and advanced glycation end products (AGEs) were 76.35, 73.78, 52.79, and 76.36%, respectively. SSCP effectively increased body weight, reduced blood glucose, and increased oral glucose tolerance in streptozotocin (STZ)-induced diabetic ratsin vivo.Furthermore, SSCP inhibited AGE formation in liver tissue and repaired pancreatic injury. The resultsin vivoandin vitroreflect that SSCP has antiglycation effects, which may be closely related to its antidiabetic effects.

Comprehensive analysis of the anti-glycation effect of peanut skin extract

Advanced glycation end-products (AGEs) are produced during protein glycation and associated with diabetic complications. Peanut skin is rich in procyanidins, which may be used as an inhibitor of glycation. This study evaluated the potential anti-glycation effect of peanut skin extract (PSE) and dissected the underlying mechanism. PSE could effectively inhibit the formation of AGEs in BSA-Glc and BSA-MGO/GO models, with 44%, 37% and 82% lower IC50 values than the positive control (AG), respectively. The inhibitory effect of PSE on BSA glycation might be ascribed to its binding interaction with BSA, attenuated formation of early glycation products and trapping of reactive dicarbonyl compounds. Notably, PSE showed a remarkably stronger inhibitory effect on Amadori products than AG. Furthermore, three new types of PSE-MGO adducts were formed as identified by UPLC-Q-TOF-MS. These findings suggest that PSE may serve as an inhibitor of glycation and provide new insights into its application.

Solid-phase synthesis of d-fructose-derived Heyns peptides utilizing N\u03b1-Fmoc-Lysin[N\u03b5-(2-deoxy-d-glucos-2-yl),N\u03b5-Boc]-OH as building block

Aldoses and ketoses can glycate proteins yielding isomeric Amadori and Heyns products, respectively. Evidently, d-fructose is more involved in glycoxidation than d-glucose favoring the formation of advanced glycation endproducts (AGEs). While Amadori products and glucation have been studied extensively, the in vivo effects of fructation are largely unknown. The characterization of isomeric Amadori and Heyns peptides requires sufficient quantities of pure peptides. Thus, the glycated building block Nα-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl),Nε-Boc]-OH (Fmoc-Lys(Glc,Boc)-OH), which was synthesized in two steps starting from unprotected d-fructose and Fmoc-l-lysine hydrochloride, was site-specifically incorporated during solid-phase peptide synthesis. The building block allowed the synthesis of a peptide identified in tryptic digests of human serum albumin containing the reported glycation site at Lys233. The structure of the glycated amino acid derivatives and the peptide was confirmed by mass spectrometry and NMR spectroscopy. Importantly, the unprotected sugar moiety showed neither notable epimerization nor undesired side reactions during peptide elongation, allowing the incorporation of epimerically pure glucosyllysine. Upon acidic treatment, the building block as well as the resin-bound peptide formed one major byproduct due to incomplete Boc-deprotection, which was well separated by reversed-phase chromatography. Expectedly, the tandem mass spectra of the fructated amino acid and peptide were dominated by signals indicating neutral losses of 18, 36, 54, 84 and 96 m/z-units generating pyrylium and furylium ions.

The Prophylactic Activity of Punica granatum L. mesocarp Protects Preadipocytes against Ribosylated BSA-Induced Toxicity

Objective It was aimed at comparing the glycating capacities of glucose and ribose in bovine serum albumin (BSA) and anti-glycation activity of pomegranate mesocarp extract (PME). The protective mechanism of PME against ribosylated BSA (BSARIB)-induced toxicity was also investigated. Methods BSA was incubated with glucose or ribose in the presence or absence of PME for 15 days. In preadipocytes pretreated with PME, cell viability, ROS production, lipid peroxidation and mitochondrial membrane potential were investigated following 1, 6, 12, 18 and 24 h exposure to BSARIB. Nuclear translocation of NFκB was assessed at 1 h and 24 h of BSARIB insult. Accumulation of oxidized proteins, activities of intrinsic antioxidant enzymes and IL-6 secretion were also determined after 24 h exposure to BSARIB. Results Ribose was a harsher glycating agent as compared to glucose and PME showed strong anti-glycation activity by suppressing (P < 0.05) the increase in levels of fluorescent AGEs, Amadori products, protein carbonyl and advanced oxidation protein products (AOPP). In preadipocytes, BSARIB potentiated pro-apoptotic activity by inhibiting the nuclear translocation of NFκB. BSARIB induced a time dependent decrease in cell viability, which was significantly suppressed (P < 0.05) by PME. The extract also significantly reduced (P < 0.05) the time dependent increase in ROS level and associated lipid peroxidation as well as loss in mitochondrial membrane potential caused by BSARIB. PME also counteracted the BSARIB-induced accumulation of oxidized proteins, decrease in intrinsic antioxidant activity and IL-6 over-secretion. Conclusions PME showed anti-glycation activity and afforded protection against BSARIB-induced toxicity, oxidative stress and inflammation in preadipocytes.

Inhibition of Protein Glycation and OH &lt;sup&gt;•&lt;/sup&gt;-Induced Protein Fragmentation by Fenugreek Phytochemicals

Glycation of proteins and generation of advanced glycation end products (AGEs) are the fundamental causes in the development of oxidative stress-induced protein modification and diabetes complications. The hyperglycemic condition causes non-enzymatic protein glycation, initiated by the reaction between the carbonyl groups of sugars with the amino groups of proteins forming the amadori products. Amadori products induce further oxidation, generating dicarbonyl compounds to form cross-linking fluorescent and non-fluorescent adduct called AGEs. In this study, the protective effect of fenugreek phytochemicals on protein glycation and oxidation-induced protein fragmentation was evaluated. Fenugreek phytochemicals (phenolics and alkaloids) protected oxidation-induced protein fragmentation and inhibited protein glycation. They also prevented the formation of fluorescent and non-fluorescent AGEs, and glycation-mediated protein conformational changes. Fenugreek phytochemicals effectively scavenged reactive carbonyl intermediates and decreased protein thiol modification. Collectively, these results suggest the potential utility of fenugreek phytochemicals as functional food ingredients for the control of diabetes and its complications. Funding Information: This work is financially supported by the planning commission and Council of Scientific and Industrial Research, Govt. India, as part of 12th- five-year plan project (BSC-0404). Declaration of Interests: The authors declare that they have no competing interests.

Identification of the Maillard reaction intermediates as divalent iron complexes in alanine/glucose/FeCl2 model system using ESI/qTOF/MS/MS and isotope labelling technique

Due to their high reactivities and short half-lives, the detection of Maillard reaction intermediates is relatively difficult to achieve in a single analytical run. In this study, the formation of Maillard reaction intermediates from heated alanine/glucose mixtures (110 ​°C for 2 ​h) was investigated through their complexation with divalent iron using electrospray ionization/quadrupole time-of-flight mass spectrometry and isotope labeling techniques. Analysis of the mixtures indicated that this approach allows the simultaneous detection of many important labile and reactive Maillard reaction intermediates along with unreacted alanine and glucose in addition to various other Maillard reaction products, such as glyceraldehyde, erythrose, ribose, acetol, glycolaldehyde, fructosamine, glucosone, osones, deoxyosones, and Amadori products. Some osones and deoxyosones also formed their corresponding Schiff bases with alanine. The above mentioned Maillard reactions intermediates were detected either as binary metal complexes with alanine or with other enediol generating species including self-complexation adducts and they formed positively charged ions such as [M + H]+, [M + Na]+, [M + K]+, [M ​+ ​Fe35Cl]+, and [M ​+ ​Fe37Cl]+, that can be detected using the positive ionization mode.

Acetoacetate enhancement of glucose mediated DNA glycation

Acetoacetate (AA) is a ketone body, which generates reactive oxygen species (ROS). ROS production is impacted by the formation of covalent bonds between amino groups of biomacromolecules and reducing sugars (glycation). Glycation can damage DNA by causing strand breaks, mutations, and changes in gene expression. DNA damage could contribute to the pathogenesis of various diseases, including neurological disorders, complications of diabetes, and aging. Here we studied the enhancement of glucose-mediated DNA glycation by AA for the first time. The effect of AA on the structural changes, Amadori and advanced glycation end products (AGEs) formation of DNA incubated with glucose for 4 weeks were investigated using various techniques. These included UV–Vis, circular dichroism (CD) and fluorescence spectroscopy, and agarose gel electrophoresis. The results of UV–Vis and fluorescence spectroscopy confirmed that AA increased the DNA-AGE formation. The NBT test showed that AA also increased Amadori product formation of glycated DNA. Based on the CD and agarose gel electrophoresis results, the structural changes of glycated DNA was increased in the presence of AA. The chemiluminescence results indicated that AA increased ROS formation. Thus AA has an activator role in DNA glycation, which could enhance the adverse effects of glycation under high glucose conditions.

Salivary Gland Dysfunction in Stroke Patients Is Associated with Increased Protein Glycoxidation and Nitrosative Stress.

Stroke is one of the leading causes of disability and death worldwide. Despite intensive medical care, many of the complaints directly threatening the patient's life marginalize their dental needs after the stroke. Recent studies indicate reduced saliva secretion in stroke patients in addition to the increased incidence of caries and periodontal disease. Since oxidative stress plays a vital role in the pathogenesis of salivary gland hypofunction and neurodegenerative disorders (including stroke), this is the first to evaluate the relationship between salivary gland activity and protein glycoxidation and nitrosative damage. The content of glycation and protein oxidation products and nitrosative stress was assessed in nonstimulated (NWS) and stimulated (SWS) whole saliva of stroke patients with normal salivary secretion and hyposalivation (reduced saliva production). The study included 30 patients in the stroke's subacute phase and 30 healthy controls matched by age and sex. We have shown that stroke patients with hyposalivation show increased contents of protein glycation (↑Amadori products and ↑advanced glycation end products), glycoxidation (↑dityrosine), and nitration (↑nitrotyrosine) products compared to stroke cases with normal salivary secretion and control group. Interestingly, higher oxidative/nitrosative stress was found in NWS, which strongly correlates with salivary flow rate, total protein content, and salivary amylase activity. Such relationships were not observed in the control group. Summarizing, oxidative and nitrosative stress may be one of the mechanisms responsible for the impairment of saliva secretion in stroke patients. However, extraglandular sources of salivary oxidative stress in stroke patients cannot be excluded. Further studies to assess salivary gland hypofunction in stroke cases are necessary.

Profiling of Low-Molecular-Weight Carbonyls and Protein Modifications in Flavored Milk.

Thermal treatments of dairy products favor oxidations, Maillard reactions, and the formation of sugar or lipid oxidation products. Additives including flavorings might enhance these reactions or even induce further reactions. Here we aimed to characterize protein modifications in four flavored milk drinks using samples along the production chain—raw milk, pasteurization, mixing with flavorings, heat treatment, and the commercial product. Therefore, milk samples were analyzed using a bottom up proteomics approach and a combination of data-independent (MSE) and data-dependent acquisition methods (DDA). Twenty-one small carbonylated lipids were identified by shotgun lipidomics triggering 13 protein modifications. Additionally, two Amadori products, 12 advanced glycation end products (AGEs), and 12 oxidation-related modifications were targeted at the protein level. The most common modifications were lactosylation, formylation, and carboxymethylation. The numbers and distribution of modification sites present in raw milk remained stable after pasteurization and mixing with flavorings, while the final heat treatment significantly increased lactosylation and hexosylation in qualitative and quantitative terms. The processing steps did not significantly affect the numbers of AGE-modified, oxidized/carbonylated, and lipid-carbonylated sites in proteins.