Tuning Human Serum Albumin (HSA) Hydrogels through Albumin Glycation.

The changes of technological properties of albumin-based hydrogels induced by increasing degrees of post-translational modification of the protein are reported. Maillard-type modification of amino acids arginine and lysine of albumin is achieved through glyoxal as an α-dicarbonyl compound. The degrees of modification are fine-tuned using different molar ratios of glyoxal. Hydrogels are thermally induced by heating highly concentrated precursor solutions above the protein's denaturation temperature. While the post-translational modifications are determined and quantified with mass spectrometry, continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy shed light on the protein fatty acid binding capacity and changes thereof in solution and in the gel state. The viscoelastic behavior is characterized as a measure of the physical strength of the hydrogels. On the nanoscopic level, the modified albumins in low concentration solution reveal lower binding capacities with increasing degrees of modification. On the contrary, in the gel state, the binding capacity remains constant at all degrees of modifications. This indicates that the loss of fatty acid binding capacity for individual albumin molecules is partially compensated by new binding sites in the gel state, potentially formed by modified amino acids. Such, albumin glycation offers a fine-tuning method of technological and nanoscopic properties of these gels.

Lipid Peroxidation Has Major Impact on Malondialdehyde-Derived but Only Minor Influence on Glyoxal and Methylglyoxal-Derived Protein Modifications in Carbohydrate-Rich Foods.

In the present work, the contribution of lipid peroxidation on modifications of lysine and arginine residues of proteins was investigated. Lipid peroxidation had a major impact on malondialdehyde-derived protein modifications; however, the influence on glyoxal and methylglyoxal-derived modifications in flat wafers was negligible. Therefore, vegetable oils (either linseed oil, sunflower oil, or coconut oil) were added to respective batters, and flat wafers were baked (150 °C, 3-10 min). Analysis of malondialdehyde indicated oxidation in linseed wafers, which was supported by the direct quantitation of three malondialdehyde protein adducts in the range of 0.09-23.5 mg/kg after enzymatic hydrolysis. In contrast, levels of free glyoxal and methylglyoxal were independent of the type of oil added, which was in line with the analysis of 13 advanced glycation end products. Comprehensive incubations of 40 mM N2-t-Boc-lysine (100 mM phosphate buffer, pH 7.4) with either 10% oil or an equimolar concentration of carbohydrates led to magnitudes higher (103-105) amounts of N6-carboxymethyl lysine, N6-glycolyl lysine, and N6-carboxyethyl lysine in the latter. Furthermore, malondialdehyde exceeded glyoxal and methylglyoxal in incubations of pure oils at 150 °C by factors of 30 and 100, respectively.

Novel Pyridinium Cross-Link Structures Derived from Glycolaldehyde and Glyoxal.

Short-chained α-hydroxycarbonyl compounds such as glycolaldehyde (GA) and its oxidized counterpart glyoxal (GX) are known as potent glycating agents. Here, a novel fluorescent lysine-lysine cross-link 1-(5-amino-5-carboxypentyl)-3-(5-amino-5-carboxy-pentylamino)pyridinium salt (meta-DLP) was synthesized and its structure unequivocally proven by 1H NMR, 13C-NMR attached proton test, and 2D NMR. Further characterization of chemical properties and mechanistic background was obtained in comparison to the known monovalent protein modification 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). Identification and quantitation in various sugar incubations with N2-t-Boc-lysine revealed a novel alternative formation pathway for both advanced glycation end products (AGEs) by the interplay of both carbonyl compounds, GA and GX, which was confirmed by isotope labeling experiments. The concentration of pyridinium AGEs was about 1000-fold lower compared to the well-established N6-carboxymethyl lysine. However, pyridinium AGEs were shown to lead to the photosensitized generation of singlet oxygen in irradiation experiments, which was verified by the detection of 3,3'-(naphthalene-1,4-diyl)-dipropionate endoperoxide. Furthermore, meta-DLP was identified in hydrolyzed potato chip proteins by collision-induced dissociation mass spectrometry after HPLC enrichment.

Glycation Alters the Fatty Acid Binding Capacity of Human Serum Albumin.

Glycation significantly alters the physicochemical and biofunctional properties of proteins in foods and in vivo. In the present study, human serum albumin (HSA) as the major transporter of fatty acids was modified with glyoxal under physiological conditions. Reversibly albumin-bound glyoxal was removed, and advanced glycation end products were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total modification of protein-bound lysine and arginine residues reached up to 4.2 and 9.6%, respectively. The impact of these modifications on the transport capacity of long-chain fatty acids was characterized by spin-labeled fatty acid probes via electron paramagnetic resonance spectroscopy. With increasing degree of glycation, the equivalence of the seven binding sites of native HSA with a dissociation constant of 0.74 ± 0.09 µM was set off with only the three high-affinity sites 2, 4, and 5 remaining (0.46 ± 0.07 µM). The other four sites were shifted to low affinities with significantly higher dissociation constants (1.32 ± 0.35 µM). Tryptic peptide mapping enabled us to relate these findings to molecular changes at specific binding sites. Modification hotspots identified were lysine 351, 286, 159 and arginine 144, 485, 117. Further investigation of plasma protein samples of uremic patients vs healthy controls gave first insights into the in vivo situation.

Analysis of Glyoxal- and Methylglyoxal-Derived Advanced Glycation End Products during Grilling of Porcine Meat.

The reaction of the N6-amino group of lysine residues and 1,2-dicarbonyl compounds during Maillard processes leads to advanced glycation end products (AGEs). In the present work, we deliver a comprehensive analysis of changes of carbohydrates, dicarbonyl structures, and 11 AGEs during the grilling of porcine meat patties. While raw meat contained mainly glyoxal-derived N6-carboxymethyl lysine (CML), grilling led to an increase of predominantly methylglyoxal-derived AGEs N6-carboxyethyl lysine (CEL), N6-lactoyl lysine, methylglyoxal lysine dimer (MOLD), and methylglyoxal lysine amide (MOLA). Additionally, we identified and quantitated a novel methylglyoxal-derived amidine compound N1,N2-di-(5-amino-5-carboxypentyl)-2-lactoylamidine (methylglyoxal lysine amide, MGLA) in heated meat. Analysis of carbohydrates suggested that approximately 50% of the methylglyoxal stemmed from the fragmentation of triosephosphates during the heat treatment. Surprisingly, N6-lactoyl lysine was the major AGE, and based on model incubations, we propose that approximately 90% must be explained by the nonenzymatic acylation of lysine through S-lactoylglutathione, which was quantitated for the first time in meat herein.

Benzothiazines as Major Intermediates in Enzymatic Browning Reactions of Catechin and Cysteine.

The course of melanin formation is yet not thoroughly resolved on a mechanistic level. With the present study, incubations of catechin (CA)- and cysteine-derived dihydro-1,4-benzothiazine carboxylic acid derivatives were investigated for colored products during enzymatic browning. Analyses by high-performance liquid chromatography (HPLC)-mass spectrometry revealed the formation of two novel decarboxylated dihydro-1,4-benzothiazine derivatives [8-(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-5-hydroxy-3,4-dihydro-2H-benzothiazine and 7-(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-5-hydroxy-3,4-dihydro-2H-benzothiazine] preferentially under acidic conditions. Furthermore, in model reactions under neutral pH, a colored phenazine dimer intermediate was isolated by high-performance countercurrent chromatography and preparative HPLC when conducting the incubations in the presence of o-phenylenediamine (OPD). Mass spectrometry and nuclear magnetic resonance spectroscopy unequivocally verified the structure as (12E)-5,5'-dioxo-11a,11a'-bis(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-3,3',4,4',5a,5a',6,6',11,11',11a,11a'-dodecahydro-2H,2'H,5H,5'H-12,12'-bi[1,4]thiazino[2,3-b]phenazine-3,3'-dicarboxylic acid. Enzymatically catalyzed incubations under aeration starting from the initial CA-cysteine adducts and their follow-up dihydro-1,4-benzothiazine carboxylic acids, respectively, proved that the unstable colored compound was a trichochrome-like reaction intermediate of the browning reaction cascade which can be trapped by postincubation with OPD, thus verifying their direct mechanistic relationship.

AGE-Rich Bread Crust Extract Boosts Oxidative Stress Interception via Stimulation of the NRF2 Pathway.

Advanced glycation end products (AGEs) result from a non-enzymatic reaction of proteins with reactive carbohydrates. Heat-processed food, such as bread, contains high amounts of AGEs. The activation of the NF-κB signaling pathway by bread crust extract (BCE) is well understood. However, it is largely unknown whether NRF2, the master regulator of oxidative stress resistance in mammalian cells, is affected by BCE. We have investigated the molecular mechanisms by which BCE induces antioxidant gene expression in cellular models. Our data showed that soluble extracts from bread crust are capable of stimulating the NRF2 signaling pathway. Furthermore, NRF2 pathway activation was confirmed by microarray and reporter-cell analyses. QRT-PCR measurements and Western blot analyses indicated an induction of antioxidative genes such as HMOX1, GCLM and NQO1 upon BCE treatment. Moreover, BCE pretreated cells had a survival advantage compared to control cells when exposed to oxidative stress. BCE induces phosphorylation of AKT and ERK kinase in EA.hy926 cells. By mass spectrometry, several new, potentially active modifications in BCE were identified. Our findings indicate that BCE activates NRF2-dependent antioxidant gene expression, thus provoking a protection mechanism against oxidative stress-mediated tissue injury. Hence, BCE can be considered as functional food with antioxidative and cardioprotective potential.

Mapping protein carboxymethylation sites provides insights into their role in proteostasis and cell proliferation.

Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devise a proteomic strategy to identify sites of carboxymethyllysine modification, one of the most abundant advanced glycation end products. We identify over 1000 sites of protein carboxymethylation in mouse and primary human cells treated with the glycating agent glyoxal. By using quantitative proteomics, we find that protein glycation triggers a proteotoxic response and indirectly affects the protein degradation machinery. In primary endothelial cells, we show that glyoxal induces cell cycle perturbation and that carboxymethyllysine modification reduces acetylation of tubulins and impairs microtubule dynamics. Our data demonstrate the relevance of carboxymethyllysine modification for cellular function and pinpoint specific protein networks that might become compromised during aging.

Novel Amidine Protein Cross-Links Formed by the Reaction of Glyoxal with Lysine.

One crucial aspect of the Maillard reaction is the formation of reactive α-dicarbonyl structures like glyoxal, which are prone toward further reactions with proteins, e.g., the N6-amino group of lysine. The initially formed labile glyoxal-imine was previously established as a key intermediate in the formation of the advanced glycation end products N6-carboxymethyl lysine (CML), glyoxal lysine amide (GOLA), glyoxal lysine dimer (GOLD), and N6-glycolyl lysine (GALA). Here, we introduce a novel amidine cross-link structure N1,N2-bis-(5-amino-5-carboxypentyl)-2-hydroxy-acetamidine (glyoxal lysine amidine, GLA), which is formed exclusively from glyoxal through the same isomerization cascade. After independent synthesis of the authentic reference standard, we were able to quantitate this cross-link in incubations of 40 mM N2-t-Boc-lysine with glyoxal and various sugars (40-100 mM) under mild conditions (pH 7.4, 37 °C) using an HPLC-MS/MS method. Furthermore, incubations of proteins (6 mg/mL) with 50 mM glyoxal confirmed the cross-linking by GLA, which was additionally identified in acidic hydrolyzed proteins of butter biscuits after HPLC enrichment.

Advanced glycation end products in human diabetic lens capsules.

Advanced glycation end products (AGEs) accumulate with age in human lens capsules. AGEs in lens capsules potentiate the transforming growth factor beta-2-mediated mesenchymal transition of lens epithelial cells, which suggests that they play a role in posterior capsule opacification after cataract surgery. We measured AGEs by liquid chromatography-mass spectrometry in capsulorhexis specimens obtained during cataract surgery from nondiabetic and diabetic patients with and without established retinopathy. Our data showed that the levels of most AGEs (12 out of 13 measured) were unaltered in diabetic patients and diabetic patients with retinopathy compared to nondiabetic patients. There was one exception: glucosepane, which was significantly higher in diabetic patients, both with (6.85 pmol/µmol OH-proline) and without retinopathy (8.32 pmol/µmol OH-proline), than in nondiabetic patients (4.01 pmol/µmol OH-proline). Our study provides an explanation for the similar incidence of posterior capsule opacification between nondiabetic and diabetic cataract patients observed in several studies.

Pathways of Non-enzymatic Lysine Acylation.

Posttranslational protein modification by lysine acylation is an emerging mechanism of cellular regulation and fine-tunes metabolic processes to environmental changes. In this review we focus on recently discovered pathways of non-enzymatic lysine acylation by reactive acyl-CoA species, acyl phosphates, and α-dicarbonyls. We summarize the metabolic sources of these highly reactive intermediates, demonstrate their reaction mechanisms, give an overview of the resulting acyl lysine modifications, and evaluate the consequences for cellular regulatory processes. Finally, we discuss interferences between lysine acylation and lysine ubiquitylation as a potential molecular mechanism of dysregulated protein homeostasis in aging and related diseases.

Comprehensive Analyses of Carbohydrates, 1,2-Dicarbonyl Compounds, and Advanced Glycation End Products in Industrial Bread Making.

The technology of bread making is characterized by three major steps: dough mixing, proofing, and baking. To follow the course of Maillard processes in an authentic food matrix, the complete manufacturing process of wheat bread rolls was assessed along all production steps with the quantitation of sugars, furfurals, 1,2-dicarbonyl compounds, and advanced glycation end products (AGEs). As a result, the AGE profile was significantly enlarged to more than 12 structures, and comprehensive mechanistic insights were provided. The analyses of five major German bread types including wheat, brown, rye bread, pumpernickel, and crispbreads led to AGE contents of 69-149 mg/kg bread or 984-1857 mg/kg protein. Major lysine protein modifications were carboxymethyl, carboxyethyl, and formyl lysine and pyrraline. Arginine was mainly modified by methylglyoxal (MGO) to give imidazolinones. A major part of MGO was confirmed to stem from microbial metabolism.

Glycation-mediated protein crosslinking and stiffening in mouse lenses are inhibited by carboxitin in vitro.

Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.

Transient elevation of temperature promotes cross-linking of α-crystallin-client proteins through formation of advanced glycation endproducts: A potential role in presbyopia and cataracts.

The chaperone activity of α-crystallin is important for maintaining the transparency of the human lens. αB-crystallin (αBC) is a long-lived protein in the lens that accumulates chemical modifications during aging. The formation of advanced glycation end products (AGEs) through glycation is one such modification. αBC is a small heat shock protein that exhibits chaperone activity. We have previously shown that αBC-client protein complexes can undergo AGE-mediated interprotein cross-linking. Here, we demonstrate that short-term (1 h) exposure to elevated temperatures and methylglyoxal (MGO) during the chaperoning of client proteins by αBC promotes AGE-mediated interprotein cross-linking. Liquid chromatography/mass spectrometry (LC-MS/MS) analyses revealed the rapid formation of AGEs by MGO. Interestingly, we found that despite protein cross-linking, the chaperone activity of αBC increased during the transient elevation of temperature in the presence of MGO. Together, these results imply that transient and subtle elevation of temperature in the lens of the eye can promote protein cross-linking through AGEs, and if this phenomenon recurs over a period of many years, it could lead to early onset of presbyopia and age-related cataracts.

Comprehensive analysis of posttranslational protein modifications in aging of subcellular compartments.

Enzymatic and non-enzymatic posttranslational protein modifications by oxidation, glycation and acylation are key regulatory mechanisms in hallmarks of aging like inflammation, altered epigenetics and decline in proteostasis. In this study a mouse cohort was used to monitor changes of posttranslational modifications in the aging process. A protocol for the extraction of histones, cytosolic and mitochondrial proteins from mouse liver was developed and validated. In total, 6 lysine acylation structures, 7 advanced glycation endproducts, 6 oxidative stress markers, and citrullination were quantitated in proteins of subcellular compartments using HPLC-MS/MS. Methionine sulfoxide, acetylation, formylation, and citrullination were the most abundant modifications. Histone proteins were extraordinary high modified and non-enzymatic modifications accumulated in all subcellular compartments during the aging process. Compared to acetylation of histone proteins which gave between 350 and 305 µmol/mol leucine equivalents in young and old animals, modifications like acylation, glycation, and citrullination raised to 43%, 20%, and 18% of acetylation, respectively. On the other hand there was an age related increase of selected oxidative stress markers by up to 150%. The data and patterns measured in this study are mandatory for further studies and will strongly facilitate understanding of the molecular mechanisms in aging.

Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin-γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens-epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone-client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone-client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin-client complexes could contribute to lens aging and presbyopia.

Influence of ß-Carotene and Lycopene on Oxidation of Ethyl Linoleate in One- and Disperse-Phased Model Systems.

The induction period (IP) of ethyl linoleate stressed at 60 °C was monitored via the formation of hydroperoxides. The addition of lycopene (1% w/w) increased the IP from 7.0 to 10.0 h to prove the strong antioxidative potential in contrast to ß-carotene with pro-oxidative effects (IP: 6.0 h), both showing strong scavenging activity under fast degradation. When peroxidation was induced by singlet oxygen, both carotenoids effectively inhibited the formation of hydroperoxides, with quenching activity only observed at low singlet oxygen concentrations, while scavenging still dominated. Thus, carotenoids did not interact with the introduced singlet oxygen but rather with the radical intermediates of fat oxidation. These experiments were then transferred to lecithin-based micelles more related to biological systems, where singlet oxygen was generated in the outer aqueous phase. Lycopene and ß-carotene delayed or inhibited lipid peroxidation depending on concentration. In this setup, ß-carotene showed exclusively quenching activity, while lycopene was additionally degraded to about 70%.

Oxidative Fragmentation of Aspalathin Leads to the Formation of Dihydrocaffeic Acid and the Related Lysine Amide Adduct.

In the present study, the degradation of C-glucosidic dihydrochalcone aspalathin as the major phenolic compound in rooibos (Aspalathus linearis) was investigated. Analyses by gas chromatography-mass spectrometry of aqueous aspalathin-lysine incubations after silylation showed the formation of dihydrocaffeic acid [3-(3,4-dihydroxyphenyl)-propionic acid] under oxidative conditions as a novel degradation product up to 10 mol %. High-performance liquid chromatography analyses revealed the concurrent formation of the dihydrocaffeic acid lysine amide at about 30-fold lower concentrations, which was unequivocally verified by synthesis of an authentic reference standard. The amide was also verified in aspalathin-protein incubations after enzymatic hydrolysis by high-performance liquid chromatography-tandem mass spectrometry analyses. Thus, the covalent interaction of phenolic plant compounds with proteins under mild conditions (ambient temperatures and neutral pH) was confirmed for the first time. Acid and free amide were also quantitated in rooibos teas with significantly higher values in fermented varieties. The mechanism of formation was clarified to be initiated by singlet oxygen and to include a rearrangement-fragmentation mechanism with 1,2,3,5-tetrahydroxybenzene as the counterpart.

Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?

Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.

Increased Expression of Immature Mannose-Containing Glycoproteins and Sialic Acid in Aged Mouse Brains.

Aging represents the accumulation of changes in an individual over time, encompassing physical, psychological, and social changes. Posttranslational modifications of proteins such as glycosylation, including sialylation or glycation, are proposed to be involved in this process, since they modulate a variety of molecular and cellular functions. In this study, we analyzed selected posttranslational modifications and the respective proteins on which they occur in young and old mouse brains. The expression of neural cell adhesion molecule (NCAM), receptor for advanced glycation endproducts (RAGE), as well as the carbohydrate-epitopes paucimannose and high-mannose, polysialic acid, and O-GlcNAc were examined. We demonstrated that mannose-containing glycans increased on glycoproteins in aged mouse brains and identified synapsin-1 as one major carrier of paucimannose in aged brains. In addition, we found an accumulation of so-called advanced glycation endproducts, which are generated by non-enzymatic reactions and interfere with protein function. Furthermore, we analyzed the expression of sialic acid and found also an increase during aging.