High hydrostatic pressure processing of human milk preserves milk oligosaccharides and avoids formation of Maillard reaction products.

BACKGROUND & AIMS: High hydrostatic pressure (HHP) processing is a non-thermal method proposed as an alternative to Holder pasteurization (HoP) for the treatment of human milk. HHP preserves numerous milk bioactive components that are degraded by HoP, but no data are available for milk oligosaccharides (HMOs) or the formation of Maillard reaction products, which may be deleterious for preterm newborns. METHODS: We evaluated the impact of HHP processing of human milk on 22 HMOs measured by liquid chromatography with fluorescence detection and on furosine, lactuloselysine, carboxymethyllysine (CML) and carboxyethyllysine (CEL) measured by liquid chromatography with tandem mass spectrometric detection (LC-MS/MS), four established indicators of the Maillard reaction. Human raw milk was sterilized by HoP (62.5 °C for 30 min) or processed by HHP (350 MPa at 38 °C). RESULTS: Neither HHP nor HoP processing affected the concentration of HMOs, but HoP significantly increased furosine, lactuloselysine, CML and CEL levels in milk. CONCLUSIONS: Our findings demonstrate that HPP treatment preserves HMOs and avoids formation of Maillard reaction products. Our study confirms and extends previous findings that HHP treatment of human milk provides safe milk, with fewer detrimental effects on the biochemically active milk components than HoP.

Glycated peptides obtained from cultured crocodile meat hydrolysates via Maillard reaction and the anti-aging effects on Drosophila in vivo.

With the aging problems increasing, the discovery of anti-aging compounds has become a popular research direction. Accumulation of free radicals and the consequent oxidative stress are the chief culprit of aging. Given this, cultured crocodile meat peptides-Maillard reaction product (CMP-MRP) with remarkable antioxidant activity was obtained via Maillard reaction of cultured crocodile meat hydrolysates and xylose. The antioxidant activity in vitro and anti-aging activity in vivo of CMP-MRP were investigated. Results indicated that the lifespan and the athletic ability of Drosophila were significantly improved after the administration of CMP-MRP in natural aging, H2O2- and paraquat-induced models. Furthermore, the antioxidant enzyme activities of Drosophila treated with CMP-MRP were enhanced while the levels of malondialdehyde (MDA) and protein carbonyl (PCO) were reduced in three Drosophila models. With the supplement of 5 mg/mL CMP-MRP in natural aging Drosophila model, the maximum lifespan increased from 61 days to 73 days, athletic ability raised by 95.45%, MDA and PCO reduced by 52.72% and 47.43%, respectively. Taken together, CMP-MRP exhibited outstanding antioxidant and anti-aging capacities in Drosophila models, suggesting that CMP-MRP possesses great potential in the health food and biomedicine fields as a food-derived anti-aging agent.

The Genotoxic and Pro-Apoptotic Activities of Advanced Glycation End-Products (MAGE) Measured with Micronuclei Assay Are Inhibited by Their Low Molecular Mass Counterparts.

An association between the cancer invasive activities of cells and their exposure to advanced glycation end-products (AGEs) was described early in some reports. An incubation of cells with BSA-AGE (bovine serum albumin-AGE), BSA-carboxymethyllysine and BSA-methylglyoxal (BSA-MG) resulted in a significant increase in DNA damage. We examined the genotoxic activity of new products synthesized under nonaqueous conditions. These were high molecular mass MAGEs (HMW-MAGEs) formed from protein and melibiose and low molecular mass MAGEs (LMW-MAGEs) obtained from the melibiose and N-α-acetyllysine and N-α-acetylarginine. We have observed by measuring of micronuclei in human lymphocytes in vitro that the studied HMW-MAGEs expressed the genotoxicity. The number of micronuclei (MN) in lymphocytes reached 40.22 ± 5.34 promille (MN/1000CBL), compared to 28.80 ± 6.50 MN/1000 CBL for the reference BSA-MG, whereas a control value was 20.66 ± 1.39 MN/1000CBL. However, the LMW-MAGE fractions did not induce micronuclei formation in the culture of lymphocytes and partially protected DNA against damage in the cells irradiated with X-ray. Human melanoma and all other studied cells, such as bronchial epithelial cells, lung cancer cells and colorectal cancer cells, are susceptible to the genotoxic effects of HMW-MAGEs. The LMW-MAGEs are not genotoxic, while they inhibit HMW-MAGE genotoxic activity. With regard to apoptosis, it is induced with the HMW-MAGE compounds, in the p53 independent way, whereas the low molecular mass product inhibits the apoptosis induction. Further investigations will potentially indicate beneficial apoptotic effect on cancer cells.

Generation and Characterization of Anti-Glucosepane Antibodies Enabling Direct Detection of Glucosepane in Retinal Tissue.

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

Galangin inhibits α-glucosidase activity and formation of non-enzymatic glycation products.

Inhibition of α-glucosidase and non-enzymatic glycation is considered as an effective approach to treat type 2 diabetes. Herein, multispectroscopic techniques and molecular docking analysis were used to investigate the inhibition of galangin on α-glucosidase and non-enzymatic glycation. Galangin showed a reversible inhibition on α-glucosidase activity in a mixed-type manner through a monophasic kinetic process, and induced the fluorescence quenching and conformational changes of α-glucosidase by forming α-glucosidase-galgangin complex. Molecular docking revealed that galangin primarily interacted with the amino acid residues within the active site of α-glucosidase, which may prevent the entrance of substrate resulting in a decrease in catalytic efficiency of α-glucosidase. Moreover, galangin moderately inhibited the formation of intermediates of non-enzymatic glycation, fructosamine and α-dicarbonyl compounds and strongly inhibited the formation of advanced glycation end products.

Trapping of glyoxal by propyl, octyl and dodecyl gallates and their mono-glyoxal adducts.

Glyoxal (GO) is one of the major toxic intermediates generated during lipid oxidation and degradation. We investigated the inhibitory activities and mechanisms of propyl, octyl, and dodecyl gallates (PG, OG, and DG) on the formation of GO in buffer and during thermo-processing of corn oil, and the anti-carbonyl and antioxidative activities of the mono-GO adducts of PG, OG, and DG. Our results suggested that alkyl gallates could more effectively trap GO than gallic acid. The major mono-GO adducts of PG, OG, and DG were purified and their structures were elucidated based on their 1H, 13C, 2D-NMR, and HRMS data. We further demonstrated that the mono-GO (MG) adducts retained the anti-carbonyl and antioxidative activities. This is the first study to demonstrate that alkyl gallates, the popular food additives, could prevent not only food oxidation, but also the formation of toxic reactive carbonyl species and their corresponding advanced glycation end products (AGEs) during food processing.

Inhibition of Methylglyoxal-Induced Histone H1 Nε-Carboxymethyllysine Formation by (+)-Catechin.

Reactive dicarbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are common intermediates in protein damage, leading to the formation of advanced glycation end products (AGEs) through nonenzymatic glycation. (+)-Catechin, a natural plant extract from tea, has been evaluated for its ability in trapping GO and MGO. However, (+)-catechin is also reported to have both antioxidant ability and pro-oxidant properties. Until now, whether (+)-catechin can inhibit the formation of nonenzymatic glycation and the mechanism of the inhibition in nucleoprotein nonenzymatic glycation is still unclear. In the present study, histone H1 and MGO were used to establish an in vitro (100 mM phosphate buffer solution (PBS), pH 7.4, 37 °C) protein glycation model to study the trapping ability of (+)-catechin. Our data show that MGO caused dose-dependent protein damage, and the content of MGO-induced Schiff base formation was inhibited by (+)-catechin when the molecular ratio of catechin:MGO was 1:6. The formation of Nε-carboxymethyllysine (CML) was reduced significantly when the ratio of (+)-catechin and MGO was 1:1, which was similar to the inhibition effect of aminoguanidine (AG). The formation of CML under in vitro conditions can be inhibited by low concentration (12.5-100 µM) of (+)-catechin but not with high concentration (200-800 µM) of (+)-catechin. The reason is that the high concentration of (+)-catechin did not inhibit CML formations due to H2O2 produced by (+)-catechin. In the presence of catalase, catechin can inhibit MGO-induced CML formation. In conclusion, the trapping ability of (+)-catechin may be more effective at the early stage of nonenzymatic glycation. However, a high concentration (200-800 µM) of (+)-catechin may not inhibit the formation of CML because it induced the increase of H2O2 formation.

Generation of Advanced Glycation End-Products (AGEs) by glycoxidation mediated by copper and ROS in a human serum albumin (HSA) model peptide: reaction mechanism and damage in motor neuron cells.

Glucose, in the presence of reactive oxygen species (ROS), acts as an as an oxidative agent and drives deleterious processes in Diabetes Mellitus. We have studied the mechanism and the toxicological effects of glucose-dependent glycoxidation reactions driven by copper and ROS, using a model peptide based on the exposed sequence of Human Serum Albumin (HSA) and containing a lysine residue susceptible to copper complexation. The main products of these reactions are Advanced Glycation End-products (AGEs). Carboxymethyl lysine and pyrraline condensed on the model peptide, generating a Modified Peptide (MP). These products were isolated, purified, and tested on cultured motor neuron cells. We observed DNA damage, enhancement of membrane roughness, and formation of domes. We evaluated nuclear abnormalities by the cytokinesis-blocked micronucleus assay and we measured cytostatic and cytotoxic effects, chromosomal breakage, nuclear abnormalities, and cell death. AGEs formed by glycoxidation caused large micronucleus aberrations, apoptosis, and large-scale nuclear abnormalities, even at low concentrations.

Arginine side-chain modification that occurs during copper-catalysed azide-alkyne click reactions resembles an advanced glycation end product.

Dehydroascorbate is a by-product of copper-catalysed azide-alkyne click (CuAAC) reactions and also forms advanced glycation end products (AGEs) in tissues undergoing oxidative stress. Here we isolate and characterize an arginine-dehydroascorbate adduct formed during CuAAC reactions, investigate strategies for preventing its formation, and propose its biological relevance as an AGE.

Carboxymethyl-lysine: thirty years of investigation in the field of AGE formation.

In 1985 carboxymethyl-lysine (CML), the first glycoxidation product, was discovered by Dr Ahmed while trying to identify the major products formed in reactions of glucose with lysine under physiological conditions. From that moment, a significant number of researchers have joined efforts to study its formation routes both in foods and in living beings, and the possibility of the existence of an additive action between food-occurring and in vivo produced CML and to explore all the implications associated with its appearance in the biological systems, regardless of its origin. This review presents interesting information on the latest advances in the research on CML sources, mitigation strategies, intake, metabolism and body fluid and tissue delivery, its possible in vivo synergy with highly modified advanced glycation end products-protein, and the physio-pathological implications derived from the presence of this compound in body fluids and tissues.

Concise total synthesis of glucosepane.

Glucosepane is a structurally complex protein posttranslational modification that is believed to exist in all living organisms. Research in humans suggests that glucosepane plays a critical role in the pathophysiology of both diabetes and human aging, yet comprehensive biological investigations of this metabolite have been hindered by a scarcity of chemically homogeneous material available for study. Here we report the total synthesis of glucosepane, enabled by the development of a one-pot method for preparation of the nonaromatic 4H-imidazole tautomer in the core. Our synthesis is concise (eight steps starting from commercial materials), convergent, high-yielding (12% overall), and enantioselective. We expect that these results will prove useful in the art and practice of heterocyclic chemistry and beneficial for the study of glucosepane and its role in human health and disease.

Generation and characterization of antibodies against arginine-derived advanced glycation endproducts.

Although antibodies reagents have been widely employed for studying advanced glycation end-products (AGEs), these materials have been produced using complex mixtures of immunogens. Consequently, their epitope specificity remains unknown. Here we have generated the first antibodies capable of recognizing each of the three isomers of the methylglyoxal hydroimidazolones (MG-Hs) by using chemical synthesis to create homogenous immunogens. Furthermore, we have thoroughly characterized the epitope specificity of both our antibodies and that of two existing monoclonals by implementing a direct ELISA protocol employing synthetic MG-H antigens. Finally, we employed the reported anti-MG-H antibodies to the detection of MG-Hs in cellular systems using immunofluorescence microscopy. These studies have demonstrated that anti-MG-H1 and anti-MG-H3 staining is concentrated within the nucleus, while anti-MG-H2 affords only minimal signal. These observations are consistent with reported formation preferences for MG-Hs, and may suggest novel nuclear targets for non-enzymatic posttranslational modification. The antibody reagents reported herein, as well as the strategy employed for their creation, are likely to prove useful for the immunochemical study of AGEs in biological systems.

Liquid chromatography-tandem mass spectrometry of phosphatidylserine advanced glycated end products.

Phosphatidylserine (PS) is an aminophospholipid that is prone to glycation. In oxidative conditions, glycated PS may lead to the formation of Amadori compounds and advanced glycated end products (AGEs), which are known to accumulate in diabetic patients. Nevertheless, there have been no studies that identified products from the oxidative reaction of glycated PS. In this study, glycated 1-palmitoyl-2-oleoyl-PS was synthesized and further oxidized by Fenton reagent. The AGES formed were structurally characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in negative mode. The oxidation products from glycated PS that we have found include products arising from the oxidation of the fatty acyl chains (hydroperoxides, hydroxides and keto derivatives), and arising from oxidative cleavage of serine polar head and lyso-glycated PS. Oxidation in C6 of glucose lead to the formation of glucuronyl-PS. In addition, new products arising from oxidative cleavage of glucose moiety (between C1C2, C2C3 and C3C4 bonds) were identified as PS-AGES. The current findings add substantially to the best of our knowledge of PS glycoxidation products, opening new perspectives for the detection of these products in complex biological matrices.

Synthesis of stable isotope-labelled monolysyl advanced glycation endproducts.

Advanced Glycation Endproducts (AGEs) are modified amino acids that form on proteins and are known to be implicated in the pathogenesis of diabetes and related diseases. Ready access to synthetic stable isotope-labelled AGEs allows for quantitative mass spectrometry studies to be undertaken, providing key insights into the roles AGEs play in the progression of such diseases. However, the majority of current syntheses of these compounds suffer from poor yields and lengthy procedures and are not suitable for the purposes required here. Here, we report robust syntheses of stable isotope-labelled monolysyl AGEs, N(ε)-(carboxymethyl)lysine, N(ε)-(carboxyethyl)lysine and pyrraline, that provide straightforward access to these compounds for quantitative amino acid analysis. This work will facilitate future investigations with these compounds and lead to a better understanding of the roles they play in diabetes and related diseases.

Formation and inhibition of Nε-(carboxymethyl)lysine in saccharide-lysine model systems during microwave heating.

N(ε)-(carboxymethyl) lysine (CML) is the most abundant advanced glycation end product (AGE), and frequently selected as an AGEs marker in laboratory studies. In this paper, the formation and inhibition of N(ε)-(carboxymethyl)lysine in saccharide-lysine model systems during microwave heating have been studied. The microwave heating treatment significantly promoted the formation of CML during Maillard reactions, which was related to the reaction temperature, time and type of saccharide. The order of CML formation for different saccharides was lactose > glucose > sucrose. Then, the inhibition effect on CML by five inhibitors was further examined. According to the results, ascorbic acid and tocopherol did not affect inhibition of CML, in contrast, thiamin, rutin and quercetin inhibited CML formation, and the inhibitory effects were concentration dependent.

Synthesis of monolysyl advanced glycation endproducts and their incorporation into collagen model peptides.

The synthesis of advanced glycation endproducts (AGEs), CML, CEL, and pyrraline and their incorporation into collagen model peptides is reported. AGEs are modified amino acids that form on proteins such as collagen and are thought to play a significant role in the pathogenesis of many diseases, particularly diabetes. The synthesis and incorporation of these compounds into synthetic peptides is a key step in developing model systems with which to investigate AGE-modified proteins.

The role of ribosylated-BSA in regulating PC12 cell viability.

Glycation, one of the post-translational modifications, is known to influence protein structure and biological function. Advanced glycation end products (AGEs) have been shown to cause pathologies of diabetes. Glycation levels in patients with Alzheimer's disease (AD) are higher than in normal people. However, whether the glycation of susceptible proteins is a triggering event for cell damage or simply a result remains to be elucidated. In this study, we demonstrated that ribose-conjugated BSA (Rib-BSA) directly induces PC12 cell death in a dose- and time-dependent manner. The IC(50) is 4.6 µM. Unlike glucose-incubated BSA, Rib-BSA rapidly forms cytotoxic AGEs. PC12 is vulnerable to Rib-BSA. However, fructose can induce AGE formation, although no effect on cell survival was observed. This effect of Rib-BSA is reversed by pretreatment of pioglitazone and rosiglitazone, which belongs to thiazolidinediones (TZDs) and are peroxisome proliferator-activated receptor (PPAR-γ) ligands. Moreover, Rib-BSA upregulates inducible nitric oxide synthase (iNOS), cycloxygenase 2 (COX-2) expression, and p-38 phosphorylation and leaves extracellular regulated protein1/2 (ERK1/2) phosphorylation unchanged. The Rib-BSA-induced signaling changes are blocked by rosiglitazone and confirmed by PPAR-γ small-interfering RNA transfection. The reduction of cell survival by Rib-BSA is blocked by the iNOS inhibitor and p38 inhibitor. No effect on cell survival was observed using the COX-2 inhibitor. Consequently, these results show that Rib-BSA directly inducing PC12 cell death is a triggering event and TZDs protect PC12 cell from Rib-BSA damage. Signaling molecules, such as PPAR-γ, P38, and iNOS, are involved in Rib-BSA-mediated cytotoxicity.

The in vitro glycation of human serum albumin in the presence of Zn(II).

Amino groups of human serum albumin (HSA) can react non-enzymatically with carbonyl groups of reducing sugars to form advanced glycation end products (AGEs). These AGEs contribute to many of the chronic complications of diabetes including atherosclerosis, cataract formation and renal failure. The current study focused on in vitro non-enzymatic reactivity of glyceraldehyde (GA) and methylglyoxal (MG) with HSA and evaluated the rate and extent of AGE formation in the presence of varied concentrations of Zn(II). At normal physiological conditions, GA and MG readily react with HSA. The presence of Zn(II) in HSA-GA or HSA-MG incubation mixtures reduced AGE formation. This finding was confirmed by UV and fluorescence spectrometry, HPLC techniques, and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF). HPLC studies revealed decreased adduct formation of the glycated protein in the presence of Zn(II). The inhibition of AGE formation was intense at elevated Zn(II) concentrations. The results of this study suggest that Zn(II) may prove to be a potent agent in reducing AGE formation.

Controversial behavior of aminoguanidine in the presence of either reducing sugars or soluble glycated bovine serum albumin.

The elucidation of the controversial inhibitory effect of aminoguanidine (AG) on the cross-linking and fluorescent advanced glycation end products (AGEs) formation during long-term in vitro glycation of type I collagen with 250 mM reducing sugars or 0.5mg/ml soluble glycated bovine serum albumin (AGE-BSA) was researched. Chromatographic and SDS-PAGE analyses revealed the formation of aggregates during collagen glycation. AG at all concentrations (5-80 mM) prevented the cross-linking of collagen peptides with monosaccharides but an increase in fluorescence with a maximum value at 10 mM AG was noticed. In the presence of AGE-BSA, AG prevented the cross-linking process and decreased the fluorescence levels in a concentration-dependent manner. Our results suggest that AG is an efficient inhibitor of collagen cross-linking and the highest increase in fluorescence due to reducing sugars and AG can be explained by the competition between guanidine group of AG and arginine residues of some protein-bound dideoxyosones, which could form fluorescent compounds.