Studies on the effect of Amadoriase from Aspergillus fumigatus on peptide and protein glycation in vitro.

Amadoriase I is a fructosyl amine oxidase from Aspergillus fumigatus that catalyzes the oxidation of Amadori products (APs) producing glucosone, H2O2, and the corresponding free amine. All the enzymes of this family discovered so far only deglycate small molecular weight products and are inactive toward large molecular weight substrates, such as glycated BSA or ribonuclease A. Therefore, they cannot be used to reverse protein glycation occurring in diabetes or in foods. In this paper, the effect of Amadoriase I added during the in vitro reaction between glucose and peptides having different polarities or proteins with molecular weights ranging from to 5 to 66 kDa was tested. The formation of APs was monitored by ESI-MS of intact glycated protein or peptides and by measuring the Nepsilon-(1-deoxy-d-fructos-1-yl)-L-lysine and furosine concentrations. Results showed that the formation of APs is reduced up to 80% when peptides and glucose are incubated in the presence of Amadoriase. The effect is more evident for hydrophobic peptides. In protein-glucose systems, the effect was dependent on the molecular weight and steric hindrance being negligible for BSA and at a maximum for insulin, where the formation of APs was reduced up to 60%. These findings indicate new potential applications of Amadoriase I as an efficient tool for inhibiting protein glycation in real food systems.

Glycated fibroblast growth factor-2 is quickly produced in vitro upon low-millimolar glucose treatment and detected in vivo in diabetic mice.

Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control; however, molecular mechanisms involved are still unclear. Fibroblast growth factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high-sugar exposure. Therefore, the purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot, and competitive ELISA experiments demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mm glucose exposure. In addition, FGF-2 exposed for 30 min to 12.5 mm glucose lost mitogenic and chemotactic activity in a time- and dose-dependent manner. Under similar conditions, binding affinity to FGF receptor 1 was dramatically reduced by 20-fold, as well as FGF receptor 1 and ERK-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and chorioallantoic-membrane) assays. Such glucose-induced modification was specific, because other angiogenic growth factors, namely platelet-derived growth factor BB and placental-derived growth factor were not significantly or markedly less modified. Finally, for the first time, glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic nonobese diabetic mice, in significantly higher amounts than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features, and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM.

Effect of cooking on the concentration of Vitamins B in fortified meat products.

B vitamins fortification of meat products is useful to compensate the loss of these compounds occurring during the heat treatment. The objective of this study was to evaluate the influence of heat treatments on the B vitamins concentration in fortified meat products. A rapid and reliable method for the simultaneous determination of Vitamins B(1), B(6) and B(12) in homogenized boiled ham and in various fortified burgers was set up. Extraction procedure and HPLC method ensure low detection limits, good sensitivity and resolution. Results showed that cooking processes caused a decrease in the B vitamins content both in mild (70-90 degrees C) and severe (120 degrees C) conditions. Performing a fortification of 25 microg g(-1) the residual concentration of B vitamins after cooking allow to reach the recommended daily allowance, thus suggesting that B vitamins fortification of meat product is an useful practice.

Glycation of lysine-containing dipeptides.

Protein glycation through Maillard reaction (MR) is a fundamental reaction both in foods and in the human body. The first step of the reaction is the formation of Amadori product (AP) that is converted into intermediate and advanced MR products during reaction development. Although the MR is not an enzymatic reaction, a certain degree of specificity in the glycation site has been observed. In the present study, we have monitored the glycation of different lysine-containing dipeptides to evaluate the influence on the NH(2) reactivity of the neighboring amino acid.Lysine dipeptides were reacted with glucose, galactose, lactose and maltose. The formation and identification of glycated compounds were monitored by mass spectrometry (MALDI-TOF and ESI-MS/MS) and by HPLC of their Fmoc derivatives. MS/MS analysis showed that the glucose APs formed on dipeptides have a characteristic fragmentation pattern: the fragment at [M - 84](+) due to the formation of pyrylium and furylium ion is mainly present in the monoglucosylated form, while the [M - 162](+) and the [M - 324](+) are more evident in the fragmentation pattern of the diglucosylated forms. The nature of the vicinal amino acids strongly affects lysine reactivity towards the different carbohydrates: the presence of hydrophobic residues such as Ile, Leu, Phe strongly increases lysine reactivity. Contrasting results were obtained with basic residues. The Lys-Arg dipeptide was among the most reactive while the Lys-Lys was not.

Substrate specificity of amadoriase I from Aspergillus fumigatus.

Generation of Amadori products is the major single modification by the Maillard reaction in vivo and a source of biologically active glycoxidation products leading to protein cross-linking in biological tissues. Amadoriase I from Aspergillus fumigatus cleaves Amadori products into deoxyglucosone, hydrogen peroxide, and the corresponding primary amine. It has been reported that Amadori products formed on free amino acids are a good substrate for amadoriase I, whereas the enzyme is unable to cleave Amadori products formed on whole proteins. This work aims to investigate the affinity of amadoriase I for oligopeptides and small proteins. Recombinant amadoriase I was expressed in E. coli and purified by Ni His-tag affinity chromatography. Di-, tri-, and tetrapeptides were derivatized with glucose, and the corresponding Amadori products were purified by TLC and HPLC. Glycated beta-lactoglobulin was also used as a substrate. In both cases the formation of Amadori products was monitored by electrospray ionization-mass spectrometry (ESI-MS). The Km of amadoriase for glycated-L-lysine was 4.2 mM, which is in accordance with the literature. Km decreases with the length of peptide, being slightly reduced for dipeptides, and is around 10 mM for tri- and tetrapeptides. Glycated proteins are not substrates of the enzyme; but when amadoriase I was added during the glycation reaction, a significant reduction of Amadori product formation was observed on both peptides and proteins. Data confirm the hypothesis that steric hindrance is critical for amadoriase I activity, indicating that oligopeptides up to four amino acids in length are good substrates. Moreover, these data show that, at least in some experimental conditions, amadoriase I is able to reduce the formation of protein-bound Amadori product.

Chemical characterization and antioxidant properties of coffee melanoidins.

Melanoidins, the brown polymers formed through Maillard reaction during coffee roasting, constitute up to 25% of the coffee beverages' dry matter. In this study chemical characterization of melanoidins obtained from light-, medium-, and dark-roasted coffee beans, manufactured from the same starting material, was performed. Melanoidins were separated by gel filtration chromatography and studied by MALDI-TOF mass spectrometry. Results showed that the amount of melanoidins present in the brews increased as the intensity of the thermal treatment increased, while their molecular weight decreased. The antioxidant activity of melanoidins isolated from the different brews was studied by using different methodologies. Melanoidins antiradical activity determined by ABTS(*)(+) and DMPD(*)(+) assays decreased as the intensity of roasting increased, but the ability to prevent linoleic acid peroxidation was higher in the dark-roasted samples. Data suggest that melanoidins must be carefully considered when the relevance of coffee intake in human health is studied.