Investigations on the Reaction of C3 and C6 α-Dicarbonyl Compounds with Hydroxytyrosol and Related Compounds under Competitive Conditions

α-Dicarbonyl compounds are intermediates in reactions that lead to the formation of potentially harmful advanced glycation end-products. Carbonyl-trapping capacities of antiglycative substances have been traditionally limited to C2 and C3 α-dicarbonyl structures. Glyoxal (GO)-, methylglyoxal (MGO)-, 3-deoxyglucosone (3-DG)-, 3-deoxygalactosone (3-DGal)-, 3,4-dideoxyglucoson-3-ene-, and glucosone-trapping capacities of hydroxytyrosol (HT), hydroxytyrosol acetate (HTA), and 3,4-dihydroxyphenylacetic acid (DOPAC) in simple (phenolic/dicarbonyl) and competitive model systems (phenolic/dicarbonyl/dicarbonyl) were investigated. HT and HTA were more effective for MGO than 3-DG and 3-DGal. Furthermore, DOPAC exerted higher trapping capacity than HT and HTA for C3 and C6 α-dicarbonyl compounds. In the competitive systems, HT-related substances did not show preference for trapping 3-DG or 3-DGal and behaved as in the simple systems. In the presence of MGO, however, HT-related substances were more effective for trapping MGO than C6 structures. The results demonstrate the C6 α-dicarbonyl-trapping capacities of HT, HTA, and DOPAC, with DOPAC exerting the highest activity.Peer Reviewe

Identification of potential bioactive peptides in sheep milk kefir through peptidomic analysis at different fermentation times

Sheep farming is an important socioeconomic activity in most Mediterranean countries, particularly Spain, where it contributes added value to rural areas. Sheep milk is used in Spain mainly for making cheese, but it can be used also for making other dairy products, such as the lactic-alcoholic fermentation product known as kefir. Dairy products have health benefits because, among other reasons, they contain molecules with biological activity. In this work, we performed a proteomics strategy to identify the peptidome, i.e., the set of peptides contained in sheep milk kefir fermented for four different periods of time, aiming to understand changes in the pattern of digestion of milk proteins, as well as to identify potential bioactive peptides. In total, we identified 1942 peptides coming from 11 different proteins, and found that the unique peptides differed qualitatively among samples and their numbers increased along the fermentation time. These changes were supported by the increase in ethanol, lactic acid, and D-galactose concentrations, as well as proteolytic activity, as the fermentation progressed. By searching in databases, we found that 78 of the identified peptides, all belonging to caseins, had potential biological activity. Of these, 62 were not previously found in any milk kefir from other animal species. This is the first peptidomic study of sheep milk kefir comprising time-course comparison

Methionine sulfoxide profiling of milk proteins to assess the influence of lipids on protein oxidation in milk

Thermal treatment of milk and milk products leads to protein oxidation, mainly the formation of methionine sulfoxide. Reactive oxygen species, responsible for the oxidation, can be generated by Maillard reaction, autoxidation of sugars, or lipid peroxidation. The present study investigated the influence of milk fat on methionine oxidation in milk. For this purpose, quantitative methionine sulfoxide profiling of all ten methionine residues of β-lactoglobulin, α-lactalbumin, and αs1-casein was carried out by ultrahigh-performance liquid chromatography–electrospray ionization tandem mass spectrometry with scheduled multiple reaction monitoring (UHPLC–ESI–MS/MS–sMRM). Analysis of defatted and regular raw milk samples after heating for up to 8 min at 120 °C and analysis of ultrahigh-temperature milk samples with 0.1%, 1.5%, and 3.5% fat revealed that methionine oxidation of the five residues of the whey proteins and of residues M 123, M 135, and M 196 of αs1-casein was not affected or even suppressed in the presence of milk fat. Only the oxidation of residues M 54 and M 60 of αs1-casein was promoted by lipids. In evaporated milk samples, formation of methionine sulfoxide was hardly influenced by the fat content of the samples. Thus, it can be concluded that lipid oxidation products are not the major cause of methionine oxidation in milk

Identification of hydrogen peroxide as a major cytotoxic component in Maillard reaction mixtures and coffee

The cytotoxic activity of Maillard reaction products and coffee was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and the neutral red uptake (NRU) assay. Equimolar mixtures of sugars and lysine were heated at 120°C and used to stimulate bovine aorta endothelial cells for 24 h. The cytotoxic activity increased with increase in educt concentration and heating time. Mixtures containing ribose were most active, followed by lactose and glucose. Hydrogen peroxide, which was present in the Maillard mixtures in concentrations between 7 and 87 µM, was identified as one of their major cytotoxic components. H2O2-concentrations increased further up to 130 lM under cell culture conditions. Filter coffee, espresso, and green coffee extract reduced cell viability significantly to 10, 19, and 83% of PBS-treated control. The effect was largely attenuated by the addition of catalase. Nil, 33, and 41 lM H2O2was measured in green coffee extract, filter coffee, and espresso, respectively, increasing to 13, 369, and 333 µM during cell culture conditions. No additional H2O2 formation was detected when coffee was incubated for up to 5 h without further treatment. In conclusion, hydrogen peroxide is a major product in Maillard mixtures and coffee inducing cell death in vitro

DNA damage by ribose: Inhibition at high ribose concentrations

148-156Nucleobases and DNA react non-enzymatically with sugars, and generate DNA-advanced glycation end products (DNA-AGEs). Incubation of plasmid pBr322 with ribose for 3-7 days caused the transformation of the supercoiled plasmid to the open circular and linear forms. Removal of sugar after an initial 24 h incubation resulted in marked enhancement in the transformation rate. Enhancement in transformation was also observed when bovine serum albumin (BSA) exposed to ribose for short durations was incubated with plasmid in absence of the sugar. The effect on DNA was attenuated when excess ribose remained in the incubation mixture of ribose and protein. The data suggested that an increase in ribose concentration in the vicinity of DNA could be damaging and the damage exacerbated, if sugar levels fell subsequently. Incubation of herring sperm DNA with ribose resulted in a concentration and time-dependent increase of N2-carboxyethyl-2’-deoxyguanosine (CEdGA,B). The concentration of CEdGA,B, however, did not increase further when ribose was removed from the reaction mixture.</b

Aged garlic extract and S-allyl cysteine prevent formation of advanced glycation endproducts

Hyperglycaemia causes increased protein glycation and the formation of advanced glycation endproducts which underlie the complications of diabetes and ageing. Glycation is accompanied by metal-catalysed oxidation of glucose and Amadori products to form free radicals capable of protein fragmentation. Aged garlic extract is a potent antioxidant with established lipid-lowering effects attributed largely to a key ingredient called S-allyl cysteine. This study investigated the ability of aged garlic extract and S-allyl cysteine to inhibit advanced glycation in vitro. Bovine serum albumin (BSA) was glycated in the presence of Cu2+ ions and different concentrations of aged garlic extract and protein fragmentation was examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Lysozyme was glycated by glucose or methylglyoxal in the presence of different concentrations of aged garlic extract or S-allyl cysteine with subsequent analysis of glycation-derived crosslinking using SDS-PAGE. Amadori-rich protein was prepared by dialysing lysozyme that had been glycated by ribose for 24 h. This ribated lysozyme was reincubated and the effects of aged garlic extract, S-allyl cysteine and pyridoxamine on glycation-induced crosslinking was monitored. Aged garlic extract inhibited metal-catalysed protein fragmentation. Both aged garlic extract and S-allyl cysteine inhibited formation of glucose and methylglyoxal derived advanced glycation endproducts and showed potent Amadorin activity when compared to pyridoxamine. S-allyl cysteine inhibited formation of carboxymethyllysine (CML), a non-crosslinked advanced glycation endproduct derived from oxidative processes. Further studies are required to assess whether aged garlic extract and S-allyl cysteine can protect against the harmful effects of glycation and free radicals in diabetes and ageing