Eva Dorta

The ORAC (oxygen radical absorbance capacity) index does not reflect the capacity of antioxidants to trap peroxyl radicals

In the present work we demonstrate from kinetic studies that under the experimental conditions proposed for the ORAC protocol, ORAC values do not correlate with the capacity of antioxidants to trap peroxyl radicals (ROO˙), suggesting a dominant role of alkoxyl radicals (RO˙) in the assay.

Oxidation of free, peptide and protein tryptophan residues mediated by AAPH-derived free radicals: Role of alkoxyl and peroxyl radicals

The oxidation of tryptophan (Trp) residues, mediated by peroxyl radicals (ROO˙), follows a complex mechanism involving free radical intermediates, and short chain reactions. The reactivity of Trp towards ROO˙ should be strongly affected by its inclusion in peptides and proteins. To examine the latter, we investigated (by fluorescence) the kinetic of the consumption of free, peptide- and protein-Trp residues towards AAPH (2,2′-azobis(2-amidinopropane)dihydrochloride)-derived free radicals. Interestingly, the initial consumption rates (Ri) were only slightly influenced by the inclusion of Trp in small peptides and proteins (human serum albumin and human superoxide dismutase). Depending on the Trp concentration, the Riversus Trp concentration ([Trp]) plots showed three regions. At low Trp concentrations (1–10 μM), a linear dependence was observed between Ri and [Trp]; at intermediate Trp concentrations (10–50 μM), the values of Ri were nearly constant; and at high Trp concentrations (50 μM to 1 mM), a slower increase of Ri than expected for chain reactions. Similar behavior was detected for all three systems (free Trp, and Trp in peptides and proteins). For the first time we are showing that alkoxyl radicals, formed from self-reaction of ROO˙, are responsible of the Trp oxidation at low concentrations, while at high Trp concentrations, a mixture of peroxyl and alkoxyl radicals are involved in the oxidation of Trp residues.

α- and β- casein aggregation induced by riboflavin-sensitized photo-oxidation occurs via di-tyrosine cross-links and is oxygen concentration dependent

Type I photo-oxidation generates Trp-(TrpN) and Tyr-derived (TyrO) radicals in proteins which can dimerize producing cross-links, or alternatively react with O2. It was therefore hypothesized that the O2 concentration may have a significant effect on dye-photosensitized reactions. We studied photo-oxidation of α- and β-caseins induced by riboflavin (RF), a photosensitizing vitamin present in milk, under aerobic and anaerobic conditions. Triplet-state RF induced oxidative modifications on both caseins, and significant levels of cross-links. The extent of damage, and the yield of cross-links versus oxidized products, was dependent on the O2 concentration. In the absence of O2, the overall extent of damage was decreased, but the yield of cross-linked products was significantly elevated. These cross-links are consistent with inter- and intra-molecular di-Tyr or di-Trp bridges. Alternative cross-links were detected in the presence of O2, consistent with pathways involving the reaction of protein radicals with O2 or O2-.

Reaction of tetracycline with biologically relevant chloramines

Helicobacter pylori (H. pylori) infection triggers inflammatory processes with the consequent production of hypochlorous acid (HOCl), monochloramine (NH2Cl), and protein-derived chloramines. As the therapy for eradicating H. pylori is partially based on the use of tetracycline, we studied the kinetic of its consumption elicited by HOCl, NH2Cl, N-chloro-n-butylamine (NHCl-But, used as a lysine-derived chloramine model), and lysozyme-derived chloramines. In the micromolar concentration range, tetracycline reacted rapidly with HOCl, generating in the first few seconds intermediates of short half-life. In contrast, a slow tetracycline consumption was observed in the presence of high NH2Cl and NHCl-But concentrations (millimolar range). Similar chlorinated products of tetracycline were identified by mass spectrometry, in the presence of HOCl and NH2Cl. These results evidenced that tautomers of tetracycline are pivotal intermediates in all reactions. In spite of the low reactivity of chloramines towards tetracycline, it is evident that, in the concentration range where they are produced in a H. pylori infection (millimolar range), the reactions lead to oxidation and/or chlorination of tetracycline. This kind of reactions, which were also observed triggered by lysozyme-derived chloramines, could limit the efficiency of the tetracycline-based therapy.

Cytoprotective Mechanisms Mediated by Polyphenols from Chilean Native Berries against Free Radical-Induced Damage on AGS Cells

The prevalence of cytoprotective mechanisms induced by polyphenols such as activation of intracellular antioxidant responses (ICM) and direct free radical scavenging was investigated in native Chilean species of strawberries, raspberries, and currants. Human gastric epithelial cells were co- and preincubated with polyphenolic-enriched extracts (PEEs) from Chilean raspberries (Rubus geoides), strawberries (Fragaria chiloensis ssp. chiloensis f. chiloensis), and currants (Ribes magellanicum) and challenged with peroxyl and hydroxyl radicals. Cellular protection was determined in terms of cell viability, glyoxalase I and glutathione s-transferases activities, and carboxymethyl lysine (CML) and malondialdehyde levels. Our results indicate that cytoprotection induced by ICM was the prevalent mechanism for Rubus geoides and F. chiloensis. This agreed with increased levels of glyoxalase I and glutathione S-transferase activities in cells preincubated with PEEs. ORAC index indicated that F. chiloensis was the most efficient peroxyl radical scavenger. Moreover, ICM mediated by F. chiloensis was effective in protecting cells from CML accumulation in contrast to the protective effects induced by free radical scavenging. Our results indicate that although both polyphenol-mediated mechanisms can exert protective effects, ICM was the most prevalent in AGS cells. These results suggest a potential use of these native berries as functional food.

Use of Banana Peel Extract To Stabilise Antioxidant Capacity and Sensory Properties of Orange Juice During Pasteurisation and Refrigerated Storage

Banana peel extract (BPE) was added to freshly squeezed orange juices (FSJ) and orange juices from concentrate (JFC), in order to evaluate if it stabilises their antioxidant activity, sensory attributes and physicochemical characteristics after heat treatment and during refrigerated storage. Mild pasteurisation increased shelf life in refrigeration for both types of orange juice, although JFC was more stable than FSJ. Antioxidant activity, determined as ABTS•+ scavenging capacity, tends to decrease with refrigeration time. However, when BPE was added, pasteurised juices tend to have greater capacity to scavenge ABTS•+ over time. No relevant effects were observed on the capacity to inhibit lipid peroxidation. Panellists found it extremely difficult to detect differences between juices with BPE and conventional ones. Moreover, panellists found the juices with BPE were more acceptable than conventional ones, although some modifications became more evident over time. The sensory quality of the JFC remained more stable during storage than that of FSJ. Juice colour changed slightly after heat treatment, but these changes tended to disappear over time. Adding BPE increased the total soluble solid content in all FSJ and in unpasteurised JFC. Adding BPE or pasteurising did not produce significant changes in pH in either type of juice. An increase of pH and acidity was detected in all the FSJ (except in acidity of juices without BPE) with time of refrigeration.