Eduardo Pino

A Pyranine based Procedure for Evaluation of the Total Antioxidant Potential (TRAP) of Polyphenols: A Comparison with closely related Methodologies

A novel procedure for the evaluation of total reactive antioxidant potentials (TRAP) is described. The method is based on the measurement of the bleaching of pyranine by peroxyl radicals. The addition of the antioxidants produces a clear induction time whose magnitude is directly related to the antioxidant concentration. A comparison of the values obtained with those reported employing closely related methodologies shows that the results are significantly affected by the substrate employed to monitor the steady state free radical concentration. Possible sources of this dependence are discussed.

Quantitative Treatment of the Kinetics of Free‐Radical‐Mediated Damage. Protection by Free‐Radical Scavengers

Equations are derived to quantitatively describe the effect of a free-radical scavenger upon the rate of a radical-mediated process that senses the steady-state free-radical concentration. The dependence of the ratio R°/R (where R° is the rate of the process in the absence of additive) upon the additive concentration depends upon the type of reaction that determines the free-radical lifetime. Normal Stern-Volmer-like behavior is expected only when the lifetime of the radical in the absence of free-radical scavengers is determined by the concentration of the substance employed as the reporter of the free-radical concentration. These predictions are tested in a system comprised of 2,2′-azobis[2-methylpropanimidamide dihydrochloride) as the free-radical source, c-phycocyanin as the reporter molecule, and Trolox (=3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carboxylic acid) tryptophan and 4-methoxyphenol as peroxyl-radical scavengers. The data obtained with Trolox show that it behaves as a nearly ideal free-radical scavenger. On the other hand, the data obtained with tryptophan and 4-methoxyphenol as scavengers show, when plotted according to the Stern-Volmer equation, a strong downward curvature. These results are explained in terms of c-phycocyanin bleaching by scavenger-derived free radicals.

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.

Singlet Oxygen‐mediated Photobleaching of the Prosthetic Group in Hemoglobins and C‐Phycocyanin

Proteins bearing colored prosthetic groups, such as the heme group in hemoglobin or the bilin group in c‐phy‐cocyanin, quench singlet oxygen by interactions at the apoprotein and the prosthetic group levels. In both proteins, chemical modification of the chromophore constitutes only a minor reaction pathway. While total deactivation of singlet oxygen takes place with rate constants of 4.0 times 109and 4.2 times 108M‐ls‐1for hemoglobin and phycocyanin, respectively, the bleaching of the chromophore takes place with rate constants of 3.2 times 106and ˜1 times 107M‐1s‐1. Irradiation of phycocyanin with red light bleaches the chromophore with low yields (˜0.8 times 10‐4). Part of this bleaching is mediated by singlet oxygen produced by the irradiation of the bilin group. The low relevance of the singlet oxygen pathway is compatible with a low quantum yield (˜10‐3) of free singlet oxygen production after irradiation of the protein.

Antioxidant and pro-oxidant effects of red wine and its fractions on Cu(II) induced LDL oxidation evaluated by absorbance and chemiluminescence measurements

Cu(II) mediated low density lipoprotein (LDL) oxidation has been followed by the changes in absorbance at 234 nm and the emitted low level chemiluminescence (CL). The similarity of the time profiles allows us to conclude that the emitted CL is due to the decomposition of a transient product, most likely a hydroperoxide. Red wine, as well as its fractions, afford a noticeable protection when added prior to the start of the LDL oxidation process. On the other hand, when they are added after the onset of the autocatalytic oxidation phase, red wine and its fractions behave as pro-oxidants. This is particularly evidenced by a strong burst of CL (enhancement of the light by a factor ≈ 20). This burst is reduced by metal chelators (EDTA and DFO) and can be associated to a sequence of reactions such as where XOH is a phenolic compound and LOOH is a peroxide-like compound produced in the LDL oxidation.

Coumarin 314 free radical cation: formation, properties, and reactivity toward phenolic antioxidants.

We have explored the photogeneration of the coumarin 314 radical cation by using nanosecond laser excitation at wavelengths longer than 400 nm in benzene, acetonitrile, dichloromethane, and aqueous media. In addition, time-resolved absorption spectroscopy measurements allowed detection of the triplet excited state of coumarin 314 (C(314)) with a maximum absorption at 550 nm in benzene. The triplet excited state has a lifetime of 90 μs in benzene. It is readily quenched by oxygen (k(q) = 5.0 × 10(9) M(-1) s(-1)). From triplet-triplet energy transfer quenching experiments, it is shown that the energy of this triplet excited state is higher than 35 kcal/mol, in accord with the relatively large singlet oxygen quantum yield (Φ(Δ) = 0.25). However, in aqueous media, the coumarin triplet was no longer observed, and instead of that, a long-lived (160 μs in air-equilibrated solutions) free radical cation with a maximum absorbance at 370 nm was detected. The free radical cation generation, which has a quantum yield of 0.2, occurs by electron photoejection. Moreover, density functional theory (DFT) calculations indicate that at least 40% of the electronic density is placed on the nitrogen atom in aqueous media, which explains its lack of reactivity toward oxygen. On the other hand, rate constant values close to the diffusion rate limit in water (>10(9) M(-1) s(-1)) were found for the quenching of the C(314) free radical cation by phenolic antioxidants. The results have been interpreted by an electron-transfer reaction between the phenolic antioxidant and the radical cation where ion pair formation could be involved.

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.

Reaction Kinetics of Phenolic Antioxidants toward Photoinduced Pyranine Free Radicals in Biological Models

8-Hydroxy-1,3,6-pyrenetrisulfonic acid (pyranine, PyOH) free radicals were induced by laser excitation at visible wavelengths (470 nm). The photochemical process involves photoelectron ejection from PyO- to produce PyO• and PyO•- with maxima absorption at 450 and 510 nm, respectively. The kinetic rate constants for phenolic antioxidants with PyO•, determined by nanosecond time-resolved spectroscopy, were largely reliant on the ionic strength depending on the antioxidant phenol/phenolate dissociation constant. Further, the apparent rate constant measured in the presence of Triton X100 micelles was influenced by the antioxidant partition between the micelle and the dispersant aqueous media but limited by its exit rates from the micelle. Similarly, the rate reaction between ascorbic acid and PyO• was markedly affected by the presence of human serum albumin responding to the dynamic of the ascorbic acid binding to the protein.