Controversial alkoxyl and peroxyl radical scavenging activity of the tryptophan metabolite 3-hydroxy-anthranilic acid.

3-Hydroxy-anthranilic acid (3-OHAA), a tryptophan metabolite produced in the kynurenine pathway, is an efficient antioxidant towards peroxyl radicals (ROO) derived from the AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) thermolysis. However, self-reactions of ROO can give rise to alkoxyl radicals (RO), which could strongly affect the fate of scavenging reactions. In the present work, we studied the influence of RO in the scavenging activity of 3-OHAA in three different systems: i) Monitoring of the direct reaction between 3-OHAA and AAPH-derived free radicals (kinetic studies); ii) Evaluation of the protective effect of 3-OHAA on the AAPH-induced consumption of fluorescein; and, iii) Inhibition, given by 3-OHAA, of the AAPH-initiated lipid peroxidation of both, rat brain synaptosomes and homogenate preparations (assessed by chemiluminescence). For such purposes, the fraction of free radicals (f) trapped per 3-OHAA molecule was determined in each system. Kinetic results show that the oxidation of 3-OHAA follows a process dominated by ROO with a zero order kinetic limit in 3-OHAA, and a fraction (fri) equal to 0.88. From the induction times, elicited by 3-OHAA in the kinetic profiles of fluorescein consumption, a fraction (fT) of 0.28 was determined. 3-OHAA also generated induction times in the kinetic profiles of light emission during the AAPH-initiated lipid peroxidation of rat brain synaptosomes and homogenates. From such induction times, fractions of 0.61 and 0.63 were determined for rat brain synaptosomes (fsyn) and homogenates (fhom), respectively. These results show that during the incubation of 3-OHAA and AAPH, a low fraction of ROO self-reacts to generate RO. Nevertheless, when 3-OHAA is employed to protect particular targets, such as fluorescein, rat brain synaptosomes and homogenates, reactions of ROO and/or RO should be considered.

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.

Nitroxide amide-BODIPY probe behavior in fibroblasts analyzed by advanced fluorescence microscopy.

A novel synthesized nitroxide amide-BODIPY prefluorescent probe was used to study cellular redox balance that modulates nitroxide/hydroxylamine ratio in cultured human fibroblasts. FLIM quantitatively differentiated between nitroxide states of the cytoplasm-localized probe imaged by TIRF, monitoring nitroxide depletion by hydrogen peroxide; eluding incorrect interpretation if only fluorescence intensity is considered.

Role of alkoxyl radicals on the fluorescein-based ORAC (Oxygen Radical Absorbance Capacity) assay.

During the last decades the ORAC (Oxygen Radical Absorbance Capacity) assay has been widely employed to evaluate the in vitro antioxidant capacity of polyphenol-rich fruits, vegetables and beverages. The method employs fluorescein (FLH) as target molecule and AAPH (2,2'-azo-bis(2-amidinopropane)dihydrochloride) as the source of peroxyl radicals (ROO•). The protection of FLH, afforded by antioxidants (XH), is often characterized by kinetic profiles with clear lag times (LT), which are directly associated with the stoichiometry (n) of the XH-ROO• reaction. However, even for simple phenolic compounds, the LT measured imply large n values (defined as the number of ROO• moles trapped by each antioxidant molecule) which cannot be explained by a simple reaction mechanism. Nonetheless, they can be explained when considering the formation of alkoxyl radicals (RO•) from the recombination of two AAPH-derived ROO•. In the present work, we provide kinetic data showing that, in the zero order kinetic limit of FLH consumption, there is a low reaction rate incompatible with total trapping of ROO•. Thus, the consumption of FLH should be mostly related to its reaction with RO•. In addition, we present data regarding the assumption that in competitive measurements, the LT is due to efficient trapping of the ROO• by the added phenols, leading to high n values (1.7 to 23) for mono and polyphenols. These values are not in agreement with kinetic studies of the antioxidant consumption mediated by the presence of AAPH carried out by HPLC-DAD technique, which imply a competition by RO•. The results suggest that the use of FLH as probe at low concentrations give, for several antioxidants, ORAC values mainly related to their reaction towards RO• radicals instead of primary ROO•radicals.

Mechanism of pyrogallol red oxidation induced by free radicals and reactive oxidant species. A kinetic and spectroelectrochemistry study.

Pyrogallol red (PGR) presents high reactivity toward reactive (radical and nonradical) species (RS). This property of PGR, together with its characteristic spectroscopic absorption in the visible region, has allowed developing methodologies aimed at evaluating the antioxidant capacity of foods, beverages, and human fluids. These methods are based on the evaluation of the consumption of PGR induced by RS and its inhibition by antioxidants. However, at present, there are no reports regarding the degradation mechanism of PGR, limiting the extrapolation to how antioxidants behave in different systems comprising different RS. In the present study, we evaluate the kinetics of PGR consumption promoted by different RS (peroxyl radicals, peroxynitrite, nitrogen dioxide, and hypochlorite) using spectroscopic techniques and detection of product by HPLC mass spectrometry. The same pattern of oxidation and spectroscopic properties of the products is observed, independently of the RS employed. Mass analysis indicates the formation of only one product identified as a quinone derivative, excluding the formation of peroxides or hydroperoxides and/or chlorinated compounds, in agreement with FOX's assays and oxygen consumption experiments. Cyclic voltammetry, carried out at different pH's, shows an irreversible oxidation of PGR, indicating the initial formation of a phenoxy radical and a second charge transfer reaction generating an ortho-quinone derivative. Spectroelectrochemical oxidation of PGR shows oxidation products with identical UV-visible absorption properties to those observed in RS-induced oxidation.

Evaluation of solute binding to proteins and intra-protein distances from steady state fluorescence measurements.

Steady state fluorescence measurements, due to their relative simplicity and fast and easy implementation, are one of the most employed techniques for evaluating the non-covalent binding of small molecules to proteins. In the present review we discuss the main characteristics of solute binding and the experimental procedures that can be employed for evaluating both, the efficiency of the process and the number of binding sites. It is also discussed the possibility of determining the distance between endogenous fluorophores and non-covalently bound solutes. Albumins (human serum albumin and bovine serum albumin) are considered as model proteins due to their relevance as solute carriers, the extensive available data comprising binding of a large number of solutes, and the reduced number of intrinsic fluorophores which simplifies the data treatment. It is shown that, in spite of the apparent simplicity of the systems, extreme care must be exercised in data treatment and interpretation to avoid misleading results. This applies to the evaluation of binding constants, number of binding sites, and average distance between intrinsic fluorophores and non-covalently bound solutes associated to the proteins.

Photobehavior of merocyanine 540 bound to human serum albumin.

The photobehavior of merocyanine 540 (MC) was studied in homogeneous media (ethanol, buffer and glycerol), and in microheterogenous systems (Triton X-100 micelles and in the presence of human serum albumin) using stationary and time-resolved techniques. Merocyanine 540 in aqueous solution mostly forms aggregates, which in the presence of Triton X-100 or HSA are disaggregated. The extent of binding to HSA and its characteristics were estimated from dye absorption and fluorescence changes following protein addition; the Trp-214 fluorescence quenching was also employed to assess the extent of dye association, and physical separation was employed to evaluate the dye's apparent association constant. These results showed that dye adsorption on HSA takes place at both main protein-binding sites (I and II). This adsorption leads to dye monomerization, changing its photobehavior remarkably, with a noticeable increase in fluorescence and triplet lifetimes. These slower decays can be ascribed to a reduction of the dye photoisomerization rate. In addition, the adsorption of the dye partially protects it from the oxygen present in solution, thus reducing the apparent dye triplet-quenching rate constant. However, singlet oxygen and MC triplet quantum yields remain very low in all the systems tested. Finally, we found that the photoconsumption of merocyanine bound to HSA takes place predominantly by a type I mechanism, being more than seven times more efficient than that taking place in ethanol.

Importancia del manejo del ductus arterioso persistente en la XII Región al implementar el diagnóstico ecográfico y tratamiento quirúrgico a nivel local / Surgical management of persistent ductus arteriosus in the Xllth Region of Chile

Introducción: El Ductus Arterioso Persistente (DAP) representa un problema muy importante en los Recién Nacidos de Pretérmino (RNPT) menores de 1500 g. Diversos adelantos en el Servicio de Pediatría del Hospital Regional de Punta Arenas desde 1997 hicieron posible que iniciáramos la resolución local de ésta patología, tanto en Neonatos como en pacientes Pediátricos. Objetivo: Mostrar la experiencia obtenida después de 9 a±os de iniciado el diagnóstico y cirugía del DAP a nivel regional. Metodología: Evaluación prospectiva-retrospectiva de todos los pacientes operados de DAP desde Septiembre de 1997 hasta Agosto de 2006, recolectando información clínica, técnica quirúrgica y complicaciones, seguimiento y sobrevida. Se separan los pacientes en 2 grupos, los RNPT (Grupo A) y los niños de edad pediátrica (Grupo B). Resultados: Se han intervenido quirúrgicamente 16 pacientes del Grupo A y 10 pacientes del Grupo B. La edad al momento de la operación fluctuó entre 9 días y los 6 años 3 meses. En el grupo A, el peso al momento de la cirugía se encontraba entre 527 g y 2062 g. Para el diagnóstico ecocardiográfico se utilizó desde envío de imágenes por video hasta telemedicina con ecocardiografistas experimentados, hasta lograr la experiencia local. El equipo quirúrgico fue apoyado inicialmente por Cirujano Infantil con experiencia en cirugía Ductal, siendo parte del equipo en el 20 por ciento de las cirugías en el grupo A y en el 60 por ciento del grupo B. Se abordó el tórax por vía transpleural en 4 casos y extrapleural en 22 casos, sin dejar drenajes pleurales. No hubo complicaciones significativas. El tiempo de seguimiento varió entre 2 meses y 9 años, y la sobrevida fue de un 96 por ciento, con 1 paciente fallecido hasta Diciembre 2006. Conclusiones: La resolución quirúrgica de ésta patología es posible en el ámbito local, lo cual tiene gran importancia en los casos en que el traslado sea imposible o riesgoso por las condiciones de los enfermos...

Patent ductus arteriosus (PDA) accounts is a very important problem for extreme premature newborns < 1500 g due to a high associated morbi-mortality in this group of patients. After the arrival of a pediatric cardiac surgeon and training in appropriate echocardiography diagnosis, surgical treatment of this condition began at the Regional Hospital in Punta Arenas, Chile. Aim. to repon local results of PDA surgical treatment in neonates and children during a 9 year period. Method. All patients operated on for closure of PDA from September 1997 to August 2006 were included. A retrospective recollection of data, age weight at time of surgery (preterm neonates), means used to diagnose PDA, co-morbidity, surgical technique, complications and survival, was performed. A separate analysis of preterm neonates (PTN) and pediatric age patients (PA) was carried out. Results. 16 PTN and 10 PA patients were operated on. Overall, age at operation ranged from 9 days to 6 years. Weight of PTN ranged from 572 to 2062 g. Diagnosis was confirmed by echocardiography in all but one patient. Echocardiographic findings were discussed via tele-medicine with experienced specialists in many cases. The local surgical team was supported by an experienced surgeon in 20 percent of PTN and 60 percent of PA patients. A transpleural approach was used in 4 and an extrapleural approach in 22 patients; no pleural drainage was used. There were no significant complications. Only 1 patients has died in follow-up ranging from 2 months to 9 years. Conclusions. Local resolution of PDA in neonates and small children has been made possible in Punta Arenas, avoiding dangerous transfer of these patients. Assistance by trained specialists was essential to accomplish this goal. Use of tele-medicine proved to be an important factor to increase safety in the management of PDA.

Influence of the target molecule on the oxygen radical absorbance capacity index: a comparison between alizarin red- and fluorescein-based methodologies.

A comparison of alizarin red (AR) and fluorescein (FL) as target molecules in oxygen radical absorbance capacity (ORAC)-like methods is reported. Galangin, apigenin, ferulic acid, and coumaric acid decreased AR initial consumption rate, whereas quercetin, kaempferol, luteolin, caffeic acid, and sinapic acid inhibited its consumption through an induction time, associated with a repair mechanism. On the other hand, all compounds protected FL with a clear induction time. AR was more selective and provides ORAC-AR values considerably smaller for compounds of low reactivity. The ORAC-AR value for luteolin was nearly 200 times that of coumaric acid. However, the ratio of ORAC-FL values for luteolin and coumaric acid was only 1.2. This different selectivity implies that AR provides ORAC values more related to reactivity than FL. ORAC-AR values of infusions were considerably smaller than the corresponding ORAC-FL values. These differences are interpreted in terms of the capacity of FL to generate induction times, irrespective of the reactivity of the additive. It is proposed that comparison of ORAC-AR and ORAC-FL values could afford a rough estimation of the average reactivity of the antioxidants titrated by the ORAC-FL methodology.

Interaction and reactivity of urocanic acid towards peroxyl radicals.

The capacity of urocanic acid to interact with peroxyl radicals has been evaluated in several systems: oxidation in the presence of a free radical source (2,2'-azobis(2-amidinopropane; AAPH), protection of phycocyanin bleaching elicited by peroxyl radicals, and Cu(II)- and AAPH-promoted LDL oxidation. The results indicate that both isomers (cis and trans) are mild peroxyl radical scavengers. For example, trans-urocanic acid is nearly 400 times less efficient than Trolox in the protection of the peroxyl radical promoted bleaching of phycocyanin. Regarding the removal of urocanic acid by peroxyl radicals, nearly 100 muM trans-urocanic acid is required to trap half of the produced radicals under the employed conditions (10 mM AAPH, 37 degrees C). Competitive experiments show that the cis-isomer traps peroxyl radicals 30% less efficiently than the trans-isomer. Given the high concentrations that trans-urocanic acid reaches in skin, its capacity to trap peroxyl radicals could contribute to the protection of the tissue towards ROS-mediated processes. Furthermore, both isomers, and particularly the cis-isomer, protect LDL from Cu(II)-induced oxidation.

Chemiluminescence associated with amino acid oxidation mediated by hypochlorous acid.

Although most amino acids readily react with hypochlorous acid (HOCl), only the reaction involving tryptophan (Trp) produces a measurable chemiluminescence (CL). Most of this luminescence takes place after total consumption of HOCI when the process is carried out in an excess of Trp. The quantum yield of the process is relatively low (2 x 10(-8) Einstein/mol HOCl reacted). The luminescence is attributed to free radical-mediated secondary reactions of the initially produced chloramines. This is supported by experiments showing that the chloramines produced when HOCl reacts with alanine are able to induce Trp chemiluminescence, and that this luminescence is partially quenched by free radical scavengers. The spectral changes and the effect of pH upon the observed luminescence are compatible with light emission from products produced in the free radical oxidation of Trp.

Kinetics of the chemiluminescence associated to the reaction between peroxyl radicals and proteins.

Protein oxidation, mediated by peroxyl radicals derived from 2,2'-azobis(2-amidinopropane) dihydrochloride is sided by a significant visible chemiluminescence (CL). The light emission shows a complex dependence with the protein concentration and with the incubation time that cannot be interpreted in terms of peroxyl radicals recombination (Russell's mechanism). In all the systems studied, the chemiluminescent behavior requires to consider the participation of several oxidation products as precursors of the excited states. These compounds lead to the formation of excited states by competing radical and nonradical mediated pathways. These intermediates (most probably hydroperoxide-like compounds) would arise from the oxidation of Trp and Tyr residues. This conclusion is based on the similarity of the time profile of the chemiluminescence observed in the oxidation of the free amino acids and the proteins, both in the presence of and absence of free-radical scavengers.

Kinetics and mechanism of the chemiluminescence associated with the free radical-mediated oxidation of amino acids.

When amino acids are incubated in the presence of a free radical source [2,2'-azobis(2-amidinopropane) dihydrocloride], only tyrosine (Tyr) and tryptophan (Trp) produce significant chemiluminescence. The relationship between the observed light intensity, the rate of the oxidation process and the substrate concentration is complex and can not be explained in terms of the formation of excited states in termination processes involving two peroxyl radicals (Russell's mechanism). The observed increase in light emission with the incubation time, for both Trp and Tyr, would indicate the participation of more than one reaction product as intermediates in the pathways leading to the production of excited molecules. However, the fact that after product accumulation a high proportion of the observed luminescence is quenched by Trolox addition, implies that the main chemiluminescent process must involve the interaction of product(s) and free radicals. From the effect of added Ebselen, it is proposed that hydroperoxides and peroxides, formed along the reaction path, are the intermediates whose accumulation leads to the observed increase in chemiluminescence with elapsed time. The observed time profiles and the proposed mechanism strongly resemble those associated with the oxidation of complex biological systems, suggesting that protein oxidation could be one of the main sources of chemiluminescence in biological oxidations.

Acid phosphatase reaction with peroxyl radicals: inactivation mechanism and behavior of the partially modified ensemble.

Acid phosphatase (AP) is readily inactivated when exposed to the free radicals generated in the pyrolysis of 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions. On average, a large number of tryptophan groups are modified by each protein molecule that loses its catalytic activity. In spite of this, the enzyme inactivation takes place without induction times, a result that indicates either that damage is progressive or that damage of a critical target is needed to inactivate the enzyme (all-or-nothing mechanism). A Lineweaver-Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that after exposure of the native protein ensemble to the free radical source there are partially damaged molecules whose affinity for the substrate is widely different from that of the native molecules. On the other hand, the partially damaged ensemble shows a normal Michaelis-Menten behavior when the activity is measured at pH 7.0, with only a reduced value of V(M), relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation studies of the native and pretreated proteins support this proposal.

Kinetics of phycocyanine bilin groups destruction by peroxyl radicals.

Bilin groups in c-phycocyanine are readily bleached by peroxyl radicals produced in the thermolysis of 2, 2'-azobis(2-amidinopropane). From an evaluation of the bilin groups destroyed per radical that interacts with the protein, it is concluded that the bilin moiety is the main target of the radicals. Kinetic expressions are derived that allows an estimation of the substrate reactivity from the analysis of the rate of bilin group modification as a function of the protein concentration. From this analysis it is concluded that micromolar concentrations of c-phycocyanine are able to reduce the steady state concentration of the peroxyl radicals by one half, indicating a high antioxidant activity for this compound. This conclusion is confirmed by measuring the capacity of the protein to protect 1-naphthol from modification by peroxyl radicals. The results obtained show that the bilin groups have, on a molar basis, an antioxidant activity similar to that of potent antioxidants such as catechin.

A comparison of methods employed to evaluate antioxidant capabilities.

Three different methodologies frequently employed to evaluate the indexes that report the antioxidant capabilities of pure compounds and/or complex mixtures of antioxidants are applied to a series of mono- and polyphenols, as well as to two wine (red and white) samples. These methodologies are based on the bleaching of a stable radical, the effect of the additive upon luminol chemiluminescence induced by peroxyl radicals, and the effect of the additive upon the bleaching of the fluorescence from a dye molecule. Widely different responses are obtained from the different methodologies. These differences are interpreted in terms of the different factors (stoichiometric factors and/or reactivities) that determines the indexes evaluated by these different methodologies.

A comparison of methods employed to evaluate antioxidant capabilities

Three different methodologies frequently employed to evaluate the indexes that report the antioxidant capabilities of pure compounds and/or complex mixtures of antioxidants are applied to a series of mono- and polyphenols, as well as to two wine (red and white) samples. These methodologies are based on the bleaching of a stable radical, the effect of the additive upon luminol chemiluminescence induced by peroxyl radicals, and the effect of the additive upon the bleaching of the fluorescence from a dye molecule. Widely different responses are obtained from the different methodologies. These differences are interpreted in terms of the different factors (stoichiometric factors and/or reactivities) that determines the indexes evaluated by these different methodologies.

Interfacial Free Energies and Surface Areas of Cycloalkanols and alpha,omega-Alkanediols at the Air-Water Interface.

The surface area occupied at the air/water interface for cycloalkanols and alpha,omega-alkanediols has been estimated from surface tension measurements. From these data, standard free energies of transfer of the alkanols from the bulk of the solution to the interface were obtained. Cycloalkanols constitute a family whose surface excess diminishes when the size of the alkyl ring increases and whose free energies of transfer at zero surface pressure are similar to those of 1-alkanols of similar hydrophobicities. alpha,omega-Alkanediols occupy considerably larger surface areas than 1-alkanols, a result that can be interpreted in terms of a different geometrical arrangement of the molecules at the interface. In particular, for alkanediols with n > 7, the data suggest an important contribution of bent conformations to the average arrangement of the molecules in the monolayer. The need to adopt bent conformations to allow the interaction of both hydroxyl groups with the aqueous interface results in a less favorable change in free energy when they are transferred from the solution standard state to the interface at constant surface pressure. Copyright 1998 Academic Press.

Fluorescence of 8-Anilinonaphthalene-1-sulfonate and Properties of Sodium Dodecyl Sulfate Micelles in Water-Urea Mixtures

8-Anilinonaphthalene-1-sulfonate (ANS) was used as fluorescence probe to study the effects of urea on properties of sodium dodecyl sulfate micellar solutions. The critical micelle concentration (CMC) of the surfactant, the degree of counterion association to the micelles, and the partition constant of ANS between the micelles and the external medium in the vicinity of the CMC were obtained at different urea concentrations ranging from 0 to 8 M. Complementary results regarding the effects of urea on the micelle aggregation number, the ET(30) parameter, and the partitioning of ANS between n-octanol and water or water-plus-urea mixtures were also obtained to allow critical evaluation of the ANS fluorescence data. It is concluded that the effect of urea on ANS fluorescence at intermediate surfactant concentrations is due mainly to increased stability of the probe in the aqueous medium. Extrapolation of the data at total ANS incorporation into the micelles allows the conclusion that the intramicellar emission increases slightly on urea addition. This result is attributed to an increase in the microviscosity at the micellar surface resulting from urea-micelle interactions.