Analysis of radicals and radical reaction products in cell signalling and biomolecular damage:the long hard road to gold-standard measures

The field of free radical biology and medicine continues to move at a tremendous pace, with a constant flow of ground-breaking discoveries. The following collection of papers in this issue of Biochemical Society Transactions highlights several key areas of topical interest, including the crucial role of validated measurements of radicals and reactive oxygen species in underpinning nearly all research in the field, the important advances being made as a result of the overlap of free radical research with the reinvigorated field of lipidomics (driven in part by innovations in MS-based analysis), the acceleration of new insights into the role of oxidative protein modifications (particularly to cysteine residues) in modulating cell signalling, and the effects of free radicals on the functions of mitochondria, extracellular matrix and the immune system. In the present article, we provide a brief overview of these research areas, but, throughout this discussion, it must be remembered that it is the availability of reliable analytical methodologies that will be a key factor in facilitating continuing developments in this exciting research area.

Free radical reaction products and antioxidant capacity in beating heart coronary artery surgery compared to conventional bypass

Oxygen-derived free radicals are important agents of tissue injury during ischemia and reperfusion. The aim of this study was to investigate changes in protein and lipid oxidation and antioxidant status in beating heart coronary artery surgery and conventional bypass and to compare oxidative stress parameters between the two bypass methods. Serum lipid hydroperoxide, nitric oxide, protein carbonyl, nitrotyrosine, vitamin E, and β-carotene levels and total antioxidant capacity were measured in blood of 30 patients undergoing beating heart coronary artery surgery (OPCAB, off-pump coronary artery bypass grafting) and 12 patients undergoing conventional bypass (CABG, on-pump coronary artery bypass grafting). In the OPCAB group, nitric oxide and nitrotyrosine levels decreased after reperfusion. Similarly, β-carotene level and total antioxidant capacity also decreased after anesthesia and reperfusion. In the CABG group, nitric oxide and nitrotyrosine levels decreased after ischemia and reperfusion. However, protein carbonyl levels elevated after ischemia and reperfusion. Vitamin E, β-carotene, and total antioxidant capacity decreased after ischemia and reperfusion. Significantly decreased nitration and impaired antioxidant status were seen after reperfusion in both groups. Moreover, elevated protein carbonyls were found in the CABG group. The off-pump procedure is associated with lower degree of oxidative stress than on-pump coronary surgery. © 2011 Pleiades Publishing, Ltd.

Free radical reactions in atherosclerosis; An EPR spectrometry study

The copper catalysed oxidation of homocysteine has been studied by electron paramagnetic resonance (EPR) spectroscopy and spin trapping techniques to determine the nature of free radical species formed under varying experimental conditions. Three radicals; thiyl, alkyl and hydroxyl were detected with hydroxyl being predominant. A reaction mechanism is proposed involving Fenton chemistry. Inclusion of catalase to test for intermediate generation of hydrogen peroxide showed a marked reduction in amount of hydroxyl radical generated. In contrast, the addition of superoxide dismutase showed no significant effect on the level of hydroxyl radical formed. Enhanced radical formation was observed at higher levels of oxygen, an effect which has consequences for differential oxygen levels in arterial and venous systems. Implications are drawn for a higher incidence of atherosclerotic plaque formation in arteries versus veins. © 2006 - IOS Press and the authors. All rights reserved.

Atom transfer radical polymerisation - towards the synthesis of a fully-functional photorefractive polymer

Atom transfer radical polymerisation (ATRP) of styrene in xylene solution initiated with 1-phenylethyl bromide and mediated by CuBr/N-propyl-2- pyridinemethanimine catalyst complex was studied. The polymerisation was ill-controlled, yielding polymers with broad molecular weight distributions and values of number average molecular weight considerably higher than the theoretical values calculated from 100% initiator efficiency. The degree of control afforded over the polymerisation was enhanced by use of a more soluble catalyst complex, CuBr/N-octyl-2-pyridinemethanimine. Furthermore, the use of a more polar solvent, diglyme, generated a homogeneous catalyst complex that facilitated the production of polymers having narrow molecular weight distributions (1.10 < PDi < 1.20). The kinetics of the atom transfer radical polymerisation of methyl methacrylate at 90°C in diglyme solution initiated with ethyl-2-bromoisobutyrate and mediated by CuBr/N-octyl-2-pyridinemethanimine was studied and the orders of the reaction were established. The effect on the rate of polymerisation of the ratio of CuBr:N-octyl-2-pyridinemethanimine was also determined. The temperature dependencies of the rate of polymerisation of methyl methacrylate in diglyme solution and xylene solution were studied, and were found to be non-linear and dependent upon the polarity of the solvent. The use of highly polar aprotic solvents, such as N,N-dimethylformamide and dimethylsulphoxide, was found to be detrimental to the degree of control afforded over the polymerisation of methyl methacrylate. This was circumvented by use of a 5-fold excess, over that conventionally used, of catalyst complex. The atom transfer radical polymerisation of (4-nitrophenyl)-[3-[N-[2- (methacryloyloxy)ethyl]carbazolyl]]diazene in dimethyl sulphoxide solution was studied. Although homopolymerisation yielded only oligomers, copolymerisation of this monomer with methyl methacrylate was found to be readily achievable. Keywords: ATRP, Styrene; Methyl methacrylate; Polar solvents; Fully-functional photorefractive polymer. 2

The role of stable nitroxyl radical precursors as antioxidants in polyolefins

Various 2,2,6,6-tetramethyl piperidines and their N-alkyl derivatives of stable nitroxyl radical precursors containing acrylic(s) and methacrylic(s) groups were reactively processed in the presence of a peroxide as bound-antioxidant masterbatches for polyolefin stabilisation. It was found that grafting of the antioxidant monomers onto the polymer backbone was inevitably in competition with homopolymerisation of the monomers as well as melt degradation of the polymer and other side reactions. As previously reported, binding efficiency of bisacrylic nitroxyl precursor was maximum due to formation of unextractable homopolymer of the antioxidant. On the other hand, the binding efficiency of monoacrylic derivatives was low and the homopolymers were found extractable, which suggests that the bound monoacrylic derivatives are entirely grafted onto the polyolefin backbone. Application of bis and tri-functional coagents gave improved binding efficiency of the monoacrylic monomers. This may be due to copolymerisation of the antioxidants with the coagents and grafting of the copolymers onto the polymer backbone. Comparison of photostabilising activity of the fully extracted bound antioxidants to those of the corresponding unbound analogous showed lower results for the former. However, thermal stabilising activity of the bound antioxidants was higher than that of the unbound analogous due to better substantivity. Analysis using physical techniques and GPC for molecular weight distribution of masterbatches containing the bound monoacrylic antioxidants showed formation of high molecular weight products. Model reaction of a secondary amine derivative in liquid hydrocarbon and analysis of the product using FTIR and NMR spectroscopy indicated a possibility of side reaction, i.e. involvement of the amine active group (>N-H) of the antioxidant in the binding process to form the high molecular weight product. Implementation of various N-alkylated derivatives did not inhibit the side reaction. The photostabilising activity of the bound-antioxidants can be improved when applied in conjunction with small amounts of a benzophenone uv-stabiliser. The synergistic stabilising activity, however, was diminished when the uv-stabiliser was removed from the system during the service time. Nitroxyl precursors containing methacrylic group(s) gave lower binding efficiency than the corresponding acrylic derivatives. Reversible deploymerisation of the grafted methacrylic antioxidants may be responsible for this. Bis and tris-acrylic coagents improved the binding efficiency, and the presence of methacrylic group improved stabilising activity of the antioxidants. N-methyl derivatives were found to exhibit better stabilising activity than their parent secondary amine derivatives.

A spectroscope study of some semiquinone radical ions

The effect of substituents on the value of the oxidation potential of quinones is reviewed and attempts to prepare substituted diphenoquinones with high oxidation potentials are reported. Attempts to characterise the mechanism of addition and substitution in diphenoquinones by identifying the products of the Thiele acetylation of diphenoquinone are reported. The reaction proved most efficient when the incoming acetylinium ion is directed by substituents in the diphenoquinone. A 1,8-addition to diphenoquinone is reported and characterised by isolating the products of the reaction between acetyl chloride and diphenoquinone, with perchloric acid as catalyst. The alternating linewidth effects observed in e.s.r.spectra are discussed and applied to account for such effects observed in the e.s.r.spectra of diphenosemiquinone anion and cation radicals. The spectra are analysed and the intramolecular processes producing these effects are discussed. A dianion diradical where intramolecular rotation about the 1 - 1' bond is restricted is produced by the oxidation of 2,2' ,4,4' -tetra hydroxybiphenyl. Previous studies of diphenosemiquinone anions are reviewed and alkylated diphenosemiquinone anion are produced by the reduction of the parent quinone with potassium hydroxide solution, the resulting radical being stabilised by the presence of pyridine. A qualitative interpretation of the solvent-ion effect in alkylated diphenosemiquinone anions is given. Diphanosemiquinone cation radicals are reviewed and previous studies are re-examined.

Radical intermediates in chloroform reactions over triphenylphosphine-protected Au nanoparticles

Reactions of chloroform over triphenylphosphine-protected Au nanoparticles have been studied using electron paramagnetic resonance (EPR) spectroscopy and a spin trapping technique. Two competing reactions, abstraction of hydrogen and halogen atoms, were identified. The hydrogen abstraction reaction showed an inverse kinetic isotope effect. Treatment of nanoparticles with oxidizing or reducing reagents made it possible to tune the selectivity of radical formation from halogen to hydrogen (deuterium) abstraction. Treatment with PbO2 promoted the deuterium abstraction reaction followed by the loss of nanoparticle activity, whereas treatment with NaBH4 regenerated the nanoparticle activity towards Cl atom abstraction. X-ray photoelectron spectroscopy showed an increased Au:P ratio upon treatment with oxidizing reagents. This is likely due to the oxidation of some phosphine ligands to phosphine oxides which then desorb from the nanoparticle surface. © 2009 The Royal Societ of Chemistry.

Influence of reaction atmosphere (H2O, N2, H2, CO2, CO) on fluidized-bed fast pyrolysis of biomass using detailed tar vapor chemistry in computational fluid dynamics

Secondary pyrolysis in fluidized bed fast pyrolysis of biomass is the focus of this work. A novel computational fluid dynamics (CFD) model coupled with a comprehensive chemistry scheme (134 species and 4169 reactions, in CHEMKIN format) has been developed to investigate this complex phenomenon. Previous results from a transient three-dimensional model of primary pyrolysis were used for the source terms of primary products in this model. A parametric study of reaction atmospheres (H2O, N2, H2, CO2, CO) has been performed. For the N2 and H2O atmosphere, results of the model compared favorably to experimentally obtained yields after the temperature was adjusted to a value higher than that used in experiments. One notable deviation versus experiments is pyrolytic water yield and yield of higher hydrocarbons. The model suggests a not overly strong impact of the reaction atmosphere. However, both chemical and physical effects were observed. Most notably, effects could be seen on the yield of various compounds, temperature profile throughout the reactor system, residence time, radical concentration, and turbulent intensity. At the investigated temperature (873 K), turbulent intensity appeared to have the strongest influence on liquid yield. With the aid of acceleration techniques, most importantly dimension reduction, chemistry agglomeration, and in-situ tabulation, a converged solution could be obtained within a reasonable time (∼30 h). As such, a new potentially useful method has been suggested for numerical analysis of fast pyrolysis.

Hydroxyl radical generation by cactus-like copper oxide nanoporous carbon catalysts for microcystin-LR environmental remediation

Copper oxide supported on nanoporous activated carbon (CuO-NPAC) is reported for the aqueous phase catalytic degradation of cyanotoxin microcystin-LR (MC-LR). The loading and spatial distribution of CuO throughout the NPAC matrix strongly influence the catalytic efficiency. CuO-NPAC synthesis was optimized with respect to the copper loading and thermal processing, and the physicochemical properties of the resulting materials were characterized by XRD, BET, TEM, SEM, EPR, TGA, XPS and FT-IR spectroscopy. EPR spin trapping and fluorescence spectroscopy showed in situ ˙OH formation via H2O2 over CuO-NPAC as the catalytically relevant oxidant. The impact of reaction conditions, notably CuO-NPAC loading, H2O2 concentration and solution pH, is discussed in relation to the reaction kinetics for MC-LR remediation.