试论茶多酚清除生物自由基的高效性

从生物自由基出发,本文综述了茶多酚(TP)清除自由基的效能.茶多酚对多种自由基具有卓越的清除特性,并明显优于其它抗氧化剂,但茶多酚浓度、体系pH值、自由基类型、儿茶素组成与结构对其清除效能有较大影响.     

Pulsed UV Laser Generated Short-Lived free Radicals from Biological Samples

EPR characterization of the short-lived free radicals generated by pulsed UV laser ablation of biological samples has been investigated using a spin trap method. The obtained EPR spectra suggest that the trapped short-lived free radicals generated by excimer laser ablation of collagen and myocardium are identical. The obtained results are discussed in association with the production scheme of free radicals and an empirical mechanism of laser generated short-lived free radicals.     

Radical-radical reactions of superoxide: a potential route to toxicity

Superoxide reacts with many radicals, such as phenoxyl radicals, at near diffusion-controlled rates. These reactions are usually considered to be repair processes and have received little biological attention. However, addition of superoxide to give hydroperoxides and secondary oxidation products can also occur. The relative contributions of addition and repair vary depending on the properties of the phenol. With tyrosine, addition to give tyrosine hydroperoxide predominates, but in peptides the efficiency of hydroperoxide formation depends on the proximity of free amine groups. Radicals from other phenolic compounds, such as alpha-tocopherol and serotonin, also undergo addition reactions with superoxide. Physiologically, these reactions are likely to be more significant than dimerization when both radicals are generated together. They warrant attention as potential contributors to superoxide toxicity.     

Effects of Hyperthermic Conditions on the Reactivity of Oxygen Radicals

Generation and reactivity of superoxide (0₂^?) and hydroxyl (OH') radicals in enzymatic and radiolytic systems were investigated over the temperature range from 20^o-50^oC. The generation rate and reaction kinetics of both enzymatically and radiolytically produced superoxide radicals were determined by a cytochrome c reduction assay. For OH' radical reaction studies the degradation of hyaluronic acid was assayed. An increase in temperature leads to a greater reactivity of both radicals, but in the case of an enzymatic source a disproportionate increase in the rate of generation is observed. In the pulse radiolysis system, the reactivity of superoxide radicals was found to be stimulated 15-fold over the temperature range from 20^oC to 60^oC, although the activity of superoxide dismutase was only minimally increased (about 1.6-fold). The results are discussed with respect to the possible importance of active oxygen species to the biological effects of hyperthermia.     

LC/ESR/MS study of spin trapped carbon-centred radicals formed from in vitro lipoxygenase-catalysed peroxidation of γ-linolenic acid

γ-Linolenic acid (GLA) has been reported as a potential anti-cancer and anti-inflammatory agent and has received substantial attention in cancer care research. One of the many proposed mechanisms for GLA biological activity is free radical-mediated lipid peroxidation. However, no direct evidence has been obtained for the formation of GLA-derived radicals. In this study, a combination of LC/ESR and LC/MS was used with α-[4-pyridyl-1-oxide]-N-tert-butyl nitrone (POBN) to profile the carbon-centred radicals that are generated in lipoxygenase-catalysed GLA peroxidation. A total of four classes of GLA-derived radicals were characterized including GLA-alkyl, epoxyallylic, dihydroxyallylic radicals and a variety of carbon-centred radicals stemming from the β-scissions of GLA-alkoxyl radicals. By means of an internal standard in LC/MS, one also quantified each radical adduct in all its redox forms, including an ESR-active form and two ESR-silent forms. The results provided a good starting point for ongoing research in defining the possible biological effects of radicals generated from GLA peroxidation.     

Radical Scavenging by Flavonoid Antioxidants

Aroxyl radicals of fifteen structurally distinct flavonoids were generated by attack of azide radicals (N₃) on the parent compounds dissolved in aqueous solution at pH 11.5. Generation rate constants were all found to be very high (2.4 – 8.8 × 10⁹dm³mol^?1s^?1), whereas the decay rates differed considerably, ranging from 10⁵ to 108dm3mol^?1s^?1. In most cases the spectral characteristics of the transient aroxyl radicals relate to structural features of the parent compounds and according to spectral similarities they can be classed in three distinct groups (with only two exceptions).

Although the data do not conclusively prove that the biological function of flavonoids might be the scavenging of radicals, the very high rate constants of formation and the relative stability of some of the aroxyl radicals, are in support of such a hypothesis.     

The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound “crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress.

Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.     

Magnetic fields,radicals and cellular activity

Some effects of low-intensity magnetic fields on the concentration of radicals and their influence on cellular functions are reviewed. These fields have been implicated as a potential modulator of radical recombination rates. Experimental evidence has revealed a tight coupling between cellular function and radical pair chemistry from signaling pathways to damaging oxidative processes. The effects of externally applied magnetic fields on biological systems have been extensively studied, and the observed effects lack sufficient mechanistic understanding. Radical pair chemistry offers a reasonable explanation for some of the molecular effects of low-intensity magnetic fields, and changes in radical concentrations have been observed to modulate specific cellular functions. Applied external magnetic fields have been shown to induce observable cellular changes such as both inhibiting and accelerating cell growth. These and other mechanisms, such as cell membrane potential modulation, are of great interest in cancer research due to the variations between healthy and deleterious cells. Radical concentrations demonstrate similar variations and are indicative of a possible causal relationship. Radicals, therefore, present a possible mechanism for the modulation of cellular functions such as growth or regression by means of applied external magnetic fields.     

Hydroxylation of aromatic compounds as indices of hydroxyl radical production: A cautionary note revisited

While setting up an intracerebral microdialysis system to estimate the extent of oxidative stress induced by the neurotoxin, N-methylphenylpyridinium ion (MPP⁺), we encountered a problem in the use of hydroxybenzoic acids as traps of hydroxyl radicals. Using either 2-hydroxybenzoate (salicylate) or 4-hydroxybenzoate as trapping agents, we observed a nonspecific, that is, nontissue derived, production of hydroxyl radicals as measured by the hydroxylation products, 2,3- and 2,5-dihydroxybenzoate from 2-hydroxybenzoate and 3,4-dihydroxybenzoate from 4-hydroxybenzoate. This production of dihydroxybenzoates was 10 times that expected due to the administration of MPP⁺, thus making it impossible to interpret our results. Careful investigation of the various components of the microdialysis system indicated that contact of the microdialysate with metal surfaces resulted in dihydroxybenzoic acid formation. These results should serve as a reminder to perform stringent tests of the experimental system prior to experiments with biological tissues to evaluate the contribution of hydroxyl radical production from nonbiological sources. Therefore, along with the possibility of enzymatic production of dihydroxybenzoates, artefactual production by components of the experimental apparatus must be considered before assuming that one is measuring hydroxyl radical production by a biological system.     

How to Characterize a Biological Antioxidant

An antioxidant is a substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate. Many substances have been suggested to act as antioxidants in vivo, but few have been proved to do so. The present review addresses the criteria necessary to evaluate a proposed antioxidant activity. Simple methods for assessing the possibility of physiologically-feasible scavenging of important biological oxidants (superoxide, hydrogen peroxide, hydroxyl radical, hypochlorous acid, haem-associated ferryl species, radicals derived from activated phagocytes, and peroxyl radicals, both lipid-soluble and water-soluble) are presented, and the appropriate control experiments are described. Methods that may be used to gain evidence that a compound actually does function as an antioxidant in vivo are discussed. A review of the pro-oxidant and anti-oxidant properties of ascorbic acid that have been reported in the literature leads to the conclusion that this compound acts as an antioxidant in vivo under most circumstances.     

The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound “crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress.Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.     

Mechanism and regulation of peroxidase-catalyzed nitric oxide consumption in physiological fluids: Critical protective actions of ascorbate and thiocyanate

Catalytic consumption of nitric oxide (NO) by myeloperoxidase and related peroxidases is implicated as playing a key role in impairing NO bioavailability during inflammatory conditions. However, there are major gaps in our understanding of how peroxidases consume NO in physiological fluids, in which multiple reactive enzyme substrates and antioxidants are present. Notably, ascorbate has been proposed to enhance myeloperoxidase-catalyzed NO consumption by forming NO-consuming substrate radicals. However, we show that in complex biological fluids ascorbate instead plays a critical role in inhibiting NO consumption by myeloperoxidase and related peroxidases (lactoperoxidase, horseradish peroxidase) by acting as a competitive substrate for protein-bound redox intermediates and by efficiently scavenging peroxidase-derived radicals (e.g., urate radicals), yielding ascorbyl radicals that fail to consume NO. These data identify a novel mechanistic basis for how ascorbate preserves NO bioavailability during inflammation. We show that NO consumption by myeloperoxidase Compound I is significant in substrate-rich fluids and is resistant to competitive inhibition by ascorbate. However, thiocyanate effectively inhibits this process and yields hypothiocyanite at the expense of NO consumption. Hypothiocyanite can in turn form NO-consuming radicals, but thiols (albumin, glutathione) readily prevent this. Conversely, where ascorbate is absent, glutathione enhances NO consumption by urate radicals via pathways that yield S-nitrosoglutathione. Theoretical kinetic analyses provide detailed insights into the mechanisms by which ascorbate and thiocyanate exert their protective actions. We conclude that the local depletion of ascorbate and thiocyanate in inflammatory microenvironments (e.g., due to increased metabolism or dysregulated transport) will impair NO bioavailability by exacerbating peroxidase-catalyzed NO consumption.     

Gamma radiolysis as a tool to study lipoprotein oxidation mechanisms

Well-defined quantities of *OH, O2*-,HO2* or RO2*)radicals (reactive oxygen species) can be specifically produced by radiolysis of water or ethanol. Such radical species can initiate one-electron oxidation or one-electron reduction reactions on numerous biological systems. The oxidative hypothesis of atherosclerosis classically admits the involvement of the oxidation of low density lipoproteins (LDLs) but also of high density lipoproteins (HDLs) in the development of the atherosclerotic process. The initiation mechanisms of this oxidation are still incompletely defined, although free radicals are likely involved. Therefore, gamma-radiolysis appears as a method of choice for the in vitro study of the mechanisms of oxidation of LDLs and HDLs by oxygen-centred free radicals (*OH, O2*-,HO2* and RO2*). Radiolytically oxidized lipoproteins exhibited a very well defined oxidation status (radiation dose-dependent quantification of vitamin E, beta-carotene, lipid peroxidation, protein carbonylation ...). gamma-Radiolysis is a less drastic method than other oxidation procedures such as for example copper ions. Moreover, gamma-radiolysis is also especially suitable for studying the reducing properties of antioxidant compounds with regard to their scavenging capacity.     

Homolytic carbon to phosphorus bond scission of some phosphonates catalyzed by bacterial carbon-phosphorus lyase

The production of volatile degradation products of phosphonates was monitored to investigate the mechanism involved in the biodegradation of propylphosphonic acid and phenylphosphonic acid byRhizobium sp MMM101a. The biodegradation of propylphosphonic acid gave rise to the production, in decreasing order, of propane, methane, ethane, 1-butene, propene, isobutene, butane and ethene. The formation of these degradation products was strongly reduced by adding catalase to the growing cultures indicating the involvement of peroxides in the biodegradation mechanism. OH⁰ radical scavengers did not reduce the rate of biodegradation, and therefore these radicals appear not to be involved. Addition of ascorbate, a known hydroxylating agent in biological systems, increased the amount of biodegradation products. The involvement of iron in the degradation was indicated and was optimal at a concentration of 950 µM. This suggests the involvement of a metalloenzyme involving iron and peroxide. The decomposition of phenylphosphonic acid yielded benzene and biphenyl. No phenol could be detected, again suggesting that OH⁰ radicals were not involved in the biodegradation. The presence of deuterated benzene did not result in the occurrence of biphenyl consisting of one nondeuterated and a deuterated ring, which is chemically more likely. It therefore appears that the degradation of the phosphonates occurs on a multicentered enzyme. The diversity of the products generated by this bacterium from phosphonates, many of them due to rearrangement of the carbon moiety of the substrate molecule, suggests an overall involvement of superoxide radicals in the homolytic carbon to phosphorus bond scission.     

Commentary: Oxygen Radicals,A Failure Or A Success of Evolution?

Oxygen radicals are no doubt involved in the development of many pathological states. Nevertheless, the possibility that oxygen radical production was selected for during biological evolution in order to perform useful roles in relation to cellular metabolism is contemplated; previous data on this subject are briefly reviewed. The concept of an “oxygen radical cycle” is proposed as a useful theoretical model.     

The reactivity of carotenoid radicals with oxygen

The possibility that carotenoid radicals react with oxygen to form chain-carrying peroxyl radicals has been postulated to account for the reduction in antioxidant effetiveness displayed by some carotenoids at high oxygen concentrations. The primary objective of the work described in this paper was to measure the rate constants for oxygen addition to a series of carotenoid radicals and to examine any influence of carotenoid structural features on these rate constants. Laser flash photolysis has been used to generate long-lived carotenoid radicals (PhS-CAR^√) derived from radical addition reactions with phenylthiyl radicals (PhS^√) in benzene. The PhS-CAR^√ radicals are scavenged by oxygen at rates that display a moderate dependence on the number of conjugated double bonds (n_db) in the carotenoid. The rate constants range from ∼10³ to ∼10⁴ M^- 1 s^- 1 for n_db = 7-11. The data also suggest that the presence of terminal cyclic groups may cause an increase in the rate constant for oxygen addition.     

Antioxidant effect of FeAOX-6 on free radical species produced during iron catalyzed breakdown of tert-butyl hydroperoxide

There is a body of evidences demonstrating, in biological systems, a cooperative interaction between tocopherols and carotenoids. FeAOX-6 is a novel antioxidant that combines the chroman head of α-tocopherol and a fragment of the isoprenyl chain of lycopene. We have tested its antioxidant effect on different radical species generated in a chemical system, where peroxyl, alkoxyl and methyl radicals are generated by the ferrous ion-mediated decomposition of tert-butyl hydroperoxide. We found that FeAOX-6 has the same effectiveness of α-tocopherol in quenching peroxyl radical with no contribution by lycopene. The antioxidant activity of FeAOX-6 on alkoxyl and methyl radicals is comparable to that of the equimolar mixture of the parent compounds. Lycopene is able to quench alkoxyl radical, while it has no effect on peroxyl radical, showing a different antioxidant activity compared to other carotenoids, such as β-carotene and lutein.     

Commentary: Oxygen Radicals, A Failure Or A Success of Evolution?

Oxygen radicals are no doubt involved in the development of many pathological states. Nevertheless, the possibility that oxygen radical production was selected for during biological evolution in order to perform useful roles in relation to cellular metabolism is contemplated; previous data on this subject are briefly reviewed. The concept of an “oxygen radical cycle” is proposed as a useful theoretical model.     

EPR spin-trapping of protein radicals to investigate biological oxidative mechanisms

Summary. Presently, free radicals and oxidants are considered to mediate from signaling circuits involved in physiology and pathology to cell and tissue injury. The elucidation of these many inter-related processes requires a better understanding of cellular oxidative mechanisms many of which are mediated by protein radicals. Here, we will discuss the potentialities of EPR spin-trapping of protein radicals to unravel oxidative mechanisms. An overview of the methodology and its application to identify protein residues that are the target of specific oxidants, characterize emerging oxidants, and discriminate radical from non radical mechanisms will be presented. The examples are based on work developed in our laboratories but will be discussed in a broad scenario to emphasize that simple experiments can provide relevant insights into the biological reactivity of known and emerging biological oxidants and into signaling mechanisms.