Reactivity of ubiquinone and ubiquinol with superoxide and the hydroperoxyl radical: implications for in vivo antioxidant activity

Endogenous ubiquinones (UQ) such as coenzyme Q(10) are essential electron carriers in the mitochondrial respiratory chain, and the reduced ubiquinol form (UQH(2)) is a chain-breaking antioxidant, decreasing oxidative damage caused by lipid peroxidation within mitochondria. Consequently, exogenous UQ are used as therapies to decrease mitochondrial oxidative damage. The proximal radical produced during mitochondrial oxidative stress is superoxide (O(2)(.-)) and the reaction between UQ and O(2)(.-) to form the ubisemiquinone radical anion (UQ(.-)) may also be important for the scavenging of O(2)(.-) by exogenous UQ. The situation in vivo is that many UQ are predominantly located in the hydrophobic membrane core, from which O(2)(.-) will be excluded but its conjugate acid, HOO(.), can enter. The reactivity of UQ or UQH(2) with HOO(.) has not been reported previously. Here a pulse radiolysis study on the reactions between UQ/UQH(2) and O(2)(.-)/HOO(.) in water and in solvent systems mimicking the surface and core of biological membranes has been undertaken. O(2)(.-) reacts very rapidly with UQ, suggesting that this may contribute to the scavenging of O(2)(.-) in vivo. In contrast, UQH(2) reacts relatively slowly with HOO(.), but rapidly with other oxygen- and carbon-centered radicals, indicating that the antioxidant role of UQH(2) is mainly in preventing lipid peroxidation.     

Obesity-induced tissue free radical generation: an in vivo immuno-spin trapping study

Assessment of tissue free radical production is routinely accomplished by measuring secondary by-products of redox reactions and/or diminution of key antioxidants such as reduced thiols. However, immuno-spin trapping, a newly developed immunohistochemical technique for detection of free radical formation, is garnering considerable interest as it allows for the visualization of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-adducted molecules. Yet, to date, immuno-spin trapping reports have utilized in vivo models in which successful detection of free radical adducts required exposure to lethal levels of oxidative stress not reflective of chronic inflammatory disease. To study the extents and anatomic locations of more clinically relevant levels of radical formation, we examined tissues from high-fat (HF) diet-fed mice, a model of low-grade chronic inflammation known to demonstrate enhanced rates of reactive species production. Mice subjected to 20 weeks of HF diet displayed increased free radical formation (anti-DMPO mean fluorescence staining) in skeletal muscle (0.863±0.06 units vs 0.512±0.07 units), kidney (0.076±0.0036 vs 0.043±0.0025), and liver (0.275±0.012 vs 0.135±0.014) compared to control mice fed normal laboratory chow (NC). Western blot analysis of tissue homogenates confirmed these results showing enhanced DMPO immunoreactivity in HF mice compared to NC samples. The obesity-related results were confirmed in a rat model of pulmonary hypertension and right heart failure in which intense immunodetectable radical formation was observed in the lung and right ventricle of monocrotaline-treated rats compared to saline-treated controls. Combined, these data affirm the utility of immuno-spin trapping as a tool for in vivo assessment of altered extents of macromolecule oxidation to radical intermediates under chronic inflammatory conditions.     

Radiation inactivation of ribonucleotide reductase, an enzyme with a stable free radical

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of approximately 210 kDa and the other of approximately 20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.     

Reevaluation of the 2-deoxyribose assay for determination of free radical formation

Background

The 2-deoxyribose (2-DR) degradation assay is a widely used test for determining anti/pro-oxidant properties of molecules and plant extracts. Most reports use reaction blanks omitting 2-DR or thiobarbituric acid (TBA). However, when studying Fe(II)-mediated reactions, we verified that these blanks are not appropriate. Fe(III) – a product of these reactions – causes a relevant artifact in the assay, where 2-DR is oxidized by Fe(III).

Method

2-DR degradation was determined at 532 nm as TBA-reactive substances.

Results and conclusion

HPLC determinations indicated that Fe(III) added after or before TBA generates considerable amounts of malondialdehyde (2-DR degradation product) in comparison with assays employing Fenton reagents or Fe(II) autoxidation. Addition of catalase and thiourea has no effect on Fe(III)-induced 2-DR degradation indicating lack of ROS involvement. This Fe(III)-mediated 2-DR damage is dependent on iron and 2-DR concentrations, but not on H₂O₂, buffer composition or iron-chelators. Depending on the assay conditions Fe(III)-interference accounts for 20% to 90% of 2-DR degradation mediated by Fe(II).

Significance

A new reaction blank is proposed herein–based on the use of Fe(III)–for the assay. The lack of such correction has caused the underestimation of antioxidant capacity of various compounds in many studies in the last 2 decades.     

An analysis of the role of coenzyme Q in free radical generation and as an antioxidant.

The vital role of coenzyme Q in mitochondrial electron transfer and its regulation, and in energy conservation, is well established. However, the role of coenzyme Q in free oxyradical formation and as an antioxidant remains controversial. Demonstration of the existence of the semiquinone form of coenzyme Q during electron transport, coupled with recent evidence that hydrogen peroxide (but not molecular oxygen) may act as an oxidant of the semiquinone, suggests that the highly reactive OH. radical may be formed from the semiquinone. On the other hand, data exist implicating the Fe-S species as the source of electron transfer chain, free radical production. Additional data exist suggesting instead that the unpaired electron of the coenzyme Q semiquinone most likely dismutases superoxide radicals. These concepts and those arising from observations at several levels of organization including subcellular systems, intact animals, and human subjects in the clinical setting, supporting the concept of reduced coenzyme Q as an antioxidant, will be presented. The results of recent studies on the interaction between the two-electron quinone reductase--DT diaphorase and coenzyme Q10 will be presented. The possibility that superoxide dismutase may interact with reduced coenzyme Q, in conjunction with DT diaphorase inhibiting its autoxidation, will be described. The regulation of cellular coenzyme Q concentrations during oxidative stress accompanying aerobic exercise, resulting in increased protection from free radical damage, will also be presented.     

Effect of O-glycosilation on the antioxidant activity and free radical reactions of a plant flavonoid,chrysoeriol

Chrysoeriol and its glycoside (chrysoeriol-6-O-acetyl-4'-beta-D-glucoside) are two natural flavonoids extracted from the tropical plant Coronopus didymus. The aqueous solutions of both the flavonoids were tested for their ability to inhibit lipid peroxidation induced by gamma-radiation, Fe (III) and Fe (II). In all these assays chrysoeriol showed better protecting effect than the glycoside. The compounds were also found to inhibit enzymatically produced superoxide anion by xanthine/xanthine oxidase system; here the glycoside is more effective than the aglycone. The rate constants for the reaction of the compounds with superoxide anion determined by using stopped-flow spectrometer were found to be nearly same. Chrysoeriol glycoside reacts with DPPH radicals at millimolar concentration, but the aglycone showed no reaction. Using nanosecond pulse radiolysis technique, reactions of these compounds with hydroxyl, azide, haloperoxyl radicals and hydrated electron were studied. The bimolecular rate constants for these reactions and the transient spectra of the one-electron oxidized species indicated that the site of oxidation for the two compounds is different. Reaction of hydrated electron with the two compounds was carried out at pH 7, where similar reactivity was observed with both the compounds. Based on all these studies it is concluded that chrysoeriol exhibits potent antioxidant activity. O-glycosylation of chrysoeriol decreases its ability to inhibit lipid peroxidation and reaction with peroxyl radicals. However the glycoside is a more efficient scavenger of DPPH radicals and a better inhibitor of xanthine/xanthine oxidase than the aglycone.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.     

Screening of free radical scavengers from Erigeron breviscapus using on-line HPLC-ABTS/DPPH based assay and mass spectrometer detection

Erigeron breviscapus is a well-known traditional Chinese herbal medicine. In this study, on-line HPLC-ABTS/DPPH assay coupled with MS detection were applied to screen and identify the free radical scavengers in 70% methanol extracts of E. breviscapus. Using on-line HPLC-ABTS-MS and HPLC-DPPH-MS assay, 13 radical scavengers (including 4-O-caffeoylquinic acid (4-CQA) (1), 9-caffeoyl-2,7-anhydro-2-octulosonic acid (9-COA) (2), 3-caffeoyl-2,7-anhydro-3-deoxy-2-octulopyranosonic acid (3-CDOA) (3), erigeside I (4), quercetin-3-O-glucuronide (5), eriodictyol-7-O-glucuronide (6), scutellarin (7), 1,4-di-O-caffeoylquinic acid (1,4-di-CQA) (8), 3,5-di-CQA (9), 1-malonyl-3,5-di-CQA (10), erigoster B (11), 4,5-di-CQA (12) and 4,9-di-CDOA (13)) and 9 radical scavengers (including 1, 4, 7, 8, 9, 10, 11, 12 and 13) were discovered, respectively. Furthermore, the anti-oxidative activities of 4 compounds, including 7, 9, 11 and 12 were evaluated. Reverse anti-oxidative activity order of scutellarin and 3,5-di-CQA was observed in on-line HPLC-ABTS assay and on-line HPLC-DPPH assay. To validate their anti-oxidative activities, the off-line ABTS and DPPH assays were performed. Given sufficient reaction time, 3,5-di-CQA showed higher activity than scutellarin, which was consistent with the order obtained in on-line HPLC-ABTS assay. These results revealed that on-line HPLC-ABTS assay is a more sensitive method for screening and determining free radical scavengers, especially more suitable for those compounds with slower reaction kinetics.     

Screening of free radical scavengers from Erigeron breviscapus using on-line HPLC-ABTS/DPPH based assay and mass spectrometer detection

Erigeron breviscapus is a well-known traditional Chinese herbal medicine. In this study, on-line HPLC-ABTS/DPPH assay coupled with MS detection were applied to screen and identify the free radical scavengers in 70% methanol extracts of E. breviscapus. Using on-line HPLC-ABTS-MS and HPLC-DPPH-MS assay, 13 radical scavengers (including 4-O-caffeoylquinic acid (4-CQA) (1), 9-caffeoyl-2,7-anhydro-2-octulosonic acid (9-COA) (2), 3-caffeoyl-2,7-anhydro-3-deoxy-2-octulopyranosonic acid (3-CDOA) (3), erigeside I (4), quercetin-3-O-glucuronide (5), eriodictyol-7-O-glucuronide (6), scutellarin (7), 1,4-di-O-caffeoylquinic acid (1,4-di-CQA) (8), 3,5-di-CQA (9), 1-malonyl-3,5-di-CQA (10), erigoster B (11), 4,5-di-CQA (12) and 4,9-di-CDOA (13)) and 9 radical scavengers (including 1, 4, 7, 8, 9, 10, 11, 12 and 13) were discovered, respectively. Furthermore, the anti-oxidative activities of 4 compounds, including 7, 9, 11 and 12 were evaluated. Reverse anti-oxidative activity order of scutellarin and 3,5-di-CQA was observed in on-line HPLC-ABTS assay and on-line HPLC-DPPH assay. To validate their anti-oxidative activities, the off-line ABTS and DPPH assays were performed. Given sufficient reaction time, 3,5-di-CQA showed higher activity than scutellarin, which was consistent with the order obtained in on-line HPLC-ABTS assay. These results revealed that on-line HPLC-ABTS assay is a more sensitive method for screening and determining free radical scavengers, especially more suitable for those compounds with slower reaction kinetics.     

Determination of poultry feed-through insecticide activity in an in vivo anticoccidial assay

The ability of an in vivo anticoccidial assay to identify potential feed-through insecticides was demonstrated using first-instar Lucilia sericata on droppings from chicks fed medicated diets. Cyromazine, a commercially available feed-through insect growth regulator, ivermectin, diflubenzuron, fipronil, permethrin, and 2 experimental compounds were effective in varying degrees in killing L. sericata larvae. Eleven coccidiostats were effective against the protozoan parasites (Eimeria spp.) at commercially used levels, whereas they were ineffective against Lucilia larvae.     

Lipoperoxides as an index of free radical activity in bone marrow transplant recipients

It has been previously demonstrated that the conditioning therapy given to bone marrow transplant (BMT) recipients creates a high oxidant stress, resulting in a measured reduction in antioxidants, such as glutathione peroxidase (GSH-Px), vitamin E, and cell peroxide fragilities. As part of a current intervention trial of antioxidant therapy in BMT recipients, plasma thiobarbituric acid reactive substances (TBARS) were measured to assess peroxidation and free radical activity. Measurements were performed before and after conditioning therapy, and then at weekly intervals for a period of 6 wk after transplantation in 20 patients (10 controls and 10 antioxidant therapy [AOT] recipients). The TBARS results were compared with concurrent measurements of more specific elements of the antioxidant pathways, such as red blood cell glutathione peroxidase (RBC-GSH-Px), plasma vitamin C, and serum vitamin E. In all cases, TBARS concentration was significantly increased after conditioning compared with baseline levels (p<0.001), an increase that correlated inversely with RBC-GSH-Px (r=?0.81;p<0.01). The TBARS concentration fell gradually after conditioning in all patients. The fall in the AOT group was more rapid than in the control group, and it paralleled the gradual return toward normal levels of the other antioxidants. The change in TBARS concentration occurred faster than changes in other indices, suggesting that TBARS might be a better index of overall free radical activity. Although the patient numbers are small, there is some evidence to suggest that MDA may act as a prognostic marker. In 70% of the patients with a poor postoperative course and who eventually died, the peak TBARS concentration was significantly higher than that in the successful transplants. Also, the fall in TBARS concentration was much slower (if at all). This point requires further investigation and a more detailed analysis on a larger number of patients.     

Basal protein phosphorylation is decreased and phosphatase activity increased by an antioxidant and a free radical trap in primary rat glia.

Reversible protein phosphorylation regulates a wide array of cellular functions. Cells respond to cytokines and various stressors via phosphorylation and thus activation of one or more of the mitogen-activated protein kinase (MAPK) pathways. Involvement of these signal transduction pathways has been implicated in numerous pathologies, including inflammation. Using a primary glia cell culture, we show here that the antioxidant N-acetylcysteine (NAC) and the nitrone-based free radical trap, alpha-phenyl-N-tert-butyl nitrone (PBN), reduce total basal protein phosphorylation in a concentration-dependent manner as assessed by phosphotyrosine analysis and by [gamma-32P]ATP transfer radioassay. In addition we show that NAC inhibits H2O2-induced phosphatase inactivation in glia cell lysate. The PBN- and NAC-induced reduction in protein phosphorylation is accompanied by an increase in phosphatase activity, suggesting that PBN and NAC reduce protein phosphorylation by globally augmenting oxidant-sensitive phosphatase activities. These results partly explain why certain antioxidants also possess anti-inflammatory actions.     

Quantitative structure-activity relationship analyses of antioxidant and free radical scavenging activities for hydroxybenzalacetones

Antioxidant activities for a series of hydroxybenzalacetones, OH-BZ, were evaluated by measuring inhibitory potencies of OH-BZ against lipid peroxidation induced by t-BuOOH or gamma-irradiation. Their quantitative structure-activity relationship (QSAR) studies indicated that the activities are mainly governed by electronic and steric factors. To rationalize these results, we also performed QSAR analyses for DPPH radical scavenging activities of OH-BZ, which indicated that antioxidant and radical scavenging activities could be expressed by the same physicochemical parameters but the hydrogen bonding behavior of phenolic OH varies with the reaction medium.     

Protective effect of oxygen-derived free radical scavengers on the endothelium in vivo

The endothelo-protective activity of a series of low-molecular oxygen-derived free radical scavengers (OFRS) was tested in rats. A model of endothelaemia provoked by intravenous administration of hydrogen peroxide was used. With each OFRS the activity in the hydrogen peroxide model was compared with that in the less specific model using the provocation by citrate as a calcium chelating agent. Relatively unspecific but biologically important OFRS, ascorbic acid, tocopherol, troxerutin and glutathione were tested in the first phase of the study. A marked optimum of endothelo-protective activity was shown with all agents, the optimum against hydrogen peroxide having been observed at doses from 3 to 50 times lower than against citrate. Ascorbic acid, troxerutin and the combination of both were also tested in another model based on leg ischaemia produced by ligature of the common femoral artery. Without OFRS, a marked increase of endothelaemia was observed after 30-60 min ischaemia showing a second peak after the release of the ligature. This second peak was completely abolished by the preventive administration of OFRS in a dose which was also effective in the hydrogen peroxide model.     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

On-line antioxidant activity determination: comparison of hydrophilic and lipophilic antioxidant activity using the ABTS*+ assay

The ABTS/H(2)O(2)/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples -- conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) -- provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.     

Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical scavenging assay for physiological studies of total antioxidant activity in woody plant germplasm.

A robust spectroscopic method for determining total antioxidant activity in aqueous extractions has been applied to tissues from diverse woody plant species, including seeds of Coffea arabica and in vitro shoots from Ribes nigrum, Picea sitchensis and Shorea leprosula. The assay involves scavenging of an ABTS [2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)] radical generated by the reaction of potassium persulphate with ABTS to produce an ABTS*(+) chromophore (lambda=734 nm). Antioxidants reduce ABTS*(+) back to ABTS with a concomitant decrease in absorbance. Aqueous extractions from C. arabica and S. leprosula had considerably higher (110-205 micromol Trolox eq. g(-1) FW) total antioxidant activities than P. sitchensis and R. nigrum (6-11 micromol Trolox eq. g(-1) FW). Further studies in two of these species showed that the inclusion of water-insoluble polyvinylpyrrolidone during aqueous tissue extraction enabled the combined phenolic and alkaloid antioxidant activity to be determined. These fractions accounted for 85% and 60% of total antioxidant activity for C. arabica seeds and R. nigrum shoots, respectively. The ABTS radical scavenging assay is presented herein as a robust method for determining total antioxidant activity in germplasm from diverse woody plant tissues and species. Its applicability to study oxidative stress in tissue cultures and germplasm employed in plant biotechnology, breeding and stress physiology programmes is discussed.     

Phenylpropanoid glycoside analogues: enzymatic synthesis, antioxidant activity and theoretical study of their free radical scavenger mechanism

Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase. A sequence comparison

The small subunit of ribonucleoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center. An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula solidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important. Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved. We propose that Tyr-122 is responsible for radical stabilization and that His-118 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center.