Repair of Protein Radicals by Antioxidants

In vivo, proteins are the main targets for radicals and other reactive species. Their reactions result in formation of amino acid radicals on the protein surface that often yield tryptophan and tyrosyl radicals or, in the presence of O₂, protein peroxyl radicals and hydroperoxides. All these species may propagate damage to biomolecules. Low molecular weight antioxidants, such as ascorbate, urate, and glutathione, are part of the defense system and function by repairing damaged proteins. We briefly review the existing knowledge about protein and amino acid radicals and their repair by antioxidants, including results of our investigations. The main question addressed is whether the antioxidants ascorbate, urate, and glutathione are able to repair amino acid radicals in model compounds and in proteins in vitro by pulse radiolysis. We show that ascorbate and urate repair tryptophan and tyrosyl radicals efficiently and inhibit proton-coupled electron transfer from tyrosine residues to tryptophan radicals in a number of proteins. In contrast, repair by glutathione is much slower. Ascorbate also rapidly reduces the peroxyl radicals of the N-acetylamide derivatives of glycine, alanine, and proline, whereas glutathione reduces peroxyl radicals in lysozyme. In vivo urate, ascorbate, and glutathione may prevent biological damage or, at least, reduce its rate, because they: (a) repair tryptophan and tyrosyl radicals in proteins and (b) reduce protein peroxyl radicals to the corresponding protein hydroperoxides. Most likely, in vivo, ascorbate and glutathione do not inhibit the reaction of C-centered amino acid radicals with O₂. Glutathione is less efficient that urate and ascorbate in repairing protein radicals; furthermore, the resulting glutathiyl radical is harmful. Ascorbate may be the more important repair agent in cells and tissues characterized by high ascorbate concentrations, such as the lens and brain; urate may be mainly responsible for repair in tissue compartments with higher urate concentrations, such as in plasma and saliva.     

Matrix Extension Validation of AOCS Ce 2c-11 for Omega-3 Polyunsaturated Fatty Acids in Conventional Foods and Dietary Supplements Containing Added Marine Oil

Marine oils are commonly added to conventional foods and dietary supplements to enhance their contents of omega-3 polyunsaturated fatty acids (PUFA), including eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), which have been associated with numerous potential health benefits. This study compared American Oil Chemists’ Society (AOCS) Official Methods Ce 2b-11 and Ce 2c-11 for determining EPA and DHA in foods and dietary supplements and found that AOCS Ce 2c-11 produces significantly higher analyzed values, which could be attributed to a more comprehensive breakdown of the sample matrix and derivatization of fatty acids. Our subsequent food matrix extension validation of AOCS Ce 2c-11 demonstrated that the method produces true, accurate, sensitive, and precise determinations of EPA, DHA, and total omega-3 PUFA in foods and dietary supplements containing added marine oil, including those formulated with emulsified and microencapsulated oils. The method detection limits for EPA and DHA were 0.012 ± 0.002 and 0.011 ± 0.003 mg g⁻¹, respectively (means ± SD). The analyzed contents of EPA (1.26–386 mg serving⁻¹), DHA (1.37–563 mg serving⁻¹), and total omega-3 PUFA (2.69–1270 mg serving⁻¹) were reported for 27 conventional food and dietary supplement products. Eighteen products declared contents of DHA, EPA + DHA, or total omega-3 PUFA on product labels, and the analyzed contents of those fatty acids varied from 95 to 162% of label declarations for all but two of the products.     

The protective effect of antioxidants to a phototoxin-sensitive insect herbivore,Manduca sexta

Photo-activated plant secondary compounds have been shown to be toxic to many organisms including insects. Insect defenses include behavioral mechanisms such as light avoidance, as well as specific biochemical defenses such as antioxidants and antioxidant enzymes. These antioxidant defenses eliminate or quench the deleterious singlet oxygen and free radicals formed by these phototoxins. In this paper we examined the role of dietary antioxidants in protecting the phototoxin-sensitive insect herbivoreManduca sexta. Elevated dietary levels of the lipid-soluble antioxidants-carotene and vitamin E resulted in a concentration-dependent reduction in the mortality associated with treatment ofM. sexta larvae with the phototoxic thiophene-terthienyl. Elevated levels of dietary ascorbic acid had no effect, whereas reduced levels greatly increased the toxicity of-terthienyl. Tissue levels of antioxidants were shown to increase substantially in larvae fed antioxidant-supplemented diets. The results suggest that the ability to absorb and utilize plant-derived antioxidants could be an important defense against photo-activated plant secondary compounds and may have allowed some insects to exploit phototoxic plants.     

Assessment of potential prooxidant and antioxidant actions

Suggestions that oxidative stress plays a role in human diseases have led to the proposal that health might be improved by increased dietary intake of antioxidants. Plant-derived antioxidants, such as flavonoids or rosemary extracts, are increasingly proposed as important dietary antioxidant factors, and foods rich in antioxidants are also receiving attention. Before widescale usage of natural and synthetic antioxidants can be suggested, it is necessary to establish the properties of such molecules. Assays for characterizing the potential prooxidant/antioxidant actions of food additives, antioxidant supplements, antioxidant drug molecules, and nutrient components have been developed for this purpose. Based on a paper presented at the 86th AOCS Annual Meeting & Expo, San Antonio, Texas, May 1995.     

Application of Infrared Spectroscopy for Characterization of Dietary Omega‐3 Oil Supplements

Fish oil dietary supplements have been linked with health benefits, due to high omega‐3 concentration. The sources of these effects, polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, are almost exclusively found in seafood products. Our objectives were to characterize the composition of commercial omega‐3 dietary supplements dietary supplements and to generate partial least square regression (PLSR) models using infrared spectroscopy and chemometrics. Fatty acid (FA) composition of oils was determined by FA methyl ester gas chromatography. The supplements encompassed a wide range of FA profiles and delivery methods. Infrared spectral data were collected by portable mid‐infrared Fourier transform infrared (MID FT‐IR) equipment. Principal components analysis (PCA) separated samples based on the type of ester present in the fish oil dietary supplements, showing a strong influence of the 1038 cm^?1 band, which is typically associated with C=C and C–O stretching vibrations. In addition, PLSR was used to correlate the spectra data with GC‐FAME results. PCA using the spectroscopy data allowed for tight clustering of fish oil into distinct classes, depending on the source and processing. PLSR using MID FT‐IR spectra and FA composition generated multivariate models with high correlation coefficient (R ≥ 0.93), and SEP between 0.53 and 2.13 g of FA per 100 g of oil. Our results indicate that IR spectroscopy combined with chemometrics provides for robust screening of FA composition of fish oil supplements, and discriminate types of FAs esterification.     

Ascorbate and phenolic antioxidant interactions in prevention of liposomal oxidation

Efficient prevention of membrane lipid peroxidation by vitamin E (α-tocopherol) may involve its regeneration by vitamin C (ascorbate). Conceivably, the efficacy of antioxidants designed as therapeutic agents could be enhanced if a similar regeneration were favorable; thus, a model membrane system was developed which allowed assessment of interaction of phenolic antioxidants with ascorbate and ascorbyl-6-palmitate. Ascorbate alone (50–200 μM) potentiated oxidation of soybean phosphatidylcholine liposomes by Fe²⁺/histidine-Fe³⁺, an effect which was temporally related to reduction of Fe³⁺ generated during oxidation. Addition of 200 μM ascorbate to α-tocopherol-containing liposomes (0.1 mol%) resulted in marked, synergistic protection. Accordingly, in the presence but not absence of ascorbate, α-tocopherol levels were maintained relatively constant during Fe²⁺/histidine-Fe³⁺ exposure. Probucol (4,4′-[(1-methylethylidine)bis(thio)]bis[2,6-bis(1,1-dimethylethyl)]phenol), and antioxidant which prevents oxidation of low density lipoproteins, and its analogues MDL 27,968 (4,4′-[(1-methylethylidene)bis(thio)]-bis[2,6-dimethyl]phenol) and MDL 28,881 (2,6-bis(1,1-dimethylethyl)-4-[(3,7,11-trimethyldodecyl)thio]phenol) prevented oxidation but exhibited no synergy with ascorbate. Ascorbyl-6-palmitate itself was an effective antioxidant but did not interact synergistically with any of the phenolic antioxidants. Differential scanning calorimetry revealed significant differences among the antioxidants in their effect on the liquid-crystalline phase transition of dipalmitoyl phosphatidylcholine (DPPC) liposomes. Both α-tocopherol and MDL 27,968 significantly reduced the phase transition temperature and the enthalpy of the transition. MDL 28,881 had no effect while probucol was intermediate. The potential for ascorbate or its analogues to interact with phenolic antioxidants to provide a more effective antioxidant system appears to be dictated by structural features and by the location of the antioxidants in the membrane.     

Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains

Cholesterol and α-tocopherol oxidations were studied in brain subcellular fractions isolated from cerebral hemispheres of 4-month-old, male Fischer 344 rats. The fractions were suspended in buffered media (pH 7.4, 37°C) and oxidized by adding (i) ferrous iron (Fe²⁺) with or without ascorbate or (ii) peroxynitrite (an endogenous oxidant produced by the reaction of superoxide and nitric oxide). Treatment of subcellular fractions with Fe²⁺ in the presence or absence of ascorbate produced primarily 7-keto- and 7-hydroxy-cholesterols and small amounts of 5α,6α-epoxycholesterol. Since brain contains high levels of ascorbate, any release of iron could result in oxysterol formation. Peroxynitrite oxidized α-tocopherol but not cholesterol. Hence, the toxicity of peroxynitrite or nitric oxide could not be due to cytotoxic oxysterols. When synaptosomes were incubated for 5 min in the presence of 0.5 to 2 μM Fe²⁺ and ascorbate, α-tocopherol was oxidized while cholesterol remained unchanged. Thus, α-tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA dit not. A significant percentage of mitochondrial cholesterol was oxidized by treatment with Fe²⁺ and ascorbate. Hence, mitochondrial membrane properties dependent on cholesterol could be particularly susceptible to oxidation. The oxysterols formed were retained within the membranes of synaptosomes and mitochondria. The 7-oxysterols produced are known to be inhibitors of membrane enzymes and also can modify membrane permeability. Hence, oxysterols may play an important role in brain tissue damage during oxidative stress.     

Dietary Supplements and Sports Performance: Metabolites, Constituents, and Extracts

This is the fifth in a series of six articles to discuss the major classes of dietary supplements (vitamins; minerals; amino acids; herbs or botanicals; and metabolites/constituents/extracts). The major focus is on efficacy of such dietary supplements to enhance exercise or sports performance.     

Dietary Supplements and Sports Performance: Amino Acids

This is the third in a series of six articles to discuss the major classes of dietary supplements (vitamins; minerals; amino acids; herbs or botanicals; metabolites, constituents/extracts, or combinations). The major focus is on efficacy of such dietary supplements to enhance exercise or sport performance.     

Dietary Supplements and Sports Performance: Herbals

This is the fourth in a series of six articles to discuss the major classes of dietary supplements (vitamins; minerals; amino acids; herbs or botanicals; metabolites, constituents/extracts, or combinations). The major focus is on efficacy of such dietary supplements to enhance exercise or sport performance.     

Catalytic oxidation of alcohols using molecular oxygen mediated by poly(ethylene glycol)-supported nitroxyl radicals

The selective catalytic oxidation of alcohols over a mixture of copper(I) chloride and a number of linear ‘linker-less’ or ‘branched’ poly(ethylene glycol)-supported nitroxyl radicals of the 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) family as a catalyst system has been investigated in the presence of molecular oxygen in a batch reactor. It is found that the activity profile of the polymer-supported nitroxyl radicals is in good agreement with that of low-molecular weight nitroxyl catalysts, for example, allylic and benzylic alcohols are oxidised faster than aliphatic alcohols. The oxidations can be tuned to be highly selective such that aldehydes are the only oxidation products observed in the oxidation of primary alcohols and the oxidations of secondary alcohols yield the corresponding ketones. A strong structural effect of the polymeric nitroxyl species on catalytic activity that is dependent upon their spatial orientation of the nitroxyl radicals is particularly noted. The new soluble macromolecular catalysts can be recovered readily from the reaction mixture by solvent precipitation and filtration. In addition, the recycled catalysts demonstrate a similar selectivity with only a small decrease in activity compared to the fresh catalyst even after five repetitive cycles.     

Direct Determination of Antioxidants in Whole Olive Oil Using the SIFT-MS-TOSC Assay

A modified Selected Ion Flow Tube Mass Spectrometry Total Oxyradical Scavenging (SIFT-MS-TOSC) assay has been used to assess the antioxidants present in olive oil samples. The assay compares antioxidants in emulsified olive oil competing with added 2-keto-4-methylthiobutanoic acid (KMBA) for peroxyl radicals formed from thermal decomposition of 2,2′-azobis (2-amidinopropane) hydrochloride. No solvent extraction from the oil is required. The assay reaction is monitored by analysis of ethene, a break down product of KMBA, from attack by peroxyl radicals. A new simpler modification to the TOSC assay was found to produce reliable results requiring measurements of just a single concentration of emulsion. The results of the SIFT-MS-TOSC assay show correlation with results from the Folin Ciocalteu assay of total phenol content and HPLC analysis of several standard antioxidants. The use of hydroxyl radicals generated by the Fenton reaction of Fe³⁺ with ascorbic acid was also investigated, but with less repeatable results. A wide range of antioxidant levels differing by more than a factor of 10 were found in 20 different olive oils examined.     

Potential role of ascorbate oxidase as a plant defense protein against insect herbivory

Ascorbic acid is essential for both nutritive and antioxidant functions in phytophagous insects; however, maintaining sufficient quantities of reduced ascorbate may be problematical for them. In this investigation, we show that the plant enzyme ascorbate oxidase retains activity in the digestive system of the herbivoreHelicoverpa zea. High levels of the enzyme are present in several host plants ofH. zea, including cotton, tomato, soybean, crimson clover, and vetch. The enzyme oxidizesL-ascorbic acid to dehydro-L-ascorbic acid, a potentially toxic product. The oxidation of ascorbic acid also produces active oxygen species such as the highly reactive hydroxyl radical. The nutritional quality of protein for larvalH. zea was significantly reduced by treatment with ascorbate and ascorbate oxidase. Oxidative damage to the protein was indicated by decreased lysine content, increased carbonyl formation, and the occurrence of protein fragmentation and polymerization. Furthermore, the oxidative loss of ascorbate in the herbivore's digestive system prevents ascorbate from functioning as an important antioxidant against a plethora of dietary prooxidants.     

Low‐density lipoprotein is oxidized by phospholipase A2 and lipoxygenase in xanthoma lesions

Oxidized LDL has been obtained by incubation with copper ions (Cu‐LDL) or various kinds of cells. LDL incubated with xanthoma tissues (x‐LDL) is considered a model of in vivo oxidized LDL that has extravasated into xanthoma lesions. To investigate the mechanism of x‐LDL formation, we studied the effects of various enzyme inhibitors or antioxidants on the oxidation process of LDL. Thiobarbituric acid‐reactive substance (TBARS) levels, electrophoretic mobility and spectrophotometric pattern of the oxidized LDL were examined. Antioxidants suppressed TBARS formation in both x‐LDL and Cu‐LDL. Enzyme inhibitors inhibited TBARS levels in x‐LDL, but not in Cu‐LDL. All the enzyme inhibitors and antioxidants, except for the cyclooxygenase inhibitor, inhibited the anodic electrophoretic mobility of x‐LDL. The anodic electrophoretic mobility of Cu‐LDL was suppressed only with antioxidants. Spectrophotometry indicated that an increase in the absorbance at 240 nm was observed in Cu‐LDL, but not in x‐LDL. x‐LDL oxidation is primarily catalyzed by phospholipase A₂, and subsequently generated polyunsaturated free fatty acids propagate the peroxidation. Fatty acid hydroperoxides conjugated with dienes are not synthesized in x‐LDL. On the other hand, non‐enzymatic oxidants, such as superoxide anion and hydroxyl radicals generate Cu‐LDL with diene‐conjugated fatty acid hydroperoxides.     

Catalytic oxidation of alcohols using molecular oxygen mediated by poly(ethylene glycol)-supported nitroxyl radicals

The selective catalytic oxidation of alcohols over a mixture of copper(I) chloride and a number of linear ‘linker-less’ or ‘branched’ poly(ethylene glycol)-supported nitroxyl radicals of the 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) family as a catalyst system has been investigated in the presence of molecular oxygen in a batch reactor. It is found that the activity profile of the polymer-supported nitroxyl radicals is in good agreement with that of low-molecular weight nitroxyl catalysts, for example, allylic and benzylic alcohols are oxidised faster than aliphatic alcohols. The oxidations can be tuned to be highly selective such that aldehydes are the only oxidation products observed in the oxidation of primary alcohols and the oxidations of secondary alcohols yield the corresponding ketones. A strong structural effect of the polymeric nitroxyl species on catalytic activity that is dependent upon their spatial orientation of the nitroxyl radicals is particularly noted. The new soluble macromolecular catalysts can be recovered readily from the reaction mixture by solvent precipitation and filtration. In addition, the recycled catalysts demonstrate a similar selectivity with only a small decrease in activity compared to the fresh catalyst even after five repetitive cycles.     

The use of a commercial vegetable juice as a practical means to increase vegetable intake: a randomized controlled trial

Background

A plant-based diet protects against chronic oxidative stress-related diseases. Dietary plants contain variable chemical families and amounts of antioxidants. It has been hypothesized that plant antioxidants may contribute to the beneficial health effects of dietary plants. Our objective was to develop a comprehensive food database consisting of the total antioxidant content of typical foods as well as other dietary items such as traditional medicine plants, herbs and spices and dietary supplements. This database is intended for use in a wide range of nutritional research, from in vitro and cell and animal studies, to clinical trials and nutritional epidemiological studies.

Methods

We procured samples from countries worldwide and assayed the samples for their total antioxidant content using a modified version of the FRAP assay. Results and sample information (such as country of origin, product and/or brand name) were registered for each individual food sample and constitute the Antioxidant Food Table.

Results

The results demonstrate that there are several thousand-fold differences in antioxidant content of foods. Spices, herbs and supplements include the most antioxidant rich products in our study, some exceptionally high. Berries, fruits, nuts, chocolate, vegetables and products thereof constitute common foods and beverages with high antioxidant values.

Conclusions

This database is to our best knowledge the most comprehensive Antioxidant Food Database published and it shows that plant-based foods introduce significantly more antioxidants into human diet than non-plant foods. Because of the large variations observed between otherwise comparable food samples the study emphasizes the importance of using a comprehensive database combined with a detailed system for food registration in clinical and epidemiological studies. The present antioxidant database is therefore an essential research tool to further elucidate the potential health effects of phytochemical antioxidants in diet.     

Copper Transfer from Cu–Aβ to Human Serum Albumin Inhibits Aggregation,Radical Production and Reduces Aβ Toxicity

Amyloid‐β peptides (Aβ) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Aβ and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Aβ aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper‐exchange reaction between Aβ and HSA or the tetrapeptide DAHK (N‐terminal Cu‐binding domain of HSA) and the consequence of this exchange on Aβ‐induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu^II from Aβ rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu‐induced aggregation of Aβ, 3) HSA and DAHK suppressed the catalytic HO^. production in vitro and ROS production in neuroblastoma cells generated by Cu–Aβ and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu–Aβ with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu^II–HSA than Cu–DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu^II–Aβ interaction. The repercussion of the competition between HSA and Aβ to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.     

Inhibition of soybean lipoxygenase-1 by chain-breaking antioxidants

The aim of this investigation was to determine whether chain-breaking antioxidants able to prevent lipid peroxidation can inhibit lipoxygenase-1 (EC 1.13.11.12). Therefore, the effects of ascorbic acid, 6-palmitoylascorbic acid and trolox on the enzyme activity were analyzed by means of Lineweaver-Burk double reciprocal plots and Yoshino's graphical method. The effect of these compounds on the formation of free radicals during lipoxygenase-1 reaction was investigated as well, by monitoring the enzymic formation of oxodienes. We present evidence that the chain-breaking antioxidants ascorbic acid, 6-palmitoylascorbic acid and trolox inhibit soybean lipoxygenase-1 in the micromolar concentration range (Ki 27, 3 and 18 μM, respectively). The inhibition is competitive, complete and reversible. All three compounds trap the free radicals formed during the lipoxygenase-catalyzed reaction, which might substantially contribute to their inhibitory ability. These findings can have physiological significance in the light of the lipoxygenase involvement in biomembrane remodelling.     

Production and properties of chlorogenic acid lipophilic esters

Chlorogenic acid is a dietary polyphenol with important bioactivities. However, its high hydrophilicity prevents its applications as an antioxidant for lipid protection in a complex matrix such as food. Grafting an aliphatic chain onto chlorogenic acid, so‐called lipophilisation, via lipase‐catalysed esterification represents an efficient means of increasing its lipophilicity. A series of alkyl esters with various chain lengths were produced and used to explore the structure‐antioxidant activity relationship of amphiphilic antioxidants. Data obtained from complex systems (oil‐in‐water emulsion and cell culture) concluded that short or medium chain lengths enhance the antioxidant activity while longer chains tend to decrease the antioxidant ability of chlorogenic acid. This phenomenon remains to be fully explained. Acyl esters, in which the carboxylic acid group remains free were also produced and were shown to be interesting antioxidant candidates as well as useful tools for the understanding of antioxidant activities.