•HODEs and HETEs produced in the plasma oxidation by multiple oxidants were measured.•9- and 13-(E,E)-HODE were produced only by free radicals and peroxynitrite.•15-Lipoxygenase forms 13-(S)-HODE selectively and hypochlorite produces little HODE.•10- and 12-HODEs are specific biomarker for singlet oxygen oxidation.•Total HETEs and isoprostanes produced were less than HODEs.Free and ester forms of unsaturated fatty acids and cholesterol are oxidized in vivo by multiple oxidants to give diverse products. Some lipid oxidation is mediated by enzymes to selectively give specific products, while others proceed randomly to produce mixtures of many kinds of regioisomers and stereoisomers. The efficacy of antioxidants against lipid oxidation depends on the nature of the oxidants and therefore the identification of oxidant is important for understanding the roles and effects of lipid oxidation and antioxidants in vivo. In the present study, the isomer distribution of hydro(pero)xyoctadecadienoates (H(p)ODEs) and hydro(pero)xyeicosatetraenoates (H(p)ETEs), the most abundant lipid oxidation products found in human plasma, produced in the oxidation of plasma by peroxyl radicals, peroxynitrite, hypochlorite, 15-lipoxygenase, and singlet oxygen were examined. It was shown that 9- and 13-(E,E)-HODEs, 13(S)-(Z,E)-HODE, and 10- and 12-(Z,E)-HODEs were specific lipid oxidation products by free radical, 15-lipoxygenase, and singlet oxygen, respectively. The isomer distribution of HODEs produced by peroxynitrite was similar to that by peroxyl radical, suggesting that the peroxynitrite mediated lipid oxidation proceeds by free radical mechanisms. The production of HODEs and HETEs by hypochlorite was very small. HODEs may be a better biomarker than HETEs since linoleates are oxidized by simpler mechanisms than arachidonates and all the HODEs isomers can be quantified more easily. These products may be used as specific biomarkers for the identification of responsible oxidants and for the assessment of oxidant-specific lipid oxidation levels and effects of antioxidants in vivo.Download high-res image (206KB)Download full-size image

•Antioxidant effect of fermented grain food supplement was assessed in plasma solution.•Rate and amount of scavenging peroxyl radical were measured separately.•Inhibition of plasma lipid oxidation by biologically relevant oxidants was assessed.•Lipid oxidation by peroxyl radical, hypochlorite, and singlet oxygen were suppressed.•This method may be useful for screening antioxidant capacity of foods and supplement.Unregulated oxidative modification of biological molecules induced by multiple oxidants in vivo has been implicated in the pathogenesis of various diseases. Accordingly, the role of antioxidants contained in foods in the maintenance of health and prevention of diseases has received much attention. The efficacy of antioxidants against oxidative stress depends on the nature of oxidants. In the present study, the antioxidant action of fermented grain food supplement, Antioxidant Biofactor (AOB), for scavenging peroxyl radical and inhibition of plasma lipid oxidation induced by multiple oxidants was measured. The antioxidant efficacy against lipid oxidation was assessed by the level of lipid hydroperoxides produced using diphenyl-1-pyrenylphosphine, which is not fluorescent per se but reacts with lipid hydroperoxides stoichiometrically to yield highly fluorescent diphenyl-1-pyrenylphosphine oxide. AOB acted as a potent peroxyl radical scavenger and suppressed lipid oxidation induced by peroxyl radical, peroxynitrite, hypochlorite, and singlet oxygen, but not by 15-lipoxygenase.

•Plasma oxidation by five kinds of biological oxidants produced lipid hydroperoxides.•Antioxidant effects of six clinical drugs were determined by nature of oxidants.•Probucol and edaravone suppressed plasma oxidation by peroxynitrite and hypochlorite.•Troglitazone and edaravone inhibited 15-lipoxygenase mediated plasma lipid oxidation.•Multiple antioxidants with different specificity and reactivity are required in vivo.With increasing evidence showing the involvement of oxidative stress in the pathogenesis of various diseases, the effects of clinical drugs possessing antioxidant functions have received much attention. The unregulated oxidative modification of biological molecules leading to diseases is mediated by multiple oxidants including free radicals, peroxynitrite, hypochlorite, lipoxygenase, and singlet oxygen. The capacity of antioxidants to scavenge or quench oxidants depends on the nature of oxidants. In the present study, the antioxidant effects of several clinical drugs against plasma lipid oxidation induced by the aforementioned five kinds of oxidants were investigated from the production of lipid hydroperoxides, which have been implicated in the pathogenesis of various diseases. Troglitazone acted as a potent peroxyl radical scavenger, whereas probucol and edaravone showed only moderate reactivity and carvedilol, pentoxifylline, and ebselen did not act as radical scavenger. Probucol and edaravone suppressed plasma oxidation mediated by peroxynitrite and hypochlorite. Troglitazone and edaravone inhibited 15-lipoxygenase mediated plasma lipid oxidation, the IC50 being 20 and 34 μM respectively. None of the drugs used in this study suppressed plasma lipid oxidation by singlet oxygen. This study shows that the antioxidant effects of drugs depend on the nature of oxidants and that antioxidants against multiple oxidants are required to cope with oxidative stress in vivo.

The assessment of the radical scavenging capacity of antioxidants has been the subject of extensive studies and controversy. The aim of this study is to develop a simple and inexpensive method for the assessment of the radical scavenging capacity of antioxidants contained in foods and beverages in plasma solution, a biologically relevant heterogeneous medium. Three types of probes, hydrophilic pyranine, with low reactivity, hydrophilic pyrogallol red (PGR), with high reactivity, and lipophilic BODIPY, with moderate reactivity, were separately used to measure the amount and rate of peroxyl radical scavenging. The amount of radicals scavenged by antioxidants was assessed from the lag phase produced by antioxidants in the decay of pyranine and BODIPY, while the reactivity of the antioxidants was assessed from their effect on the decay rate of PGR. Two liquid and two solid samples were tested. Commercial bottled green tea and vegetable juice were found to scavenge 15.6 and 3.45 mmol radicals L−1 and the former scavenged peroxyl radicals 81 times faster than the latter. As for the solid samples, instant coffee powder was found to scavenge several times more radicals and more rapidly than green tea powder. This method may be applied to the assessment of the radical scavenging capacity of antioxidants contained in foods, beverages, and supplements in biologically relevant heterogeneous media.

Carotenoids have received much attention as natural antioxidants. The role and action of carotenoids as singlet oxygen quenchers have been well demonstrated. The radical scavenging is another function of carotenoids as antioxidants, but the method to assess the radical scavenging capacity has not been established and previous studies have given inconsistent results. Carotenoids have strong absorptions in the visible light region and, unlike phenolic antioxidants, they do not have a reactive hydrogen to donate to radicals, which make it difficult to use conventional probes for the assessment of their radical scavenging capacity. In the present study, a method for the assessment of peroxyl radical scavenging capacity by carotenoids was re-examined and applied for β-carotene, lycopene, and commercial tomato juice. The capacity for scavenging peroxyl radicals was assessed from the effect of α-tocopherol on the bleaching of carotenoids being tested. Total content of carotenoids was also assessed from the rate of bleaching under constant flux of peroxyl radicals. The peroxyl radical scavenging capacity of β-carotene and lycopene exhibited was about one-tenth of that observed for α-tocopherol, and the efficacy of lipid peroxidation inhibition was much smaller.

The role of antioxidants contained in foods, beverages, and supplements against oxidative stress has received much attention. The capacity of antioxidants has been assessed by various methods. In this study, the antioxidant capacity of a complex mixture of fermented grains has been assessed by using two probes, pyranine and pyrogallol red (PGR). A supplement commercialised as Antioxidant Biofactor, AOB, was used as a substrate. The extracts from AOB obtained with water and dimethylsulphoxide (DMSO) inhibited the free radical-induced consumption of pyranine and PGR in a concentration-dependent manner. They also suppressed the free radical-induced oxidation of human plasma. It was estimated that 1 g of AOB contained, as Trolox equivalent, roughly 0.13 and 0.24 mmol (2.5 and 4.7 wt%) antioxidants, which could be extracted by water and DMSO, respectively. This study shows that the combination of the above two probes is useful for assessing the total content and activity of antioxidants contained in complex mixtures.

The action of C60 fullerene and its derivatives as a radical-scavenging antioxidant has received much attention, but their reactivity toward free radicals and antioxidant capacity have not been well elucidated yet. In the present study, the reactivity of the two types of water-soluble, sugar-pendant C60 fullerenes, C60-1S and C60-2S, toward peroxyl radical and their effect against human plasma lipid peroxidation were measured. The rate constants for the reaction of C60-1S and C60-2S with peroxyl radicals were obtained from their effect on the bleaching of β-carotene in lipid-SDS micelle system as 4.6 × 103 and 8.0 × 103 M−1 s−1 at 37 °C, respectively. They inhibited the free radical-induced lipid peroxidation in human plasma in a concentration-dependent manner. These results suggest that the sugar-pendant fullerenes C60-1S and C60-2S act as a radical-scavenging antioxidant with the activity similar to the phenolic antioxidants.The sugar-pendant fullerene C60 derivatives inhibited human plasma lipid peroxidation by scavenging radicals.

The radical-scavenging antioxidants play an important role against oxidative stress in the defense system in vivo. The beneficial effects of antioxidants contained in foods and beverages have been well-accepted, and their antioxidant capacity has been assessed by various methods. In the present study, a simple method is proposed in which the total radical scavenging capacity is assessed from the bleaching of pyranine and pyrogallol red induced by free radicals generated from azo initiator. The total content of antioxidants contained in red wine, green tea, and cassis drink and their reactivities toward peroxyl radicals were measured from the lag phase and rate of bleaching using pyranine and pyrogallol red as a probe, respectively. It was found that this method to follow the bleaching of two probes by visible light spectrophotometer is convenient and applicable for assessment of total radical scavenging capacity of both content and activity of the antioxidants contained in beverages.

The role of radical scavenging antioxidants against oxidative stress has received much attention, and the antioxidant capacity has been assessed by various methods. Among them, a method that measures the effect of antioxidant on decay of the probe is one of the most widely used methods. The present study was performed to compare the two methods to assess the antioxidant capacity, one to follow the decay of the probe and the other to measure lipid peroxidation products in human plasma. It was shown that the method following probe decay was suitable for assessment of radical scavenging capacity of antioxidant, but not for the capacity to inhibit lipid peroxidation in plasma. This is true whether a hydrophilic or lipophilic probe is used. Such different results arise from the fact that the efficacy of inhibition of lipid peroxidation by antioxidants depends on the fate of antioxidant-derived radical and interaction between antioxidants as well as the capacity of free radical scavenging. Thus, the capacity of antioxidants for inhibition of lipid peroxidation should be assessed from the effect on the extent of oxidation, not from the effect on probe decay.

•Multiple oxidants oxidize plasma to give lipid hydroperoxides.•Peroxyl radicals, peroxynitrite, hypochlorite and lipoxygenase were studied.•Plasma lipid hydroperoxides react with DPPP to give highly fluorescent DPPP oxide.•Production of lipid hydroperoxide was followed by increase in fluorescence intensity.•This system was applied to assessment of antioxidant capacity with microplate reader.Lipid oxidation has been implicated in the pathogenesis of many diseases. Lipids are oxidized in vivo by several different oxidants to give diverse products, in general lipid hydroperoxides as the major primary product. In the present study, the production of lipid hydroperoxides in the oxidation of mouse plasma induced by multiple oxidants was measured using diphenyl-1-pyrenylphosphine (DPPP) as a probe. DPPP itself is not fluorescent, but it reacts with lipid hydroperoxides stochiometrically to give highly fluorescent DPPP oxide and lipid hydroxides. The production of lipid hydroperoxides could be followed continuously in the oxidation of plasma induced by peroxynitrite, hypochlorite, 15-lipoxygenase, and peroxyl radicals with a microplate reader. A clear lag phase was observed in the plasma oxidation mediated by aqueous peroxyl radicals and peroxynitrite, but not in the oxidation induced by hypochlorite and lipoxygenase. The effects of several antioxidants against lipid oxidation induced by the above oxidants were assessed. The efficacy of antioxidants was dependent markedly on the type of oxidants. α-Tocopherol exerted potent antioxidant effects against peroxyl radical-mediated lipid peroxidation, but it did not inhibit lipid oxidation induced by peroxynitrite, hypochlorite, and 15-lipoxygenase efficiently, suggesting that multiple antioxidants with different selectivities are required for the inhibition of plasma lipid oxidation in vivo. This is a novel, simple and most high throughput method to follow plasma lipid oxidation induced by different oxidants and also to assess the antioxidant effects in biologically relevant settings.Download full-size image

Glutamate plays a critical role in pathological cell death within the nervous system. Vitamin E is known to protect cells from glutamate cytotoxicity, either by direct antioxidant action or by indirect nonantioxidant action. Further, α-tocotrienol (α-T3) has been reported to be more effective against glutamate-induced cytotoxicity than α-tocopherol (α-T). To shed more light on the function of vitamin E against glutamate toxicity, the protective effects of eight vitamin E homologues and related compounds, 2,2,5,7,8-pentamethyl-6-chromanol (PMC) and 2-carboxy-2,5,7,8-pentamethyl-6-chromanol (Trolox), against glutamate-induced cytotoxicity on immature primary cortical neurons were examined using different protocols. Glutamate induced the depletion of glutathione and generation of reactive oxygen species and lipid hydroperoxides, leading to cell death. α-, β-, γ-, and δ-T and -T3; PMC; and Trolox all exerted cytoprotective effects against glutamate-induced cytotoxicity, and a longer preincubation time increased both the cellular content and the cytoprotective effects of T more significantly than those of T3, the effect of preincubation being relatively small for T3 and PMC. The protective effect of Trolox was less potent than that of PMC. The cytoprotective effects of α-T and α-T3 corresponded to their intracellular content. Further, lipid peroxidation products were measured after reduction with triphenylphosphine followed by saponification with potassium hydroxide. It was found that glutamate treatment increased the formation of hydroxyeicosatetraenoic acid, hydroxyoctadecadienoic acid, and 8-F2-isoprostane 2α, which was suppressed by α-T. This study shows that vitamin E protects cells from glutamate-induced toxicity primarily by direct antioxidant action and that the apparent higher capacity of T3 compared to T is ascribed to the faster uptake of T3 compared to T into the cells. It is suggested that, considering the bioavailability, α-T should be more effective than α-T3 against glutamate toxicity in vivo.

Lipopolysaccharide (LPS) induces host inflammatory responses and tissue injury and has been implicated in the pathogenesis of various age-related diseases such as acute respiratory distress syndrome, vascular diseases, and periodontal disease. Antioxidants, particularly vitamin E, have been shown to suppress oxidative stress induced by LPS, but the previous studies with different vitamin E isoforms gave inconsistent results. In the present study, the protective effects of α- and γ-tocopherols and α- and γ-tocotrienols on the oxidative stress induced by LPS against human lung carcinoma A549 cells were studied. They suppressed intracellular reactive oxygen formation, lipid peroxidation, induction of inflammatory mediator cytokines, and cell death. Tocopherols were incorporated into cultured cells much slower than tocotrienols but could suppress LPS-induced oxidative stress at much lower intracellular concentration than tocotrienols. Considering the bioavailability, it was concluded that α-tocopherol may exhibit the highest protective capacity among the vitamin E isoforms against LPS-induced oxidative stress.Graphical abstractDownload full-size imageHighlights► Lipopolysaccharide (LPS) induces oxidative stress in cultured cells. ► Protective effect of four vitamin E isoforms against LPS cytotoxicity was studied. ► α- and γ-tocopherols and tocotrienols suppressed oxidative damage and cell death. ► Tocotorienols were incorporated into cells much faster than tocopherols. ► α-Tocopherol may exert the highest capacity to inhibit LPS-induced cytotoxicity.

Oxidative modification of biologically essential molecules by reactive oxygen and nitrogen species (ROS/RNS) has been implicated in the pathogenesis of various diseases. At the same time, roles of ROS/RNS as physiological signaling messenger have been established. Lipid oxidation products also have two faces. It is argued that the radical scavenging antioxidants taken from diet or supplement may impair such beneficial effects of ROS/RNS and lipid oxidation products. However, it is unlikely that antioxidants impair physiologically important signaling, since the antioxidants do not scavenge signaling ROS/RNS nor do they inhibit the formation of signaling molecules. Lipid peroxidation products are not produced on purpose and inhibition of lipid peroxidation by antioxidants should be beneficial for maintenance of health and reducing disease risk.Highlights► Reactive oxygen/nitrogen species, ROS/RNS, have dual functions. ► Lipid oxidation products exert both harmful and beneficial effects. ► Lipid peroxidaton (LPO) products may induce adaptive response. ► Radical scavenging antioxidants do not impair physiological signaling. ► Inhibition of LPO by antioxidants should be beneficial for human health.

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported undergo oxidation preferentially at the 106th cysteine residue (Cys-106) under oxidative stress. Recently, it has been found that the levels of oxidized DJ-1 in erythrocytes of unmedicated PD patients are markedly higher than those in medicated PD patients and healthy subjects. In the present study, we examined the changes in oxidized DJ-1 levels in the brain and erythrocytes of PD animal models using specific antibodies against Cys-106-oxidized DJ-1. Treatment with PD model compounds such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine significantly elevated the levels of oxidized DJ-1 in erythrocytes. Immunohistochemical analysis also revealed that the number of oxidized DJ-1 antibody-positive cells in the substantia nigra of MPTP-treated mouse increased in a dose-dependent manner. These results suggest that the oxidative modification of DJ-1 in the brain and erythrocytes is involved in the pathogenesis of PD in animal models.

Free radical-mediated lipid peroxidation has been implicated in the pathogenesis of various diseases. Lipid peroxidation products are cytotoxic and they modify proteins and DNA bases, leading eventually to degenerative disorders. Various synthetic antioxidants have been developed and assessed for their capacity to inhibit lipid peroxidation and oxidative stress induced by free radicals. In this study, the capacity of novel 6-amino-2,4,5-trimethyl-3-pyridinols for scavenging peroxyl radicals, inhibiting plasma lipid peroxidation in vitro, and preventing cytotoxicity induced by glutamate, 6-hydroxydopamine, 1-methyl-4-phenylpyridium (MPP+ ), and hydroperoxyoctadecadienoic acid was assessed. It was found that they exerted higher reactivity toward peroxyl radicals and more potent activity for inhibiting the above oxidative stress than α-tocopherol, the most potent natural antioxidant, except against the cytotoxicity induced by MPP+. These results suggest that the novel 6-amino-3-pyridinols may be potent antioxidants against oxidative stress.

α-Tocopheryl phosphate (α-TP), a water-soluble analogue of α-tocopherol, is found in humans, animals, and plants. α-TP is resistant to both acid and alkaline hydrolysis and may exert its own function in this form in vivo. In this study, the uptake, hydrolysis, and antioxidant action of α-TP were measured using α-TP with a deuterated methyl group, CD3, at position 5 of the chroman ring (α-TP(CD3)). The hydrolysis of α-TP(CD3) was followed by measuring α-tocopherol containing the CD3 group, α-T(CD3), in comparison to unlabeled α-tocopherol, α-T(CH3). α-TP(CD3) was incubated with cultured cells, and the intracellular α-T(CD3) formed was measured with HPLC–ECD and GC–MS. α-TP(CD3) was also administered to mice for 4 weeks by mixing in the diet, and α-T(CD3) was measured in plasma, liver, brain, heart, and testis to compare with endogenous unlabeled α-T(CH3). It was found that α-TP(CD3) was taken in and hydrolyzed readily to α-T(CD3) in cultured cells and in mice. The hydrolysis of α-TP(CD3) in cell culture medium was not observed. α-TP protected primary cortical neuronal cells from glutamate-induced cytotoxicity, and α-TP given to mice reduced the levels of lipid peroxidation products in plasma and liver. These results suggest that α-TP is readily hydrolyzed in vivo to α-T, which acts as an antioxidant, and that α-TP may be used as a water-soluble α-T precursor in intravenous fluids, in eye drops, or as a dietary supplement.

The role and beneficial effects of antioxidants against various disorders and diseases induced by oxidative stress have received much attention. Many types of antioxidants with different functions play their role in the defense network in vivo. The free radical scavenging antioxidants are one of the important classes of antioxidants and the assessment of their capacity has been the subject of extensive studies and argument. Various methods have been developed and applied in different systems, but many available methods result in inconsistent results. There is no simple universal method by which antioxidant capacity can be assessed accurately and quantitatively. In this review article, the available methods are critically reviewed on the basis of the mechanisms and dynamics of antioxidant action, and the methods are proposed to assess the capacity of radical scavenging and inhibition of lipid peroxidation both in vitro and in vivo. It is emphasized that the prevailing competition methods such as oxygen radical absorption capacity (ORAC) using a reference probe may be useful for assessing the capacity for scavenging free radicals but that such methods do not evaluate the characteristics of antioxidants and do not necessarily show the capacity to suppress the oxidation, that is, antioxidation. It is recommended that the capacity of antioxidant compounds and their mixtures for antioxidation should be assessed from their effect on the levels of plasma lipid peroxidation in vitro and biomarkers of oxidative stress in vivo.

Lipid peroxidation (LPO) has been shown to induce disturbance of membrane organization and functional loss and modification of proteins and DNA bases, and it has been implicated in the pathogenesis of various diseases. At the same time, LPO products have been shown to act as redox signaling mediators. Free and ester forms of both polyunsaturated fatty acids and cholesterol are important substrates for LPO in vivo and they are oxidized by both enzymatic and nonenzymatic mechanisms to give a variety of products. The results of numerous studies reported in the literatures show that the levels of LPO products in plasma of healthy human subjects are below 1 μM and that the molar ratios of LPO products to the respective parent lipids are below 1/1000, that is, below 0.1%. The levels of LPO products in human erythrocytes were found to be higher than those in plasma. Considerable levels of cholesterol oxidation products were observed. Although many LPO products exert cyctotoxicity, sublethal concentrations of LPO products induce cellular adaptive responses and enhance tolerance against subsequent oxidative stress through upregulation of antioxidant compounds and enzymes. This adaptive response is observed not only for chemically reactive α,β-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal and 15-deoxy-delta-12,14-prostaglandin J2 but also for chemically stable compounds such as hydroxyoctadecadienoic acid, hydroxylcholesterol, and lysophosphatidylcholine. Such opposite dual functions of LPO products imply that LPO, and probably oxidative stress in general, may exert both deleterious and beneficial effects in vivo. LPO as well as reactive oxygen and nitrogen species has been shown to play an important role as a regulator of gene expression and cellular signaling messenger. In order to exert physiologically important functions as a regulator of gene expression and mediator of cellular signaling, the formation of LPO products must be strictly controlled and programmed. In contrast to LPO products by enzymatic oxidation, it appears difficult to regulate the formation of free radical-mediated LPO products. Even such unregulated LPO products may exert beneficial effects at low levels, but excessive unregulated LPO may lead to pathological disorders and diseases.