Evaluation of antioxidants: Scope, limitations and relevance of assays

Peroxidation of lipids, particularly polyunsaturated fatty acid residues (PUFA) of phospholipids and cholesterol esters, is a process of marked implications: it shortens the shelf-life of food and drugs, it causes fragmentation of DNA, it damages cellular membranes and it promotes the genesis of many human diseases. Much effort is therefore devoted to a search for "potent antioxidants", both synthetic and from natural sources, mostly plants. This, in turn, requires a reliable, simple, preferably high throughput assay of the activity of alleged antioxidants. The most commonly used assays are based on measurements of the total antioxidant capacity (TAC) of a solution, as evaluated either by determining the rate of oxidation of the antioxidant or by measuring the protection of an easily determined indicator against oxidation by the antioxidants. The commonly used assays utilized for ranking antioxidants share three common problems: (i) They usually evaluate the effects of those antioxidants that quench free radicals, which constitute only a part of the body's antioxidative network, in which enzymes play the central role. (ii) Both the capacity and potency of antioxidants, as obtained by various methods, do not necessarily correlate with each other. (iii) Most estimates are based on methods conducted in solution and are therefore not necessarily relevant to processes that occur at the lipid-water interfaces in both membranes and micro emulsions (e.g. lipoproteins). Given this "state of art", many researchers, including us, try to develop a method based on the formation of hydroperoxides (LOOH) upon peroxidation of PUFA in lipoproteins or in model membranes, such as liposomes. In these systems, as well as in lipoproteins, the most apparent effect of antioxidants is prolongation of the lag time preceding the propagation of a free radical chain reaction. In fact, under certain conditions both water soluble antioxidants (e.g. vitamin C and urate) and the lipid soluble antioxidant tocopherol (vitamin E), promote or even induce peroxidation. Based on the published data, including our results, we conclude that terms such as 'antioxidative capacity' or 'antioxidative potency' are context-dependent. Furthermore, criteria of the efficacy of antioxidants based on oxidation in solution are not necessarily relevant to the effects of antioxidants on peroxidation in biological systems or model lipid assemblies, because the latter processes occur at water/lipid interfaces. We think that evaluation of antioxidants requires kinetic studies of the biomarker used and that the most relevant characteristic of 'oxidative stress' in the biological context is the kinetics of ex vivo peroxidation of lipids. We therefore propose studying the kinetics of lipid-peroxidation in the absence of the studied antioxidant and in its presence at different antioxidant concentrations. These protocols mean that antioxidants are assayed by methods commonly used to evaluate oxidative stress. The advantage of such evaluation is that it enables quantization of the antioxidants' efficacy in a model of relevance to biological systems. In view of the sensitivity of the lag time preceding peroxidation, we propose studying how much antioxidant is required to double the lag observed prior to rapid peroxidation. The latter quantity (C(2lag)) can be used to express the strength of antioxidants in the relevant system (e.g. LDL, serum or liposomes).     

Hypoxia and oxidation levels of DNA and lipids in humans and animal experimental models

The objective of this review was to evaluate the association between hypoxia and oxidative damage to DNA and lipids. Evaluation criteria encompassed specificity and validation status of the biomarkers, study design, strength of the association, dose-response relationship, biological plausibility, analogous exposures, and effect modification by intervention. The collective interpretation indicates persuasive evidence from the studies in humans for an association between hypoxia and elevated levels of oxidative damage to DNA and lipids. The levels of oxidatively generated DNA lesions and lipid peroxidation products depend on both the duration and severity of the exposure to hypoxia. Largest effects are observed with exposure to hypoxia at high altitude, but other factors, including ultraviolet light, exercise, exertion, and low intake of antioxidants, might contribute to the effect observed in subjects at high altitude. Most of the animal experimental models should be interpreted with caution because the assays for assessment of lipid peroxidation products have suboptimal validity.     

Oxidative stress and aberrant signaling in aging and cognitive decline

Brain aging is associated with a progressive imbalance between antioxidant defenses and intracellular concentrations of reactive oxygen species (ROS) as exemplified by increases in products of lipid peroxidation, protein oxidation, and DNA oxidation. Oxidative conditions cause not only structural damage but also changes in the set points of redox-sensitive signaling processes including the insulin receptor signaling pathway. In the absence of insulin, the otherwise low insulin receptor signaling is strongly enhanced by oxidative conditions. Autophagic proteolysis and sirtuin activity, in turn, are downregulated by the insulin signaling pathway, and impaired autophagic activity has been associated with neurodegeneration. In genetic studies, impairment of insulin receptor signaling causes spectacular lifespan extension in nematodes, fruit flies, and mice. The predicted effects of age-related oxidative stress on sirtuins and autophagic activity and the corresponding effects of antioxidants remain to be tested experimentally. However, several correlates of aging have been shown to be ameliorated by antioxidants. Oxidative damage to mitochondrial DNA and the electron transport chain, perturbations in brain iron and calcium homeostasis, and changes in plasma cysteine homeostasis may altogether represent causes and consequences of increased oxidative stress. Aging and cognitive decline thus appear to involve changes at multiple nodes within a complex regulatory network.     

Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid beta-peptide.

Amyloid beta-peptide (Abeta) is heavily deposited in the brains of Alzheimer's disease (AD) patients. Free-radical oxidative stress, particularly of neuronal lipids, proteins and DNA, is extensive in those AD brain areas in which Abeta is abundant. Recent research suggests that these observations might be linked, and it is postulated that Abeta-induced oxidative stress leads to neurodegeneration in AD brain. Consonant with this postulate, Abeta leads to neuronal lipid peroxidation, protein oxidation and DNA oxidation by means that are inhibited by free-radical antioxidants. Here, we summarize current research on phospholipid peroxidation, as well as protein and DNA oxidation, in AD brain, and discuss the potential role of Abeta in this oxidative stress.     

Postprandial oxidative stress

Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma concentration of lipid hydroperoxides, detectable by a sensitive chemiluminescence procedure. This is associated with increased susceptibility of LDL to oxidation, apparently due a structural perturbation at the particle surface brought about by lipid oxidation products. The postprandial modification of LDL is at least partially accounted for by an increase of LDL-, a subfraction containing lipid oxidation products where apoprotein-B-100 (apoB-100) is denatured. Consuming the meal with a suitable source of antioxidants, such as those found in red wine, minimizes this postprandial oxidative stress. The inhibition of peroxidation of lipids present in the meal during digestion is a possible mechanism for the observed protection of LDL. The in vivo oxidatively modified LDL- has numerous features that correspond to the atherogenic minimally modified LDL produced in vitro. These modified particles could account for a relevant link between nutrition and early biological processes that foster the development of atherosclerosis.     

Changes in the physico-chemical characteristics of mouse liver lipids after administration of a mixture of unsaturated fatty acids

The effects of a mixture of unsaturated fatty acids (UFA) on changes in the lipid oxidation substrate, a system of natural antioxidants and the functioning of a system controlling lipid peroxidation were studied. It was shown that arachidonate induces the incorporation of fatty acids into individual lipid fractions and the dilution of lipids by UFA esters, as a result of which the relative amount of natural antioxidants diminishes. Under these conditions, the oxidative capacity of lipids decreases, which manifests itself in a reduction of the rate of natural antioxidants utilization and an increase in their number. It was shown that purposeful modification of the lipid substrate does not interfere with the interaction between individual parameters of the system controlling lipid peroxidation.     

Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma

Oxidative stress and the role of antioxidants are currently one of the most important subjects in the field of life science. In the present study, we assessed the oxidation of plasma lipids induced by free radicals and its inhibition by antioxidants with a fluorescence probe BODIPY. Vitamin E and C-depleted plasma was used to evaluate the inherent action of several antioxidants. BODIPY reacted with free radicals in plasma to emit fluorescence (ex. 510 nm, em. 520 nm), which was suppressed by the antioxidants in a concentration-dependent manner. However, the suppression of fluorescence emission by antioxidants did not always correlate quantitatively with the suppression of lipid peroxidation. For example, alpha-tocopherol suppressed BODIPY fluorescence but enhanced the peroxidation of plasma lipids in the absence of ascorbic acid. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without a phytyl side chain, almost completely suppressed both fluorescence emission and lipid peroxidation in the plasma. These results show that BODIPY can be used as a convenient probe for radical scavenging, but that care should be taken for the evaluation of antioxidant capacity.     

Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid β-peptide

Amyloid β-peptide (Aβ) is heavily deposited in the brains of Alzheimer's disease (AD) patients. Free-radical oxidative stress, particularly of neuronal lipids, proteins and DNA, is extensive in those AD brain areas in which Aβ is abundant. Recent research suggests that these observations might be linked, and it is postulated that Aβ-induced oxidative stress leads to neurodegeneration in AD brain. Consonant with this postulate, Aβ leads to neuronal lipid peroxidation, protein oxidation and DNA oxidation by means that are inhibited by free-radical antioxidants. Here, we summarize current research on phospholipid peroxidation, as well as protein and DNA oxidation, in AD brain, and discuss the potential role of Aβ in this oxidative stress.     

Inhibition of antioxidants and hyperthermia enhance bleomycin-induced cytotoxicity and lipid peroxidation in Chinese hamster ovary cells.

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. Heat enhances the cytotoxic effect of certain anticancer agents such as bleomycin, but the mechanisms involved in cell killing are currently unknown. Bleomycin generates reactive oxygen species. It is likely that hyperthermia itself also increases oxidative stress in cells. We evaluate whether oxidative stress has a role in the mechanism of cell death caused by bleomycin and heat in Chinese hamster ovary cells. Heat (41 to 44 degrees C) increased cytotoxicity of bleomycin, evaluated by clonogenic cell survival. Decreased levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus enhancing cytotoxic responses to heat and to oxidant-generating drugs. We determine the involvement of four major cellular antioxidant defenses, superoxide dismutase (SOD), the glutathione redox cycle (GSH cycle), catalase, and glutathione S-transferase (GST), in cellular sensitivity to bleomycin, alone or combined with hyperthermia. These cellular defenses were inhibited by diethyldithiocarbamate, l-buthionine sulfoximine, aminotriazole, and ethacrynic acid, respectively. We show that levels of antioxidants (SOD, GSH cycle, and GST) affect cellular cytotoxic responses to bleomycin, at normal and elevated temperatures (41 to 44 degrees C), suggesting the involvement of oxidative stress. Bleomycin and iron caused oxidative damage to membrane lipids in intact cells, at 37 and 43 degrees C. Lipid peroxidation was evaluated by fluorescence detection of thiobarbituric acid-reactive products. There was an increase in damage to membrane lipids when the antioxidant defenses, SOD and catalase, were inhibited. The differing effects of antioxidant inhibitors on bleomycin-induced cytotoxicity and membrane lipid damage suggest that different mechanisms are involved in these two processes. However, free radicals appear to be involved in both cases. The marked sensitization of cells by diethyldithiocarbamate, to both bleomycin-induced cytotoxicity and lipid peroxidation, suggests that superoxide could be involved in both of these processes.     

The Correlation between Selenium-Dependent Glutathione Peroxidase Activity and Oxidant/Antioxidant Balance in Sera of Diabetic Patients with Nephropathy

Background:Oxidative stress is an imbalance between free radical''s production and the body''s ability to counteract or detoxify their harmful effects through neutralization by antioxidants, oxidative stress is thought to be involved in the pathogenesis of diabetic nephropathy. One of the key enzymatic antioxidants is glutathione peroxidase (GPx), which plays an important protective function in diabetes complications, by reducing the rising state of oxidative stress and removing toxicity from peroxides and converting them into a non-toxic substance. The objective of this research was to evaluate the rule of glutathione peroxidase in regulate oxidants/antioxidants levels diabetic patients with nephropathy.Methods:In a case-control study, we assessed serum GPx activity (Se-Dependent, non-selenium dependent and total GPx), total oxidant, lipid peroxidation, total antioxidant, and catalase in healthy control subjects (group 1), in diabetic patients without diabetic nephropathy (group 2) and diabetic patients with nephropathy (group 3).Results:GPx activity was significantly lower in T2D patients with and without nephropathy compared to healthy subject’s control. Total oxidants and lipids peroxidation have a negative correlation with the GPx and other antioxidants.Conclusion:Decreased GPx activity indicate a relationship between GPx activity and diabetic nephropathy.Key Words: Diabetic Nephropathy, Glutathione Peroxidase, Oxidative Stress, Reactive Oxygen Species, Selenium     

The effects of Maras powder (smokeless tobacco) on oxidative stress in users

Maras Powder (MP) is a special kind of smokeless tobacco widely used in the southeast region of Turkey especially in Kahramanmaras and Gaziantep and other southeastern cities. It is obtained from a tobacco species, Nicotiana rustica L and ash of oak or grapevine wood. Tobacco may increase oxidative stress, which is related to the products of the oxygen metabolism taking place in all cells. Cellular antioxidants, e.g. catalase (CAT), superoxide dismutase (SOD) and glucose 6-phosphate dehydrogenase (G6PD) protect the cell against oxidative damage. An imbalance between the ROS and antioxidants in favour of ROS is described as oxidative stress. In this study, we aimed to investigate the effect of MP on antioxidant enzyme levels and lipid peroxidation. We measured malondialdehyde (MDA), CAT, SOD and G6PD levels in blood of 68 MP users and 30 healthy controls who did not use MP. CAT, SOD and G6PD levels were lower in MP users than in the controls. On the other hand, lipid peroxidation levels (MDA), one of the best indicators of cytological damage, was increased in MP users compared with the controls. The present study showed that MP increases oxidative stress, which may cause many systemic disorders, including arteriosclerosis.     

Involvement of free radicals and oxidative stress in NAFLD/NASH

Abstract

Non-alcoholic fatty liver disease (NAFLD) is now the most common liver disease affecting high proportion of the population worldwide. NAFLD encompasses a large spectrum of conditions ranging from fatty liver to non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and cancer. NAFLD is considered as a multifactorial disease in relation to the pathogenic mechanisms. Oxidative stress has been implicated in the pathogenesis of NAFLD and NASH and the involvement of reactive oxygen species (ROS) has been suggested. Many studies show the association between the levels of lipid oxidation products and disease state. However, often neither oxidative stress nor ROS has been characterized, despite oxidative stress is mediated by multiple active species by different mechanisms and the same lipid oxidation products are produced by different active species. Further, the effects of various antioxidants have been assessed in human and animal studies, but the effects of drugs are determined by the type of active species, suggesting the importance of characterizing the active species involved. This review article is focused on the role of free radicals and free radical-mediated lipid peroxidation in the pathogenesis of NAFLD and NASH, taking characteristic features of free radical-mediated oxidation into consideration. The detailed analysis of lipid oxidation products shows the involvement of free radicals in the pathogenesis of NAFLD and NASH. Potential beneficial effects of antioxidants such as vitamin E are discussed.     

Nicotinamide (vitamin B3) as an effective antioxidant against oxidative damage in rat brain mitochondria

Nicotinamide (vitamin B3) an endogenous metabolite, showed significant inhibition of oxidative damage induced by reactive oxygen species (ROS) generated by ascorbate-Fe2+ and photosensitization systems in rat brain mitochondria. It protected against both protein oxidation and lipid peroxidation, at millimolar concentrations. Inhibition was more pronounced against oxidation of proteins than peroxidation of lipids. Chemically related endogenous compounds, tryptophan and isonicotinic acid, showed comparable inhibitory properties. The protective effect observed, at biologically relevant concentrations, with nicotinamide was more than that of the endogenous antioxidants ascorbic acid and alpha-tocopherol. Hence our studies suggest that nicotinamide (vitamin B3) can be considered as a potent antioxidant capable of protecting the cellular membranes in brain, which is highly susceptible to prooxidants, against oxidative damage induced by ROS.     

Dynamics of estrogen-induced oxidative stress

The objective of this study was to assess the dynamics of oxidative damage to cellular macromolecules such as proteins, lipids and DNA under conditions of oxidative stress triggering early stages of estrogen-dependent carcinogenesis. A rodent model of carcinogenesis was used. Syrian hamsters were sacrificed after 1, 3, 5 h and one month from the initial implantation of estradiol. Matching control groups were used. Kidneys as target organs for estradiol-mediated oxidative stress were excised and homogenized for biochemical assays. Subcellular fractions were isolated. Carbonyl groups (as a marker of protein oxidation) and lipid hydroxyperoxides were assessed. DNA was isolated and 8-oxodGuo was assessed. Electron paramagnetic resonance spectroscopy was used to confirm the results for lipid peroxidation. Exposition to estradiol in the rodent model leads to damage of macromolecules of the cell, including proteins and DNA, but not lipids. Proteins appear to be the primary target of the damage but are closely followed by DNA. It has previously been speculated that protein peroxides can increase DNA modifications. This time sequence was observed in our study. Nevertheless, the direct relation between protein and DNA damage still remains unsolved.     

Oxidative stress markers during a course of hyperthyroidism

INTRODUCTION: Previous studies have shown the presence of oxidative stress in hyperthyroid patients. The aim of this study was to evaluate the influence of hyperthyroidism on lipid peroxidation, plasma lipoprotein oxidation and antioxidant status. We have estimated the clinical utility of the biochemical parameters analysed as markers of oxidative stress in hyperthyroidism. MATERIAL AND METHODS: Twenty five patients with overt hyperthyroidism because of Graves' disease or toxic multinodular goitre and 20 healthy subjects were included in the study. Lipid peroxidation was evaluated by measurement of peroxides and malondialdehyde with 4-hydroxynonenal (MDA + 4-HNE) concentrations. Autoantibodies against oxidised LDL (anti-oxLDL) were assayed as a marker of lipoprotein oxidation. Changes in the antioxidant defence system were estimated by measurement of total antioxidant status in serum (TAS) and erythrocyte superoxide dismutase activity (SOD). RESULTS: A significant increase in serum concentration of peroxides and MDA + 4-HNE was observed in patients with hyperthyroidism. However, no difference was found in anti-oxLDL concentration and antioxidant status parameters (TAS, SOD) between the control group and the patient group. CONCLUSIONS: Our results indicate an intensification of the oxidative processes caused by an excess of thyroid hormones, which is not accompanied by a response from the antioxidant system. Elevated concentrations of lipid peroxidation products in serum, both peroxides and malondialdehyde with 4-hydroxynonenal, may be useful as markers of oxidative stress during the course of hyperthyroidism.     

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F2-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F2alpha (PGF2alpha), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF2alpha in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF2alpha levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of "oxidative stress" or "oxidative damage" in vivo.     

Lipid peroxidation products in biological tissues

Over the past twenty-years of lipid peroxidation research in this laboratory, considerable effort has gone into development of new methods, with emphasis on measurement of lipid-soluble fluorophores and the volatile hydrocarbons ethane and pentane. Application of these and other methods has been made to biological materials and living animals. Although the various methodologies used in lipid peroxidation research do not necessarily measure the same class of products, and although agreement of results is not always 100%, there is substantial documentation of good correlations between measurements; for example, of trace volatile hydrocarbons with thiobarbituric acid-reacting substances, of pentane production with dietary and/or tissue vitamin E content, and of pentane production with lipid-soluble fluorophores accumulated in spleen as a function of oxidant stress. Individual methodologies do have their inherent limitations. However, measurements of multiple products and their correlations have added significantly to the base of information on biological damage and protection by dietary antioxidants against nutritional and toxicological insults to tissues, cells, and macromolecules as a result of peroxidative and oxidative reactions.     

Diquat-induced oxidative damage in hepatic microsomes: effects of antioxidants.

The ability of the redox cycling compound, diquat, to induce lipid peroxidation and oxidative damage was investigated using hepatic microsomes. Antioxidants, with demonstrated efficacy in physical models of oxidative stress, were examined in a diquat model. Diquat (10 microM-3 mM) induced lipid peroxidation (TBARS) in hepatic microsomes prepared from Fischer 344 rats. Diquat (1 mM) also increased protein carbonyl formation, NADPH oxidation and superoxide anion radical production (acetylated cytochrome c reduction). The novel antioxidants U-74,006F, U-78,517G and the known antioxidant, DPPD, decreased diquat-induced lipid peroxidation to levels below that of the control. These antioxidants also decreased protein carbonyl formation caused by diquat. U-74,006F and U-78,517G reduced NADPH oxidation slightly; although this inhibition was statistically significant, the biological significance is questionable. DPPD had no effect on this parameter. U-78,517G inhibited the reduction of acetylated cytochrome c slightly, whereas the other antioxidants had little effect. Thus overall, the increase in NADPH oxidation and the production of superoxide anion by redox cycling of diquat were not substantially affected by antioxidants. Neither did the test compounds show evidence of activity as iron chelators. This leads to the suggestion that antioxidants are preventing diquat-induced oxidative damage by scavenging lipid peroxyl radicals and preventing the propagation of the lipid peroxidation process.     

Studies of phospholipid oxidation by electrospray mass spectrometry: from analysis in cells to biological effects

The oxidation of lipids is important in many pathological conditions and lipid peroxidation products such as 4-hydroxynonenal (HNE) and other aldehydes are commonly measured as biomarkers of oxidative stress. However, it is often useful to complement this with analysis of the original oxidized phospholipid. Electrospray mass spectrometry (ESMS) provides an informative method for detecting oxidative alterations to phospholipids, and has been used to investigate oxidative damage to cells, and low-density lipoprotein, as well as for the analysis of oxidized phosphatidylcholines present in atherosclerotic plaque material. There is increasing evidence that intact oxidized phospholipids have biological effects; in particular, oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) have been found to cause inflammatory responses, which could be potentially important in the progression of atherosclerosis. The effects of chlorohydrin derivatives of lipids have been much less studied, but it is clear that free fatty acid chlorohydrins and phosphatidylcholine chlorohydrins are toxic to cells at concentrations above 10 micromolar, a range comparable to that of HNE and oxidized PAPC. There is some evidence that chlorohydrins have biological effects that may be relevant to atherosclerosis, but further work is needed to elucidate their pro-inflammatory properties, and to understand the mechanisms and balance of biological effects that could result from oxidation of complex mixtures of lipids in a pathophysiological situation.     

Lipid peroxidation: mechanisms, inhibition, and biological effects

In the last 50 years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipids are oxidized by three distinct mechanisms; enzymatic oxidation, non-enzymatic, free radical-mediated oxidation, and non-enzymatic, non-radical oxidation. Each oxidation mechanism yields specific products. The oxidation of linoleates and cholesterol is discussed in some detail. The relative susceptibilities of lipids to oxidation depend on the reaction milieu as well as their inherent structure. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. Phospholipid hydroperoxides, for example, are reduced to the corresponding hydroxides by selenoproteins in vivo. Various kinds of antioxidants with different functions inhibit lipid peroxidation and the deleterious effects caused by the lipid peroxidation products. Furthermore, the biological role of lipid peroxidation products has recently received a great deal of attention, but its physiological significance must be demonstrated in future studies.