Comparison of LDL trap assay to other tests of antioxidant capacity; effect of vitamin E and lovastatin treatment

Oxidized low density lipoprotein (LDL) has a major impact in the development of atherosclerosis. Risk for oxidative modification of LDL is usually determined indirectly by measuring the capability of LDL to resist radical insult. We compared three different methods quantifying the antioxidative capacity of LDL ex vivo in dyslipidemic patients with coronary heart disease. Plasma samples were obtained from two double-blinded cross-over trials. The duration of all interventions (placebo, lovastatin 60 mg/day, RRR-alpha-tocopherol 300 mg/day and lovastatin + RRR-alpha-tocopherol combined) was 6 weeks. The total radical capturing capacity of LDL (TRAP) in plasma was determined using 2,2-azo-bis(2,4-dimethyl-valeronitrile) (AMVN) -induced oxidation, and measuring the extinction time of chemiluminescence. TRAP was compared to the variables characterizing formation of conjugated dienes in copper-induced oxidation. Also the initial concentrations and consumption times of reduced alpha-tocopherol (alpha-TOH) and ubiquinol in AMVN-induced oxidation were determined. Repeatability of TRAP was comparable to that of the lag time in conjugated diene formation. Coefficient of variation within TRAP assay was 4.4% and between TRAP assays 5.9%. Tocopherol supplementation produced statistically significant changes in all antioxidant variables except those related to LDL ubiquinol. TRAP increased by 57%, the lag time in conjugated diene formation by 34% and consumption time of alpha-TOH by 88%. When data of all interventions were included in the analyses, TRAP correlated with the lag time (r = 0.75, p < 10(-6)), with LDL alpha-TOH (r = 0.50, p < 0.001) and with the consumption time of alpha-TOH (r = 0.58, p < 0.0001). In the baseline data, the associations between different antioxidant variables were weaker. TRAP correlated with the lag time (r = 0.55, p < 0.001) and alpha-TOH consumption time (r = 0.48, p < 0.05), and inversely with apolipoprotein Al (r = -0.51, p < 0.05). Lag time at the baseline did not correlate with ubiquinol or tocopherol parameters, or with any plasma lipid or lipoprotein levels analyzed. Lovastatin treatment did not significantly affect the antioxidant capacity of LDL. In conclusion, TRAP reflects slightly different properties of LDL compared to the lag time. Thus, LDL TRAP assay may complement the other methods used to quantify the antioxidant capacity of LDL. However, TRAP and the lag time react similarly to vitamin E supplementation.     

Application of optimized chemiluminescence assay for determination of the antioxidant capacity of herbal extracts

A chemiluminescence (CL) assay for the determination of antioxidant capacity (AOC) has been optimized and applied to analyses of herbal extracts in the present study. The optimal concentrations of reagents (luminol, H₂O₂, horseradish peroxidase) have been determined, as well as the optimal reaction conditions (wavelength, pH, temperature, sample volume). All of the measurements were performed at the emission maximum of the oxidized form of luminol (425 nm). The optimal concentrations of the reagents were determined as follows: 1.6 mmol/L luminol, 7.5 mmol/L H₂O₂ and 0.14 U/mL horseradish peroxidase activity in the reaction mixture. Analyses were carried out in phosphate buffer, pH 7.4, at room temperature. With the optimized CL assay, the AOCs of various water and methanol herbal extracts were determined (dog rose hips, plantain leaves and coltsfoot and thyme flowers) and the results were compared to those obtained by other classical methods for the evaluation of antioxidants. Strong correlations (r > 0.9) with the Folin–Ciocalteau assay and the 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH)^● assay are confirmed, although there is no correlation between AOC and the concentration of ascorbic acid in the samples analysed. This optimized CL assay is simple, rapid and reliable, and it represents a good alternative to classical methods (Folin–Ciocalteau, DPPH^●) for the determination of AOC of herbal extracts and other food samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Relationship between DNA damage, total antioxidant capacity and coronary artery disease

OBJECTIVE: It has been known that there was a relation between the levels of DNA damage and the severity of the coronary artery disease (CAD). However, little is known about association of DNA damage with total antioxidant capacity (TAC) and CAD. The aim of this study is to evaluate the relationship between DNA damage, TAC and CAD. METHODS: We used the comet assay to measure DNA damage from 53 patients with angiographically documented CAD and 42 patients with angiographically documented normal coronary vessel. The extent and severity of CAD was calculated to Gensini score index. TAC of plasma was determined using a novel automated measurement method. RESULTS: Mean values of DNA damage were significantly higher in CAD patients than in the control group (p<0.001). There was a positive correlation between Gensini score index and DNA damage (r=0.590, p<0.001). Additionally, significantly positive correlations between score of DNA damage, and diabetes, smoking, obesity and hyperlipidemia were found (p<0.05). There was also a negative correlation between TAC and DNA damage (r=-0.711, p<0.001). The DNA damage was significantly higher in diabetic, smoker, hyperlipidemic and obese individuals than those without these conditions (p=0.001, p=0.006, p=0.001, p=0.004, respectively). CONCLUSIONS: These data indicate that the level of DNA damage is increased and TAC level is decreased in CAD. DNA damage is correlated with the severity of the CAD, and levels of TAC.     

Evaluation of the copper(II) reduction assay using bathocuproinedisulfonic acid disodium salt for the total antioxidant capacity assessment: The CUPRAC-BCS assay

There is heightened interest in determining antioxidant status of individuals in experimental and clinical studies investigating progression of diseases or diverse aspects of oxidative stress, among others. The aim of this study was to evaluate the copper(II) reduction assay with bathocuproinedisulfonic acid disodium salt as chelating agent (the CUPRAC-BCS assay) for the total antioxidant capacity (TAC) assessment in human plasma and urine. Samples from 20 individuals were determined with four spectrophotometric assays—CUPRAC-BCS, ferric reducing ability of plasma (FRAP), trolox equivalent antioxidant capacity (TEAC), and 1,1-diphenyl-2-picrylhydrazyl assay (DPPH)—to compare these methods. CUPRAC-BCS was significantly correlated with FRAP and TEAC for plasma and urine samples (r > 0.5, P < 0.05 for all) and with DPPH for urine samples (r = 0.925, P < 0.001) but not with DPPH for plasma samples (r = 0.366, P = 0.112). However, the four methods do not agree given that lines of equality and regression were not matched up. The imprecision of the method is less than 6%, the detection limit is 41.8 μmol trolox equivalents/L, it is linear up to 2 mM trolox, and ethylenediaminetetraacetic acid dihydrate disodium salt (EDTA) binds to Cu(II), avoiding the formation of Cu(I)-BCS complex. This study shows that CUPRAC-BCS is a simple, fast, inexpensive, and suitable method for TAC assessment in human urine and heparinized plasma samples.     

The effects of vitamin C supplementation on oxidative stress and antioxidant content in the brains of chronically exercised rats

The purpose of this study was to ascertain whether vitamin C supplementation during chronic exercise training alters rat brain antioxidant content. Female Wistar albino rats were exercised on a treadmill for 30 min/day for 6.5 weeks and were administered daily intraperitoneal injections of vitamin C (20 mg/kg). After the training period, chronically exercised rats showed no significant changes in total brain thiobarbituric acid reactive substances (TBARS) levels. In contrast, rats supplemented with vitamin C during the training period showed significantly elevated brain TBARS levels. If such results were extrapolated to man, where vitamin supplementation is a common practice, this would indicate that vitamin C supplementation may not protect brain tissue against exercise-induced oxidative damage, in such circumstances, this water-soluble antioxidant behaves as a pro-oxidant. (Mol Cell Biochem xxx: 135–138, 2005)     

Maternal intake of vitamin E and birth defects,national birth defects prevention study, 1997 to 2005

BACKGROUND In a recent study, high maternal periconceptional intake of vitamin E was found to be associated with risk of congenital heart defects (CHDs). To explore this association further, we investigated the association between total daily vitamin E intake and selected birth defects. METHODS: We analyzed data from 4525 controls and 8665 cases from the 1997 to 2005 National Birth Defects Prevention Study. We categorized estimated periconceptional energy‐adjusted total daily vitamin E intake from diet and supplements into quartiles (referent, lowest quartile). Associations between quartiles of energy‐adjusted vitamin E intake and selected birth defects were adjusted for demographic, lifestyle, and nutritional factors. RESULTS: We observed a statistically significant association with the third quartile of vitamin E intake (odds ratio [OR], 1.17; 95% confidence interval [CI], 1.01–1.35) and all CHDs combined. Among CHD sub‐types, we observed associations with left ventricular outflow tract obstruction defects, and its sub‐type, coarctation of the aorta and the third quartile of vitamin E intake. Among defects other than CHDs, we observed associations between anorectal atresia and the third quartile of vitamin E intake (OR, 1.66; 95% CI, 1.01–2.72) and hypospadias and the fourth quartile of vitamin E intake (OR, 1.42; 95% CI, 1.09–1.87). CONCLUSION: Selected quartiles of energy‐adjusted estimated total daily vitamin E intake were associated with selected birth defects. However, because these few associations did not exhibit exposure‐response patterns consistent with increasing risk associated with increasing intake of vitamin E, further studies are warranted to corroborate our findings. Birth Defects Research (Part A), 100:647–657, 2014. © 2014 Wiley Periodicals, Inc.     

Increased hepatic lipid soluble antioxidant capacity as compared to other organs of streptozotocin-induced diabetic rats: A cyclic voltammetry study

It has been suggested that oxidative stress plays an important role in the chronic complications of diabetes. The experimental findings regarding the changes in tissue antioxidant enzymes and lipid peroxidation of diabetic tissues have been inconsistent. Previous studies in our laboratory demonstrated that the reducing power of a specific tissue correlates with its low molecular weight antioxidant (LMWA) capacity. In the present study, the overall LMWA capacity (reducing equivalents) of plasma and tissues of streptozotocin (STZ)-induced diabetic rats (1–4 weeks) and insulin treated diabetic rats were measured by cyclic voltammetry. Levels of water and lipid soluble LMWA capacity progressively decreased in the diabetic plasma, kidney, heart and brain, while the diabetic liver, at 2, 3 and 4 weeks after STZ injection, showed a significant increase in the overall lipid soluble LMWA capacity (p < 0.001). Subsequently, analysis of specific components by high pressure liquid chromatography (electrochemical detection) showed decreased levels of ascorbic acid in plasma, kidney, heart and brain of diabetic animals. The α-tocopherol level dropped in all tissues, except for the liver in which there was a significant increase (p < 0.01 and p < 0.001 at 2–4 weeks). Lipid peroxidation was assessed by conjugated diene levels, which increased significantly in all diabetic tissues except the liver. Insulin treatment that was started after 3 weeks of diabetes and continued for 3 weeks showed no change in the conjugated dienes and in the overall LMWA capacity in all organs. Our results suggest a unique behavior of the liver in the STZ-induced diabetic rats to the stress and indicate its higher capacity to cope with oxidative stress as compared to other organs.     

Comparison of in vitro tests for antioxidant and immunomodulatory capacities of compounds

Oxidative stress is considered to be critically involved in the normal aging process but also in the development and progression of various human pathologies like cardiovascular and neurodegenerative diseases, as well as of infections and malignant tumors. These pathological conditions involve an overwhelming production of reactive oxygen species (ROS), which are released as part of an anti-proliferative strategy during pro-inflammatory immune responses. Moreover, ROS themselves are autocrine forward regulators of the immune response.Most of the beneficial effects of antioxidants are considered to derive from their influence on the immune system. Due to their antioxidant and/or radical scavenging nature, phytochemicals, botanicals and herbal preparations can be of great importance to prevent oxidation processes and to counteract the activation of redox-regulated signaling pathways. Antioxidants can antagonize the activation of T-cells and macrophages during the immune response and this anti-inflammatory activity could be of utmost importance for the treatment of above-mentioned disorders and for the development of immunotolerance.Herein, we provide an overview of in vitro assays for the measurement of antioxidant and anti-inflammatory activities of plant-derived substances and extracts, by discussing possibilities and limitations of these methods. To determine the capacity of antioxidants, the oxygen radical absorbance capacity (ORAC) assay and the cell-based antioxidant activity (CAA) assay are widely applied. To examine the influence of compounds on the human immune response more closely, the model of mitogen stimulated human peripheral blood mononuclear (PBMC) cells can be applied, and the production of the inflammatory marker neopterin as well as the breakdown of the amino acid tryptophan in culture supernatants can be used as readout to indicate an immunomodulatory potential of the tested compound. These two biomarkers of immune system activation are robust and correlate with the course of cardiovascular, neurodegenerative and malignant tumor diseases, but also with the normal aging process, and they are strongly predictive. Thus, while the simpler ORAC and CAA assays provide insight into one peculiar chemical aspect, namely the neutralization of peroxyl radicals, the more complex PBMC assay is closer to the in vivo conditions as the assay comprehensively enlights several properties of immunomodulatory test compounds.     

The role of ascorbate in antioxidant protection of biomembranes: Interaction with vitamin E and coenzyme Q

One of the vital roles of ascorbic acid (vitamin C) is to act as an antioxidant to protect cellular components from free radical damage. Ascorbic acid has been shown to scavenge free radicals directly in the aqueous phases of cells and the circulatory system. Ascorbic acid has also been proven to protect membrane and other hydrophobic compartments from such damage by regenerating the antioxidant form of vitamin E. In addition, reduced coenzyme Q, also a resident of hydrophobic compartments, interacts with vitamin E to regenerate its antioxidant form. The mechanism of vitamin C antioxidant function, the myriad of pathologies resulting from its clinical deficiency, and the many health benefits it provides, are reviewed.     

Comparison of melatonin, vitamin E and L-carnitine in the treatment of neuro- and hepatotoxicity induced by thioacetamide

This study was designed to evaluate and compare the effect of melatonin, vitamin E and L-carnitine on brain and liver oxidative stress and liver damage. Oxidative stress and hepatic failure were produced by a single dose of thioacetamide (TAA) (150 mg kg(-1)) in Wistar rats. A dose of either melatonin (3 mg kg(-1)) vitamin E (20 mg kg(-1) ) or L-carnitine (100 mg kg(-1)) was used. Blood samples were taken from the neck vasculature in order to determine ammonium, blood urea nitrogen (BUN) and liver enzymes. Lipid peroxidation products, glutathione (GSH) content and antioxidative enzymes were determined in cerebral and hepatic homogenates. The results showed a decrease in BUN and in the antioxidant enzymes activities and GSH in the brain and liver. Likewise, TAA induced significant enhancement of lipid peroxidation products levels in both liver and brain, as well as in ammonia values. Melatonin, vitamin E and L-carnitine, although melatonin more significantly, decreased the intensity of the changes produced by the administration of TAA alone. Furthermore melatonin combined with TAA, decreased the ammonia levels and increased the BUN values compared with TAA animals. Also it was more effective than vitamin E or L-carnitine in these actions. These data show the protective effect of these agents, especially melatonin, against oxidative stress and hepatic damage present in fulminant hepatic failure.     

Euglena gracilis as source of the antioxidant vitamin E. Effects of culture conditions in the wild strain and in the natural mutant WZSL

The photosynthetic wild type and the spontaneous non-photosynthetic WZSL mutant of the unicellular flagellate Euglena gracilis were grown to investigate the influence of photoheterotrophic and heterotrophic conditions on α-tocopherol (vitamin E) content. HPLC analysis demonstrated a marked enhancement (almost 100%) of tocopherol content in the light in both strains, independent of the presence of chloroplasts. These findings indicate that the formation of vitamin E occurs inside both the mitochondrial and chloroplastic compartment, and that the correlation between light and vitamin E production is not linked to the existence of chlorophyll. This revised version was published online in August 2006 with corrections to the Cover Date.     

The reduction of nitroblue tetrazolium by red blood cells: a measure of red cell membrane antioxidant capacity and hemoglobin-membrane binding sites

The reduction of nitroblue tetrazolium (NBT) with intact Red Blood Cells (RBCs) is biphasic with an initial rapid reduction followed by a slower second phase. This biphasic kinetics has been explained with the initial rapid phase attributed to antioxidants in the red cell which reduce membrane bound NBT and the slower phase associated with the reaction of NBT with membrane bound hemoglobin. This model has been confirmed by a utilization of a number of red cell modifications which either increase the red cell antioxidants (vitamin C and vitamin E) or damage the red cell membrane (cumene hydroperoxide and N-ethylmaleimide). The utilization of this assay for human blood samples was investigated by studying a series of 20 human subjects ranging between 34 and 87 years of age. It was possible to fit all of these samples with two adjustable parameters which reflect the red cell membrane antioxidant capacity (x) and the hemoglobin membrane interactions (m). The antioxidant capacity shows a significant (p < 002; R = -.67) decrease with age. This finding is consistent with a decrease in the level of antioxidants in aged subjects. In addition, the number of hemoglobin membrane sites are negatively correlated with the antioxidant capacity (p < .02; R = -.52) suggesting that the oxidative stress associated with reduced antioxidants results in increased hemoglobin-membrane interactions.     

Assessment of DNA strand breakage by the alkaline COMET assay in dialysis patients and the role of Vitamin E supplementation

Although the role of reactive oxygen species (ROS) in chronic renal failure (CRF) is not definitely demonstrated, a consistent number of observations has provided evidence for the presence of oxidative stress in uremic patients undergoing maintenance dialysis. In order to investigate this hypothesis further and to understand the role of antioxidant supplementation, peripheral blood lymphocytes were taken from 36 dialysis patients before and after Vitamin E supplementation in a dosage of 600 mg per day (2×300 mg) for 14 weeks and examined in the alkaline Comet assay for DNA strand breakage. The results were also compared with those of 36 controls with comparable age, sex, and smoking habits, and with no history of renal disease. The DNA breakage observed in the lymphocytes of patients before Vitamin E supplementation was significantly higher than in the controls (P<0.001) but a clear protective effect of Vitamin E supplementation were observed after 14 weeks of therapy.     

Antioxidant enzyme activity and lipid peroxidation in liver of female rats co-exposed to lead and cadmium: effects of vitamin E and Mn2+

The oxidative status of liver of female rats exposed to lead acetate and cadmium acetate either alone or in combination at a dose of 0.05 mg/kg body wt intraperitoneally for 15 days was studied. After the administration of lead alone, the activity of superoxide dismutase (SOD) decreased in liver, whereas no changes were observed in catalase (CAT) activity, and glutathione (GSH) and thiobarbituric acid (TBARS) levels. Cadmium exposure and combined exposure to lead and cadmium led to decrease in GSH content and increased TBARS levels. Moreover, animals exposed to either cadmium alone or in combination with lead showed a decrease in SOD activity and an increase in CAT activity. The in vitro experiments showed that vitamin E failed to restore the antioxidant enzyme activities in metal treated postmitochondrial supernatant fraction of liver. But Mn²⁺ ions protected the mitochondria from lipid peroxidation and could completely restore Mn-superoxide dismutase (Mn-SOD) activity following metal intoxication. The results of this study indicate that despite the ability of lead and cadmium to induce oxidative stress the effect in liver is not intensified by combined exposure to both lead and cadmium. The observed changes in various oxidative stress parameters in the liver of rats co-exposed to lead and cadmium may result from an independent effect of lead and /cadmium and also from their interaction such as changes in metal accumulation and content of essential elements like Cu, Zn and Fe. These results suggest that when lead and cadmium are present together in similar concentrations, cadmium mediates major effects due to its more reactive nature.     

Antioxidant enzyme activity and lipid peroxidation in liver of female rats co-exposed to lead and cadmium: Effects of vitamin E and Mn2+

The oxidative status of liver of female rats exposed to lead acetate and cadmium acetate either alone or in combination at a dose of 0.05?mg/kg body wt intraperitoneally for 15 days was studied. After the administration of lead alone, the activity of superoxide dismutase (SOD) decreased in liver, whereas no changes were observed in catalase (CAT) activity, and glutathione (GSH) and thiobarbituric acid (TBARS) levels. Cadmium exposure and combined exposure to lead and cadmium led to decrease in GSH content and increased TBARS levels. Moreover, animals exposed to either cadmium alone or in combination with lead showed a decrease in SOD activity and an increase in CAT activity. The in vitro experiments showed that vitamin E failed to restore the antioxidant enzyme activities in metal treated postmitochondrial supernatant fraction of liver. But Mn²⁺ ions protected the mitochondria from lipid peroxidation and could completely restore Mn-superoxide dismutase (Mn-SOD) activity following metal intoxication. The results of this study indicate that despite the ability of lead and cadmium to induce oxidative stress the effect in liver is not intensified by combined exposure to both lead and cadmium. The observed changes in various oxidative stress parameters in the liver of rats co-exposed to lead and cadmium may result from an independent effect of lead and /cadmium and also from their interaction such as changes in metal accumulation and content of essential elements like Cu, Zn and Fe. These results suggest that when lead and cadmium are present together in similar concentrations, cadmium mediates major effects due to its more reactive nature.     

Using chemiluminescence to determine whole blood antioxidant capacity in rheumatoid arthritis and Parkinson's disease patients

The consequences of oxidative stress and inflammation are implicated in a wide range of diseases, including rheumatoid arthritis and Parkinson's disease. The status of antioxidant capacity in rheumatoid arthritis and Parkinson's disease remains unclear, in part due to common practice of assaying erythrocytes separately to plasma. This method removes any synergistic interactions between plasma and erythrocyte‐based antioxidants. The experiments in this report tested antioxidant capacity in whole blood, erythrocytes and plasma by group and disease stage. Medically diagnosed patients were recruited along with appropriate control group participants. Fasting venous blood was assayed using chemiluminescence methods for: time to maximum light emitted, maximum light emitted, and plasma antioxidant capacity in vitamin E analogue units. Here we demonstrate that whole blood exhibits higher antioxidant capacity than either plasma or erythrocytes assayed separately. We report increased oxidative stress in the blood of rheumatoid arthritis patients by group (p = 0.018, p = 0.049). We show increased antioxidant capacity in Parkinson's disease patients by group (p < 0.001). For later stage Parkinson's disease patients, we report reduced oxidative stress (p = 0.025), and increased antioxidant capacity and for erythrocytes (p < 0.001, p = 0.004) and whole blood (p < 0.001, p = 0.003). Early stage Parkinson's disease showed higher antioxidant capacity on only one measure (p = 0.008). Whole blood chemiluminescence is a useful technique for determining redox status in disease and might help clarify the role of oxidative stress in rheumatoid arthritis and Parkinson's disease.     

Sialic acid and oxidizability of lipid and proteins and antioxidant status in patients with coronary artery disease

The aim of this study was to investigate the possible relationship between serum total sialic acid (TSA) concentration, recently shown to be a cardiovascular risk factor, and lipid and protein oxidation and antioxidant status and the severity of coronary artery disease (CAD) according to the obstructive vessel number in patients. The study was carried out on a total of 200 patients (142 men and 58 women) who were hospitalized for elective coronary angiographic evaluation with complaint of typical angina pectoris. According to the results of angiography, 150 patients had angiographically proven CAD (CAD group) and 50 patients had a history suggestive of angina pectoris but normal coronary angiograms (control group). The CAD group was further divided into single-, double- and triple-vessel disease groups according to the number of vessels involved. Lipid parameters were determined by routine laboratory methods. Plasma malondialdehyde (MDA) and vitamin E concentrations were determined by high-performance liquid chromatography. TSA and other oxidant and antioxidant parameters were studied spectrophotometrically. Our results demonstrated significant increases both in TSA levels and in indicators of oxidative stress in the patients with CAD compared with the controls. However, antioxidant parameters were decreased in the patients with CAD. We found strong positive correlations between TSA and plasma MDA, Delta-MDA which represents the degree of oxidative modification of apolipoprotein B-containing lipoproteins, serum protein carbonyls and apolipoprotein B and weak correlations between TSA and low density lipoprotein cholesterol, triacylglycerol, paraoxonase, glutathione peroxidase (GPx), vitamin C and vitamin E. In conclusion, TSA is related to markers of lipid and protein oxidation, paraoxonase and GPx activities, vitamin C and E levels and the severity of CAD.     

Effect of physical restraint on oxidative stress in mice fed a selenium and vitamin E deficient diet

Physical restraint has been associated with increased oxidative damage to lipid, protein, and DNA. The purpose of this experiment was to determine whether physical restraint would further exacerbate oxidative stress in mice fed a selenium (Se) and vitamin E (VE) deficient diet. Three-week-old mice were fed a Torula yeast diet containing adequate or deficient Se and VE. Menhaden oil was added to the deficient diet to impose an additional oxidative stress. After 4 wk feeding, half the mice in each group were restrained for 5 d in well-ventilated conical tubes for 8 h daily. Mice fed the Se and VE deficient diets had increased liver thiobarbituric acid-reactive substance (TBARS) levels and decreased liver glutathione peroxidase (GPX1) activity and α-tocopherol levels. Plasma corticosterone levels were elevated in restrained mice fed the deficient diet compared to unrestrained mice fed the adequate diet. Restraint had no effect on liver TBARS or α-tocopherol levels. Liver GPX1 activity, however, was lower in restrained mice fed the adequate diet. In addition, liver superoxide dismutase (SOD) activity was lower in the restrained mice fed the adequate or deficient diet. Thus, under our conditions, Se and VE deficient diet, but not restraint, increased lipid peroxidation in mice. Restraint, however, decreased antioxidant protection in mice due to decreased activities of GPX1 and SOD enzymes.     

Ascorbate-Fe2+ lipid-peroxidation of rat liver microsomes: Effect of vitamin E and cytosolic proteins

In the present study, we examined the effect of the intraperitoneal administration of vitamin E (100 mg/kg weight/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of rat liver microsomes . We also analyzed the effect of hepatic cytosolic proteins on this process. The results indicate that the ascorbate induced light emission was 76% lower in microsomes (1 mg protein) obtained from vitamin E treated animals when compared with controls. In the presence of cytosolic protein (1 mg) the chemiluminescence of control microsomes diminished 55.8 and 59.5% when cytosol from controls and treated animals was used, respectively. The chemiluminescence of vitamin E microsomes diminished 25.03 and 22.08% when both types of cytosol were added to the medium. Dialyzed or treated at 70°C cytosol was also able to inhibit the lipid peroxidation of either control or vitamin E rat liver microsomes. By means of gas chromatography we analyzed the fatty acid composition of native and peroxidated microsomes from both animal groups. The peroxidation affected principally arachidonic acid and its diminution was more evident in the control microsomes than in the microsomes from the vitamin E treated group. By HPLC we analyzed the vitamin E content in all subcellular fractions employed. In microsomes from the vitamin E-group, the content of vitamin was 11 times higher than in the control ones (0.678 ± 0.1038 vs. 0.062 ± 0.0045 g -tocopherol/mg protein, respectively), while levels in the cytosol from the vitamin E-group were only 2 times higher than in the control cytosol (0.057 ± 0.0051 vs. 0.025 ± 0.0015 g -tocopherol/mg protein, respectively).