User talk:Mixedtocopherol

Mixed Tocopherols Have a Stronger Inhibitory Effect on LipidPeroxidation Than -Tocopherol Alone

Meilin Liu, Rolf Wallin, Agneta Wallmon, and Tom Saldeen Department of Surgery, Section of Forensic Medicine, University of Uppsala, Uppsala, Sweden

Summary: Intake of vitamin E with food (mixed tocopherols) has been found to counteract the development of atherosclerotic cardiovascular disease, whereas intake of large amounts of pure �-tocopherol has shown only a slight or no effect in clinical studies. This study was designed to investigate the effects of �-tocopherol alone and a mixed tocopherol preparation (�-, �-, and �-tocopherol) on hydrogen peroxide–induced lipid peroxidation in human erythrocytes. Erythrocytes were incubated with different concentrations of �-tocopherol or mixed tocopherols and then exposed to hydrogen peroxide. Tocopherol levels and malondialdehyde-thiobarbituric acid–reactive substances were determined by high-performance liquid chromatography and fatty acids by gas chromatography. Incubation of erythrocytes with tocopherols (30–120 �M) increased the tocopherol level in a concentrationdependent manner. The uptake of �- and �-tocopherols was much higher than that of �-tocopherol. Hydrogen peroxide strongly increased lipid peroxidation and decreased polyunsaturated fatty acids in erythrocytes. Both �-tocopherol and the tocopherol mixture protected the cells from lipid peroxidation, the mixture being much more potent than �-tocopherol alone. This study indicates that a mixture of tocopherols has a stronger inhibitory effect on lipid peroxidation induced in human erythrocytes than �-tocopherol alone, due to higher uptake of �- and �-tocopherol in the cells. Key Words: Hydrogen peroxide— Lipid peroxidation—Malondialdehyde—Tocopherols—Vitamin E.

Vitamin E is the most effective chain-breaking lipidsoluble anti-oxidant in biologic membranes. It contributes to membrane stability and protects critical cell structures against damage from free radicals and reactive products of lipid peroxidation (1). There are eight different tocopherols in the diet: �-, �-, �-, and �-tocopherol, and �-, �-, �-, and �-tocotrienol, with different biologic activities. �- and �-tocopherol are the most common forms in nature. Previous studies (2,3) have shown that �-tocopherol has the highest biologic activity and it is generally considered the most important antioxidant. However, recent studies have demonstrated that �-tocopherol is a more effective free radical scavenger than �-tocopherol (4,5). The results of several studies have indicated that people who eat food with a high vitamin E content have a lower risk of degenerative diseases, such as cardiovascular disease, cancer, and diabetes mellitus (6–9). However, clinical trials have yielded conflicting results of tocopherol treatment.

MIXED TOCOPHEROLS INHIBIT LIPID PEROXIDATION: association was found between the �-tocopherol level and risk of myocardial infarction. Although in the Cambridge Heart Antioxidant Study (CHAOS) trial (11) patients randomly assigned to receive �-tocopherol supplementation showed a significant reduction in the risk of nonfatal myocardial infarction, there was no decrease in mortality from cardiovascular causes. Recently, two large studies together enrolling > 10,000 patients, the Heart Outcomes Prevention Evaluation (HOPE) study (12) and the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico Prevention (GISSI) trial (13), failed to demonstrate any beneficial effect of �-tocopherol on mortality or cardiovascular events. This discrepancy between the observed effect of food with a high vitamin E content and the results of clinical trials of �-tocopherol supplementation may be explained by the fact that vitamin E in the food consists of a mixture of different tocopherols, mainly �-, �-, and �-tocopherols, whereas in the clinical studies mentioned, preparations containing �-tocopherol alone were used. Öhrvall et al. (14) found that only serum levels of �-tocopherol, and not those of �-tocopherol, are reduced in patients with coronary heart disease. In line with these results, studies from our laboratory showed that a �-tocopherol- rich preparation of mixed tocopherols was more effective in preventing lipid peroxidative damage than �-tocopherol alone (15). Previous studies have demonstrated that peroxides play an important role in oxidative cell damage. Erythrocytes have been used extensively as a model for investigating oxidative damage. Hydrogen peroxide (H2O2) induces lipid peroxidation, causing erythrocyte damage (16). In the current study we therefore examined the effects of �-tocopherol alone and of a mixed tocopherol preparation in which the major component is �-tocopherol on H2O2-induced injury in human erythrocytes. METHODS Reagents Natural �-tocopherol (Covitol F-1000) was purchased from Henkel (Dusseldorf, Germany). Mixed tocopherols (Cardi-E) were obtained from Cardinova (Uppsala, Sweden); Cardi-E contains 64% �-tocopherol, 24% �-tocopherol, and 12% �-tocopherol (all natural). A stock solution of �-tocopherol or mixed tocopherols was prepared (480 �M in 100% ethanol). The stock solution was diluted with 100% ethanol to appropriate concentrations before addition to the erythrocytes. H2O2, trichloroacetic acid, and 2-thiobarbituric acid (TBA) were purchased from Merck (Whitehouse Station, NJ, U.S.A.). Butylated hydroxytoluene (BHT) was purchased from Sigma (St. Louis, MO, U.S.A.). Preparation of erythrocyte suspension Fresh human blood was collected from healthy volunteers into tubes containing 0.129 M sodium citrate. The subjects, aged 30–45 (mean 35 ± 5) years, were not taking any medication. Erythrocytes were separated by centrifugation at 120 g at 4°C for 15 min. After removal of the plasma and buffy coat, the erythrocytes were washed three times with phosphate-buffered saline (PBS, pH 7.45) containing 4 mM sodium azide (17), which was used to inhibit catalase activity to prevent the destruction of added H2O2. Erythrocytes (0.1 ml) washed with PBS were prepared to measure normal levels of tocopherol. The hematocrit of the washed erythrocytes was adjusted to 10% in PBS containing 4 mM sodium azide. Ethanol 2 �l containing 30, 60, 120, 240, or 480 mM of tocopherols was added to 1-ml suspensions of erythrocytes. The final concentrations of �-tocopherol or mixed tocopherols in the suspension of erythrocytes were 30, 60, 120, 240, and 480 �M. The erythrocytes with tocopherols, as well as control samples without tocopherols and with or without ethanol, were incubated for 90 min at 37°C in a shaking water bath. The erythrocytes were washed twice with PBS buffer containing 4 mM sodium azide to remove nonbound tocopherol for tocopherol measurement and H2O2 incubation, respectively. Erythrocyte samples in duplicate were exposed to H2O2 (5 or 10 mM) in PBS containing 4 mM sodium azide for 60 min at 37°C in a shaking water bath. In one set of experiments, erythrocytes were incubated with a mixture containing one part �-tocopherol and one part mixed tocopherols (final concentrations: 120 �M) for 90 min for tocopherol measurement. In other experiments, erythrocytes were incubated with 120 �M �-tocopherol or mixed tocopherols or without tocopherols for 90 min and then stimulated with 10 mM H2O2 for 60 min at 37°C. At the end of the experiments, fatty acids were measured. Tocopherol measurement Tocopherol concentrations in erythrocytes were measured by high-performance liquid chromatography (HPLC) with fluorescence detection, as described earlier (18). In brief, the assay mixture consisted of 0.1 ml erythrocytes incubated with tocopherols, 0.4 ml PBS, 0.5 ml ethanol containing 0.005% BHT, and 2 ml hexane. The samples were completely mixed and centrifuged at 2,000 g for 10 min, and 1 ml of the hexane layer was then dried with nitrogen and dissolved in 1 ml methanol. The solution was analyzed with HPLC at 292 nm, with a flow rate of 1 ml/min. Standards containing 1–10 �g/ml �-, �-, and �-tocopherol (Sigma) were used. Results are given as �g/ml packed erythrocytes. Measurement of lipid peroxidation Malondialdehyde-thiobarbituric acid (MDA-TBA) formation was used as an index of lipid peroxidation. MDA-TBA analysis was determined according to the TBA reaction, as described earlier (19). The H2O2- induced injurious reaction was stopped by addition of 1 ml 20% (w/v) trichloroacetic acid. The suspension was centrifuged at 2,000 g for 5 min, and the supernatant was transferred into an Eppendorf tube with 1 mM BHT and stored at −70°C pending MDA-TBA measurement. For measurement of MDA-TBA, 0.5 ml of the supernatant was mixed with 0.75 ml of 0.15 M phosphoric acid solution and 0.25 ml of 42 mM TBA. The mixtures were boiled at 95–100°C for 60 min, cooled, and extracted with methanol-NaOH. After centrifugation at 23,000 g for 10 min, the solvent layer containing the TBA-reactive substances (TBARS) was determined at 532 nm by HPLC (Gilson, Middleton, Wl, U.S.A.); Nova-Pak column, C18, 60A 4 �M, 3.9 × 150 mm (Waters Corp., Milford, MA, U.S.A.). Various amounts of MDA (0, 0.25, 0.5, 0.75, 1.0, 1.5, and 2.0 �M) were used as external standards. To determine tocopherols or MDA-TBA in the samples, the external standard solutions were used and peak-area ratios were used for calculations. The concentrations of tocopherol and MDA-TBA were always within the range of the calibration curves. Fatty acid measurement. Lipids of erythrocytes were extracted with chloroform/methanol with 0.005% BHT as described earlier (20). In brief, the phospholipids were separated by thin-layer chromatography and after transmethylation the fatty acid methyl esters were separated by gas-lipid chromatography. The results are given as area percentage of all fatty acids detected. Statistical analysis The SPSS 10.1 statistical package (SPSS Inc., Chicago, IL, U.S.A.) was used. Data are presented as mean ± SE in figures and as mean ± SD in tables. One-way analysis of variance (ANOVA) (tocopherol and fatty acid results) and two-way ANOVA (MDA-TBA results) followed by the post hoc least significant difference (LSD) test were used in multiple comparisons. The Pearson correlation coefficient was used in correlation analyses and the transformed regression model was used in logarithmic trendline). A p value < 0.05 was considered significant. RESULTS Tocopherol levels in erythrocytes The erythrocyte tocopherol levels before incubation were � 0.09 ± 0.10 and � 1.34 ± 0.41 �g/ml. After incubation (without addition of tocopherols), the tocopherol levels were very low and only �-tocopherol in the range 0–1.62 (mean, 0.64 ± 0.83) �g/ml was detected. Incubation of erythrocytes with tocopherols increased the erythrocyte levels of �-tocopherol and mixed tocopherols in a concentration-dependent manner (up to 120 �M). Incubation with the higher concentrations (240, 480 �M) of tocopherol did not further increase the tocopherol levels in the erythrocytes, which decreased. The uptake of mixed tocopherols was much higher than that of �-tocopherol after incubation with the two respective preparations at the same concentration (Fig. 1). After incubation with mixed tocopherols at 120 �M, the relative proportions of tocopherols (� 61%, � 32%, � 7%) in the erythrocytes were consistent with those in the added preparation (� 64%, � 24%, � 12%). After incubation with the other concentrations of mixed tocopherols, the �-tocopherol levels were very low and could not be quantified by HPLC. After incubation of erythrocytes with a mixture containing one part �-tocopherol and one part mixed tocopherols, erythrocytes took up more �- and �-tocopherol than �-tocopherol (Fig. 2). Lipid peroxidation MDA-TBA was detected in very low concentrations in the erythrocytes before H2O2 stimulation, and incubation with 2 �l of ethanol did not alter the MDA-TBA concentrations. The MDA-TBA concentration was increased in erythrocytes incubated with 5 or 10 mM H2O2 (Fig. 3) (p < 0.0001 in both cases versus control erythrocytes, 5-mM data not shown). Lipid peroxidation was more increased after stimulation with 10 mM H2O2 than with 5 mM (p < 0.0001). Incubation of erythrocytes with �-tocopherol or mixed tocopherols prior to H2O2 stimulation inhibited the MDA-TBA increase. Mixed tocopherols had stronger effects than �-tocopherol. There was an inverse relationship between tocopherol levels and MDA-TBA formation (Fig. 4), the correlation coefficients being −0.93 and −0.91, after incubation of erythrocytes with mixed tocopherols and �-tocopherol, respectively.