Vitamin E is the most effective chain-breaking lipid-soluble 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: α-, β-, γ-, and δ-tocopherol, and α-, β-, γ-, and δ-tocotrienol, with different biologic activities. α- and γ-tocopherol are the most common forms in nature. Previous studies (2,3) have shown that α-tocopherol has the highest biologic activity and it is generally considered the most important anti-oxidant. However, recent studies have demonstrated that γ-tocopherol is a more effective free radical scavenger than α-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. In the Rotterdam study (10) no association was found between the α-tocopherol level and risk of myocardial infarction. Although in the Cambridge Heart Antioxidant Study (CHAOS) trial (11) patients randomly assigned to receive α-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 α-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 α-tocopherol supplementation may be explained by the fact that vitamin E in the food consists of a mixture of different tocopherols, mainly γ-, δ-, and α-tocopherols, whereas in the clinical studies mentioned, preparations containing α-tocopherol alone were used.
Öhrvall et al. (14) found that only serum levels of γ-tocopherol, and not those of α-tocopherol, are reduced in patients with coronary heart disease. In line with these results, studies from our laboratory showed that a γ-tocopherol-rich preparation of mixed tocopherols was more effective in preventing lipid peroxidative damage than α-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 α-tocopherol alone and of a mixed tocopherol preparation in which the major component is γ-tocopherol on H2O2-induced injury in human erythrocytes.
Natural α-tocopherol (Covitol F-1000) was purchased from Henkel (Dusseldorf, Germany). Mixed tocopherols (Cardi-E) were obtained from Cardinova (Uppsala, Sweden); Cardi-E contains 64% γ-tocopherol, 24% δ-tocopherol, and 12% α-tocopherol (all natural). A stock solution of α-tocopherol or mixed tocopherols was prepared (480 μM 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 m M 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 m M sodium azide. Ethanol 2 μl containing 30, 60, 120, 240, or 480 m M of tocopherols was added to 1-ml suspensions of erythrocytes. The final concentrations of α-tocopherol or mixed tocopherols in the suspension of erythrocytes were 30, 60, 120, 240, and 480 μM. 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 m M sodium azide to remove nonbound tocopherol for tocopherol measurement and H2O2 incubation, respectively. Erythrocyte samples in duplicate were exposed to H2O2 (5 or 10 m M) in PBS containing 4 m M 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 α-tocopherol and one part mixed tocopherols (final concentrations: 120 μM) for 90 min for tocopherol measurement.
In other experiments, erythrocytes were incubated with 120 μM α-tocopherol or mixed tocopherols or without tocopherols for 90 min and then stimulated with 10 m M H2O2 for 60 min at 37°C. At the end of the experiments, fatty acids were measured.
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 μg/ml α-, γ-, and δ-tocopherol (Sigma) were used. Results are given as μg/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 m M 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 m M 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 μM, 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 μM) 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.
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.
Tocopherol levels in erythrocytes
The erythrocyte tocopherol levels before incubation were γ 0.09 ± 0.10 and α 1.34 ± 0.41 μg/ml. After incubation (without addition of tocopherols), the tocopherol levels were very low and only α-tocopherol in the range 0–1.62 (mean, 0.64 ± 0.83) μg/ml was detected.
Incubation of erythrocytes with tocopherols increased the erythrocyte levels of α-tocopherol and mixed tocopherols in a concentration-dependent manner (up to 120 μM). Incubation with the higher concentrations (240, 480 μM) 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 α-tocopherol after incubation with the two respective preparations at the same concentration (Fig. 1).
After incubation with mixed tocopherols at 120 μM, the relative proportions of tocopherols (γ 61%, δ 32%, α 7%) in the erythrocytes were consistent with those in the added preparation (γ 64%, δ 24%, α 12%). After incubation with the other concentrations of mixed tocopherols, the α-tocopherol levels were very low and could not be quantified by HPLC.
After incubation of erythrocytes with a mixture containing one part α-tocopherol and one part mixed tocopherols, erythrocytes took up more γ- and δ-tocopherol than α-tocopherol (Fig. 2).
MDA-TBA was detected in very low concentrations in the erythrocytes before H2O2 stimulation, and incubation with 2 μl of ethanol did not alter the MDA-TBA concentrations.
The MDA-TBA concentration was increased in erythrocytes incubated with 5 or 10 m M H2O2 (Fig. 3) (p < 0.0001 in both cases versus control erythrocytes, 5-m M data not shown). Lipid peroxidation was more increased after stimulation with 10 m M H2O2 than with 5 m M (p < 0.0001). Incubation of erythrocytes with α-tocopherol or mixed tocopherols prior to H2O2 stimulation inhibited the MDA-TBA increase. Mixed tocopherols had stronger effects than α-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 α-tocopherol, respectively.
Effects of tocopherols on fatty acids are shown in Table 1. H2O2 decreased polyunsaturated fatty acids (PUFAs) in erythrocytes (p < 0.05 versus control). Pretreatment with tocopherols inhibited the decrease in PUFA induced by H2O2 (p < 0.05 in the α-tocopherol group, p < 0.01 in the mixed tocopherol group versus the H2O2 group). PUFAs tended to be slightly higher after incubation with mixed tocopherols, but the difference was not significant compared with α-tocopherol.
Incubation of erythrocytes with α-tocopherol or mixed tocopherols increased the tocopherol levels in the erythrocytes in a concentration-dependent manner (up to 120 μM). The tocopherols significantly inhibited H2O2-induced erythrocyte lipid peroxidation and prevented the loss of PUFAs. There was an inverse correlation between the tocopherol content and H2O2-induced injury in erythrocytes. Importantly, we found that the uptake of tocopherols in erythrocytes was much higher after incubation with mixed tocopherols than after incubation with α-tocopherol alone and that mixed tocopherols had a greater protective effect than α-tocopherol alone against H2O2-induced erythrocyte injury. Both α-tocopherol and mixed tocopherols prevented fatty acid oxidation.
Uptake of tocopherol in erythrocytes
Previous studies have demonstrated that there is a selective tocopherol uptake in human erythrocytes. The existence of α-tocopherol binding sites and α-tocopherol binding protein in erythrocytes may regulate the α-tocopherol level (21,22). A 15-kD protein is responsible for intracellular transport, distribution, and metabolism of α-tocopherol in cells. α-Tocopherol binding protein, owing to a lack of specificity for binding of α-tocopherol, may enable erythrocytes to use other vitamin E forms to prevent oxidative damage (23). There may be a competition for the binding sites among the different tocopherols. The finding in the present investigation that the uptake of tocopherols by erythrocytes was much higher after incubation with mixed tocopherols is very interesting. The explanation is not known, but the results may indicate a higher affinity of γ- and δ-tocopherol for the tocopherol binding sites. Supplementation with α-tocopherol decreases the γ-tocopherol concentration in plasma in patients (24). Also, γ- and δ-tocopherol can practically substitute for α-tocopherol as an anti-oxidant in vitro and partially in the resorption gestation assay, the differences being primarily due to the α-tocopherol-specific sorting system. γ-Tocopherol can act as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mustagens, forming stable carbon-centered adducts through its nucleophilic 5-position, which is blocked in α-tocopherol (25).
Protective effect of tocopherol against erythrocyte injury
Oxidative stress is believed to play a critical role in the development of different pathologic processes. H2O2 is produced in vivo as a consequence of normal cellular metabolism, with physiologic and nontoxic levels < 1 m M. In physiologic conditions, erythrocytes remove extracellular H2O2 and protect surrounding tissue against oxidative damage mediated by H2O2. However, in pathologic conditions, erythrocytes are exposed to much higher concentrations of H2O2, which cause oxidative damage in the erythrocytes, such as lipid peroxidation, metabolic imbalance, and even hemolysis (26–28). Studies have shown that vitamin E–deficient cells are much more susceptible to oxidative damage than vitamin E–supplemented cells (29).
In the current study, the tocopherol levels in erythrocytes after incubation (without addition of tocopherols) were lower than before incubation. A possible explanation is that some tocopherols were oxidized during the process of incubation. We found that both α- and mixed tocopherols exerted protective effects against H2O2-induced lipid peroxidation and fatty acid consumption in the erythrocytes. These effects on erythrocytes were concentration dependent within a limited concentration range. We found that a high concentration of α-tocopherol and of mixed tocopherols (480 μM) decreased the uptake of tocopherols in erythrocytes and did not reduce H2O2-induced injury to those cells. The reason for this phenomenon is not clear. We suppose that large lipophilic substances may be formed at higher concentrations of tocopherol and may not be incorporated into the erythrocyte membranes. Other possible mechanisms may be saturation of tocopherol binding sites and toxicity of high concentrations of tocopherols. The observation suggests that large amounts of tocopherol are not useful and may even be harmful. Reports that high doses of α-tocopherol worsen endothelial vasodilator function in vivo (30) and that higher concentrations of β-, δ-, and γ-tocopherol reduced viability of mouse splenocytes (31) seem to support this suggestion.
Mixed tocopherols have a more potent effect than α-tocopherol alone
As noted previously, several clinical trials have failed to show any benefits of commercial α-tocopherol preparations in prevention of cardiovascular death, whereas tocopherols in the food seem to be beneficial. This discrepancy may be explained by the fact that the vitamin E in the food is a mixture of different tocopherols, mainly γ-, δ-, and α-tocopherols, which may be superior to α-tocopherol alone in preventing lipid peroxidation. Recent studies by our group (15) showed that a preparation of mixed tocopherols, rich in γ-tocopherol, given to rats markedly attenuated platelet aggregation and arterial thrombosis. These effects were associated with a decrease in superoxide anion generation and low-density lipoprotein oxidation and an increase in protein expression and activity of endogenous superoxide dismutase and constitutive nitric oxide synthase. In the current study we have shown that the mixed tocopherol preparation has a more potent protective effect than α-tocopherol alone on H2O2-induced lipid peroxidation in erythrocytes. This may be due to the higher uptake of tocopherols by the erythrocytes after incubation with mixed tocopherols resulting in an increased activity of endogenous superoxide dismutase (15).
The present study shows that γ- and δ-tocopherol in mixed tocopherol are taken up by erythrocytes more readily than α-tocopherol. Importantly, we found that the use of mixed tocopherol protects erythrocytes more effectively from lipid peroxidation than α-tocopherol alone. This observation may explain why the administrations of α-tocopherol in clinical trials have failed. Previous studies (32) have shown that the α-tocopherol level in plasma is about 19–63 μM in normal healthy adults. The tocopherol concentrations used in our study were higher than the physiologic concentration, but they were not unrealistically high.
In summary, this study indicates that mixed tocopherols have more potent anti-oxidation effects in erythrocytes than α-tocopherol alone. Mixed tocopherols may thus play an important role in the suppression of free radical–induced lipid peroxidation. The results may have clinical implications.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
Hydrogen peroxide; Lipid peroxidation; Malondialdehyde; Tocopherols; Vitamin E