Vitamin E is a generic term that refers to a class of compounds that are divided into two subgroups called tocopherols and tocotrienols. The major sources of dietary tocopherols are plant oils such as wheat-germ oil, safflower-seed oil, maize oil and soyabean oil as well as nuts, for example, almonds, groundnuts and pistachios, while plant oils such as palm oil, rice bran oil, palm kernel oil, coconut oil, and cereal grains such as oats, barley, and rye are the main sources of tocotrienols(Reference Ong, Packer and Fuchs1). Both forms of vitamin E are powerful lipid-soluble antioxidants(Reference Hughes2) that can prevent lipid peroxidation(Reference Macdonald, Galley and Webster3), scavenge peroxyl radicals(Reference Packer, Weber and Rimbach4, Reference Constantinescu, Han and Packer5) and protect thalassaemic erythrocytes from undergoing premature lysis due to oxidative stress(Reference Das, Chowdhury, Chattopadhyay and Datta6). However, both groups also differ in their pharmacokinetics(Reference Yap, Yean and Wong7) and biological properties(Reference Prasad, Kumar, Yan, Hanson and Cole8, Reference Gao, Wilde, Lichtenstein, Bermudez and Tucker9). Recent reports show that tocotrienols may have more potent biological effects than α-tocopherol as these are more readily transferred between and incorporated into the cell membranes(Reference Yoshida, Niki and Noguchi10–Reference Meydani, Barklund, Liu, Meydani, Miller, Cannon, Morrow, Rocklin and Blumberg12). In addition, only tocotrienols could exert direct inhibitory and apoptotic effects on in vitro human breast cancer cell lines(Reference Carroll, Guthrie, Nesaretnam, Gapor, Chambers, Ong, Niki and Packer13–Reference McIntyre, Briski, Gapor and Sylvester15) and in vivo animal models(Reference Gu, Wakizono, Sunada, Hung, Nonaka, Sugano and Yamada16, Reference Nesaretnam, Ambra, Selvaduray, Radhakrishnan, Reimann, Razak and Virgili17). The present scientific literature provides a body of evidence to support the idea that vitamin E plays an important role in human immunity(Reference Macdonald, Galley and Webster3, Reference Del Rio, Ruedas, Medina, Victor and De la Fuente18–Reference Penn, Purkins, Kelleher, Heatley, Mascie-Taylor and Belfield20). Recently, it has been shown that the vitamin E concentration in lymphocytes is ten-times higher than in erythrocytes, underlining its importance to the immune system(Reference Lee and Wan21).
Most of the clinical studies on the effect of vitamin E supplementation on the human immune system have been using α-tocopherol. The present study is a pilot study aimed at comparing the effect of short-term supplementation of α-tocopherol or a tocotrienol-rich fraction (TRF) on immune modulation in healthy young Asian subjects. Currently, data regarding immune modulation by vitamin E on Asian populations are scarce. Studies based on Asians and ethnicity are also needed, as various ethnic populations have been reported to show differences in their immune subsets(Reference Jiang, Kang, Lu, Pan, Lin, Pan, Xue, Weng and Tang22–Reference Prince, Hirji, Waldbeser, Plaeger-Marshall, Kleinman and Lanier24). To date, no studies on the effect of tocotrienols on human immune modulation in vivo have been published.
Subjects and methods
Study design and protocol
The present study is a double-blinded, randomised and placebo-controlled trial that was conducted in accordance with good clinical practice guidelines. Ethical approval for the study was obtained from the Ethics Committee of the International Medical University. Informed written consent was obtained from all volunteers participating in the study.
Healthy men (n 19) and women (n 34) aged 20–50 years were recruited. The subjects were informed of the objective and design of the study and the level of commitment required. All subjects had to give signed informed consent. The inclusion criteria for volunteer selection included age between 20–50 years; to be healthy and free of diseases; not to have organ failures; not on any treatment/medication; not taking vitamin A, C or E supplements in the last 3 months; non-smoker; not pregnant. The exclusion criteria included age below 20 years or above 50 years; to have health-related problems; have organ failures; on some treatment or medication; currently on vitamin A, C or E supplements; to be pregnant; have smoked within the past 1 month. Individuals whose blood pressure and BMI exceeded 120/80 mmHg and 30 kg/m2, respectively, were also excluded from the study. These criteria are important as it has been reported that lymphocyte subsets, degenerative diseases and oxidative DNA damage can be influenced by smoking, sex differences, BMI and stress(Reference Loft, Vistisen, Ewertz, Thonneland, Overvad and Poulsen25).
The subjects were randomly assigned to three groups. Overnight fasting blood samples were obtained from all subjects to establish pre-supplementation baseline levels of all the parameters that were to be tested in the present study. Subjects in one experimental group received 200 mg TRF/d (Tocovid SupraBio; Hovid Sdn Bhd, Ipoh, Malaysia) while the subjects in the second experimental group were given 200 mg α-tocopherol/d (Natural vitamin E; 21st Century HealthCare, Inc., Tempe, AZ, USA). The TRF contains 70 % tocotrienol (113 mg α-tocotrienol/g, 91 mg γ-tocotrienol/g, 36 mg δ-tocotrienol/g, 10 mg β-tocotrienol/g) and 30 % tocopherols (78 mg α-tocopherol/g, 0·5 mg β-tocopherol/g). The third group received placebo tablets. These doses were chosen based on a literature review on the safety of these supplements(Reference Mustad, Smith, Ruey, Edens and DeMichele26) and also on the availability of drugs that have been approved by the National Drug Control Authority of Malaysia. All subjects were asked to take one capsule daily after dinner for 56 d. Overnight fasting blood samples were also drawn from all subjects after 28 and 56 d of supplementation for investigations related to the present study.
Sample collection and analysis
Overnight fasting blood samples from all subjects who had fasted overnight were collected and processed within 2 h on days 0, 28 and 56. On days 0 and 56, 10 ml was withdrawn while only 5 ml was taken on day 28. Half of the blood drawn on days 0 and 56 was sent to an accredited pathology laboratory (PATHLAB, Kuala Lumpur, Malaysia) for various biochemical tests such as full blood count, Hb, blood cholesterol, blood lipids, blood sugar, liver function test and renal function test. The remaining 5 ml of blood collected in heparinised tubes were used for lymphocyte culture, flow-cytometry and plasma vitamin E analysis.
High-performance liquid chromatography
About 4·5 ml of the heparinised blood were centrifuged at 1500 rpm (10 min, 4°C) to separate plasma and cellular components. Plasma was stored at − 30°C before analysis. About 1 ml of plasma taken on days 0, 28 and 56 was analysed by HPLC to determine plasma levels of α-tocopherol, tocotrienol and total vitamin E. Plasma levels of other isomers of vitamin E were also determined (data not shown). As described previously(Reference Nesaretnam, Ambra, Selvaduray, Radhakrishnan, Canali and Virgili27), a normal-phase HPLC system equipped with a silica column using Shimadzu LC-10AT (Shimadzu Corporation, Kyota, Japan) HPLC equipment was used.
Immunophenotyping by flow-cytometry analysis
About 500 μl of fresh heparinised blood were used for staining with the TriTEST reagents (2054; Becton Dickinson, NJ, USA) as recommended by the manufacturers. The percentages of lymphocyte subsets (total T-lymphocytes (CD3+), CD4+ and CD8+T-lymphocytes, B-lymphocytes (CD19+), natural killer cells (CD16+ and/or CD56+)) were obtained using a FACSCalibur flow cytometer located at the Department of Immunology, Institute for Medical Research, Kuala Lumpur. Data were analysed using the Multiset program that is used for this equipment.
Lymphocyte isolation and culture
The cellular component of blood obtained from centrifugation was treated with G-Dex™ II RBC Lysis Buffer (iNtRON Biotechnology, Seoul, South Korea) as recommended by the manufacturers to remove the erythrocytes. The leucocytes recovered by centrifugation (1000 rpm × 10 min; 4°C) were cultured in ninety-six-well flat-bottomed MICROTEST™ tissue culture plates (Falcon 3075; Becton Dickinson, NJ, USA) at 5 × 105 cells/per well in the presence of concanavalin A (Sigma-Aldrich, Inc., St Louis, MO, USA), a mitogen, at 50 μg/well for 72 h. Cultured cells were frozen ( − 30°C) until the analysis of cytokine production by ELISA was performed on the culture supernatant fraction.
Cytokine production by enzyme-linked immunosorbent assay
The frozen tissue culture plates were thawed and the contents transferred into sterile 1·5 ml microfuge tubes. The tubes were centrifuged at 1000 rpm for 10 min at 4°C. The supernatant fraction was transferred to fresh microfuge tubes. The pellet was discarded. The supernatant fraction was analysed to quantify the amount of IL-4 and interferon-γ (IFN-γ) using commercial ELISA kits for human IL-4 and IFN-γ (eBioscience, San Diego, CA, USA) as recommended by the manufacturers. The limit of sensitivity for the detection of IFN-γ and IL-4 was 8 and 4 pg/ml respectively.
Statistical analysis
Data are presented as the mean values and standard deviations. All statistical analyses were carried out using SPSS v. 11.5 for Windows software (SPSS Inc., Chicago, IL, USA). Data were first entered into Microsoft® Excel (Microsoft Corp., Berkeley, CA, USA) and the database was converted into SPSS files for treatment (removal) of outliers, assumptions testing (for example, for normality, homogeneity of inter-correlations, sphericity of data) and descriptive statistical analyses. Finally, either split-plot ANOVA or one-way ANOVA was used to analyse the data for differences between the three experimental groups (TRF, α-tocopherol or placebo).
Results
Effect of vitamin E supplementation on plasma levels of α-tocopherol, tocotrienol and total vitamin E
The baseline levels of total plasma vitamin E and plasma α-tocopherol amongst the volunteers, though high, were similar (Table 1). There was a significantly higher (P < 0·001) increase in the total vitamin E levels in the plasma of volunteers who took either α-tocopherol or TRF supplementation when compared with those taking the placebo. The highest total plasma vitamin E levels were found to be in the volunteers supplemented with the TRF (Table 1). Plasma α-tocopherol levels also significantly (P < 0·001) increased from day 0 to day 56 in the volunteers taking the α-tocopherol supplementation (Table 1). The group that was taking the TRF had higher levels of α-tocopherol compared with those taking the placebo. There was a gradual decline of plasma tocotrienol levels in the placebo-supplemented volunteers. In contrast, plasma levels of tocotrienol in the volunteers who took either α-tocopherol or TRF supplementation increased when the blood was analysed on day 28. In both these groups, plasma levels of tocotrienol dropped slightly (from 0·62 to 0·5 pg/ml) on day 56. Post hoc testing for differences in plasma total vitamin E between experimental groups showed that there was a significant (P < 0·001) difference between the placebo and the two vitamin E groups. However, there was no significant (P>0·05) difference between the two experimental groups. There was a significant (P < 0·05) difference in plasma α-tocopherol levels between the α-tocopherol supplementation and placebo (P < 0·001) and TRF-supplemented group (P>0·05). In contrast, there was no significant difference in plasma tocotrienol between the two experimental groups.
(Mean values and standard deviations)
TRF, tocotrienol-rich fraction.
* Mauchly's test tests the null hypothesis that the error covariance matrix of the orthonormalised transformed dependent variables is proportional to an identity matrix. The sphericity assumption is not violated if the within-subjects effect of time is not significant (P>0·05).
† Results were considered significant if F>1 and if P < 0·05. The P value for the between-groups effect is >0·05, i.e. statistically not significant.
Effect of vitamin E supplementation on T-lymphocytes, B-lymphocyte and natural killer cell count
There was no measurable difference in the CD4, CD8, B-cell or natural killer cell count between volunteers who received placebo, α-tocopherol or TRF supplementation (Table 2). When the percentages of these cells on days 0, 28 and 56 were compared, again there was no measurable difference. The standard deviation amongst the groups is rather wide, making it difficult to draw useful conclusions from the data. There was no difference in the ratio of CD4:CD8 cells amongst all the three groups. It should be noted here that normality tests performed on the data showed that the distribution of lymphocytes (T-, B-, CD4+ or CD8+) as a percentage of leucocytes data were normal (AK Radhakrishnan, AL Lee and K Nesaretnam, unpublished results). Skewness and kurtosis of the data were within the normal range of − 1 and 1. In addition, Box's M had a P value that was not significant (P = 0·283), therefore there was homogeneity of data between the groups.
(Mean values and standard deviations)
* Analysis was performed on heparinised blood taken on days 0, 28 and 56, using the FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using the Multiset program.
† CD56+ and/or CD16+.
Effect of vitamin E supplementation on the production of cytokines by mitogen-stimulated lymphocytes in culture
Lymphocytes isolated from the peripheral blood of all volunteers on days 0, 28 and 56 were cultured in the presence of concanavalin A for 72 h as described in the Subjects and methods section. The lymphocytes were cultured for 72 h based on results from preliminary studies to determine the optimal duration for culture (AK Radhakrishnan, AL Lee and K Nesaretnam, unpublished results). There was a reduction in IL-4 production by the concanavalin A-stimulated lymphocytes taken from the volunteers of all three groups (Fig. 1 (a)). This reduction was maintained even at day 56. In contrast there was a sharp increase in the levels of IL-10 (Fig. 1 (b)) and IFN-γ (Fig. 1 (c)) produced by the concanavalin A-stimulated lymphocytes following 72 h of culture between days 0 and 28. However, the levels of both cytokines decreased on day 56. It was of interest to note that the amount of IFN-γ produced by the mitogen-stimulated lymphocytes from the volunteers supplemented with just the placebo also rose on day 28 and dropped on day 56.
The levels of (a) IL-4, (b) IL-10 and (c) interferon-γ (IFN-γ) in the culture supernatant fraction obtained from the concanavalin A-stimulated culture of lymphocytes isolated from the peripheral blood of human volunteers who received α-tocopherol (■), a tocotrienol-rich fraction (▲) or a placebo (♦) for 56 d were determined using ELISA. Analysis was performed on the supernatant fraction obtained following 72 h of culture on the lymphocytes isolated from heparinised blood taken on days 0, 28 and 56. Values are means, with 95 % CI represented by vertical bars. Statistical analysis showed that the ‘within-subject effect’ (time) for IFN-γ, IL-4 and IL-10 production was statistically significant (P < 0·001) while the ‘between-subjects effect’ (group) and ‘interaction effect’ (time v. groups) were not statistically significant.
Discussion
The analysis of plasma levels of total vitamin E, α-tocopherol and TRF by HPLC in the volunteers showed that there was compliance amongst the volunteers in taking their supplements. The highest plasma α-tocopherol levels were observed in the α-tocopherol-supplemented volunteers (6·58 pg/ml on day 0 to 16·53 pg/ml on day 56). There was a modest increase in the plasma levels of α-tocopherol in the volunteers who took TRF supplementation (6·41 pg/ml on day 0 to 11·66 pg/ml on day 56). Similar observations have been previously reported(Reference Nesaretnam, Ambra, Selvaduray, Radhakrishnan, Canali and Virgili27, Reference Mensink, van Houwelingen, Kromhout and Hornstra28). The analysis also showed that time had a significant effect, with the levels in the plasma increasing over time following supplementation, which were further confirmed by the highly significant (P < 0·05) interaction effects. Thus there was compliance in the α-tocopherol test subjects. As the TRF supplements also contained a substantial proportion of α-tocopherol (30 %), a rise in plasma α-tocopherol was also observed in this group. Hence, it was suggestive that there was high compliance amongst the TRF group. The increase in plasma tocotrienol levels in the TRF-supplemented volunteers was not statistically significant (P>0·05). This finding was in agreement with a report by Mustad et al. (Reference Mustad, Smith, Ruey, Edens and DeMichele29) but in contrast with the findings of Mensink et al. (Reference Mensink, van Houwelingen, Kromhout and Hornstra28) who reported an increased tocotrienol concentrations following supplementation. Tocotrienols have been reported to have a short elimination half-life and low bioavailability(Reference Yap, Yean and Wong7). So, it is not surprising that we did not observe increased levels of plasma tocotrienols. Studies in the rat have shown that there is a preferential deposition of tocotrienols in the tissues, especially adipose tissues(Reference Masaaki, Mariko, Ikeda, Tachibana and Yamada30).
Flow-cytometry analysis showed that neither TRF nor α-tocopherol supplementation had a statistically significant effect on the percentages of different blood leucocytes (lymphocytes, T-cells, T-helper cells, CD8 T-cells, B-cells and natural killer cells). These results were not in agreement with the findings reported by Lee & Wan(Reference Lee and Wan21) and Penn et al. (Reference Penn, Purkins, Kelleher, Heatley, Mascie-Taylor and Belfield20), who both showed that α-tocopherol supplementation enhanced the proliferation of total T-cells, T-helper cells, as well as the CD4:CD8 ratio but in agreement with Meydani et al. (Reference Meydani, Barklund, Liu, Meydani, Miller, Cannon, Morrow, Rocklin and Blumberg12), who reported that enhancement of the immune response following α-tocopherol supplementation could only demonstrated in the elderly population. So, it was not surprising that the present results differed from Penn et al. (Reference Penn, Purkins, Kelleher, Heatley, Mascie-Taylor and Belfield20) as the volunteers in the present study were young healthy volunteers. As our sample size was similar to Lee & Wan(Reference Lee and Wan21), the difference in results could be due to different population or differences in diet or immune status.
Further analysis of the data using split-plot ANOVA suggested that the type of supplementation did influence the proportion of lymphocytes in the leucocytes; but this difference was not significant statistically. The split-plot ANOVA analyses also showed that time had a very strong effect on all three cytokines, influencing their concentrations whereas the effects of any interaction between the experimental group variable and time were all not significant.
Taken together, these results show that vitamin E did not have any significant effect on immune modulation in healthy young subjects, unlike as reported in the Lee & Wan(Reference Lee and Wan21) study. The sample number used in the present study is similar, if not higher, than that used in the Lee & Wan(Reference Lee and Wan21) study. However, the lack of statistical significance could still be due to sample size, differences in the population or nutrition amongst the volunteers. In addition, each volunteer may be exposed to different types of foreign substances and this makes it difficult to observe statistically significant changes to the immune system, as in the current model there is no common specific antigens to which specific immune responses could be measured. For future studies, we propose that a specific immunogenic challenge such as a vaccine should be administered to volunteers and the specific immune response to the vaccine be measured. This approach might provide clearer answers to the present research question.
In conclusion, the present study shows that there is no significant difference in immune modulation between healthy Asian volunteers supplemented daily with either α-tocopherol or a TRF.
Acknowledgements
The authors thank Dr Jasbir Singh Dhaliwal for allowing us to use the flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA, USA) located at the Institute for Medical Research (IMR), Kuala Lumpur, Malaysia, Mr Ghazali of the Malaysian Palm Oil Board (MPOB) for his technical assistance with the HPLC analysis and Dr R. Krishnan of the International Medical University for her help with blood-taking from the volunteers. The present study was supported by research grants from the International Medical University and the MPOB. The soft-gel TRF and placebo capsules were a gift of Tocovid SupraBio, Hovid Sdn. Bhd., Ipoh, Malaysia.
A. K. R and K. N. conceived and designed the study while A. L. L., A. K. R., P. F. W., J. K. and H. A. implemented the study. A. R. was involved in project coordination and performed the flow-cytometry analysis; A. L. L., P. F. W. and H. A. were responsible for volunteer screening, i.e. physical examination and blood-taking as well as volunteer enrolment and monitoring; A. L. L. performed most of the lymphocyte culture, ELISA and data analysis; K. N. coordinated measurements of plasma vitamin E levels by HPLC; A. R. supervised the data management; A. L. L. analysed the data; A. R. wrote the manuscript; and K. N. contributed to the writing and editing of the manuscript. None of the authors had any personal or financial conflicts of interest.
