Human ageing is affected by both genetic factors and lifestyle-related factors such as diet. Dietary intervention is feasible, as nutrients can affect the rate of ageing by altering the type and quantity of proteins synthesized  by modulating gene expression , thereby altering the oxidative status of individuals .
Our results indicate that daily supplementation for up to 6 months with TRF raised plasma HDL cholesterol levels as early as 3 months, thereby increasing the HDL-cholesterol/total cholesterol ratio. This ratio reflects the proportion of anti-atherogenic to atherogenic lipids and has been suggested as a better predictor of cardiovascular disease risk than the individual lipoprotein values . TRF might thus help to reduce the risk of coronary heart disease (CHD) in healthy older adults. In fact, HDL cholesterol increases of the magnitude observed in this study have been associated with a 22.5% reduced risk of cardiovascular events . Raising plasma HDL cholesterol and thus the HDL-cholesterol/total cholesterol is recommended by the American Diabetes Association (ADA) guidelines together with lowering plasma triglycerides for high-risk individuals particularly older adults as major mortality cases of CHD were 65 years old or older .
Conflicting data have been reported regarding effects of vitamin E supplementation which were mainly α-tocopherol on HDL cholesterol. Increased HDL cholesterol after α-tocopherol supplementation has been reported by some investigators [43–45] and disputed by others [46, 47]. It should be noted that the supplement used in this study was high in tocotrienols, which has been reported to have different effect from α-tocopherol . Tocotrienol but not tocopherol increases HDL cholesterol by inhibiting HMG-CoA reductase through signalling, thereby regulating cholesterol biosynthesis . Tocotrienol may increase HDL in this study by modulating signal transduction and gene expression; specifically, and may normalize any aberrant gene expression incurred by aging . Increases in HDL could be attained by increasing physical exercise [51, 52] but similar effects by supplementation of vitamins have not been reported in human.
Compliance of the subjects was indicated by the observed increase in plasma lipid-corrected total tocotrienol and tocopherol concentration. Standardization of plasma vitamin E levels to total cholesterol was necessary to control for age-related changes in baseline cholesterol levels as vitamin E is transported by the lipoproteins. The finding that tocotrienol levels were increased is of particular interest, as tocotrienol is now reported to have functions distinct from α-tocopherol as reviewed by Sen et al.. The marked increase in tocotrienol in the > 50 age group is interesting and suggests an increased bioavailability and possibly the need for tocotrienol with aging. The level of total tocotrienol was slightly lower in the older adults as opposed to the younger group. Supplementation of older subjects with TRF restored plasma vitamin E availability to near the levels of in the controls of the younger group. We speculate that a steady state plasma vitamin E concentration was achieved after 6 months of supplementation, as plasma concentrations were similar to those in the younger group were seen at that time point. Some studies have reported the achievement of steady-state plasma vitamin E levels after 10 to 15 days of supplementation with either natural or synthetic forms of α-tocopherol at much higher dosages [54–56]. Considering the well-documented preferential absorption and transportation of different vitamin E isomers in the body and by taking into account the tocotrienol-rich composition in the TRF, such a slow but steady increment is reasonable.
The tocotrienols are found in a wide variety of foods and it has been suggested recently that all 8 isomers of vitamin E may be necessary for optimum health . This requirement maybe more crucial for the older individuals where digestion and absorption may not be as efficient resulting in the potential benefits of supplementation. Differences noted in the plasma levels of tocotrienols detected in various human studies are mainly due to the different daily fat diet. Asians consume higher levels of palm oil rich in tocotrienols. Therefore, a comparatively higher absorption of tocotrienol and thus better level found in circulation.
The elevated plasma vitamin C level detected in the present study was most likely absorbed from the diet, as the supplement is not a source of vitamin C. Indeed, we found a positive correlation between vitamin C intake and plasma vitamin C levels (r = 0.308, p = 0.048). This is in accordance with findings by Padayatty et al. who had reported that small changes in oral intake of vitamin C resulted in large changes in plasma vitamin C concentration. The increase in plasma vitamin C might also result from a complementary effect by vitamin E in an interlinking antioxidant network. The involvement of vitamin C in regenerating vitamin E directly from its tocotrienoxyl or tocopheroxyl radical back to tocotrienol and tocopherol respectively has been well documented . Increased levels of vitamin E might reflect increased reactions with reactive free radicals, additional formation of tocotrienoxyl or tocopheroxyl radical, and a further increased need for vitamin C.
The variation observed in enzyme activity might have been due to the different roles of the analysed antioxidant enzymes. The functional roles of these enzymes are well established, with SOD acting upstream by dismutating reactive superoxide anion radicals into more stable hydrogen peroxide (H2O2) whereas catalase and GPx function downstream by converting H2O2 into water and oxygen in apparently parallel pathways. Changes in antioxidant enzymes activity observed were clearly in favour of the > 50 years old group. Reduction in SOD activity with TRF supplementation after 3 and 6 months in the older group possibly due to lesser formation of radicals as a result of radical scavenging effect by tocotrienol and tocopherol. On the other hand, increase in GPx activity in both placebo and TRF group after 6 months possibly due to higher need for H2O2 removal. Increased level of tocotrienol and tocopherol attained by TRF supplementation might reflect more radical scavenging activity, followed by increased H2O2 formation and therefore increased requirement for its removal. As for the placebo group, increased intake of dietary vitamin C might results in a similar increase in radical scavenging activity and the subsequent reactions involving formation and detoxification of H2O2. Given a decline in the catalase activity in the placebo group, the action of removing H2O2 was predominantly done by GPx. In the current study, the enzymes activity measured was evidently influenced by TRF supplementation as shown by the shifted correlation patterns with age.
Long term supplementation of TRF for 6 months caused the increase in plasma vitamin E availability (both tocopherol and tocotrienol) observed in the current study, accompanied by changes in the oxidative stress biomarkers measured. Protein carbonyls have been described as oxidized amino acids resulting from direct oxidation of protein by reactive oxygen species . Proteins are also modified indirectly by glycation or glycoxidation of amino groups with the eventual formation of the advanced glycosylation end products (AGEs) . Consistent with the fall in plasma concentrations of carbonylated protein with TRF supplementation, a sharp decrease in serum AGE was observed. When the effect of age was factored out of the statistical model, it was found that the interaction between supplementation and duration was significant for the older individuals, indicating a favourable gain in the older group. Figures 4, 5 and 6 give individual presentations of the changes in these oxidative markers with age. Ascending trends of protein damage and lipid peroxidation accumulation during ageing as shown by the correlative data were reduced, even reversed by TRF supplementation. These findings confirm that nutritional intervention can exert cumulative effects on oxidative stress in healthy individuals in the long term . The unique combination of vitamin E isomers used in the study might have acted synergistically to provide the beneficial effect.
The reduced levels of oxidative markers were mainly observed in the older group, for whom the lower cut-off point was 50 years of age. This is of interest as previous studies typically evaluated older subjects, mostly 60 years old and over. It is also noteworthy that in the present study the treatment was administered to healthy individuals for a lengthy period of 6 months and studies involving supplementation of this duration are fairly limited. This may then results in the observed changes in oxidative status as measured by protein carbonyl and AGE. Although some of the younger subjects in the study also showed increased levels of antioxidant vitamins, the magnitude of changes was less evident as compared to the older individuals. The absence or lack of response by the younger age group might reflect a well-maintained antioxidant level, more effective maintenance of oxidative balance, and better defence against spontaneous oxidative injury. It is thus possible that TRF supplementation did not provide any further improvement. Although baseline antioxidant levels in the > 50 year-old group were similar to those in the < 50 year-old group, baseline oxidative marker levels were higher in the older group, suggesting a higher level of oxidative damage. However, the amount of damaged lipids and proteins in this group was reduced by supplementation, probably due to the increased requirement for antioxidants in older individuals. It is possible that an antioxidant threshold for optimum performance exists and that this threshold (and therefore the requirement for antioxidants) could increase with ageing, thus allowing supplementation to generate an effect in the present study. A long-term prospective study will be required to test this hypothesis, particularly at the molecular level.
Compelling evidence suggests a new level of action for vitamin E under the non-antioxidative control in protection against disease . Therefore, TRF might not only act directly or solely as an antioxidant, but it may actually also act through signalling pathways and specific signal-regulated protein reaction as suggested by Sen et al.[14, 61]. Tocotrienol was shown to provide complete neuroprotection via antioxidant-independent mechanism with the protective property reported not only limited in response to non-oxidative challenges but also to oxidative insults [14, 15]. Further studies of the effects of tocotrienols in a cell model are currently underway in our laboratory.