The use of vitamins C and vitamin E as dietary supplements due to their supposed benefits in health and disease is widespread. Both are antioxidants, and as such is thought to reduce oxidative stress and its implications for the human body Breilmann J et al.,2010. One of such implications is atherosclerosis, of which hypercholesterolaemia is a risk factor. Low density lipoproteins (LDL) that are elevated in hypercholesterolaemia are prone to oxidization Palinski et al., 1989, thus initiating the cascade of atheroma formation by way of activating specific genes involved Maziere and Maziere, 2009.

Elevated oxidized LDL (oxLDL) level is independently related to the risk of developing atherosclerosis and subsequent coronary heart disease Toshima et al., 2000. Furthermore, it also contributes to atheroscle rotic plaque instability Nishi et al., 2002Okura et al., 2000, leading to thrombus formation and acute coronary events. Inflammation is considered to be the pathway by which oxLDL exerts its adverse cardiovascular effects Bieghs et al., 2013Shen et al., 2013. Additionally, oxLDL also promotes endothelial dysfunction via upregulation of its markers, such as the intercellular and vascular cell adhesion molecules (ICAM and VCAM, respectively) Huang et al., 2013.

Efforts have been made to lower LDL levels by traditional medicine and/or medications to acceptable values for patients at risk of developing cardiovascular diseases, and the use of antioxidants to quench the oxidation of LDLs could prove to be a novel approach to this problem. However, research in this area is still not provided conclusive results to support their clinical use.

Tocotrienols and tocopherols are members of the vitamin E family. Tocotrienol has been proven to be a more potent antioxidant than tocopherols Maniam et al., 2008. Furthermore, tocotrienol has been found to exert other effects outside of its antioxidant capabilities, such as cardioprotective Das et al., 2007, neuroregenerative Khanna et al., 2005 and anti-cancer properties Aggarwal et al., 2010. It was also found to directly inhibit HMG-CoA reductase, the predominant enzyme in cholesterol metabolism Khor and Ng, 2000. Vitamin C is an established antioxidant and has been demonstrated to reduce oxidative stress induced by multiple mechanisms El-Gendy et al., 2010Tsovolas et al., 2008. The combination of vitamins C and E has been shown to be beneficial in combating oxidative stress induced by intense exercise Naziroglu et al., 2010 and X-ray Kayan et al., 2009.

Due to the potential benefit of tocotrienol in hypercholesterolaemia, we investigated the effects of tocotrienol- tocopherol mixed fraction (TTMF) of palm oil and vitamin C, either alone or in combination, on lipid profile and biomarkers of inflammation and endothelial activation in patients at low risk of cardiovascular disease.

After 12 months of treatment with TTMF, either alone or in combination, we found that despite the encouraging trends, TTMF had a neutral effect on the lipid parameters when compared to placebo. The utilisation of a mixture of tocotrienol and tocopherol, rather than a pure tocotrienol compound, could be the reason behind this observation. Although other human studies have demonstrated the benefit of this mixed fraction in lowering plasma lipids Qureshi et al., 2001Qureshi et al., 2002, the fraction of tocopherol in the mixture used by them yielded a lower tocopherol fraction, compared to our TTMF (8% vs 26%, respectively).

The effect of tocopherol on plasma lipids has been positively established de Oliveira et al., 2011. However, its utilization in a mixture with tocotrienol has yielded conflicting results Fu et al., 2014. One study suggested that tocopherol has a negative impact on the lipid lowering property of tocotrienols Qureshi et al., 1995, via its stimulating effect on HMG-CoA reductase, the rate-limiting enzyme in the cholesterol synthesis pathway, thus attenuating the inhibitory effect exerted by tocotrienols Qureshi et al., 1996. It was determined that in a mixed preparation of tocotrienol and tocopherol, 20% or more of the latter leads to attenuation of the former’s hypocholesterolaemic effects Qureshi et al., 1995Qureshi et al., 1991.

A worrying observation is the consequence of HDL lowering with TTMF in this study. HDL, which is a plasma cholesterol scavenger, is positively correlated with a reduction of adverse cardiovascular (CV) risk and CV mortality Moradi et al., 2014. Whilst there is another study that reported a similar observation Tan et al., 1991, most reported a neutral effect of tocotrienols on HDL levels Baliarsingh et al., 2005Mensink et al.,1999Qureshi et al., 1995. Another study demonstrated that the same TTMF preparation was able to significantly increase plasma HDL after 6 months of treatment when compared to baseline levels Chin et al., 2011. However, the same study showed that the HDL increment was only observed to be significantly better than placebo in the older age group (>50 years) compared to those aged between 35 – 49 years, a group where most of our subjects were located in.

Despite this observation with regard to the HDL levels, a bigger percentage of subjects on TTMF (alone or in combination with vitamin C) improved their lipid profiles. This trend is encouraging, and supports the notion that tocotrienols should be an adjunct therapy for attenuating hypercholesterolaemia. This is due to the inhibitory effect that tocotrienol has on HMG-CoA reductase Khor and Ng, 2000, the same enzyme that is inhibited by the statin group of drugs. Owing to their synergistic interaction, the combination of lovastatin and tocotrienol-rich fraction (TRF) has been shown to be effective in lowering plasma lipid parameters Qureshi et al., 2001.OxLDL predisposes to inflammation Bieghs et al., 2013 and endothelial dysfunction Huang et al., 2013. In our study, we demonstrated that TTMF treatment had a neutral effect on hsCRP and TNF-ɑ, and a tendency for lowering of IL6 levels, thus proving to be potentially beneficial in quenching oxLDL-mediated inflammation. Similarly, one study has reported that TRF has a neutral effect on CRP and IL6 in haemodialysis patients Daud et al., 2013, but another reported that the level of CRP precursor protein was down-regulated in healthy individuals treated with TRF Heng et al., 2013.

TTMF was also found to have a neutral effect on biomarkers of endothelial activation in the current study. Even though many in-vitro studies have reported tocotrienol’s beneficial effect on adhesion molecules expression Ahn et al., 2007Naito et al., 2005Theriault et al., 2002, ours is the only study that evaluated its role in human. However, the homocysteine elevation seen with TTMF treatment when compared to the baseline is in disagreement with a study in animals, which have demonstrated a beneficial lowering of homocysteine with tocotrienol treatment Norsidah et al., 2013. One study in humans yielded a neutral effect of tocopherol, rather than tocotrienol, on homocysteine levels Breilmann et al., 2010. Although the effect observed here is minimal, due to the correlation of homocysteine to cardiovascular risk Nygård et al.,1995, further studies should aim to confirm this finding and look at the possible mechanisms involved, if any.

Treatment for 12- months with TTMF, either alone or in combination with vitamin C supplementations have neutral effects on lipid profiles and biochemical markers of inflammation and endothelial activation in low risk subjects, suggesting that they offer no added advantages in the low cardiovascular risk group. The utilization of TTMF with its high tocopherol fraction (26%) has probably impeded the improvement observed, due to antagonistic interaction between tocopherols and tocotrienols, although worrying, the negative impact of TTMF on plasma HDL and homocysteine is quite minimal. In the future, more studies will be needed to assess the effect of tocotrienols in human, especially on biomarkers for endothelial activation or dysfunction.