Vitamin E is the generic name for a group of 8 plant-derived, lipid soluble substances ("tocols") including four tocopherol (T) and four tocotrienol (T3) derivatives. T3 are similar to T in molecular structure, except that they have an isoprenoid tail with three unsaturation points instead of a saturated phytyl chain. Vitamin E is a recognized antioxidant and thought beneficial for human health. There have been several indications that T3 may result in superior therapeutic properties compared to T 1 2 3 4 5 6 7 8 .

The absorption and biokinetics of T3 in humans are however not fully understood. Inter alia, the above issues related to the absorption and biokinetics have been linked to several findings relating the physiological outcomes of T3 9 10 11 12 . In comparison to α-T, the metabolic pathways relevant to T3 have hardly been elucidated and optimized. Several human studies have investigated the absorption of T3 into circulating plasma 9 10 11 13 14 15 16 17 18 19 20 21 and lipoproteins 2 19 20 . The detection of T3 in plasma and lipoprotein fractions has proven difficult, possibly due to its low occurrence. In comparison to α-T, concentrations of T3 were significantly lower. Rapid disappearance of these T3 has raised questions about their potential as potent lipid-soluble antioxidant. This is most probably one of the reasons why T3 are given a low score for their biological vitamin E activity compared to α-T 22 .

T as well as T3 are transported within lipoprotein particles while circulating in the blood, but their distribution in lipoproteins has been documented only occasionally. Most studies investigating absorption of T3 in the human circulatory system focused on the plasma content of T3. From these limited human studies 2 19 20 , distribution of T3 in lipoproteins was significantly lower than that of α-T. Following postprandial intervention, T3 transport in lipoproteins appears to follow complex biochemically mediated pathways within the lipoprotein cascade 19 . The mechanism of T3 transport in human lipoproteins has not been conclusively investigated and discussed. Although laboratory evidence has been very promising 5 8 , T3 supplementation in humans has produced inconsistent results 6 7 . In addition, most studies investigating the response of T3 supplements, investigated only total plasma concentration of T3 9 10 11 12 13 16 17 and rarely in various lipoprotein fractions 19 20 .

Response of plasma and lipoproteins to T3 may be determined by the dose of T3 supplementation. This additionally could influence the fasting T3 level in blood 14 . Since supplementation with high dose of T3 has demonstrated that T3 were detected in plasma, TRP, LDL, and HDL following 8-h of postprandial challenge 19 , it is intriguing to investigate whether lower dose of T3 supplementation would also resulted a similar observation. Since α-T is the most bio-active form of vitamin E 22 , it is crucial to refer to α-T when comparing the biological activity of other isomers of vitamin E including T3. With this in mind, we investigated the metabolic fate of T3 as well as α-T in plasma and lipoprotein fractions in normolipemic humans through the current postprandial study.

Following both vitamin E treatments in the current study, α-T was the major vitamin E detected in circulating plasma and lipoproteins. All vitamin E isomers from dietary sources (including supplements) are absorbed and delivered to the liver, although only α-T is preferentially recognized by the α-tocopherol transfer protein (α-TTP) for incorporation into circulating plasma 25 . Other T (γ-T, δ-T) and T3 isomers (α-T3, γ-T3, δ-T3) are not preferentially utilized and are mostly excreted from circulation 26 . This is the main reason why α-T is the only vitamin E isomer that is currently used as the standard to estimate human vitamin E requirements 22 . However it is increasingly acknowledged that T3 and T serve different biological functions and bench marking only α-T to estimate human vitamin E requirements may no longer be the most accurate measure 3 5 8 .

In the current study, supplementation with α-T or TRF resulted in significantly increased plasma α-T concentration compared to the baseline value. Furthermore, plasma total circulating α-T for the 24 h postprandial duration (described as AUC) was significantly higher after α-T treatment. This observation was however anticipated, due to the higher content of α-T administered (537 mg of α-T) from the α-T treatment compared to that of TRF (only 167 mg α-T).

In most human clinical and bioavailability studies of vitamin E, only the plasma and lipoprotein concentrations of α-T have been reported 27 . It would therefore be ideal if the concentrations of individual T and T3 are measured to gain new insight into the physiological roles of these vitamin E isomers in humans 27 . Evaluation of the metabolic response following T3 supplementation through plasma or serum concentration of T3 and α-T is advocated. Our present study shows that all T3 isomers (α-T3, γ-T3, δ-T3) were detected in plasma and lipoproteins following supplementation of TRF, although their concentration was significantly lower compared to that of α-T. These findings are in agreement with our previous postprandial observation 19 and several other human studies that examined the bio-kinetics 2 13 16 17 20 or physiological effects 9 10 11 12 14 15 21 of T3 supplementation.

Detection of individual vitamin E isomers in plasma, following postprandial challenge could assist in elucidating their preferential absorption into circulating blood. However, this may not be true for α-T, since the liver actively secretes α-T into circulating plasma and impacts final plasma concentration of this vitamin E isomer. α-T was detected in both fasted and postprandial states in the current study. T3 isomers, including α-T3, the major T3 isomer in TRF, however was not detected in the fasted state. Their occurrence throughout the postprandial state was apparent, only in significantly lower levels compared to α-T. Despite these observations, we note that T3 have been demonstrated to have biological functions well below plasma concentrations noted in this study (5, 8). Among the T3 isomers, the absorption rates appear in the order α-T3 > γ-T3 > δ-T3. These findings might explain the possibility of bio-discrimination between T and T3 isomers in humans. Such bio-discrimination has also been demonstrated in several animal studies. Ikeda et al. 28 demonstrated that α-T3 is preferentially absorbed into the lymphatic circulation compared to γ-T3 and δ-T3. Similar observations were found by Yap et al. 29 who investigated the influence of route of administration on the absorption and disposition of α-T3, γ-T3 and δ-T3 in rats. Of the 3 isomers, α-T3 achieved the highest concentration and AUC after an oral ingestion of T3. This was followed by γ-T3 and δ-T3. In humans, plasma concentrations of α-T3 were 2-fold higher than that of γ-T3, and almost 10 times higher than δ-T3 after supplementation with the same dose of T3 preparations 10 . Similar observations were also demonstrated in hypercholesterolemic subjects who received a high γ-T3 supplements that contained ≈4-fold concentration of γ-T3 than α-T3 11 .

Distribution of T3 isomers in lipoproteins also provides a better explanation of T3 absorption and transport in circulating plasma. In agreement with our previous observation 19 , T3 were transported in TRP (chylomicrons + VLDL), LDL and HDL. Several mechanisms have been postulated to explain this observations 19 including the selectivity and affinity of hepatic α-TTP 30 , the function of a specific protein carrier in transporting α-T3 in the intestinal cells 28 , and differences in the methyl groups in the chromanol rings of T3 29 that influenced the absorption rate of each T3 isomers 10 . Following its hepatic uptake, it would be intriguing to know whether nascent VLDL or HDL generated from the liver, is readily enriched in T3 from the liver itself. The role of HDL in transporting vitamin E has recently been identified as one of the primary mechanisms in vitamin E absorption in the fasted states 31 .

The competitive uptake between isomers is only initiated following the hepatic uptake of vitamin E from chylomicrons, where the selectivity role of α-TTP is significant in transferring vitamin E into circulating VLDL 25 . The relative affinity of vitamin E isomers towards α-TTP has been demonstrated to be in the order of α-T (100%) > α-T3 (12%) > γ-T (9%) > δ-T (2%) 30 . This mechanism explains the occurrence of α-T as the major vitamin E isomer detected in TRP, LDL, and HDL, and the rapid disappearance of α-T3, γ-T3 and δ-T3 from circulating plasma and lipoproteins. Other physiological factors such as bile, urinary and fecal excretion that may influence the rapid disappearance of T3 has also been postulated 19 32 . The exchange of T3 between circulating chylomicron, VLDL, LDL, and HDL has also been suggested to explain their distribution in the lipoproteins 19 20 .

There is no bio-discrimination between T and T3 during intestinal absorption after dietary intake of vitamin E 26 33 34 35 . However, the rapid disappearance of T3 may be associated with its preferential utilization in humans (8, 25, 34). In the current and previous 19 20 studies, the amount of T3 absorbed into TRP was very low. This observation may indicate the possibility of bio-discrimination of T3, prior to the intestinal absorption. Although mechanism for the preferential absorption of T3 is difficult to describe, it has been suggested that the complexity of T3 absorption is probably due to the difference in their micellar solubility, affinity for intestinal brush border membranes, transport in enterocytes, incorporation into chylomicrons, or a combination of these processes 28 . Besides, there might be variability in the mucosal handling of vitamin E that could affect their intestinal absorption 36 . Although we did not separate chylomicrons and VLDL from TRP fraction to differentiate the T3 uptake from intestine by chylomicrons and from liver by VLDL, recent findings from Abuasal et al. 37 demonstrated that there was an inverse relationship between intestinal uptake of γ-T3 and their concentration in the intestinal lumen. Therefore, any elevation of γ-T3 concentration in the lumen would likely reduce the amount of γ-T3 transported into the enterocytes. However, no investigations on other T3 isomer were carried out. The intestinal absorption of T3, as well as T still merits further investigations, since their mechanism has not been fully described 25 . In rats, dietary vitamin E including T3 are converted to their metabolite by CYP-dependent pathway in the intestine during absorption. This could likely regulate T3 concentration in plasma and tissue 38 . Yet, excess intake of T3 has been observed to lead excretion of α-T3 and γ-T3 into bile, before both T3 isomers were metabolized into α- and γ-CEHC derivatives 32 .

The postprandial dose response effect of vitamin E in humans has basically been evaluated from the plasma and lipoproteins profiles of α-T and γ-T 39 40 41 . Surprisingly, no such evidence exists for T3, although T3 always positively imaged as a superior antioxidant compared to T 2 8 . In the previous study 19 , we investigated the postprandial response after 1011 mg TRF supplementation. In fact, this dose used was higher than the Tolerable Upper Limit Intake (UL) for vitamin E 22 . One of the rationale of conducting the current study was to investigate whether supplementation with 526 mg TRF would resulted a similar postprandial response, in comparison to the dose used in the previous study 19 , since concentrations of vitamin E in plasma can only be raised maximally two to three-fold after supplementation 39 . Plasma α-T3, γ-T3 and δ-T3 response after TRF treatment in the current study were not significantly different from the previous study. Additionally, α-T and γ-T concentrations in plasma, TRP, LDL and HDL were not apparent between both TRF treatments. However, observations in lipoprotein fractions still remains to be elucidated. In HDL, starting from 4 h to 6 h postprandial, α-T3 concentration after 526 mg TRF treatment were significantly lower compared to the 1011 mg TRF treatment. These observations merits further investigation since the transportation of vitamin E by HDL may possibly be influenced by supplementation dose and was not affected by amount of dietary fat intake 31 . In both postprandial studies, the amount of dietary fat in the test breakfast consumed before TRF supplementation was standardized.

Several studies have suggested the effectiveness of T3 as a hypocholesterolemic agent in lowering plasma or serum total cholesterol in humans 15 42 . Nevertheless, it is questionable why the effectiveness of T3 in lowering plasma total cholesterol has not been compared with α-T, since α-T has been recognized as the only form utilized to estimate human vitamin E requirements. Furthermore, the effectiveness of T3 in humans was only compared with a placebo treatment in most studies 10 15 18 21 42 43 . Although in several studies, physiological effects of T3 was compared with α-T, the concentration of α-T in the control preparations or supplements was very low 9 11 44 Unlike our previous observation 19 where supplementation with 1011 mg palm TRF or 1074 mg α-T resulted in significant lowering of plasma postprandial total cholesterol, supplementation with 526 mg palm TRF or 537 mg α-T in the current study did not demonstrated any hypocholesterolemic effect. Several postulations have been discussed to explain the inability of T3 to lower plasma or serum cholesterol in humans such as the higher content of T in the T3 supplements, in vivo bio-conversion of T3 to α-T, and very low concentration of T3 that did not reach the pharmacologically effective level in plasma 45 .

In conclusions, T3 isomers (α-T3, γ-T3, and δ-T3) were present in the circulating plasma and lipoproteins (TRP, LDL, and HDL) after T3 supplementation. As postulated, T3 concentrations were significantly lower than α-T. Low absorption into the circulation could affect the physiological effects of T3, as indicated by their inability to lower plasma cholesterol in the current postprandial study. However, it is somewhat reassuring that even at the low concentration of circulating T3 in plasma (approximately 4 nM) T3 could still have beneficial biological functions including that of neuroprotection as demonstrated by other workers 46 .

AUC: area under the curve; CEHC: carboxy-ethyl-hydroxy-chroman; CYP: cytochrome P450; d: day; h: hour; MANOVA: multiple analysis of variance; T: tocopherols; T3: tocotrienols; TRF: tocotrienol-rich fraction; UL: upper tolerable intake; α-T: alpha-tocopherol; α-T3: alpha-tocotrienol; β-T3: beta-tocotrienol; δ-T3: delta-tocotrienol; γ-T: gamma-tocopherol; γ-T3: gamma-tocotrienol.

The authors declare that they have no competing interests.

The contribution of each author was as follows: SF undertook the overall management of the study and most of the laboratory and statistical analysis and drafting of the manuscript. RMN developed the methods for vitamin E HPLC analysis and other related analytic procedures. HMC contributed to the design and subsequent finalization of the manuscript. KS was the overall researcher in charge of the study, having designed the study protocols, primed the laboratory and statistical techniques, and contributed intellectually to the final manuscript. All authors read and approved the final manuscript.