Pharmacokinetics and Bioavailability of Annatto δ-tocotrienol in Healthy Fed Subjects

Background: Although, α-tocopherol is the most bioavailable form of vitamin E, but several animal and clinical studies have demonstrated tocotrienol bioavailability to various tissues. There are few reports on bioavailability of tocotrienols in humans. Most studies were carried out with mixtures of tocotrienols + tocopherols rather than pure tocotrienols. Moreover, dietary α-tocopherol interferes with the bioavailability of tocotrienols, and prevents absorption and delivery to organs and tissues.


Introduction
Several studies have reported the antioxidant, anti-inflammatory, anticancer, hypocholesterolemic and neuroprotective properties of tocotrienols in different cell lines, animal models, and in humans [1][2][3][4][5][6][7][8]. However, question on the bioavailability of pure tocotrienols remained unanswered. Therefore, it is important to understand the absorption and bioavailability mechanism of tocotrienols before carrying out investigations into the therapeutic efficacy in humans. The bioavailability of naturally-occurring tocotrienols differ considerably in their absorption, therfore therapeutic uses of tocotrienols remain controversial. It was reported that after feeding rats mixed tocotrienols, the oral bioavailability of α-tocotrienol was 28% compared to 9% of γ-tocotrienol and δ-tocotrienol [9]. Tocotrienols in humans were detected in postprandial plasma [10,11], and they were found enriched in triacylglycerol-rich particles, HDL, and LDL after administration of palm tocotrienol-rich fraction (mixture of 68% tocotrienol + 32%

Supplies for HPLC
Fisher brand disposable borosilicate glass tubes Screw cap, 20 × 150 mm; Borosilicate screw cap with conical shape-10 ml; Screw caps with Teflon insert; Disposable glass pipettes 1 ml, Rubber teats; Plastic test tube holders for above glass tubes; Test tube Shaker; Buchner test tubes Vacuum Evaporator (48 centrifuge test tube 10 ml) holder; Water vacuum Aspirator; Solvent dispensing bottles units (1000 ml).
It was also reported that the bio-discrimination of α-tocopherol (vitamin E) influences the rate of tocotrienol absorption, due to high affinity of α-tocopherol with "α-tocopherol transfer protein" (α-TTP), which mediates secretion of α-tocopherol (100%) from the liver into the circulatory system, and is much higher than α-tocotrienol (12%) or other tocotrienols [12]. Moreover, α-tocopherol has been reported to attenuate the cholesterol-lowering effect of tocotrienols through activation of the HMG-CoA reductase activity (whereas tocotrienols have a desirable inhibiting effect on its activity) [12,13]. Also α-tocopherol interferences with tocotrienol functions such as attenuation of cancer inhibition [14][15][16], exacerbation of stroke injury [17], inhibition of absorption [18], and induction of tocotrienol catabolism [19]. Therefore, dietary supplementation of tocotrienol preparations with minimal concentrations of tocopherol (<20%) in the mixture has been recommended for the inhibition of several biological activities [12,20]. All the isomers of tocols were detected in plasma as well as in various tissues of rats (liver, heart, and adipose) [21] and humans (skin, adipose, brain, cardiac muscle, and liver) [22]. Moreover, high doses of tocotrienols may be useful for cancer chemoprevention and treatment. Large doses of δ-tocotrienol up to 3200 mg/d have been used to treat patients suffering from resectable pancreatic cancer [23].
The biological properties of tocotrienols have been underestimated despite several studies showing their positive effects on physiological and biological functions [1][2][3][4][5][6][7][8]. The scientific recognition for tocotrienols remains limited as compared to tocopherols due to their low concentrations in most food products. Although they are present in high concentrations in palm oil and annatto seeds, the latter being the richest source of δ-tocotrienol without any tocopherols. In addition, there is a lack of information on the bioavailability and metabolism of tocotrienols in humans. The absorption of tocotrienols was negligible via intraperitoneal and intramuscular route, while incomplete absorption was observed when given via the oral route in rats [9]. Tocotrienols were also administered intravenously (tail vein injection) by encapsulation into transferrin-bearing vesicles [24]. Moreover, when tested in a tumor-targeted vesicle system, tocotrienols are very promising as a potential therapeutic system to eradicate human epithelial tumors and melanoma tumors in murine xenografts [25]. There is a limited amount of literature describing the bioavailability of tocotrienol, particularly

Study subjects
The present investigation was a single-center, open-label, randomized study to determine the systemic pharmacokinetics and bioavailability of annatto-based δ-tocotrienol after oral supplementation to 33 healthy subjects under fed condition, using doses of 125 mg, 250 mg, and 500 mg (11 subjects/dose). The study was conducted in accordance with the current Good Clinical Practices (FDA, 1996) and the Declaration of Helsinki (WMA, 2008). The study protocol was approved by the institutional review board (IRB) of the Pakistan Ordinance Factory (POF) Hospital, Wah Cantt, Pakistan. The study was carried out under FDA approved "Investigational New Drug" (IND) number 36906.
Thirty three adult healthy male subjects were recruited for the study according to the guidelines provided by the United States Food and Drug Administration (FDA, 2003) from Wah Cantt, Pakistan. All participants of study signed an informed consent. All subjects were male between 18-50 years and weighed within 20% of normal body weight according to the Metropolitan Life Assurance Tables. Clinical history and physical examination of all participants was carried out by a consultant physician in the hospital. Systolic and diastolic blood pressures were measured at rest position. All relevant investigations, including fasting plasma glucose, complete liver function tests (LFTs), and serum urea, were analyzed for screening of participants. The human subjects who had acute or chronic disease, malabsorption, cholecystectomy, or were currently taking vitamin E supplements were excluded from the study.

Study design
An open-label, randomized study was carried out to determine the pharmacokinetics and bioavailability of annatto-based δ-tocotrienol after oral supplementation in 33 healthy male subjects under fed condition. All the subjects were fed Pakistani heavy breakfast comprising of fruit cocktail, halwa, puri, paratha, omelet, orange juice and tea. The volunteers were randomly assigned to one of the tocotrienol soft gel dose levels (125 mg, 250 mg, and 500 mg), which they received once after breakfast. Restricted foods included nuts, cereals and vegetable oils. The food was prepared in "Army Mess" under strictly hygienic conditions. These conditions are known to be optimal in securing the best results. In addition to breakfast, participants received lunch that included chicken biryani, mutton (lamb) qurma, sheermal/nan, and gajar ka halva (desert), and dinner that included fried Basmati rice, kabab (chicken), rogni nan, shahi tukray, and kasmiri tea with pistachios. Fruit cocktail was served with every meal.

Blood Sample Collection and Extraction
On the study day each subject consumed one dose of δ-tocotrienol immediately after consuming a typical heavy Pakistani breakfast. During the experimental period (0 h -10 h), subjects consumed a good lunch, evening tea, and dinner as described above. All subjects were allowed to drink water freely. For the determination of the pharmacokinetic profile of tocotrienols in plasma, venous blood samples (2 × 5 ml) were collected in EDTA glazed tubes at pre-dose (0 h) and at post-dose (1 h, 2 h, 3 h, 4 h, 6 h, 8 h and 10 h). The samples were then centrifuged at 3000 × g for ten minutes. Processed plasma samples were stored in Eppendroff tubes at -80°C till further analysis.

High Performance Liquid Chromatography Analysis
Highest purity HPLC grade solvents and reagents were used. The reagents consisted of absolute ethanol and methanol (Fisher Scientific, Pittsburgh, PA). Hexane, Ascorbic Acid and butylated hydroxyl toluene (BHT) were obtained from Sigma Chemical Co. Inc. (St. Louis, MO). The various tocopherol (α-, β-, γ-, δ-) and tocotrienol (α-, β-, γ-, δ-) standards were obtained from ChromaDex Inc. (10005 Muirland Blvd, Suite G, Irvine, CA). The working standards were prepared by mixing appropriate amounts of the stock solutions. The working standards used for repeated determinations were stored at -20°C for a maximum period of 24 h.
Following solutions were prepared for HPLC analysis of various tocols.

Estimation of human plasma cytokines/proteins and miRNA
The various plasma cytokines/proteins were estimated by using Human Cytokine Elisa Plate Array I (chemiluminescence), Catalog number EA-4001 (Signosis, Inc., Santa Clara, CA, 95054). Assays for estimating the plasma cytokines/protein were carried out according to the protocols provided by Signosis, Inc. The incubation of each assay mixture at various temperatures was carried out by using Enviro-Genie Shaker/incubator (Enviro-Genie Industries, Bohemia, NY). The intensity of chemiluminescence was detected using a Microplate Luminometer (GloMax Promega, Madison, WI) at 500 nm, and luminescence was monitored over 20 min period. Estimation of circulating miRNAs was carried out using "Customized miRNA Direct Hybridization Plate Array", chemiluminescence; Catalog Number Inv-00465 (Signosis, Inc., 1700 Wyatt Drive Suite 10-12. Santa Clara, CA).

Pharmacokinetic analysis
The computer software PKSolver 2.0 was used for calculation of pharmacokinetic parameters through standard two compartmental analysis of each subject in each group for area under the curve (AUC t0 -t10 , AUC t0-∞ ), maximum plasma concentration (C max ), time to reach maximum concentration (T max ), half-life time (t 1/2 ; h), and time of clearance (Cl-T). The values for these parameters were based on mean concentrations of each time point of each isomer (α, β, γ, δ) of tocotrienols (T3) and tocopherols (T) and also using the values of each subject in each group. Data of plasma tocotrienol concentrations versus time was used to calculate pharmacokinetic parameters. AUC from 0 h-time to 10 h-time (AUC t0 -t10 h) for plasma tocotrienols was calculated by trapezoidal rule, where t is the last measured time point. The C max and T max were obtained directly by inspecting each individual plasma level-time curve, and also by GraphPad Prism 5. The AUC values for each isomer were also checked by using following equation:

Statistical Analysis
Data was analyzed using SPSS 22 version (SPSS Inc, Chicago). Descriptive statistics comprising mean and SD were calculated. Percent difference was calculated from baseline value of each analyte. Repeated two-way ANOVA was applied on dose response data. P value of <0.05 was considered significant. Normal distributed variables were summarized as means ± SD, and percent differences were calculated from baseline values of each inflammatory marker by one-way variance. Paired Student's t-test was applied for normally distributed variables for percentage values of cytokines/proteins, and miRNAs. A two tailed P value <0.05 was considered significant. Data are reported as mean ± SD (Standard Deviation).

Results
The physical characteristics of all the participants were reported in Table 1. The ages, heights, weights, systolic, diastolic blood pressures, and other parameters of participants in each dose were closely similar to avoid variation in the results due to differences in the characteristics of each dose treatment ( Table 1).
The pharmacokinetics and bioavailability of a compound required estimations of plasma total area under the concentration-time curve (AUC 0 -t10 or ∞ ), plasma maximum concentration (C max ), time to reach maximum plasma concentration (T max ), the apparent volume of distribution (Vd/l), time of clearance (Cl-T, Cl/h), half-life time (t 1/2 ; t-h), and elimination rate constant (ke; h -1 ). Among them, general emphasis was focused on three parameters [(AUC 0-∞ ), (C max ), and (T max )] to establish the pharmacokinetics and bioavailability of the compound.
Significant differences were observed between logarithmic converted values of AUC t0 -t10 , AUC t0-t∞ , C max , and T max for δ-, γ-, β-, α-tocotrienol and δ-, γ-, β-, α-tocopherol. The estimation of these parameters were based on the quantitative estimation of eight isomers of each subject's plasma tocols (α-, β-, γ-, δ-tocotrienol and α-, β-, γ-, δ-tocopherol) separated by HPLC at 0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h and 10 h time for dose of 125 mg (n=11), 250 mg (n=11), and 500 mg (n=11). This is the first report describing the quantitative estimation of The HPLC was carried out by using normal phase silica and reverse phase C18 columns of plasma samples collected at these intervals (8 samples/subject) for the determination of pharmacokinetics of various tocols. The separation of all isomers of tocols was obtained as baseline of all the samples on a normal phase silica column under present conditions ( Figure 2).
In HPLC analyses, at 0 h and 1 h time period, no peaks of δ-tocotrienol and δ-tocopherol were observed in plasma samples of 125 mg dose participants (Table 2). Similarly, plasma samples of 250 mg and 500 mg doses of δ-tocotrienol and δ-tocopherol at 0 h showed no peaks, but at 1 h time period, out of 11 samples of each group, only 3 samples of the 250 mg dose and 4 samples of the 500 mg dose showed small concentrations of δ-tocotrienol, α-tocotrienol and δ-tocopherol ( Table 2). For 0 h only α-tocopherol, β-tocopherol, γ-tocopherol and β-tocotrienols were observed for the samples of all three doses (Tables  2A-2C). It is clear from Figure 2A that δ-tocotrienol appeared after 2 h with all three doses, reached maximum at 3 h, and that δ-tocopherol also appeared at 3 h. Both of these levels started declining between 3 h and 6 h (Table 2 and Figure 2B). Similar patterns of HPLC profile were noted throughout HPLC analyses of all the samples (Figures 2A  and 2B). The results obtained with the reverse phase C18 column were comparable to those of the normal phase silica column. However, the elusion profiles of all subjects showed better baseline separation with normal phase silica column [30] (data was not shown).
The effects of δ-tocopherol, γ-tocopherol, β-tocopherol, and α-tocopherol were also reported on half-life time (t 1/2 ; h), time of clearance (Cl-T, Cl/h), volume of distribution (Vd/L), and elimination rate constant ((Ke; h -1 ), respectively, for doses of 125 mg, 250 mg and 500 mg (Tables 4A-D). The values for these parameters showed dosedependent decreases, which were significantly different with P<0.01 as reported in Tables 3A-D Table  5. The highest plasma concentrations were obtained for α-tocopherol as shown in Table 5 and Figure 3.

Effects of δ-tocotrienol on human plasma cytokines/proteins and microRNAs
We have reported earlier the influence of δ-tocotrienol on several key cytokines/proteins involved in various diseases [5,6]. In the present paper, only those plasma cytokines/proteins are described which are important for inflammation, cardiovascular disease, cancer, and aging. The values of each cytokine/protein were based on percentages of predose values of 0 h plasma samples of 125 mg and 500 mg δ-tocotrienol (regarded as 100%) versus post-dose values of δ-tocotrienol at 3 h (125 mg dose), 3 h (250 mg dose) and 6 h (500 mg dose) plasma samples, respectively. The importance of these 11 cytokines/proteins is reported in Table 6.

Effects of annatto-based δ-tocotrienol on plasma miRNAs (miRNAs)
Recently, microRNAs (miRNAs) were discovered to play an important role in various diseases like cancer, diabetes, cardiovascular diseases and neurological disorders. Plasma samples from 0 h and 3h (125 mg dose) and 0 h and 6 h (500 mg dose) of δ-tocotrienol were analyzed for miRNA analysis. miR-34a which is normally found to be  increased during bipolar disorder, was downregulated by δ-tocotrienol feeding (Table 7), whereas the expression of miR-107, miR-122a, and miR-132, whose levels are low in Alzheimer's patients, were upregulated by δ-tocotrienol treatment (Table 7).

Discussion
The present study described the pharmacokinetics and bioavailability of various doses of annatto-based δ-tocotrienol (without α-tocopherol) in thirty three (n=33) well fed healthy participants. This is the first study to report the effects of δ-tocotrienol on the pharmacokinetic parameters of all eight isomers of the vitamin E family (α-, β-, γ-, δ-tocotrienols and α-, β-, γ-, δ-tocopherols). The bioavailability of δ-tocotrienol resulted in dose-dependent increases of plasma AUC t0-t10 , AUC t0-t∞ , C max , and T max that varies between 3 h-4 h for isomers of tocotrienols and 3 h-6 h for isomers of tocopherols at 125 mg, 250 mg, 500 mg doses. The t 1/2 (h) of these three main parameters peaked from 1.39 h to 4.39 h for the tocotrienol group (Table 3) as compared to 1.82 h to 5.22 h for the tocopherol group (Table 4), indicating a longer time of excretion for tocopherols compared to tocotrienols which further supports that tocotrienols have better bioavailability than tocopherols as reported recently [26].
The effects of all isomers of tocols were also reported on Cl-T, Cl/h, Vd, and Ke; h -1 for doses of 125 mg, 250 mg and 500 mg. These parameters were significantly different (P<0.001 -0.01) from each other for all three doses. After administering doses of 750 mg and 1000 mg of δ-tocotrienol under the same conditions, higher dose-dependent increases for AUC t0-t10 and C max , were observed than for the 125 mg, 250 mg, and 500 mg doses (data is not shown). These results confirmed the preliminary findings of pharmacokinetics of δ-tocotrienol reported in pancreatic cancer patients, where δ-tocotrienol was well tolerated at doses up to 800 mg daily without toxicity [23].
Most of the previous studies on pharmacokinetics of tocotrienols were carried out by using tocotrienol mixtures containing α-tocopherol. It was indicated that the bioavailability of α-tocotrienol is significantly affected due to the presence of α-tocopherol in the mixture [18]. Moreover, bio-discrimination between tocols in humans reduces the rate of tocotrienol absorption, and thus the desirable physiological

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It induces proliferation/activation of certain natural-killer cells,also an HIV-suppressive factor. Adipocyte-secreted insulin-sentizing and antiatherosclrrotic hormone. Its low level resulted decline in insulin sensitivity in humans.  effects of tocotrienol may not be achieved [28]. In rats, small amounts of orally administered α-tocopherol were found to have greater effects on α-tocotrienol serum concentrations [28]. In this study, 8 h of feeding tocotrienol mixture plus α-tocopherol (10 mg α-tocotrienol + 14 mg γ-tocotrienol + α-tocopherol 1 or 10 mg) resulted in significant reduction of plasma total α-tocotrienol concentration by 60% and 90% compared with the same mixture without α-tocopherol. It was also reported that bio-discrimination occurs in the absorption of various tocotrienols and they were absorbed in the order of α-tocotrienol > γ-tocotrienol > δ-tocotrienol [10].

III Cytokines involved in cancer
Further improvement in oral bioavailability and pharmacokinetics of tocotrienols was achieved by using novel self-emulsifying drug delivery systems (SEDDS), which resulted in 2.5-4.5 times higher plasma tocotrienol (C max ) and also enhanced plasma tocotrienol (AUC) [24]. For example, in an oral bioavailability study in rats, an SEDDS of δ-+ γ-tocotrienol from annatto was up to seven-fold more bioavailable than the same tocotrienol mixture without SEDDS [32]. This study, however, showed that as the concentration of synthetic emulsifiers increased, the bioavailability of tocotrienols decreased, hence demonstrating nonlinear kinetics. A new formulation of γ-tocotrienol (75%) + δ-tocotrienol (25%) versus palm oil tocotrienol-rich fraction (TRF with similar percentages of γ-+ δ-tocotrienol) tested in humans indicated better bioavailability for γ-tocotrienol than δ-tocotrienol and TRF [33], although the study failed to test γ-tocotrienol and δ-tocotrienol separately versus a mixture of the same tocotrienols and TRF from palm oil [33]. Meganathan et al. reported the HPLC profiles of only standard compounds but not of experimental plasma samples, and it was not clear from their data whether any other tocols eluted from experimental samples, particularly α-tocopherol isomer [33].
It was reported that average time to reach the highest maximum concentration (t max ) of α-tocopherol peaked later in both γ-tocotrienol and α-tocopherol groups [34]. In this study, the first step in the absorption of tocols is mainly attributed to passive diffusion taking place in the intestine [34]. The oral administration of γ-tocotrienol or α-tocopherol (10 mg/kg diet) to rats resulted in plasma γ-tocotrienol concentrations peaking considerably earlier (2.4 h) than the plasma concentrations of α-tocopherol (9.5 h), although α-tocopherol had a higher overall intestinal permeability and absorption rate [34]. The rapid disappearance of tocotrienols in plasma (causing the early tocotrienol plasma T max in comparison to that of α-tocopherol) is also thought to be due to its preferential utilization in humans [11]. These findings are consistent with those of the present and other studies, as plasma tocotrienols peaked earlier than plasma tocopherols [10,11]. However, several relevant papers presenting human pharmacokinetic results indicated a T max of up to 5 h for α-tocotrienol and γ-tocotrienol [10,11,27]. This T max is independent of the fed or fasted food status [27]. Our present results also supported all these findings (T max 3 h-4 h for δ-, γ-, β-tocotrienols compared T max for α-tocopherol 6 h).  This is the first report to describe the quantitative determination of all eight isomers of tocols (four tocotrienols and four tocopherols) separated from human plasma samples at 0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 10 h after administering either 125 mg, or 250 mg, or 500 mg doses of annatto-based δ-tocotrienol (without tocopherol). Importantly, our HPLC results indicated that after 2 h, γ-, β-, α-tocotrienol and δ-,γ-,β-,α-tocopherol rapidly appeared as presented in Figure 5. It is assumed that δ-tocotrienol was metabolically converted to these tocotrienols and tocopherols. A stepwise conversion of δ-tocotrienol to α-tocopherol may be occuring. In order to understand the importance of these findings, it would be necessary first to understand the biosynthesis pathways of tocotrienols and tocopherols (tocols) in nature.
The preferred proposed pathway for the biosynthesis of tocotrienols and tocopherols consists of a prenylation reaction with a polyprenyl phosphate that takes place on homogenistic acid, which is derived from p-hydroxyphenyl pyruvic acid, followed by the decarboxylation of homogenistic acid, and further attachment of the geranylgeranyl group at the position meta to the methyl group and cyclization. This leads to the formation of δ-tocotrienol, which is the first compound synthesized in the biosynthesis pathway of tocols as described in 1971 [35]. The  The delta-based δ-tocotrienol 125 mg was administered to well-fed subjects for pharcokinetic study. After 2 h of consumption, δ-tocotrienol was appeared , which gives rise to γ-tocotrienol, β-tocotrienol, α-tocotrienol by successive C-methylation, and further leads to successive reduction to give rise to δ-tocopherol, γ-tocopherol, β-tocopherol and α-tocopherol. The end product is α-tocopherol (vitamin) as reported for biosynthesis of tocotrienols and tocopherols [35]. δ-tocotrienol then metabolized to the γ-, β-, and α-tocotrienols by successive C-methylation, which leads to successive reduction to metabolize to δ-, γ-, β-, and α-tocopherol [35]. The end product of the biosynthesis pathway in nature is α-tocopherol (vitamin E) [36].
The phenomena of bioconversion of tocotrienols to tocopherols have been discussed in earlier publications [4][5][6]29]. It was reported that failure of large doses of palm TRF and rice bran TRF25 to lower lipid parameters in hypercholesterolemic subjects maybe due to their conversion to tocopherols, because the plasma concentrations of tocopherols were 2-to 4-fold higher than tocotrienols as compared to placebo group. This conversion of tocotrienol to α-tocopherol earlier was demonstrated by the conversion of radioactive γ-[4-3 H] tocotrienol to α-tocopherol. The radioactive synthetic γ-[4-3 H]-tocotrienol was fed to chickens for 4 weeks, and serum was subjected to HPLC analysis to separate individual tocopherols and tocotrienols [29]. Radioactivity was found in α-tocopherol, β-tocopherol, γ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, and not in δ-tocopherol or δ-tocotrienol [29]. The present study demonstrated that after supplementing δ-tocotrienol, this first compound synthesized in nature was converted stepwise to other tocotrienols and tocopherols as presented in Figure 5.
The correlation between plasma levels of several pro-inflammatory and anti-inflammatory cytokines affected by δ-tocotrienol treatment has been reported in our recent publications [4,5,37]. Most of the cytokines (eight out of eleven) were down-regulated in a dosedependent manner by δ-tocotrienol treatment, except adiponectin, tumor growth factor β1 and leptin ( Table 6). The key functions of all of these cytokines were reported in Table 6.
There were twenty seven miRNAs reported in Table 7. Seven were involved in inflammation, and δ-tocotrienol treatment resulted downregulation of miR-9, miR-34a, 181a and up-regulation of miR-107, miR-122a, miR-132, miR-148a. Further, miR-24 and miR-19b -associated with cardiovascular diseases -were down-regulated. Dysregulation of miR-34a is increased in neural development and a genetic risk factor for bipolar disorder (BD) patients. δ-tocotrienol treatment of participants in the present study resulted in significant down-regulation of miR-34a, suggesting that this supplement may have a beneficial effect in the treatment of BD patients [38]. Expression of miR-107, miR-122a, and miR-132 decreases during the early stage of Alzheimer's disease, and disease progression is accelerated through regulation of b-site amyloid precursor protein-cleaving enzyme-1. These miRNAs were up-regulated by δ-tocotrienol treatment in the present study, whereas other studies suggest that this type of up-regulation may be beneficial in Alzheimer's disease [39][40][41].
Another eighteen miRNAs (miR-19a, miR-26a, miR-106a, miR-182, miR-192, miR-194, miR-196a. miR-199a, miR-204, miR-205, miR-22 and miR-342) tested in this study were associated with various types of cancers, and were down-regulated by δ-tocotrienol treatment, except miR-15b and miR-17-5p, which were up-regulated by the tocotrienol treatment. miR-15b was significantly up-regulated in cisplatin-resistant lung adenocarcinoma A549/CDDP cells compared with parental A549 cells [42]. miR-17-5p, on the other hand, was a key regulator of the GI/s phase cell cycle transition, and acted as an oncogene and a tumor suppressor in different cellular contexts [43]. In summary, key important functions of up-regulation or down-regulation of each miRNA are reported in Table 7.

Conclusion
This study has established the pharmacokinetics of δ-tocotrienol that demonstrated pharmacokinetics and bioavailability of all eight isomers of tocotrienols and tocopherols in humans. Tocotrienols in general have lower T max and t 1/2 compared to tocopherols, which results in superior bioavailability of tocotrienols compared to tocopherols. This data also established that δ-tocotrienol is converted stepwise to other tocotrienols and tocopherols. Moreover δ-tocotrienol treatment resulted in down-regulation of eight cytokines and therefore may be useful in the treatment of bipolar disorder (BD) and Alzheimer's disease because of its beneficial effects on miRNA's involved in these diseases. The bioavailability of δ-tocotrienol for the first time established in this study-supports this dietary supplement's use in the prevention of various age-related and chronic illnesses.