QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Proteggente, A. R. Articles by Lodge, J. K. Search for Related Content PubMed PubMed Citation Articles by Proteggente, A. R. Articles by Lodge, J. K.

---

Human Nutrition and Metabolism

Noncompetitive Plasma Biokinetics of Deuterium-Labeled Natural and Synthetic -Tocopherol in Healthy Men with an apoE4 Genotype^1,2

Anna R. Proteggente, Rufus Turner^*, Jonathan Majewicz^*, Gerald Rimbach^*, Anne Marie Minihane^*, Klaus Krämer and John K. Lodge³

Centre for Nutrition and Food Safety, School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK; ^* Hugh Sinclair Human Nutrition Unit, School of Food Biosciences, University of Reading, Reading RG6 6AP, UK; and BASF Aktiengesellschaft, Fine Chemicals, 67056 Ludwigshafen, Germany

³To whom correspondence should be addressed. E-mail: j.lodge{at}surrey.ac.uk.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Previous studies comparing the biokinetics of deuterated natural (RRR) and synthetic (all-rac) -tocopherol (vitamin E) used a simultaneous ingestion or competitive uptake approach and reported relative bioavailability ratios close to 2:1, higher than the accepted biopotency ratio of 1.36:1. The aim of the current study was to compare the biokinetics of deuterated natural and synthetic vitamin E using a noncompetitive uptake model both before and after vitamin E supplementation in a distinct population. Healthy men (n = 10) carrying the apolipoprotein (apo)E4 genotype completed a randomized crossover study, comprised of two 4-wk treatments with 400 mg/d (RRR--tocopheryl and all-rac--tocopheryl acetate) with a 12-wk washout period between treatments. Before and after each treatment period, the subjects consumed a capsule containing 150 mg deuterated -tocopheryl acetate in either the RRR or all-rac form depending on their treatment regimen. Blood was obtained up to 48 h after ingestion, and tocopherols analyzed by LC/MS. After deuterated all-rac administration, plasma deuterated tocopherol maximum concentrations and area under the concentration vs. time curves (AUC) were lower than those following RRR administration. The RRR:all-rac ratios determined from the deuterated biokinetic profiles (maximum concentration; C_max) and AUCs were 1.35:1 ± 0.17 and 1.33:1 ± 0.18, respectively. The 4-wk supplementation with either RRR or all-rac significantly increased plasma -tocopherol concentrations (P < 0.001), but decreased the plasma response to newly absorbed deuterated RRR or all-rac -tocopherol. Using a noncompetitive uptake approach, the relative bioavailability of natural to synthetic vitamin E in apoE4 males was close to the currently accepted biopotency ratio of 1.36:1.

KEY WORDS: • tocopherol • natural • synthetic • biokinetics • bioavailability • apoE4

-Tocopherol is the most biologically active form of vitamin E, comprising 90% of vitamin E in the body (1). The potentially beneficial role of vitamin E in human health, especially with respect to heart disease, has received much attention (2–4). However, a full understanding of -tocopherol bioavailability and the range of dietary and/or supplemental intakes of vitamin E associated with optimum health benefits in subgroups of the population is lacking.

Carriers of the apolipoprotein (apo)E4 allele, 25% of Western populations, are thought to be at significantly higher risk of cardiovascular disease relative to the common E3/E3 subgroup (5,6). Traditionally, this risk differential has been attributed to higher circulating plasma cholesterol (7) and apoB concentrations (8) in this subgroup. Data generated from the second Northwick Park Heart Study indicated hazard ratios (HR)⁴ for coronary heart disease (CHD) risk of 2.79, 0.85, and 1.47 in E4 smokers, E2 smokers, and E3 smokers, respectively, relative to a "never smoked" combined group, after correction for blood lipids and other CHD risk indicators (9). The authors suggested that the increase in HR in E4 smokers was potentially attributable to higher oxidative stress in this group, with the E4 protein having a lower antioxidant capacity than the E3 and E2 isoforms. Thus, apoE4 carriers represent a population that could potentially benefit from antioxidant supplementation (10) and merit investigation with respect to vitamin E bioavailability and requirements.

The -tocopherol molecule consists of a trimethylated chromanol ring and a saturated phytyl side chain. There are 3 chiral centers at positions 2, 4', and 8'. -Tocopherol is available from various food sources. In nature, e.g., vegetable oils and nuts, -tocopherol is found as a single stereoisomer, RRR--tocopherol. Commercially available RRR--tocopherol is derived from deodorizer distillate, a by-product of soybean oil production, comprising primarily - and -tocopherol, which are chemically modified (by methylation) to RRR--tocopherol, or natural-source -tocopherol. -Tocopherol can also be synthesized chemically, yielding -tocopherol consisting of 8 stereoisomers in equal proportions (RRR, RRS, RSR, RSS, SSS, SRR, SRS, and SSR) and is designated all-racemic -tocopherol (all-rac). This form, or natural-source -tocopherol, is commonly found in commercially available vitamin E supplements and fortified products. The relative biopotencies for each individual -tocopherol stereoisomer were determined using the rat fetal resorption model (11) and range from 100% for RRR down to 21% for SSR. Based on animal studies, the biopotencies of natural and synthetic -tocopherol have derived a ratio of 1.36:1 RRR:all-rac (11–13). Thus synthetic -tocopherol is estimated to be 73% as active as natural, with 1 mg of natural -tocopherol equivalent to 1.36 mg of synthetic -tocopherol in preventing rat fetal resorption.

Regulation of plasma -tocopherol is under the control of the hepatic -tocopherol transfer protein (-TTP) (14), whose affinity is closely related to the biological activity of each vitamin E homologue (14). This protein has high affinity for 2R forms of -tocopherol, which explains the preferential systemic distribution of 2R forms of -tocopherol in deuterium-labeled biokinetic studies (15–17). These studies confirmed that there is no discrimination between the forms of tocopherol during absorption, but there is selective enrichment of VLDL with 2R forms of -tocopherol (15–17).

Human vitamin E biopotency data are lacking. Instead, biomarkers of bioavailability have been used. Bioavailability represents the rate and extent of a compound’s appearance in the blood for which absorption is an important factor. This differs from biopotency, which is a measure of the biological effects exerted by an active compound. To monitor directly the specific form of vitamin E administered, stable-isotope labeled vitamin E is commonly used. These so-called biokinetic studies involve sampling over a period of time after administration of the label, and bioavailability is measured by monitoring the plasma response as maximum concentration (C_max) and area under the concentration vs. time curve (AUC). Previously, such human studies consistently used a competitive uptake approach whereby both deuterated RRR- and all-rac--tocopheryl acetate were administered simultaneously (18–23). Using this competitive approach, these studies indicated bioavailability ratios of RRR:all-rac to be close to 2:1 (24), leading to proposals that the accepted biopotency ratio of 1.36:1 be changed to 2:1 (25). However, such studies were criticized due to bias toward the natural form when the 2 forms were administered simultaneously (26).

With this in mind, we performed a noncompetitive -tocopherol uptake study using deuterium-labeled -tocopheryl acetate in either the RRR or all-rac form administered separately. We chose to investigate this both before and after a period of vitamin E supplementation sufficient to saturate plasma vitamin E in healthy men carrying the apoE4 genotype. The aims of this study were therefore to compare the bioavailability of natural and synthetic vitamin E using a noncompetitive approach, and to compare the bioavailability of these forms of vitamin E both before and after plasma vitamin E saturation.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Subjects. Two equal cohorts were recruited by advertisement from the Universities and local communities in the Guildford and Reading area [20 miles (32 km) apart]. Selection criteria were as follows: an apoE4 genotype (E3/E4, E4/E4); 25–55 y old; BMI between 20 and 32 kg/m²; alcohol consumption 21 U/wk (where 1 U corresponds to 7.9 g alcohol); exercise 3 x 30 min aerobic sessions/wk; no diagnosed heart disease, diabetes, liver, or other endocrine dysfunction; no abnormalities of fat metabolism; and no use of antioxidant supplementation in the 6 mo before the start of the study. After screening, we recruited 10 subjects into the study. The study was approved by the University of Surrey and the University of Reading ethics committees and all participants gave written consent before participation.

    ApoE4 genotyping. Genomic DNA was isolated from the white cell layer (buffy coats) of 10 mL EDTA blood using the Blood Spin^TM DNA purification kit by Qiagen. The 2 ApoE polymorphic sites were amplified by PCR and digested with the HhaI restriction enzyme. The resultant fragments were characterized by gel electrophoresis as described by Hixon and Vernier (27).

    Study protocol. The study was a randomized, double-blind, controlled, crossover trial (Fig. 1). Subjects consumed vitamin C (60 mg/d) for 4 wk before the start of the study and continued to do so throughout the study to standardize vitamin C status. After the 4-wk "run-in" with vitamin C only, the study subjects were randomly assigned to receive either natural (RRR-) or synthetic (all-rac-) -tocopheryl acetate in the first arm and underwent a kinetic protocol with that deuterated form of -tocopheryl acetate. The kinetic protocol lasted 48 h. On the morning of d 1, the men reported to the clinical investigation unit after an overnight fast (12 h), and a cannula was inserted in the antecubital vein of the forearm. A fasting baseline blood sample was collected and the subjects consumed a capsule containing 150 mg of deuterium-labeled RRR- (hexadeuterated) or all-rac- (trideuterated) -tocopheryl acetate with a standard breakfast containing 40 g of fat to facilitate vitamin E absorption. Blood samples were subsequently taken at 3, 6, 9, 12, 24, and 48 h after the ingestion of the capsule. On d 1, standard meals and drinks were provided but for d 2 of the kinetic protocol, the subjects were free-living and consumed their normal diets. At the completion of the kinetic protocol, subjects started a 4-wk supplementation period with 400 mg/d of either RRR- or all-rac--tocopheryl acetate depending on their randomization, followed by a second kinetic protocol carried out as described with the assigned form of deuterated -tocopheryl acetate. The subjects then underwent a 12-wk washout period after which 2 more kinetic protocols and a 4-wk supplementation regimen were carried out with the other form of -tocopheryl acetate. Compliance was assessed by capsule count.

View larger version (20K): [in this window] [in a new window]   FIGURE 1 The study design of a randomized, double blind, controlled, crossover trial in men. After a "run-in" period of vitamin C only, the men were randomly assigned to either natural or synthetic -tocopheryl acetate for 4 wk. After a 12-wk washout period, participants crossed over to the opposite treatment. At the beginning (pre-) and end (post-) of each 4-wk supplementation period, the men underwent a 48-h biokinetic protocol with the corresponding deuterated form of -tocopheryl acetate.

    Vitamin E and C capsules. Gelatin capsules containing 400 mg of RRR- or all-rac--tocopheryl acetate in vitamin E–stripped corn oil were purchased from Eurocaps. Chewable vitamin C capsules (60 mg) were purchased from a national supermarket chain (Tesco®). Subjects were told to consume these capsules with breakfast.

    Sample preparation. Plasma was separated from whole blood by centrifugation at 1750 x g for 10 min in a refrigerated centrifuge (4°C). Plasma for tocopherol analysis was pipetted (1 mL) into cryovials containing 10 µL BHT (1000 g/L in absolute ethanol) and snap-frozen in liquid nitrogen to prevent oxidation. Plasma for ascorbic acid analysis was stabilized by the addition of an equal volume of 10% metaphosphoric acid in diethylenetriaminepentaacetic acid (0.1 mol/L) and snap-frozen in liquid nitrogen. All plasma samples were stored at –80°C until analysis was carried out.

    Tocopherol extraction and analysis. Plasma aliquots were first diluted 1:5 with PBS (10 µmol/L, pH 7.4) to fit into the dynamic range of the LC-MS (28). Tocopherols were extracted from these diluted aliquots by a combination of SDS (0.1 mol/L), absolute ethanol, and hexane in the presence of BHT, as previously described (29), and reconstituted into 500 µL absolute ethanol. Samples were analyzed using a Micromass LCT^TM Time-Of-Flight Mass Spectrometer (Micromass) with negative ion mode electrospray ionization using a method we developed recently (28). Amounts of deuterium-labeled and unlabeled tocopherols in extracted plasma samples were calculated from standard curves using the Mass Lynx version 4.0 software. Labeled -tocopherol concentrations were corrected for the contribution of isotopic contamination and isotopic purity essentially as described (30).

    Blood lipids. Plasma total cholesterol and triacylglycerides were determined using Randox® enzymatic kits on an automated SPACE biochemical analyzer.

    Ascorbic acid analysis. Plasma ascorbic acid was determined by paired-ion reversed-phase HPLC coupled with electrochemical detection as described (31).

    Pharmacokinetics analysis. Noncompartmental pharmacokinetic parameters associated with plasma concentration-time data after single oral dosing were determined using the PK Solution Version 2.0 software (Summit Research Services). Parameters obtained were C_max, maximum time, AUC, rate of elimination, half-life, and volume of distribution (Vd).

    Statistical analysis. This biokinetic study was part of a larger study comparing markers of antioxidant status, and monocyte gene expression in smokers and nonsmokers; thus the sample size calculations were performed for the whole study. Using a within-group variation of 20% [which corresponds to variation in biokinetic studies, e.g., (32)], and a difference to detect between treatments of 20%, a sample size of 16 would allow us to demonstrate a significant difference between groups with 0.05 probability and 80% power. In total, 10 subjects were recruited for the biokinetic part of the study. The software package GraphPad InStat (GraphPad Software) was used for all statistical analysis. This program automatically tests for normality. All data were found to be normally distributed; thus, parametric tests were used. Repeated-measures ANOVA was used to test for differences in deuterated tocopherol concentration over time and between treatments (natural vs. synthetic). Tukey’s post hoc test was used when a significant result was found (P < 0.05). Unpaired and paired t tests were used to test differences in kinetic parameters between natural and synthetic and pre- and postsupplementation. Results were considered significant at the 95% confidence level (P < 0.05). Values are shown as means ± SEM.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Subjects. At the start of the study, the subjects’ age, BMI, total cholesterol, total triacylglycerides, and plasma ascorbic acid were 46 ± 3.5 y, 26.5 ± 1.3 kg/m², 7.3 ± 0.4 mmol/L, 2.2 ± 0.3 mmol/L, and 50.8 ± 4.6 µmol/L, respectively. These variables were not affected during the study.

    Baseline plasma endogenous -tocopherol. Plasma -tocopherol concentrations before and after the 4-wk ascorbic acid supplementation regimen were 29.6 ± 1.7 and 30.7 ± 1.6 µmol/L, respectively. After the 4-wk supplementation with 400 mg/d of natural -tocopheryl acetate, plasma -tocopherol concentrations increased from 31.6 ± 1.6 to 45.5 ± 2.9 µmol/L (P < 0.001). After the 4-wk supplementation with 400 mg/d of synthetic -tocopheryl acetate, plasma -tocopherol concentrations also increased from 30.4 ± 1.3 to 38.8 ± 2.4 µmol/L (P < 0.01). Plasma -tocopherol concentration postsupplementation did not differ between natural or synthetic -tocopheryl acetate ingestion.

    Plasma deuterium labeled -tocopherol. After ingestion of deuterium-labeled natural (RRR) and synthetic (all-rac) -tocopheryl acetate (Fig. 2) there was a significant increase in labeled -tocopherols with time (P < 0.001). Deuterated -tocopherols appeared in plasma 3 h after dosing, and reached a maximum plasma concentration at 12 h presupplementation (RRR and all-rac) and 9 and 6 h for RRR and all-rac postsupplementation, respectively. The plasma C_max of deuterated tocopherols was higher after RRR than with all-rac both pre- and postsupplementation; it was 11.2 and 8.8 µmol/L with RRR and all-rac, respectively, at 12 h presupplementation. At the same time point postsupplementation, there was a significant difference between the concentration of deuterated RRR and all-rac (9.2 ± 1.3 vs. 5.8 ± 0.8 µmol/L, P < 0.05).

View larger version (29K): [in this window] [in a new window]   FIGURE 2 Plasma unlabeled and deuterium labeled -tocopherol concentration vs. time profiles in men after administration of either deuterium-labeled natural (RRR) or synthetic (all-rac) -tocopheryl acetate, both pre- (A) and post- (B) 4 wk supplementation with either natural or synthetic -tocopheryl acetate. Values are means ± SEM, n = 10.

    Percentage of -tocopherol in the labeled form. After ingestion of deuterated RRR, the percentage of labeled -tocopherol had increased to 30% at maximum (12 h) presupplementation, but was only 22% postsupplementation (Fig. 3A). The percentage of labeled -tocopherol postsupplementation at 12 and 24 h was lower (P < 0.05) than that before supplementation. Similarly, after ingestion of deuterated all-rac (Fig. 3B), the percentage of labeled -tocopherol increased to 23% at maximum (12 h) presupplementation, but was only 17% postsupplementation (6 h).

View larger version (17K): [in this window] [in a new window]   FIGURE 3 Percentage of deuterated RRR- and all-rac--tocopherol over time in men after administration of either deuterium labeled (A) natural (RRR) or (B) synthetic (all-rac) -tocopheryl acetate, both pre- and post- 4 wk supplementation with either natural or synthetic -tocopheryl acetate. Values are means ± SEM, n = 10.

View this table: [in this window] [in a new window]   TABLE 1 Noncompartmental kinetic parameters for deuterated RRR and all-rac, and endogenous undeuterated -tocopherol derived from concentration vs. time profiles after administration to men of labeled -tocopheryl acetate at baseline (pre-) and after 4 wk supplementation (post-) with natural (N) and synthetic (S) -tocopheryl acetate1

    Ratio of natural to synthetic -tocopherol.

View larger version (19K): [in this window] [in a new window]   FIGURE 4 Ratio in men of natural to synthetic -tocopherol (RRR:all-rac) from deuterium labeled -tocopherol concentration:time profiles. Ratios are shown for both pre and post -tocopheryl acetate supplementation. Values are means ± SEM, n = 10.

View this table: [in this window] [in a new window]   TABLE 2 Ratio of RRR:all-rac obtained from deuterated -tocopherol kinetic parameters after administration to men of deuterium-labeled natural and synthetic -tocopheryl acetates at baseline (pre-) and after 4 wk supplementation (post-) with natural and synthetic -tocopheryl acetates1

View this table: [in this window] [in a new window]   TABLE 3 Individual values for maximum deuterated -tocopherol concentration (Cmax) and relative bioavailability ratios obtained from Cmax and area under the deuterated -tocopherol concentration vs. time curves (AUC) after administration to men of deuterated natural (RRR) and synthetic (all-rac) -tocopheryl acetates before supplementation

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

It has been argued that the competitive uptake model results in bias against all-rac, because when RRR and all-rac are given simultaneously in equal amounts, the actual dose would contain 75% of 2R-forms to compete with 25% of 2S-forms (24,26). Because the 2R forms are preferentially selected by -TTP in the liver (no discrimination at the site of absorption) for systemic distribution (14), increasing the abundance of 2R-forms lowers the probability of -TTP binding to 2S-forms (26), leading to discrimination against all-rac (24,26). However in the present study, in which all-rac was administered separately from RRR, there would be 50% each of the 2R- to 2S-forms in all-rac, and hence no such discrimination would occur. This could explain our findings of a mean ratio of 1.34:1 using this noncompetitive model, whereas ratios close to 2:1 were found in competitive uptake studies (19,20,23). Similarly, it may explain the similar kinetic properties of RRR and all-rac when administered separately because in competitive studies, the 2S form was eliminated faster than RRR (35). In their review of the literature, Hoppe and Krennrich (24) found that the ratio in plasma responses comparing supplement doses of unlabeled RRR- or all-rac--tocopherol in 9 studies matched the presently accepted biopotency ratio of 1.36, whereas only recent bioavailability studies applying the competitive approach of simultaneous administration with deuterated -tocopheryl acetates, found ratios > 1.36:1, and closer to 2:1 (24).

The ratio RRR:all-rac increased to a maximum at 12 h. At time points before this, ratios close to 1:1 represent nondiscrimination between forms of vitamin E during absorption and chylomicron transport before first pass through the liver (15). Hepatic processing of vitamin E forms represents the major route of discrimination; however, because vitamin E can transfer between lipoproteins (36), especially during chylomicron metabolism, then it is possible that some forms bypass this discriminatory process and persist in the plasma, adding variation to the results.

It is important to note that in the present study, we chose to investigate subjects carrying the apoE4 genotype. This common polymorphism, present in 25% of the population, is associated with increased risk of cardiovascular disease (5,6) which has been attributed in part to a reduced antioxidant status in these individuals. It was suggested that there is value in genotyping subjects for apoE in relation to vitamin E supplementation because apoE4 carriers could potentially benefit from such supplementation (10). We initially hypothesized that prospectively choosing only apoE4 individuals would have the advantage of potentially limiting interindividual variation in vitamin E responses. However, large interindividual variation remained in individual biokinetic responses and bioavailability ratios, with certain subjects displaying similar or even increased bioavailability for the synthetic form. It is likely that a number of physiologic and/or genetic factors are important in influencing vitamin E status as we suggested recently (37,38).

The present study consisted of two 4-wk supplementation regimens with either natural or synthetic -tocopheryl acetate (400 mg/d) in a crossover design, separated by a 12-wk washout period. This washout period was clearly sufficient, given that the baseline unlabeled concentrations were similar before each regimen (Table 1). After supplementation, the total plasma unlabeled -tocopherol concentration increased significantly after both RRR- and all-rac--tocopheryl acetate supplementation. However, postsupplementation -tocopherol concentrations did not differ. Plasma vitamin E concentrations are limiting, in that they reach saturation kinetics and cannot be raised further than 2- to 3-fold regardless of the supplementation regimen (34,39,40). This can be explained in part by the rapid displacement of existing -tocopherol by newly absorbed -tocopherol (34). This was shown in the present study as a decrease in unlabeled -tocopherol concentrations in response to the increase in deuterated -tocopherol concentration (Fig. 2). The present study design allows, for the first time, a direct comparison of the uptake of newly absorbed -tocopherol both at baseline and after vitamin E supplementation. The supplementation regimen used (400 mg/d for 4 wk) should be sufficient to demonstrate steady-state plasma vitamin E concentrations after plasma vitamin E saturation, which reaches C_max after 5 half-lives or 10–15 d (24). For example, Dimitrov et al. (40) supplemented subjects with 440, 880, or 1320 mg/d of synthetic -tocopheryl acetate consecutively for 28 d. Steady-state plasma -tocopherol saturation occurred after 10, 5, and 5 d, respectively. Biokinetic studies supplementing 300 mg/d of a combination of natural and synthetic -tocopheryl acetate for either 8 (20) or 10 (19) consecutive days resulted in steady-state concentrations after 6 d (19,20). Even though a half-life of 34–45 h, as found in the present study, appears to represent a slow plasma turnover of -tocopherol, Traber et al. (35) demonstrated that there is rapid recycling of -tocopherol due to the constant resecretion in VLDL which is on the order of 1.4 pools/d (74 µmol/d) and this results in the longer half-life (35). We found that after vitamin E supplementation, the plasma has a diminished ability to take up newly absorbed -tocopherol. This was shown by the decreased deuterated -tocopherol concentrations postsupplementation, the decreased extent of labeling postsupplementation, and the decreased area under the labeled -tocopherol concentration vs. time curves (Table 1).

It is clear that -TTP plays an important role in regulating plasma concentrations of vitamin E. It is likely that the decreased bioavailability of newly absorbed -tocopherol after vitamin E supplementation observed in the current study is due to the saturation of -TTP, such that less is available for systemic distribution via VLDL, and presumably more is metabolized. Because this effect was observed after both natural and synthetic supplementation and biokinetics, this shows that synthetic vitamin E contained enough of the preferred 2R-forms to saturate -TTP. Indeed, this explains the observation that supplementation with synthetic vitamin E can result in similar plasma vitamin E concentrations compared with supplementation with natural source vitamin E, as observed in the present study and by others (41,42). However, because the ratio of RRR:all-rac increased to 1.5:1 postsupplementation with vitamin E, it appears that the body discriminates favorably toward natural -tocopherol in saturating conditions, i.e., at high intakes. It is also possible that the dose of deuterated -tocopherol administered influences biokinetics. Traber et al. (34) found a linear relation in labeled -tocopherol dose (up to 150 mg) and AUC, suggesting that increasing dose does not decrease incorporation into plasma. However, higher doses may influence incorporation into plasma if the dose saturates -TTP. This would also reduce the amount of newly absorbed released into the circulation as was observed after the supplementation regimen in the present study.

Alternatively, the concept that vitamin E status regulates -TTP protein concentrations or activity might also explain this observation. In vitamin E–deficient rats, supplementation with both - and -tocopherol increased hepatic concentrations of -TTP mRNA (43). Unfortunately, there is very little information to date on the regulation of tocopherol-binding proteins, but it is certainly an area for further investigation.

In summary, our findings demonstrate that when studied with a noncompetitive approach under basal conditions, natural and synthetic -tocopherol are kinetically equivalent and their relative bioavailability conforms to the currently accepted biopotency ratio of 1.36:1 in men with an apoE4 genotype. We also demonstrated that vitamin E biokinetics are influenced by vitamin E status, which becomes limiting after high vitamin E intakes, independently of the form used. In light of the findings of our study and those of others, we feel that the noncompetitive approach is an appropriate method with which to study the relative bioavailabilities of natural and synthetic -tocopherol under normal conditions. However, caution should be observed in drawing conclusions on relative biopotency based on relative bioavailability rather than on in vivo surrogate markers of vitamin E status.

	ACKNOWLEDGMENTS

 
Deuterated tocopheryl acetates were kind gifts from Dr. Christine Gärtner and Dr. James Clark of Cognis Nutrition and Health (Düsseldorf, Germany and LaGrange, IL, respectively).

	FOOTNOTES

 
¹ Presented in part at the Nutrition Society Summer Meeting, July 5–8, Dublin, Ireland [Proteggente, A. R., Turner, R., Majewicz, J., Rimbach, G., Minihane, A. M., Krämer, K. and Lodge, J. K. (2004) Plasma biokinetics of natural and synthetic -tocopherol in healthy apoE4 male smokers and non-smokers].

² Supported by BASF Aktiengesellschaft.

⁴ Abbreviations used: apo, apolipoprotein; -TTP, -tocopherol transfer protein; AUC, area under the concentration vs. time curve; CHD, coronary heart disease; C_max, maximum concentration; HR, hazard ratio; Vd, volume of distribution.

Manuscript received 13 January 2005. Initial review completed 31 January 2005. Revision accepted 10 February 2005.

	LITERATURE CITED

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Traber, M. G. & Kayden, H. J. (1989) Preferential incorporation of -tocopherol vs -tocopherol in human lipoproteins. Am. J. Clin. Nutr. 49:517-526.[Abstract/Free Full Text]

2. Gey, K. F. (1991) Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am. J. Clin. Nutr. 53:326S-334S.[Abstract/Free Full Text]

3. Brigelius-Flohe, R., Kelly, F. J., Salonen, J. T., Neuzil, J., Zingg, J. M. & Azzi, A. (2002) The European perspective on vitamin E: current knowledge and future research. Am. J. Clin. Nutr. 76:703-716.[Abstract/Free Full Text]

4. Pryor, W. A. (2000) Vitamin E and heart disease: basic science to clinical intervention trials. Free Radic. Biol. Med. 28:141-164.[Medline]

5. Lahoz, C., Schaefer, E. J., Cupples, L. A., Wilson, P.W.F., Levy, D., Osgood, D., Parpos, S., Pedro-Botet, J., Daly, J. A. & Ordovas, J. M. (2001) Apolipoprotein E genotype and cardiovascular disease in the Framingham Heart Study. Atherosclerosis 154:529-537.[Medline]

6. Scuteri, A., Bos, A. J., Zonderman, A. B., Brant, L. J., Lakatta, E. G. & Fleg, J. L. (2001) Is the apoE4 allele an independent predictor of coronary events?. Am. J. Med. 110:28-32.[Medline]

7. Minihane, A.-M., Khan, S., Leigh-Firbank, E. C., Talmud, P., Wright, J. W., Murphy, M. C., Griffin, B. A. & Williams, C. M. (2000) ApoE polymorphism and fish oil supplementation in subjects with an atherogenic lipoprotein phenotype. Arterioscler. Thromb. Vasc. Biol. 20:1990-1997.[Abstract/Free Full Text]

8. Welty, F. K., Lichenstein, A. H., Barrett, P.H.R., Jenner, J. L., Dolnikowski, G. G. & Schaefer, E. J. (2000) Effects of apoE genotype on apoB48 and apoB100 kinetics with stable isotopes in humans. Arterioscler. Thromb. Vasc. Biol. 20:1807-1810.[Abstract/Free Full Text]

9. Humphries, S. E., Talmud, P. J., Hawe, E., Bolla, M., Day, I. N. & Miller, G. J. (2001) Apolipoprotein E4 and coronary heart disease in middle-aged men who smoke: a prospective study. Lancet 358:115-119.[Medline]

10. Peroutka, S. J. & Dreon, D. M. (2000) The value of genotyping for apolipoprotein E alleles in relation to vitamin E supplementation. Eur. J. Pharmacol. 410:161-163.[Medline]

11. Weiser, H. & Vecchi, M. (1982) Stereoisomers of alpha-tocopheryl acetate. II. Biopotencies of all eight stereoisomers, individually or in mixtures, as determined by rat resorption-gestation tests. Int. J. Vitam. Nutr. Res. 52:351-370.[Medline]

12. Anonymous (1981) Stereoisomers of alpha-tocopheryl acetate–characterization of the samples by physico-chemical methods and determination of biological activities in the rat resorption-gestation test. Int. J. Vitam. Nutr. Res. 51:100-113.[Medline]

13. Weiser, H., Vecchi, M. & Schlachter, M. (1985) Stereoisomers of alpha-tocopheryl acetate. III. Simultaneous determination of resorption-gestation and myopathy in rats as a means of evaluating biopotency ratios of all-rac- and RRR-alpha-tocopheryl acetate. Int. J. Vitam. Nutr. Res. 55:149-158.[Medline]

14. Hosomi, A., Arita, M., Sato, Y., Kiyose, C., Ueda, T., Igarashi, O., Arai, H. & Inoue, K. (1997) Affinity for alpha-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett. 409:105-108.[Medline]

15. Traber, M. G., Burton, G. W., Ingold, K. U. & Kayden, H. J. (1990) RRR- and SRR--tocopherols are secreted without discrimination in human chylomicrons, but RRR--tocopherol is preferentially secreted in very low density lipoproteins. J. Lipid Res. 31:675-685.[Abstract]

16. Traber, M. G. & Kayden, H. J. (1989) -Tocopherol as compared with -tocopherol is preferentially secreted in human lipoproteins. Ann. N.Y. Acad. Sci. 570:95-108.[Medline]

17. Traber, M. G., Rudel, L. L., Burton, G. W., Hughes, L., Ingold, K. U. & Kayden, H. J. (1990) Nascent VLDL from liver perfusions of cynomolgus monkeys are preferentially enriched in RRR- compared with SRR- tocopherol: studies using deuterated tocopherols. J. Lipid Res. 31:687-694.[Abstract]

18. Traber, M. G., Burton, G. W., Hughes, L., Ingold, K. U., Hidaka, H., Malloy, M., Kane, J., Hyams, J. & Kayden, H. J. (1992) Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism. J. Lipid Res. 33:1171-1182.[Abstract]

19. Acuff, R. V., Thedford, S. S., Hidiroglou, N. N., Papas, A. M. & Odom, T. A., Jr (1994) Relative bioavailability of RRR- and all-rac-alpha-tocopheryl acetate in humans: studies using deuterated compounds. Am. J. Clin. Nutr. 60:397-402.[Abstract/Free Full Text]

20. Burton, G. W., Traber, M. G., Acuff, R. V., Walters, D. N., Kayden, H., Hughes, L. & Ingold, K. U. (1998) Human plasma and tissue alpha-tocopherol concentrations in response to supplementation with deuterated natural and synthetic vitamin E. Am. J. Clin. Nutr. 67:669-684.[Abstract]

21. Traber, M. G., Elsner, A. & Brigelius-Flohe, R. (1998) Synthetic as compared with natural vitamin E is preferentially excreted as alpha-CEHC in human urine: studies using deuterated alpha-tocopheryl acetates. FEBS Lett. 437:145-148.[Medline]

22. Traber, M. G., Winklhofer-Roob, B. M., Roob, J. M., Khoschsorur, G., Aigner, R., Cross, C., Ramakrishnan, R. & Brigelius-Flohe, R. (2001) Vitamin E kinetics in smokers and nonsmokers. Free Radic. Biol. Med. 31:1368-1374.[Medline]

23. Acuff, R. V., Dunworth, R. G., Webb, L. W. & Lane, J. R. (1998) Transport of deuterium-labeled tocopherols during pregnancy. Am. J. Clin. Nutr. 67:459-464.[Abstract]

24. Hoppe, P. P. & Krennrich, G. (2000) Bioavailability and potency of natural-source and all-racemic alpha-tocopherol in the human: a dispute. Eur. J. Nutr. 39:183-193.[Medline]

25. Food and Nutrition Board, I. o. M (2000) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids 2000 National Academy Press Washington, DC.

26. Cohn, W. (1999) Evaluation of vitamin E potency. Am. J. Clin. Nutr. 69:156-158.[Free Full Text]

27. Hixson, J. E. & Vernier, D. T. (1990) Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J. Lipid Res. 31:545-548.[Abstract]

28. Hall, W. L., Jeanes, Y. M., Pugh, J. & Lodge, J. K. (2003) Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components. Rapid Commun. Mass Spectrom. 17:2797-2803.[Medline]

29. Burton, G. W., Webb, A. & Ingold, K. U. (1985) A mild, rapid, and efficient method of lipid extraction for use in determining vitamin E/lipid ratios. Lipids 20:29-39.[Medline]

30. Burton, G. W. & Daroszewska, M. (1996) Deuterated vitamin E: measurement in tissues and body fluids. Punchard, N. A. Kelly, F. J. eds. Free Radicals. A Practical Approach 1996:257-270 Oxford University Press Oxford, UK. .

31. Cammack, J., Oke, A. & Adams, R. N. (1991) Simultaneous high-performance liquid chromatographic determination of ascorbic acid and dehydroascorbic acid in biological samples. J. Chromatogr. 565:529-532.[Medline]

32. Roxborough, H. E., Burton, G. W. & Kelly, F. J. (2000) Inter- and intra-individual variation in plasma and red blood cell vitamin E after supplementation. Free Radic. Res. 33:437-445.[Medline]

33. Birkett, D. J. (1999) Pharmacokinetics Made Easy 1999 McGraw-Hill Roseville, Australia.

34. Traber, M. G., Rader, D., Acuff, R. V., Ramakrishnan, R., Brewer, H. B. & Kayden, H. J. (1998) Vitamin E dose-response studies in humans with use of deuterated RRR-alpha-tocopherol. Am. J. Clin. Nutr. 68:847-853.[Abstract]

35. Traber, M. G., Ramakrishnan, R. & Kayden, K. J. (1994) Human plasma vitamin E kinetics demonstrate rapid recycling of plasma RRR--tocopherol. Proc. Natl. Acad. Sci. U.S.A. 91:10005-10008.[Abstract/Free Full Text]

36. Traber, M. G., Lane, J. C., Lagmay, N. & Kayden, H. J. (1992) Studies on the transfer of tocopherol between lipoproteins. Lipids 27:657-663.[Medline]

37. Doring, F., Rimbach, G. & Lodge, J. K. (2005) In silico search for single nucleotide polymorphisms in genes important in vitamin E homeostasis. IUBMB Life 56:615-620.

38. Lodge, J. K., Hall, W. L., Jeanes, Y. M. & Proteggente, A. R. (2005) Physiological factors influencing vitamin E biokinetics. Ann. N.Y. Acad. Sci. 1031:60-73.

39. Jialal, I., Fuller, C. J. & Huet, B. A. (1995) The effect of alpha-tocopherol supplementation on LDL oxidation. A dose-response study. Arterioscler. Thromb. Vasc. Biol. 15:190-198.[Abstract/Free Full Text]

40. Dimitrov, N. V., Meyer, C., Gilliland, D., Ruppenthal, M., Chenoweth, W. & Malone, W. (1991) Plasma tocopherol concentrations in response to supplemental vitamin E. Am. J. Clin. Nutr. 53:723-729.[Abstract/Free Full Text]

41. Devaraj, S., Adams-Huet, B., Fuller, C. J. & Jialal, I. (1997) Dose-response comparison of RRR-alpha-tocopherol and all-racemic alpha-tocopherol on LDL oxidation. Arterioscler. Thromb. Vasc. Biol. 17:2273-2279.[Abstract/Free Full Text]

42. Chopra, R. K. & Bhagavan, H. N. (1999) Relative bioavailabilities of natural and synthetic vitamin E formulations containing mixed tocopherols in human subjects. Int. J. Vitam. Nutr. Res. 69:92-95.[Medline]

43. Fechner, H., Schlame, M., Guthmann, F., Stevens, P. A. & Rustow, B. (1998) alpha- and delta-tocopherol induce expression of hepatic alpha-tocopherol-transfer-protein mRNA. Biochem. J. 331:577-581.

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Proteggente, A. R. Articles by Lodge, J. K. Search for Related Content PubMed PubMed Citation Articles by Proteggente, A. R. Articles by Lodge, J. K.