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1 From the Department of Food Science and Technology, Oregon State University, Corvallis, OR
2 Supported in part by a grant to MGT (NIH DK59576) and by the National Institute of Environmental Health Sciences (NIH P30 ES00210). 3 Reprints not available. Address correspondence to MG Traber, 571 Weniger Hall, Linus Pauling Institute, Oregon State University, Corvallis, OR 97331. E-mail: maret.traber{at}oregonstate.edu.
ABSTRACT
Background: Little is known about factors that modulate dietary -tocopherol bioavailability.
Objectives: The study aimed to assess the efficacy of vitamin E–fortified apples as a low-fat vitamin E delivery system, the influence of fat on vitamin E absorption, and human vitamin E requirements by using plasma -tocopherol kinetics at a dosage of -tocopherol found in food.
Design: Apples fortified with deuterium-labeled -tocopheryl acetate were consumed by 5 participants at a breakfast containing 0%, 6%, or 21% kcal from fat in 3 sequential trials. The trials were separated by a 2-wk washout period. Blood samples were obtained up to 72 h, and plasma was analyzed for labeled and unlabeled -tocopherol.
Results: Compared with observations in the 0% fat trial, the maximum observed plasma d6--tocopherol concentrations (Cmax) and the areas under the curve increased 2- and 3-fold during the 6% and 21% fat trials, respectively. The mean (±SD) estimated percentage d6--tocopherol absorbed increased from 10 ± 4% during the 0% fat trial to 20 ± 3% and 33 ± 5% during the 6% and 21% fat trials, respectively. The mean time to Cmax (9 ± 2 h), fractional disappearance rates (0.022 ± 0.003 pools/d), and half-lives (32 ± 4 h) did not differ significantly between the trials. With the use of fractional disappearance rates and baseline plasma -tocopherol concentrations, the estimated daily plasma -tocopherol efflux was 13–14 mg. The estimated rate of -tocopherol delivery to tissues was 5 mg/d.
Conclusions: Given an estimated 33% absorption, the amount of dietary vitamin E needed daily to replace irreversible losses is 15 mg. These estimates support the current human vitamin E requirements despite the claims that the median amount of vitamin E that Americans consume is 7 mg/d.
Key Words: -Tocopherol • vitamin E requirements • bioavailability • deuterium • biokinetics • dietary fat
INTRODUCTION
Because of the widespread problem of obesity (1, 2), Americans are being counseled to reduce their total energy and fat intakes. However, Maras et al (3) indicated that most persons obtain dietary vitamin E from high-energy, high-fat foods that are not particularly -tocopherol–rich. When persons are instructed to decrease their fat intake as part of weight management, they incur a 50% reduction in vitamin E intake (4). Indeed, vitamin E is one of the most difficult nutrients to obtain; only 8% of men and 2% of women in the United States had vitamin E intakes from food (3) that met the 2000 Estimated Average Requirement of 12 mg -tocopherol/d (5). Low-fat foods fortified with vitamin E might be a solution that allows consumers to eat more -tocopherol–dense foods.
Fresh-cut fruit and vegetables can be fortified with micronutrients with the use of vacuum impregnation without otherwise changing the physiochemical food properties. This is a technique used by the food industry to fortify the functional composition of high-porosity foods. We have used vacuum impregnation to incorporate micronutrients, such as -tocopherol (6) or zinc and calcium (7), into low-fat, low-energy foods.
Although vacuum impregnation of fresh apples with vitamin E incorporates substantial amounts of -tocopherol, little is known about -tocopherol bioavailability from this low-fat matrix. In most foods, vitamin E is associated with fats and oils (8). Intestinal -tocopherol absorption is dependent on the same processes that enable fat digestion, uptake into the enterocyte, and secretion in chylomicrons (9). Therefore, we hypothesized that -tocopherol bioavailability from vitamin E–fortified apples could be significantly enhanced by the simultaneous ingestion of dietary fat. To test this hypothesis, deuterium-labeled vitamin E–fortified apples, which were designed so that a serving contained approximately the vitamin E daily value (30 IU) (5), were consumed with a breakfast that contained increasing amounts of fat followed by a controlled lunch in 3 sequential trials. Importantly, because the amount of vitamin E consumed was in the range for dietary intakes, we also estimated human vitamin E requirements based on -tocopherol turnover kinetics.
SUBJECTS AND METHODS
Materials
HPLC-grade methanol was obtained from Fisher (Fair Lawn, NJ). Ascorbic acid, butylated hydroxy toluene, and potassium hydroxide were obtained from Sigma-Aldrich (St Louis, MO). Vitamin E standards, including unlabeled (d0) RRR--tocopherol, labeled (d6) RRR--tocopheryl acetate (d6--tocopheryl acetate), and d0--tocopherol, were gifts from James Clark of Cognis Nutrition and Health, LaGrange, IL. The isotopic purity of the d6--tocopheryl acetate was determined to be 89% by liquid chromatography–mass spectrometry (LC-MS); the remainder was d5--tocopheryl acetate. d9-all rac--Tocopheryl acetate, which was used as an internal standard, was provided by Carolyn Good of General Mills (Minneapolis, MN) and was synthesized by Isotec Inc (Miamisburg, OH).
Study participants and protocol
The protocol for the present study was approved by the Institutional Review Board for the Protection of Human Subjects of the Oregon State University, and all participants provided written consent. Healthy, nonsmoking, normolipidemic volunteers (n = 5; 3 men and 2 women) were selected on the basis of age (range: 18–35 y;
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TABLE 1. Dietary intakes during the first day of each trial1
After an overnight fast of 12 h and immediately before consuming the breakfast (t = 0 h), a blood sample was obtained from each participant from the antecubital vein into blood collection tubes containing 0.05 mL 15% K3 EDTA (Vacutainer; Becton Dickinson, Franklin Lakes, NJ). After breakfast, blood samples were obtained at 3, 6, 9, 12, 24, 36, 48, and 72 h. Tubes were kept on ice for <30 min before the plasma isolation. Plasma was separated by centrifugation (500 x g, 15 min, 4 °C; Beckman TJ-6, Palo Alto, CA), divided into aliquots that were put into cryovials, frozen in liquid nitrogen, and stored at –80 °C until analyzed.
Labeled (d6-) and unlabeled (d0-) -tocopherol and unlabeled -tocopherol were extracted from the plasma (10) and analyzed by LC-MS as previously described (11, 12). Plasma total cholesterol and triacylglycerol concentrations were measured with kits obtained from ThermoDMA (Louisville, CO), and the analyses were performed in accordance with the manufacturer's instructions.
Vacuum impregnation of apples with vitamin E
To emulsify and stabilize the labeled -tocopheryl acetate in the water-based vacuum impregnation solution, a mixture (1:1:0.3, by wt) of 0.8 g d6--tocopheryl acetate, 0.8 g acetylated monoglyceride (Grindsted Acetem 50–00 pk; Danisco, New Century, KS), and 0.24 g polysorbate 80 (Integra, Renton, WA) was heated to 70 °C followed by the addition of 200 g 70 °C 20% high-fructose corn syrup solution (Western Family Foods Inc, Portland, OR). The solution was then homogenized (Polytron PT 10–35; Kinematica AG, Littau, Switzerland) for 90 s at 10 000 rpm.
Fuji apples (Washington State, fall 2004 crop) were washed with distilled water, and cylindrical apple chunks (15 mm height x 15 mm diameter pieces, without the skin) were obtained with a sterile stainless steel tubular cork borer and a knife. The apple cylinders were cut in the axial direction. The pieces were immediately immersed into distilled water to avoid contact with air.
The goal was to enrich the apples with 30 mg vitamin E/100 g apples. For treatment, freshly prepared apple pieces (50 g) were immersed into 150 mL vacuum impregnation solution and placed in a chamber that was connected to a vacuum pump (Model 0211-P204; Gast Mfg Corp, Benton Harbor, MI). Vacuum pressure (100 mm Hg) was applied at room temperature for 15 min, then atmospheric pressure was restored for 30 min. The apples were removed from the solution, left to drain at room temperature for 15 min, then packed in 8" hinged clear shallow containers (Gourmet Classics DV 800S; Barrett Parkway, St Louis, MO). The packed samples were stored at 2 °C and 88% relative humidity until consumed within 48 h.
Apple pieces were pooled from various enrichment sessions and mixed thoroughly, and then aliquots were obtained for analysis. The d6-
Areas under the curve (AUCs) of plasma d6--tocopherol concentrations were calculated for each person for each trial with the trapezoidal rule. Time to maximal concentration (Tmax) and maximal concentrations (Cmax) were identified by visual inspection of the data. –Tocopherol fractional disappearance rates (FDRs) and half-lives were calculated from the plasma d6--tocopherol concentrations, as previously described (13).
Statistical comparisons between the treatments were performed with GRAPHPAD PRISM (version 4; GraphPad Software, San Diego, CA). The statistical significance of treatment effects on AUC, Tmax, and Cmax was evaluated by a one-way analysis of variance with repeated measures followed by Tukey's post hoc test when a significant main effect (P < 0.05) was observed. Data are reported as means ± SDs throughout the text; means ± SEs are shown in the figures.
The Cmax increment that was dependent on dietary fat was estimated by calculating the linear relation between the plasma d6--tocopherol Cmax and fat ingested for each participant, then the slopes and intercepts were averaged. The amount absorbed was estimated by multiplying the Cmax with the plasma volume, which converted the value from µmol to mg. The fractional absorption was estimated from the amount absorbed divided by the dose administered (22 mg d6--tocopherol).
No significant differences in plasma total cholesterol or triacylglycerol concentrations were observed between dietary treatments. Because it was suggested that plasma tocopherol concentrations should be adjusted for lipid concentrations (14), we performed the -tocopherol kinetic analyses with and without adjustment for circulating lipid concentrations. After adjustment for lipids, we nonetheless observed no significant differences in the results and, therefore, have presented the -tocopherol concentrations and analyses without adjustment for circulating lipids.
RESULTS
Dietary fat intakes
The participants consumed vitamin E–enriched apples at breakfasts that varied in amounts of fat, followed by a lunch that contained a constant amount of fat (1300 kcal, 53 g fat, 36% kcal from fat). During the first 24 h of each trial, the participants' daily fat intakes did not differ significantly, although there was some variability in the carbohydrate and protein intake between trials largely due to the differences in breakfast composition (Table 1).
Plasma unlabeled and deuterium-labeled -tocopherols
Compared with the 0% fat trial, plasma d6--tocopherol Cmax doubled and tripled during the 6% and 21% fat trials, respectively (Figure 1, Table 2). The increasing breakfast fat also increased the mean (±SD) maximum percentage plasma d6--tocopherol absorbed from the total (labeled plus unlabeled) -tocopherol consumed from 8 ± 3% during the 0% fat trial to 14 ± 2% and 22 ± 6% for the 6% and 21% fat trials, respectively.
FIGURE 1.. Plasma labeled
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TABLE 2. Plasma -tocopherol kinetics1
Mean (±SD) plasma d6-
Importantly, d6--tocopherol was absorbed from the apples in the absence of fat. The increment in Cmax that was dependent on dietary fat was also estimated (Figure 2). In the absence of fat, the average (±SD) Cmax was 2.0 ± 0.8 µmol/L, which is equivalent to 2.7 ± 1.0 mg d6--tocopherol absorbed. With each gram of fat consumed, Cmax increased by 0.33 µmol/L, which is equivalent to an increase of 0.43 mg d6--tocopherol absorbed. The data estimated from the disappearance curves were similar to the Cmax actually measured for the participants (Figure 1).
FIGURE 2.. Plasma labeled -tocopherol maximum concentration (Cmax) as a function of breakfast fat content. Plasma d6--tocopherol Cmax during each of the dietary treatments was linearly related to fat intake in each of the participants. Plasma d6--tocopherol Cmax was significantly (P < 0.0001) correlated with dietary fat consumption; each ingested gram of dietary fat resulted in a corresponding increase in plasma d6--tocopherol Cmax of 0.33 µmol/L (linear regression: y = 0.33x + 2.17; R2 = 0.72) or an increase of 0.43 mg/g fat consumed.
At baseline (t = 0 h), plasma unlabeled
DISCUSSION
The purpose of the present study was three-fold: 1) to assess the efficacy of vitamin E–fortified apples as a low-fat vitamin E delivery system, 2) to assess the influence of fat on vitamin E absorption, and 3) to assess human vitamin E requirements from plasma -tocopherol kinetics at a dosage of -tocopherol found in food.
With respect to bioavailability of the vitamin E in fortified apples, the data showed that 10% of the 22 mg d6--tocopherol was absorbed in the absence of fat or other foods. Additionally, an increase of 1 g fat consumed increased -tocopherol absorption by 0.43 mg. The 21% fat breakfast contained 11 g fat and resulted in a 33% -tocopherol absorption. This value is less than the 45% absorption rate estimated in thoracic duct-cannulated rats (17) or the 55–79% rate estimated in humans when radioactive vitamin E was used to estimate absorption (18). Thus, it is likely that the percentage absorption would have been larger with a higher fat breakfast, but additional studies are needed to estimate maximal absorption rates. These data also emphasize the relatively poor absorption of vitamin E when it is consumed without fat, as was observed when vitamin E pills were consumed without food (13). Note that the ideal method for measuring vitamin E absorption would be to feed one labeled dose and inject another labeled dose of vitamin E. The ratio of the consumed dose divided by the injected dose (which is assumed to be 100%) would yield the fraction absorbed (19). This method, however, depends on the availability of a preparation of vitamin E that is acceptable for intravenous injection in humans, which does not currently exist; the only intravenous preparation had adverse effects in premature infants (20).
The finding of increased absorption of -tocopherol in the presence of dietary fat is consistent with results of other fat-soluble nutrients. Compared with the bioavailability of lycopene administered in steamed tomatoes, the bioavailability of lycopene increased 3-fold when administered in oil (21). Carotenoid bioavailability is higher with full-fat salad dressing than with reduced-fat salad dressing (22). Similarly, ingestion of avocado significantly enhanced carotenoid absorption from salad and salsa (23). In addition, increasing dietary fat increases the absorption of vitamin E from supplements (13, 24). Roodenberg et al (25) suggested that a 3% fat intake was sufficient for optimal vitamin E bioavailability. However, they measured bioavailability as increased plasma -tocopherol concentrations after 1 wk of supplementation with 50 mg -tocopherol in 50 g of a low or high fat spread that accompanied a meal that resulted in an intake of <6.5 g fat or <45 g fat. From our study, it was apparent that the fat amount that was consumed concurrently with vitamin E was critical for its absorption. Note that the vitamin E–fortified apples were consumed with breakfast; the lunch eaten 5 h later contained 36% fat and yet had no apparent effect on absorption. The vitamin E in the study by Roodenberg et al (25) was dissolved in a spread; thus, it is difficult to estimate whether the amount of fat needed for vitamin E absorption from a low-fat food is the same amount needed to dissolve vitamin E into a micellarized form. As also noted from our study, no significant changes in total plasma -tocopherol were detectable with the low doses we used; therefore, it is imperative to use labeled -tocopherol to detect changes in bioavailability of vitamin E from dietary sources.
With respect to vitamin E kinetics, breakfast fat (0–21%) consumption had no significant influence on -tocopherol rates of disappearance, half-lives, or Tmax values during the 3 trials in normolipidemic persons who consumed daily diets containing 34–38% fat. Because the plasma vitamin E pool was essentially unchanged during each of the trials and between the trials, the absorbed d6--tocopherol dose did not significantly change plasma vitamin E kinetics. The absorbed amounts were estimated from the plasma d6--tocopherol Cmax. These estimated amounts are the minimum quantities that had to be absorbed to achieve the observed plasma d6--tocopherol concentrations and, therefore, underestimate the fractional d6--tocopherol absorption because they do not take tissue distribution into account.
The daily -tocopherol requirement for normal healthy persons was estimated by the Food and Nutrition Board (5) with data from studies that were carried out in the 1950s that evaluated the amount of dietary vitamin E necessary to prevent peroxide-induced erythrocyte hemolysis in men who were depleted of vitamin E. The Estimated Average Requirement is 12 mg -tocopherol/d (5). The mean (±SD) -tocopherol requirement is 15 ± 2 mg/d when the amount required by the tissues is 5 mg/d and absorption is 33%; when absorption is 50%, the -tocopherol requirement is 10 ± 1 mg/d. This range of the estimated dietary vitamin E requirements (10–15 mg/d) confirms that the current Daily Recommended Intakes are substantially correct. However, most Americans' vitamin E intakes are well below this amount (3), which reemphasizes the importance of enriching low-fat foods with vitamin E as a means of increasing vitamin E intakes because the long-term consequences of suboptimal vitamin E intakes are unknown.
Obesity and various chronic diseases are associated with increased oxidative stress (26). Vitamin E intakes are low in most Americans despite the obvious overconsumption of calories. We showed that it is possible to fortify low-fat fruit (apples) with vitamin E and that the vitamin E from this fruit is bioavailable; we also showed that a serving of vitamin E–fortified apples meets the calculated tissue vitamin E requirements. The present study emphasizes our lack of information about vitamin E requirements in humans—even the fractional absorption of vitamin E is not known with certainty. Appropriate studies that estimate bioavailability, such as those by Levine et al (19) for vitamin C, cannot be carried out because no injectable forms of vitamin E are available. Moreover, the present study showed that vitamin E bioavailability is greatly influenced by both the food matrix and the presence of dietary fat. Additional studies are needed to determine the amount of dietary fat necessary for optimal vitamin E absorption, because extrapolation of our data suggest that vitamin E absorption would continue to rise with the additional ingestion of dietary fat, a finding that seems highly unlikely. Furthermore, more invasive experimental approaches will be necessary to accurately determine the rates of vitamin E delivery to tissues. Collectively, these investigations will enable a more sensitive formulation of human dietary vitamin E requirements.
ACKNOWLEDGMENTS
We thank our participants and Rajasekhar Ramakrishnan for insightful discussions. We also thank the Natural Source Vitamin E Association for providing partial support for the purchase of the LC-MS. We thank James Clark of Cognis Nutrition and Health (LaGrange, IL) for the gifts of unlabeled (d0) RRR--tocopherol, d6-RRR--tocopheryl acetate, and d0--tocopherol and Carolyn Good of General Mills for providing the d9-all rac--tocopheryl acetate, which was used as an internal standard (synthesized by Isotec Inc, Miamisburg, OH).
All authors participated in the study design. SWL, S-iP, and YZ prepared the deuterium-labeled vitamin E-fortified apples. RSB and SWL carried out the sample collection and analyses. RSB and MGT wrote the initial draft of the manuscript, and all authors contributed to the editing and review of the manuscript. None of the authors had any conflicts of interest.
REFERENCES