Incorporation of deuterated RRR- or all-rac--tocopherol in plasma and tissues of -tocopherol transfer protein–null mice

¹ From the Linus Pauling Institute, Oregon State University, Corvallis (SWL and MGT); the Department of Nutrition, the University of California, Berkeley (YT); the Gladstone Institute of Cardiovascular Disease, San Francisco (YT and RVF); the Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco (RVF); and the Department of Internal Medicine, University of California, Davis, School of Medicine, Sacramento (MGT).

² Supported by grant no. NIEHS ES 00210 from the National Institute of Environmental Health Sciences and by grants to MGT from the Dairy Council of California, Nestlé, and the Tobacco Related Disease Research Program (7RT160). Unlabeled (d₀), RRR--5,7-(CD₃)₂-tocopheryl acetate, and all-rac--5-(CD₃)-tocopheryl acetate were gifts from James Clark of Cognis Health and Nutrition, LaGrange, IL. The internal standard, 2-ambo--5,7,8-(CD₃)₃-tocopherol, was a gift from Graham Burton at the National Research Council of Canada.

³ Reprints not available. Address correspondence to MG Traber, Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331. E-mail: maret.traber{at}orst.edu.

ABSTRACT  
Background: Most vitamin E supplements contain synthetic all-rac--tocopherol [2,5,7,8-tetramethyl-2RS-(4'RS,8'RS,12-trimethyltridecyl)-6-chromanol] with 8 stereoisomers; only 1 is identical to the natural stereoisomer, RRR--tocopherol [2,5,7,8-tetramethyl-2R-(4'R,8'R,12-trimethyltridecyl)-6-chromanol]. In humans, 2R--tocopherol stereoisomers are preferentially maintained in the plasma, a function that has been attributed to hepatic -tocopherol transfer protein (-TTP), but this hypothesis has not been tested.

Objective: We sought to determine the functions of -TTP by comparing mice that express -TTP with mice that are genetically unable to express -TTP.

Design: Adult -TTP null (Ttpa^-/-; n = 5), heterozygous (Ttpa^+/-; n = 7), and wild-type (Ttpa^+/+; n = 3) mice consumed equimolar RRR--[5,7-(C²H₃)₂]-(d₆)- and all-rac--[5-(C²H₃)]-(d₃)-tocopheryl acetates (30 mg/kg diet each) for 3 mo. Subsequently, we measured labeled and unlabeled -tocopherols in plasma and 17 tissues.

Results: In all mice, plasma and tissue d₆- + d₃--tocopherols represented 80–90% of total -tocopherol. In the Ttpa^-/- mice, low total -tocopherol concentrations were found in plasma (5.4%) and most other tissues (2–20%), but liver concentrations were 39% of those of Ttpa^+/+ mice. Peripheral tissue ratios of d₆- to d₃--tocopherol were 1.1 ± 0.1 and 1.8 ± 0.2 in Ttpa^-/- and Ttpa^+/+ mice, respectively (P < 0.0001), showing that -TTP preferentially selects 2R--tocopherols for secretion into plasma. This 2:1 ratio does not support the currently defined international unit of 1.36:1 RRR--tocopherol to all-rac--tocopherol.

Conclusion: Deletion of the -TTP gene in mice results in an accumulation of dietary -tocopherol in the liver and depletion of peripheral tissue -tocopherol.

Key Words: Vitamin E • deuterium-labeled -tocopherol • -TTP knockout mice • mass spectrometry • vitamin E requirement • liver • brain

INTRODUCTION  
Most vitamin E supplements contain synthetic -tocopherol: all-rac--tocopherol, or [2,5,7,8-tetramethyl-2RS-(4'RS,8'RS, 12-trimethyltridecyl)-6-chromanol]. Unlike other vitamins, synthetic -tocopherol is not identical to the only stereoisomer that is found in nature, RRR--tocopherol [2,5,7,8-tetramethyl-2R-(4'R,8'R,12-trimethyltridecyl)-6-chromanol]. The chemical synthesis of -tocopherol results in 8 different stereoisomers arising from the 3 chiral carbon-centers in -tocopherol at positions 2, 4', and 8' (1). In contrast with the carbons at the 4'- and 8'-positions in the phytyl tail, the chiral center at the 2-position (where the tail and rings meet) was shown to be the only critical chiral carbon center for biological activity (2). When RRR- and all- rac--tocopheryl acetates labeled with different amounts of deuterium were administered to humans, both plasma and tissues reflected 2:1 ratios of RRR:all-rac--tocopherol (3). These ratios reflect differences in potency that are thought to arise from differences in the affinity of the hepatic -tocopherol transfer protein (-TTP) for the various stereoisomeric forms (4).

In humans, during intestinal absorption and chylomicron secretion, all vitamin E forms are absorbed and secreted in chylomicrons (5–7). Although some dietary vitamin E is delivered to the tissues during chylomicron catabolism (8), most appears to be delivered to the liver, where RRR--tocopherol, in contrast with SRR--tocopherol or RRR--tocopherol, is preferentially secreted in VLDLs (9, 10).

Importantly, humans with defects in the -TTP gene (known as ataxia with vitamin E deficiency, or AVED) (11) are unable to secrete -tocopherol in VLDL (7, 12) and some are unable to show a preference for RRR- compared with SRR--tocopherols (7). Although gene defects have been described in humans with AVED, the functions of -TTP are still not fully understood because it is not known whether persons with AVED express defective TTP or a rapidly degraded abnormal protein. Moreover, vitamin E regulatory mechanisms in peripheral tissues are not known. Delivery of -tocopherol to tissues is largely dependent on mechanisms that deliver lipids to tissues (13–15), but these mechanisms are not specific to -tocopherol. However, a tissue -tocopherol binding protein was described (16, 17). This -tocopherol associated protein is expressed in many human tissues and it was suggested that it may play a role in tissue -tocopherol regulation (17). Thus, -tocopherol associated protein might facilitate tissue -tocopherol retention.

Previously, RRR- and SRR--tocopherols labeled with different amounts of deuterium were used in studies of AVED subjects (7). In the present study, however, deuterium-labeled SRR--tocopherol was not available; therefore, deuterium-labeled all-rac--tocopherol was used. The advantage of using this material is that this is the form of vitamin E commonly used in supplements and for food fortification. Thus, the metabolic fate of all-rac--tocopherol is highly relevant in human nutrition. Specifically, the currently used conversion factor for the definition of international units is 1.36 mg all-rac--tocopherol per 1.0 mg RRR--tocopherol. This was developed primarily on the basis of the rat fetal resorption assay (18) and does not take -TTP function into account. A factor of 2, not 1.36, was used in the latest definition of human vitamin E requirements by the Food and Nutrition Board (19). However, this factor remains controversial and has not been formally accepted by the US Pharmacopoeia.

We developed a genetic model of -TTP deficiency by disrupting the mouse -TTP gene (Ttpa) by the use of homologous recombination in embryonic stem cells (20). We used 3 groups of mice, namely -TTP-null mice, heterozygotes (expressing one-half the normal amount of -TTP), and normal -TTP expressers, to test the hypothesis that -TTP maintains plasma -tocopherol concentrations and preferentially selects 2R--tocopherol for secretion into plasma and delivery to tissues. A survey of 17 tissues was undertaken to evaluate whether specific tissues can independently regulate and thus accumulate -tocopherol despite hepatic -TTP deficiency.

MATERIALS AND METHODS  
Reagents
HPLC-grade solvents were obtained from Fisher Scientific (Fair Lawn, NJ). Ascorbic acid, potassium hydroxide, sodium dodecyl sulfate, and butylated hydroxytoluene were obtained from Sigma-Aldrich (St Louis).

Deuterated tocopherols and diet
Unlabeled (d₀), RRR--5,7-(CD₃)₂-tocopheryl acetate (d₆-RRR-), and all-rac--5-(CD₃)-tocopheryl acetate (d₃-all-rac-) were from James Clark of Cognis Health and Nutrition (LaGrange, IL). The internal standard, 2-ambo--5,7,8-(CD₃)₃-tocopherol (d₉-ambo--tocopherol), was from Graham Burton at the National Research Council of Canada. The d₆-RRR- and d₃-all-rac--tocopheryl acetates were weighed and dissolved in tocopherol-stripped corn oil (USB Corporation, Cleveland) such that the final concentration of each labeled -tocopheryl acetate in the diet was 30 mg/kg. The deuterated, tocopherol-enriched oil was then used in the manufacture of an otherwise vitamin E–deficient semipurified diet (Table 1) (TD99044; Harlan Teklad, Madison, WI). The diet was pelleted, vacuum-sealed in 1-kg quantities in plastic bags, sterilized by irradiation, and kept frozen at -20° C until used.

View this table:
TABLE 1 . Ingredients in the vitamin E–deficient diet to which deuterated tocopherols (30 mg d₆-RRR- and 30 mg d₃-all- rac--tocopheryl acetates/kg diet) were added¹  
Mice: genetics, diet, and tissue collection
This study followed the protocols approved by the Animal Care and Use Committee at the University of California, Berkeley, and the Committee on Animal Research at the University of California, San Francisco. Null (Ttpa^-/-) mice were generated as described previously (20). Ttpa^+/+ (wild-type, or control), Ttpa^+/- (heterozygous), and Ttpa^-/- female mice (strain 50% C57BL/6, 50% 129) (n = 3, 7, and 5, respectively) were used. All of the mice used for this study were littermates; thus, they had the same parents and genetic background.

All groups were weaned at 3 wk, fed a nonpurified stock diet (Picolab Mouse Diet 20; Purina Mills, Inc, St Louis) for 12 mo, and then fed the diet containing deuterated tocopheryl acetates for 3 mo. On the day the mice were killed, food was removed from the cages 4 h before the mice were anesthetized with methoxyflurane and killed. Tissues were rinsed with phosphate-buffered saline, blotted, then immediately frozen in liquid nitrogen and stored at -80°C until they were shipped to the Linus Pauling Institute for analysis. Blood was obtained by eye bleed before sacrifice on the same day that the tissue samples were collected. Blood was collected in tubes containing 10 µL 0.5 mol EDTA/L.

Plasma and tissue vitamin analysis
Tissue samples were saponified by using alcoholic potassium hydroxide and were extracted with hexane, as described previously (21). Plasma samples were extracted without saponification according to the method discussed by Burton and Daroszewska (22). A known amount of the internal standard, d₉-ambo--tocopherol, was dissolved in hexane and added to the samples. After hexane evaporation, the fractions were resuspended in 200 µL methanol:ethanol (1:1). All samples were stored at -20°C overnight before analysis with liquid chromatography– tandem mass spectrometry, as described previously (23). All samples were analyzed with an API III+ triple-quadrupole mass spectrometer (Perkin Elmer-Sciex Instruments, Thornhill, Canada) with the atmospheric pressure chemical ionization source in the positive mode. Concentrations of d₀-, d₃-, and d₆--tocopherol were calculated from the peak areas of the corresponding parent ions in the mass spectrum relative to that of the d₉--tocopherol internal standard, after corrections for the contribution of natural abundance isotopes (22).

Statistical analysis
The results are expressed as means ± SDs. The significance of differences in d₆:d₃ between genotypes was calculated with a one-way analysis of variance (STATVIEW, version 4; SAS Institute, Cary, NC). Because of the unequal group sizes, Scheffe's procedure was used for post hoc comparison of means. Differences were considered to be significant at P < 0.05.

RESULTS  
Plasma -tocopherol
After the mice were fed a diet containing only deuterium-labeled -tocopheryl acetates for 3 mo, deuterium-labeled -tocopherols represented >85% of the plasma -tocopherol in all groups (Figure 1). Plasma total (labeled plus unlabeled) -tocopherol concentrations in Ttpa^-/-mice were low and represented only 5.4% and 7.7% of the plasma concentrations in Ttpa^+/+and Ttpa^+/- mice, respectively.

View larger version (23K):
FIGURE 1. . Mean (±SD) concentrations of plasma, liver, and brain unlabeled (d₀; ), d₃- (), and d₆- () -tocopherols in -tocopherol transfer protein wild-type (Ttpa^+/+; n = 3), heterozygous (Ttpa^+/-; n = 7), and null (Ttpa^-/-; n = 5) mice fed equimolar d₆-RRR- and d₃-all-rac--tocopheryl acetates (30 mg/kg diet) for 3 mo.

 
Tissue -tocopherol
Unlike the low total -tocopherol concentrations measured in plasma, the liver concentrations in Ttpa^-/- mice were 39% of those in Ttpa^+/+ mice (Figure 2). Notably, most of the liver -tocopherol in Ttpa^-/- mice was deuterium-labeled (Figure 1). Because vitamin E is excreted in bile (24), it is not surprising that the gallbladder in Ttpa^-/- mice contained 42% of the total -tocopherol found in Ttpa^+/+ mice (Figure 2).

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FIGURE 2. . Mean total () and d₆- () RRR--tocopherol concentrations in Ttpa^-/- mice (n = 5) as a percentage of values in Ttpa^+/+ mice (n = 3).

 
Unlike the liver, most of the other tissues in Ttpa^-/- mice contained low -tocopherol concentrations compared with concentrations in the other genotype groups (Table 2). Total -tocopherol concentrations in most Ttpa^-/- mouse tissues were 2–20% of those in Ttpa^+/+ mice (Figure 2). Especially striking were the low brain -tocopherol concentrations in Ttpa^-/- mice (Figure 1). In contrast, adipose tissue and adrenal -tocopherol concentrations in the Ttpa^-/- mice were 5–40 times greater than concentrations in most of the other Ttpa^-/- tissues measured (Table 2).

View this table:
TABLE 2 . Unlabeled (d₀) and labeled (d₃ and d₆) -tocopherol concentrations in tissues of -tocopherol transfer protein (-TTP) wild-type (Ttpa^+/+; n = 3), heterozygous (Ttpa^+/-; n = 7), and null (Ttpa^-/-; n = 5) mice fed 1:1 d₆-RRR- and d₃-all-rac--tocopheryl acetates for 3 mo¹  
Ratios of d₆- to d₃--tocopherol after the study diet
After the mice ate the diet containing d₆-RRR- and d₃-all-rac--tocopheryl acetates (1:1), the mean ratios of d₆- to d₃--tocopherol in plasma and 16 tissues (excluding the liver) from Ttpa^+/+, Ttpa^+/-, and Ttpa^-/- mice were 1.8 ± 0.2, 1.9 ± 0.2, and 1.1 ± 0.1, respectively (P < 0.0001 for Ttpa^-/- compared with Ttpa^+/+ or Ttpa^+/-). In contrast, the ratio was 1 in the liver of all mice, suggesting that the liver contained substantial amounts of diet-derived tocopherols. The ratios of d₆- to d₃--tocopherols in the gallbladder were 2.2 ± 0.2 for Ttpa^+/+ and 2.3 ± 0.5 for Ttpa^+/- mice. These ratios may have been >2 because of increased metabolism of the 2S forms to the vitamin E metabolite, carboxyethyl-hydroxychroman (25).

DISCUSSION  
Disruption of the -TTP gene in mice results in extremely low plasma -tocopherol concentrations (Figure 1) (20, 26). Although studies in humans with defects in the -TTP gene suggested that -TTP is needed for maintenance of plasma -tocopherol concentrations (11), only limited data are available on human tissue -tocopherol concentrations (27). The data presented here show that virtually all of the tissues examined from Ttpa^-/- mice contained little -tocopherol (Table 2). Clearly, -TTP is needed to maintain not only plasma -tocopherol but also tissue -tocopherol.

In the diet fed to the mice, the ratio of d₆-RRR- to d₃-all-rac--tocopheryl acetate was 1. However, the ratio of d₆- to d₃--tocopherol was nearly 2 in most of the Ttpa^+/+ mouse tissues but only 1 in the Ttpa^-/- tissues. It should be emphasized that the Ttpa^+/- mice, which express half the normal amount of hepatic -TTP (20), had lower tissue -tocopherol concentrations than did the Ttpa^+/+ mice, but nonetheless had ratios of d₆- to d₃--tocopherol of nearly 2. These data suggest that plasma -tocopherol concentrations are highly dependent on the function of -TTP and that this protein preferentially selects only the 2R--tocopherol forms of all-rac--tocopherol for secretion into plasma. In mice expressing -TTP, tissue enrichment with -tocopherol derived from RRR- compared with all-rac--tocopherol appears to be a result of nonspecific uptake of -tocopherol from the plasma, which contained a 2:1 ratio of d₆ to d₃ -tocopherol. These ratios are also consistent with findings in healthy human subjects (3, 28, 29).

No tissues in Ttpa^-/- mice were enriched with d₆-RRR--tocopherol. This is surprising because -tocopherol associated protein was suggested to preferentially bind -tocopherol, be expressed in many tissues, and be involved in intercellular vitamin E transport (17).

Human and rat brains were reported to express -TTP (30, 31). The brains of Ttpa^-/- mice were particularly susceptible to vitamin E depletion; <2% of the total -tocopherol found in Ttpa^+/+ mouse brains was detected in Ttpa^-/- mice. -TTP may function in the brain to retain -tocopherol, because brains from Ttpa^+/+ and Ttpa^+/- mice contained relatively large quantities of unlabeled -tocopherol (Figure 1). Interestingly, the 2 tissues known to express -TTP have very different -tocopherol concentrations: liver -TTP apparently exports -tocopherol, whereas brain -TTP appears to prevent -tocopherol export.

Hepatic total -tocopherol concentrations in Ttpa^-/- mice were lower than those of Ttpa^+/+ and Ttpa^+/- mice. Although these data suggest that -TTP prevents catabolism of liver -tocopherol, it is also possible that the efflux of -tocopherol from the tissues to the liver accounts for a portion of the liver -tocopherol observed in the Ttpa^+/+ and Ttpa^+/- mice. Tissue -tocopherol concentrations were examined closely to evaluate whether any tissues had mechanisms for -tocopherol enrichment. However, caution must be used in evaluating the tissue data because only one time point was examined. Tissue -tocopherol concentrations are dependent on rates of delivery, uptake, and release of -tocopherol; tissue lipid concentrations; and other, as yet unidentified, factors. Moreover, it is known that various tissues have different -tocopherol turnover rates (32, 33).

Additionally, tissue -tocopherol delivery resulting from chylomicron catabolism is not dependent on -TTP. Thus, delivery of -tocopherol to adipose tissue and muscle may be similar, but the release from adipose tissue may be slower because of its high fat content. Some Ttpa^-/- mouse tissues, such as adipose tissue and adrenal glands, had relatively high total -tocopherol contents, but these concentrations appeared to result from relatively slow -tocopherol efflux on the basis of their high unlabeled -tocopherol contents and the known lipid contents of these organs. Indeed, it was suggested that adipose tissue -tocopherol is unavailable for export in guinea pigs (34), whereas in dogs fed vitamin E–deficient diets, -tocopherol efflux from adipose tissue took 120 d to reach one-half of the initial concentrations (35). In humans, adipose tissue -tocopherol concentrations were estimated to require >2 y to reach a new steady state (36). Clearly, tissue -tocopherol regulatory mechanisms remain an enigma and warrant further investigation.

In conclusion, RRR--tocopherol is twice as bioavailable as is all-rac--tocopherol, even in rodents, as a result of the function of hepatic -TTP. This study shows that -TTP deficiency leads to low plasma and tissue -tocopherol concentrations. Moreover, this study also highlights the dependence of peripheral tissues on the delivery of liver-derived lipoproteins that contain 2R--tocopherol as a result of -TTP function.

ACKNOWLEDGMENTS  
We are grateful to Alan W Taylor and Bryan Arbogast (Department of Chemistry, OSU) for technical support.

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