Human plasma and tissue a-tocopherol concentrations in
response to supplementation with deuterated natural and synthetic
vitamin E1–5
Graham W Burton, Maret G Traber, Robert V Acuff, David N Walters, Herbert Kayden, Lise Hughes, and Keith U Ingold
KEY WORDS
Vitamin E, a-tocopherol, g-tocopherol,
deuterated tocopherols, tissue concentrations of vitamin E, tocopherol supplementation, a-tocopherol isomers, adults, humans
INTRODUCTION
a-Tocopherol, vitamin E, is available commercially for use as
a dietary supplement in both its natural, single stereoisomeric
form (RRR, formerly d) and in a synthetic form (all-rac, formerly dl). The latter form consists of an approximately equimolar mixture of eight stereoisomers. Both forms of a-tocopherol
are usually sold either as acetate esters (a-tocopheryl acetate;
TAc) or, less frequently, as succinate esters. According to a spe-
cific protocol, the acetate ester of the single, natural stereoisomer RRR-a-TAc is 1.36 times more biologically potent than allrac-a-TAc in rats (1–6). This relative potency factor of 1.36 is
officially accepted (7) despite the fact that application of the
same specific protocol to measure the relative potencies of RRRa-tocopherol and RRR-a-TAc has been shown to yield results
that are irrelevant to both humans and rats under normal dietary
conditions (8).
The higher biological activity of natural compared with synthetic vitamin E does not result from differences in intrinsic
antioxidant activity. Burton and Ingold (9, 10) showed that the
chromanol structure of vitamin E (Figure 1) controls inherent
reactivity toward peroxyl radicals in vitro. Because the chromanol structure is identical in both natural and synthetic a-tocopherol, differences in the phytyl tail must determine the differences between these forms in vivo. The phytyl tail encompasses
three chiral centers, which give rise to the eight stereoisomers of
synthetic vitamin E (Figure 1). Animal assays provide evidence
that the chiral center at the 2-position, the point at which the
phytyl tail is bound to the chroman ring, is the major and possibly sole determinant of the biological differences between atocopherol stereoisomers. The lack of importance of the 49 and
89 chiral carbons is illustrated by the similar biopotencies of allrac-a-tocopherol (RRR + RRS + RSR + RSS + SRR + SRS + SSR
+ SSS) compared with 2-ambo-a-tocopherol (RRR + SRR) and of
1
From the Steacie Institute for Molecular Sciences, National Research
Council of Canada, Ottawa; the Department of Molecular and Cell Biology,
University of California, Berkeley; the Eastman Center for Nutrition
Research, East Tennessee State University (ETSU), Johnson City; Veterans’
Medical Center Hospital, Surgical Service and Department of Surgery,
ETSU; and the Department of Medicine, New York University Medical Center.
2
Issued as NRC number 39121.
3
d3-RRR-a-tocopheryl acetate and d6-all-rac-a-tocopheryl acetate were
gifts from the Natural Source Vitamin E Association.
4
Supported by the Natural Source Vitamin E Association, the National
Foundation for Cancer Research, and the Association for International Cancer Research.
5
Address reprint requests to GW Burton, National Research Council of
Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6 Canada. E-mail: gburton@ned1.sims.nrc.ca.
Received October 16, 1996.
Accepted for publication November 5, 1997.
Am J Clin Nutr 1998;67:669–84. Printed in USA. © 1998 American Society for Clinical Nutrition
669
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ABSTRACT
We report a comparison of natural and synthetic vitamin E in humans using deuterium labeling to permit the
two forms of vitamin E to be measured independently in plasma
and tissues of each subject. Differences in natural and synthetic
vitamin E concentrations were measured directly under equal
dosage conditions using an equimolar mixture of deuterated
RRR-a-tocopheryl acetate and all-rac-a-tocopheryl acetate. Two
groups of five adults took 30 mg of the mixture as a single dose
and as eight consecutive daily doses, respectively. After a 1-mo
interval the schedule was repeated but with a 10-fold higher dose
(ie, 300 mg). In each case, the ratio of plasma d3-RRR-a-tocopherol to d6-all-rac-a-tocopherol (RRR:rac) increased from
<1.5–1.8 to <2 after dosing ended. In an elective surgery study
in which 22 patients were given 150 mg/d for up to 41 d before
surgery, the RRR:rac in tissues was lower than in plasma and the
percentage of deuterated a-tocopherol was lower in all tissues
except gallbladder and liver. In a terminally ill patient given 30
mg/d for 361 d, plasma and tissue (x– ± SD) RRR-rac ratios (and
% deuterated a-tocopherol) at autopsy were 2.06 (6.3%) and
1.71 ± 0.24 (5.9 ± 2.2%), respectively. In a second terminally ill
patient given 300 mg/d for 615 d, the corresponding values were
2.11 (68%) and 2.01 ± 0.17 (65 ± 10%), respectively. The results
indicated that natural vitamin E has roughly twice the availability of synthetic vitamin E. This 2:1 ratio is significantly higher
than the currently accepted RRR:rac of 1.36:1.00. g-Tocopherol,
expressed as a fraction of total unlabeled tocopherols in 15 elective surgery patients, was 1.4–4.6 (mean: 2.6) times greater in
adipose tissue, muscle, skin, and vein than in plasma, which is
a substantially larger fraction than had been recognized previously.
Am J Clin Nutr 1998;67:669–84.
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BURTON ET AL
2R,49-ambo-89-ambo-a-tocopherol (RRR + RRS + RSR + RSS)
compared with RRR-a-tocopherol. Furthermore, significantly
higher biopotencies were found for RRR-a-tocopherol than for
2-ambo-a-tocopherol (1–3, 6). Similarly, the vitamin E activity
of the acetates of three 2RS-n-alkyl-2,5,7,8-tetramethyl-6hydroxychroman analogues of a-tocopherol have been measured
and compared directly with all-rac-a-TAc, or indirectly with
RRR-a-TAc, using the rat curative myopathy, plasma pyruvate
kinase assay (11). The analogues with alkyl chain lengths of 11
and 13 carbons have activities that do not differ significantly
from each other or from all-rac-a-TAc. Thus, methyl branching
in the phytyl tail at the 49, 89, and 129 positions has little influence on vitamin E activity.
To study how a-tocopherol stereochemistry affects plasma and
tissue concentrations, we synthesized deuterated RRR-a-tocopherols containing three or six deuterium atoms per molecule
(12–14) and 2-ambo- and all-rac-a-tocopherol with three, six, or
nine deuterium atoms per molecule (14). The deuterium atoms
are located specifically in one or more of the three, nonlabile aromatic methyl positions: 5-CD3-a-tocopherol (d3-a-tocopherol);
5,7-(CD3)2-a-tocopherol (d6-a-tocopherol); and 5,7,8-(CD3)3-atocopherol (d9-a-tocopherol) (Figure 1). The nonradioactive
nature of deuterium and its nonlabile location in the molecule
make deuterated vitamin E especially suitable for studies in
humans (15). A particularly useful feature of the deuterated vitamin E method is that a-tocopherols substituted with deuterium to
discretely different extents (eg, d3 and d6) can easily be distinguished by mass spectrometry, which allows the relative concentrations of two or more forms of vitamin E to be evaluated simultaneously. This competitive technique, using two (or more) forms
of vitamin E imbibed simultaneously, eliminates the statistical
variability that individual animals (and humans) exhibit when
they are dosed separately with different forms of vitamin E.
We previously measured vitamin E concentrations in human
plasma and in plasma and tissues of experimental animals using
gas chromatography-mass spectrometry (GC-MS) after administration of deuterated vitamin E (8, 12, 16–25). Plasma concen-
trations of stereoisomeric forms of vitamin E are apparently
dependent on the ability of the hepatic a-tocopherol transfer protein to select RRR-a-tocopherol preferentially for secretion in
nascent VLDL (12, 17–21). Catabolism of VLDL in plasma
results in the enrichment with RRR-a-tocopherol of other circulating lipoproteins and hence, eventually, the tissues. The importance of the hepatic a-tocopherol transfer protein (17–21) is further indicated by recent studies of humans with neurologic
defects (both isolated patients and a large kindred in Tunisia)
that showed that a genetic defect in this protein leads to a severe
vitamin E deficiency (26). The rat a-tocopherol transfer protein
has been purified and characterized (27) and the human form has
been cloned and its chromosomal localization reported (28).
The available experimental data (see above) suggest that the
greater biological activity of natural relative to synthetic a-tocopherol is due to the preferential enrichment of VLDL with RRRa-tocopherol and subsequently of other lipoproteins, with ultimate delivery of RRR-a-tocopherol to the tissues by these
lipoproteins. To investigate these ideas further we measured
plasma a-tocopherol (unlabeled and labeled) in healthy human
volunteers after consumption of deuterated natural and synthetic
forms of a-TAc, in the tissues from patients undergoing elective
surgery, and in two terminally ill patients who daily consumed
deuterated a-TAcs for 1 and 2 y, respectively, until death.
SUBJECTS AND METHODS
Deuterated tocopherols
RRR-a-5-(CD3)tocopheryl acetate (d3-RRR-a-TAc) and all-raca-5,7-(CD3)2tocopheryl acetate (d6-all-rac-a-TAc) were synthesized by Eastman Kodak, Rochester, NY. The two compounds
were determined by GC analysis to be 96% pure RRR- and 93%
pure all-rac-a-TAc, respectively. The isotopic purities at the nominal level of deuteration were 84% (d0: 4.0%; d1: 2.0%; and d2:
9.7%) and 86% (d0, d1: < 0.1%; d2: 0.1%; d3: 0.8%; d4: 1.3%; and
d5: 11.2%) for d3-RRR-a-TAc and d6-all-rac-a-TAc, respectively.
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FIGURE 1. The different forms of vitamin E. T, tocopherol.
NATURAL AND SYNTHETIC VITAMIN E IN HUMAN TISSUES
d3-RRR-aTAc and d6-all-rac-a-TAc were encapsulated in no.
9 “softgel” gelatin capsules as nominal 1:1 mixtures in 30-mg
(ie, 15 mg RRR-a-TAc and 15 mg all-rac-a-TAc) and 150-mg
(ie, 75 mg RRR-a-TAc and 75 mg all-rac-a-TAc) quantities
diluted with a-tocopherol-stripped corn oil. The actual ratio of
d3-RRR-a-tocopherol to d6-all-rac-a-tocopherol (RRR:rac) was
determined to be 0.98 by GC-MS.
The internal standard used for a-tocopherol analysis, 2-amboa-5,7,8-(CD3)3 tocopherol (d9-ambo-a-tocopherol), was synthesized previously at the National Research Council (NRC) of
Canada as were RRR-a-5,7-(CD3)2tocopheryl acetate (d6-RRR-aTAc) and all-rac-a-5-(CD3)tocopheryl acetate (d3-all-rac-aTAc) (8, 12–14). A deuterated g-tocopherol, d17-g-tocopherol,
synthesized earlier (14) was used as described previously (20) as
an internal standard for GC-MS determination of g-tocopherol in
some tissues and blood obtained from subjects in the elective
surgery study.
Kinetics of RRR-a-tocopherols compared with all-rac-atocopherols in human plasma
Study of elective surgery patients
This study was carried out at the Eastman Center for Nutrition
Research (ECNR), College of Medicine, East Tennessee State
University (ETSU), Johnson City, TN, and the Surgical Service,
Veterans’ Administration Medical Center Hospital, Johnson City,
TN, after approval of the Institutional Review Board (ETSU) and
the Research and Development Committee, Veterans’ Administration Medical Center Hospital. Written, informed consent was
obtained from the selected elective surgery patients. Patients with
biliary or lymphatic disease, hyperlipidemia, lipid malabsorption,
jaundice, or chronic infection were excluded from the study, as
were those who had received chemotherapy or radiation therapy.
Patient characteristics are shown in Table 1. Each of the
patients consumed one 150-mg capsule of the 1:1 mixture of
deuterated a-TAc daily with breakfast until the day before
surgery. Patients fasted 12–24 h before surgery. Subjects consumed the deuterated vitamin E for various lengths of time
before surgery (Table 1). Blood samples obtained from fasted
subjects at entry into the study and at various times before and
after surgery were collected, separated into plasma and red cell
fractions, and stored at 270 °C essentially as described earlier
(18). Tissue specimens obtained during surgery for diagnostic
purposes were placed in cold, phosphate-buffered saline and
transported to Laboratory Service for evaluation by a surgical
pathologist. Excess remaining tissue was transported to the
ECNR, where it was blotted dry, weighed, identified, and stored
at 270 °C in cryotubes until shipped with blood fractions on dry
ice to the NRC, Ottawa, for analysis. Some of these samples
were also analyzed for g-tocopherol.
Study of terminally ill patients
Two terminally ill patients, recruited specifically for the longterm study of natural compared with synthetic vitamin E uptake
into tissue approved by the Institutional Review Board of the
ETSU, gave informed, written consent for their participation. The
two patients were each provided a daily dose of the 1:1 mixture
of d3-RRR-a-TAc and d6-all-rac-a-TAc with breakfast (30 mg for
one patient and 300 mg for the other). After pronouncement of
death by a physician, the body of each patient was removed to the
morgue for storage at <5 °C. Each autopsy, carried out 10–14 °C
1–3 h after death, took 1–2 h to complete. Tissue specimens were
placed in cold, phosphate-buffered saline and transported to the
ECNR, where the tissue was blotted dry, weighed, identified, and
stored at 270 °C in cryotubes until shipped with blood fractions
on dry ice to the NRC, Ottawa, for analysis.
Extraction of tissues and fluids
Plasma tocopherols were extracted and analyzed essentially as
described previously (8, 12, 29–31). Bile and all tissues except
adipose and skin were extracted by using the sodium dodecyl
sulfate method (12, 29). Adipose tissue and skin were saponified
in alcoholic potassium hydroxide with 1% ascorbic acid before
extraction (32). The extracted tocopherol fraction was isolated
from lipid extracts by injecting the concentrated extract containing the deuterated internal standards into a Varian (Varian Associates, Palo Alto, CA) model 5000 HPLC equipped with a Varian
model 9090 autosampler, a Varian Fluorichrom fluorescence
detector (lex 220 nm, lem 350 nm), a Foxy series 2130–00 series
fraction collector (ISCO, Inc, Lincoln, NE), and a 5-mm
LiChrosorb Si 60 column (Merck, Darmstadt, Germany). The
sample was eluted isocratically at 20 °C with 90% heptane and
10% methyl t-butyl ether at a flow rate of 2 mL/min. The tocopherol fraction (a and g) eluting at 1.8–3.0 min was collected
and stored at 220 °C until required for GC-MS analysis.
Measurement of tocopherols
a-Tocopherol samples were analyzed by GC-MS, mostly as
their silyl ethers. The a-tocopherol fraction obtained after HPLC
purification was taken to dryness under a stream of nitrogen gas,
redissolved in silylation-grade pyridine (100 mL) and
bis(trimethylsilyl)trifluoroacetamide (50 mL) with 1%
trimethylchlorosilane and heated at 65 °C in a closed vial for 15
min. bis(Trimethylsilyl)trifluoroacetamide and trimethylchlorosilane were obtained from Pierce, Rockford, IL. Adipose tissue
samples and other samples containing large amounts of unlabeled
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Studies were carried out at the NRC with two groups of five
adult volunteers aged 20–59 y after informed consent had been
obtained. The studies were designed to gather information on the
effect of taking single and multiple doses in amounts that
approximate those most commonly encountered by the general
public who take supplements in the form of multivitamin pills
and vitamin E capsules. One group consumed, with their evening
meal, a single 30-mg dose of the encapsulated 1:1 mixture of d 3RRR-a-TAc and d6-all-rac-a-TAc. One month later this group
consumed a 10-fold higher dose (300 mg) provided in the form
of two capsules, each containing 150 mg of the same mixture. A
second group received this mixture in eight consecutive daily
doses of 30 mg, followed > 1 mo later by eight consecutive daily
doses of 300 mg. A third group of five subjects consumed a single 100-mg dose of deuterated vitamin E in which the labeling of
the two forms of vitamin E was reversed, ie, 50 mg d 6-RRR- and
50 mg d3-all-rac-a-TAc.
Blood samples from nonfasting subjects were drawn in the
morning into Na2EDTA-coated evacuated tubes at the Ottawa
branch of the Canadian Red Cross Society and immediately placed
on ice for transport to and extraction at the NRC laboratory in
Ottawa. During vitamin E dosing, blood was sampled daily, except
during weekends, 12–14 h after each dose. Some samples of
plasma obtained during the 8-d study with 300 mg were separated
into lipoprotein fractions and analyzed for deuterated and unlabeled a-tocopherols as described previously (17, 18, 20, 21).
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BURTON ET AL
TABLE 1
Characteristics of subjects participating in the elective surgery (groups 1–6) and terminally ill patient studies1
Group and patient
Age
Height
Weight
Plasma cholesterol
Tissues
Diagnosis
d
y
cm
kg
mmol/L
3
3
67
75
—
180
—
59
2.43
2.53
A, M, N, S
N
7
10
9
7
7
8
77
—
—
—
—
64
188
183
—
—
—
173
86
64
—
101
—
74
4.09
5.69
3.31
4.68
3.16
3.13
A, M, N, S PVD non-healing heel ulcer
A, S
Hernia
A, M
Lung cancer
M
Thyroid cancer
G,M,L
Leg contractures
A,G
Gallbladder disease
14
16
14
13
14
65
67
58
52
64
173
173
178
188
183
65
70
84
113
109
3.70
4.16
3.83
4.97
5.72
A,M
A,M
A,M,S
A,G
A
Lung cancer
Lung cancer
Hernia
Gallbladder disease
Carotid surgery for CAD
23
21
21
21
41
76
65
64
185
—
185
170
101
—
88
57
5.02
—
4.97
3.36
A,S,V
A
A,S
A
Hernia
Hernia
Hernia
Hernia
28
28
33
28
—
76
47
56
—
178
185
191
—
78
116
94
—
4.24
4.29
4.16
A
A
A,V
A
Hernia
Vascular surgery
Hernia
41
56
201
84
5.33
A
Hernia
361
644
50
69
—
—
—
—
3.91
7.14
see Table 5
see Table 5
Colon cancer
Pancreatic cancer
LLE wound infection
Above-knee amputaion
1
A, adipose tissue; CAD, coronary artery disease; G, gallbladder; L, liver; LLE, left lower extemity; M, muscle; N, nerve; PVD, peripheral vascular disease; S, skin; V, vein.
a-tocopherol (ie, d0-a-tocopherol) relative to d3-a-tocopherol
were analyzed directly as free a-tocopherols to remove the contribution to the d3 peak area of silicon isotopes present in the d0a-tocopherol silyl ether. This modification in the analysis procedure reduced the correction factor by a factor of 4, from <2.4%
to <0.6% of the d0-a-tocopherol peak area.
Derivatized and underivatized a-tocopherols were analyzed by
GC-MS with a Hewlett-Packard (Palo Alto, CA) HP 5970A Series
Mass Selective Detector linked to an HP 5890 gas chromatograph
(injection port: 300 °C; oven: 280 °C; split ratio: 30:1) equipped
with an HP 7673A autosampler and an HP Ultra 1 fused silica capillary column (12 m 3 0.2 mm internal diameter, cross-linked
methyl silicone bonded phase, column pressure 6.9 3 104 Pa)
interfaced with an HP 59970 MS Chem Station. The mass selective detector was used in the selected ion-monitoring mode. The
ions monitored were as follows: 502.4 (d0), 505.4 (d3), 508.4 (d6),
and 511.4 (d9) mass units for the silyl ethers and 430.4, 433.4,
436.4, and 439.4 mass units for the corresponding underivatized
a-tocopherols.
Concentrations of d0-a-tocopherol, d3-a-tocopherol, and d6-atocopherol were calculated from the peak areas of the corresponding parent ions in the mass spectrum relative to that of the
d9-a-tocopherol internal standard, after corrections were made
for the isotopic purities of each deuterated a-tocopherol, where
appropriate, and for the contributions of natural abundance isotopes (principally carbon and silicon). The latter correction
applies only for isotopomer pairs differing by three atomic mass
units (ie, d0 and d3, d3 and d6, and d6 and d9) and is calculated by
subtracting either 2.4% or 0.6% of the peak area of the light isotopomer from the peak area of the heavy isotopomer of the silyl
ether or the underivatized form of a-tocopherol, respectively. The
GC-MS selected ion-monitoring mode was also used for the
simultaneous analysis of a-tocopherols and d0-g-tocopherol in
some human tissue and fluid samples with d17-g-tocopherol as the
internal standard.
Statistical analyses
Data are reported in the text and in the figures as means ±
SDs. Statistical analyses were carried out with SPSS 6.1 for the
Macintosh (SPSS Inc, Chicago). Differences between concentrations of RRR-a-tocopherol and all-rac-a-tocopherol at individual time points in the human plasma kinetics study were tested
for significance by conducting paired-comparison t tests. Onesample t tests also were used to test whether plasma RRR:rac dif-
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Elective surgery
1
1
2
2
3
4
5
6
7
8
3
9
10
11
12
13
4
14
15
16
17
5
18
19
20
21
6
22
Terminally ill
1422
A6690
Duration of dosing
NATURAL AND SYNTHETIC VITAMIN E IN HUMAN TISSUES
fered significantly from the traditional value of 1.36. Tests for
significance in changes in RRR:rac, unlabeled (d0), and total
deuterated a-tocopherol, and the percentage of deuterated atocopherol over time were performed by using repeated-measures analysis of variance.
RESULTS
Kinetics of RRR-a-tocopherol compared with all-rac-atocopherol in human plasma
Changes in plasma concentrations of a-tocopherols were
studied in humans in response to supplementation with two different amounts (15 + 15 mg or 150 + 150 mg) of deuterated vitamin E (d3-RRR- and d6-all-rac-a-TAc) given once (Figure 2) or
eight times (Figure 3).
Single 30-mg dose
d3-RRR-a-tocopherol and d6-all-rac-a-tocopherol and 2% for d0a-tocopherol.
Plasma concentrations of d3-RRR-a-tocopherol were significantly greater than those of d6-all-rac-a-tocopherol at all times (P
< 0.001–0.05), with both forms of vitamin E declining steadily during the week from their maximal values at day 0 (Monday morning) of 1.5 ± 0.9 and 0.9 ± 0.5 mmol/L, respectively (Figure 2A).
Note that the statistical comparison of the two forms of vitamin E
was made by using the paired values obtained simultaneously for
each individual. This approach, made possible by the use of the two
distinctly labeled forms of vitamin E, eliminates much of the variance that arises from factors influencing individual vitamin E concentrations and, consequently, group means and SDs.
RRR:rac was significantly > 1.36 at all times (P < 0.001–0.02),
increasing from 1.63 ± 0.15 on day 0 to nearly 2 (1.96 ± 0.07) by
day 4 (P < 0.01; Figure 2C). Unlabeled a-tocopherol did not
change significantly from its initial value of 20 ± 1 mmol/L at day
0 (Figure 2B). Total a-tocopherol decreased slightly (P < 0.03),
from 22 ± 2 mmol/L on day 0 to 20 ± 1 mmol/L on day 4.
The percentage of deuterated a-tocopherol [% deuterated atocopherol = 100 3 (d3-RRR-a-tocopherol + d6-all-rac-a-tocopherol)/(d0-a-tocopherol + d3-RRR-a-tocopherol + d6-all-rac-atocopherol)] was at a maximum (11 ± 6%) on day 0, decreasing
to 4 ± 2% on day 4 (P < 0.001; Figure 2C).
FIGURE 2. Kinetics of RRR-a-tocopherol compared with all-rac-a-tocopherol in human plasma. Effect on vitamin E concentrations (days 0–4)
after a single dose of 30 mg (A-C) of a mixture of d3-RRR-a-tocopheryl acetate and d6-all-rac-a-tocopheryl acetate and after a single 300-mg dose (DF) of the same mixture given to the same group 1 mo later. Mean (± SD) plasma concentrations of d3-RRR-a-tocopherol (j) and d6-all-rac-a-tocopherol (h) are shown semilogarithmically in A and D, and d0-a-tocopherol (s) and total a-tocopherol (d) are shown in B and E. The mean percentage of deuterated a-tocopherol (D) and the ratio of d3-RRR-a-tocopherol to d6-all-rac-a-tocopherol (m; RRR:rac) are shown in C and F. T, tocopherol.
Downloaded from ajcn.nutrition.org by guest on May 29, 2014
Five subjects consumed a 30-mg dose of deuterated vitamin E
(15 mg d3-RRR-a-TAc and 15 mg d6-all-rac-a-TAc) with their
Sunday evening meal. Blood samples were obtained every morning for the next 5 d. The reproducibility and precision of the data
were estimated by analyzing four replicate samples of plasma
obtained from each of three subjects on day 2. The SD was 5% for
673
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BURTON ET AL
Single 300-mg dose
One month later the same five subjects consumed a single 300mg dose of the 1:1 deuterated vitamin E mixture with their Sunday evening meal. (This dose corresponds to 354 IU; the dose
most commonly consumed by individuals who take vitamin E
supplements is 400 IU.) Again, blood samples were taken each
morning for the next 5 d. The SDs of the a-tocopherol values
averaged over six replicate samples taken from three subjects on
day 2 were 1.6% for d3-RRR-a-tocopherol, 1.3% for d6-all-rac-atocopherol and, 1.2% for d0-a-tocopherol.
On the first morning (day 0) the plasma concentrations of d3RRR-a-tocopherol (13 ± 3 mmol/L) and d6-all-rac-a-tocopherol
(8 ± 3 mmol/L) were a substantial fraction of the total a-tocopherol (34 ± 7 mmol/L). The percentage of deuterated a-tocopherol declined from its maximal value of 55 ± 8% on day 0 to 25
± 6% on day 4 (Figure 2F; P < 0.001). Although the concentration of unlabeled a-tocopherol remained unchanged, the total atocopherol concentration in plasma decreased significantly
throughout the week because of the loss of the deuterated a-tocopherols (P < 0.001). The concentrations of d3-RRR-a-tocopherol
were significantly greater than those of d6-all-rac-a-tocopherol
on every day (P < 0.001), with RRR:rac, initially 1.57 ± 0.09 on
day 0, rising to 2.0 by the end of the week (1.97 ± 0.04 on day 4;
P < 0.001). RRR:rac values were > 1.36 at all times (P <
0.001–0.01).
Eight daily 30-mg doses
A second group of five subjects consumed a total of eight 30mg doses of deuterated vitamin E at the rate of one per day on
consecutive days, starting with their Sunday evening meal (Figure 3, A-C; day 27 to day 0). During this time the increase in
both forms of deuterated a-tocopherol (Figure 3A) was accompanied by a corresponding decrease of the unlabeled a-tocopherol (Figure 3B; P < 0.001), thereby dampening the net change
in total a-tocopherol (the trends are more sharply illustrated in
Figure 4, A-B, representing the data for one individual). At the
end of the dosing period (ie, on day 0), the concentrations of
both deuterated a-tocopherols had begun a rapid decline and, by
day 7, had declined to below the concentrations seen on day 27
(Figure 3A). The percentage of deuterated a-tocopherol
increased from 11 ± 5% on day 27 to 33 ± 6% on day 0 and
dropped back to 10 ± 2% by day 7 (Figure 3C).
The concentration of d3-RRR-a-tocopherol was always significantly greater than that of d6-all-rac-a-tocopherol (P < 0.001–0.03).
RRR:rac quickly rose from its lowest value of 1.48 ± 0.25 on day 27
to 1.9 ± 0.1 by day 0, staying at that value for the remainder of the
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FIGURE 3. Kinetics of RRR-a-tocopherol compared with all-rac-a-tocopherol in human plasma. Effect on vitamin E concentrations (day 27 to
day 11) of eight consecutive daily total doses of either 30 mg (A-C) of a mixture of deuterated a-tocopheryl acetate esters (a-TAcs) or 300 mg of the
same mixture taken by the same group > 1 mo later (D-F). A second group of five subjects (4 men, 1 woman) consumed eight consecutive daily doses
of 30 mg (A-C) and then 300 mg (D-F) d3-RRR-a-TAc + d6-all-rac-a-TAc (1:1) with an evening meal > 1 mo later on day 28. Data (x– ± SD) are from
blood samples obtained during the first 3 wk, with day 0 being the last day of dosing. Plots G-I, corresponding to plots D-F, show data for days 1-507.
Note that time is plotted on a log scale and that G is a log-log plot. Symbols are as described in Figure 2. T, tocopherol.
NATURAL AND SYNTHETIC VITAMIN E IN HUMAN TISSUES
675
study. RRR:rac exceeded 1.36 after the first day of dosing (ie, by day
26; P < 0.001–0.01). The constancy of the RRR:rac from day 1
onward indicated that the two labeled tocopherols disappeared from
plasma at the same rate after dosing ended.
Eight daily 300-mg doses
More than 1 mo after the previous study, the same group of
subjects consumed 300 mg/d of the 1:1 mixture of deuterated atocopherols for 8 d. Plasma vitamin E concentrations were followed for > 500 d (Figures 3, D-I). The percentage of deuterated
a-tocopherol reached a peak of 80 ± 5% between days 23 and 0,
which was followed by an initially rapid decline to 35 ± 8% by
day 7 (Figure 3F). However, the rate of decline thereafter slowed
substantially, with the deuterated tocopherols still comprising as
much as 2.5% of the total plasma a-tocopherol > 17 mo later (Figure 3I). The slowing of the disappearance of the deuterated tocopherols and their continued persistence are consistent with the
return of deuterated vitamin E to plasma from tissues.
At this highest dosage, the effect of the newly absorbed deuterated vitamin E on unlabeled a-tocopherol was most evident (Figure 3E), particularly on an individual basis (Figure 4E). The mean
plasma concentration of d0-a-tocopherol decreased from 17.4 ±
9.4 mmol/L on day 27 to 8.7 ± 5.3 mmol/L on day 0 (P < 0.02)
and did not return to normal until day 7. The large influx of deuterated vitamin E caused the mean total a-tocopherol to increase
from 36 ± 13 mmol/L on day 27 to 46 ± 19 mmol/L on day 23 (P
< 0.04), which was followed by a decrease to 25 ± 10 mmol/L by
day 7 (P < 0.02).
The concentration of d3-RRR-a-tocopherol was significantly
greater than that of d6-all-rac-a-tocopherol at nearly all times (P
< 0.001–0.05; P > 0.05 for days 50, 150, and 507 only). During
the dosing period RRR:rac was constant at 1.51 ± 0.11, being significantly > 1.36 from day 26 onward (P < 0.02–0.05). After supplementation ended, this ratio increased sharply to 2.01 ± 0.12 by
day 7 (Figure 3F) and stayed constant at a value of 2.00 ± 0.13 for
< 100 d (ie, up to day 106) by which time the percentage of
deuterated a-tocopherol had declined to 5% (Figure 3I).
The increase in the RRR-rac ratios immediately after dosing
ended reflects the more rapid disappearance of all-rac-a-tocopherol during this period; by day 3 the d6-all-rac-a-tocopherol
concentration had declined to the value observed on day 27,
whereas it took until day 4 before the RRR-a-tocopherol concentration had declined to its day 27 value (Figure 3D). After > 100
d RRR:rac appeared to decrease (Figure 3I), but this may have
been an experimental artifact arising from the relatively small
quantities of the deuterated a-tocopherols remaining (< 5%).
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FIGURE 4. Kinetics of RRR-a-tocopherol compared with all-rac-a-tocopherol in human plasma. Effect on vitamin E concentrations of one individual (subject NRC-1) from the group of subjects given eight daily doses of 30 mg of a mixture of deuterated a-tocopherol acetate esters (A-C) and
then 300 mg of the same mixture (D-F) > 1 mo later. Plots G-I, corresponding to plots D-F, show data for days 1-507. Note that time is plotted on a
log scale and that G is a log-log plot. Symbols are as described in Figure 2.
676
BURTON ET AL
During this study a limited opportunity arose to examine lipoprotein fractions. VLDL, LDL, and HDL were obtained on day 43 of the
8-d study with 300 mg. RRR-rac ratios and the percentage of deuterated a-tocopherol showed, for each individual, a high degree of uniformity between the lipoprotein fractions, with the RRR-rac ratios all
being close to 2.0 (Table 2). In contrast, the results obtained for
blood sampled at day 23 from an individual who was not a part of
the main study (subject NRC-11) showed a trend of increasing
enrichment of RRR-a-tocopherol compared with all-rac-a-tocopherol in the direction chylomicrons < VLDL < LDL, HDL.
Single 100-mg dose
In a third group given 100 mg of the 1:1 mixture of deuterated
vitamin E in which the deuterium labeling of the two vitamin E
forms was reversed, RRR:rac in plasma again reached a maximum value of 2:1 (data not shown). This result indicates that the
presence of the deuterium label in the molecule does not contribute in any significant way to the observed discrimination
between RRR-a-tocopherol and all-rac-a-tocopherol.
Study of elective surgery patients
TABLE 2
Plasma concentrations of unlabeled and deuterated a-tocopherols in plasma lipoproteins from subjects participating in plasma kinetic studies1
Subject and fraction
NRC-1
VLDL
LDL
HDL
NRC-2
VLDL
LDL
HDL
NRC-3
VLDL
LDL
HDL
NRC-4
VLDL
LDL
HDL
NRC-5
VLDL
LDL
HDL
NRC-11
Chylomicron
VLDL
LDL
HDL
RRR:rac2
Deuterated a-T
d0-a-T
d3-RRR-a-T
d6-all-rac-a-T
mmol/L
mmol/L
mmol/L
3.9
6.6
8.2
0.19
0.33
0.39
0.10
0.17
0.21
1.83
1.95
1.87
7.1
7.0
6.8
15.5
13.0
8.6
0.88
0.75
0.53
0.44
0.37
0.27
2.02
2.02
1.93
7.8
8.0
8.5
2.4
7.5
8.2
0.12
0.37
0.39
0.07
0.17
0.19
1.89
2.14
2.01
7.3
6.8
6.6
2.7
7.8
6.4
0.26
0.69
0.57
0.13
0.29
0.25
2.01
2.37
2.26
13
11
11
3.5
4.1
4.4
0.37
0.44
0.47
0.17
0.20
0.23
2.13
2.19
2.09
14
14
14
0.04
1.2
2.3
2.0
0.13
3.2
6.3
5.4
0.11
2.2
3.5
3.1
1.20
1.43
1.79
1.76
85
82
81
81
%
1
Subjects took 300 mg of a 1:1 mixture of d3-RRR- and d6-all-rac-a-tocopheryl acetate daily with an evening meal. Blood from subject NRC-11, a 42y-old woman who was not part of the main study, was obtained ø15 h after she took the fifth capsule (ie, on day –3). Blood from the other subjects was
obtained on day 43. T, tocopherol.
2
Ratio of d3-RRR- to d6-all-rac-a-tocopherol.
Downloaded from ajcn.nutrition.org by guest on May 29, 2014
The subjects recruited into the study were distributed into six
groups and were given 150 mg of the 1:1 mixture of deuterated
vitamin E daily for nominal periods of 3, 8, 14, 21, 29, and 41 d
before surgery, respectively. The mean plasma d 0-a-tocopherol
concentration just before dosing began; the mean d 0-a-tocopherol and total a-tocopherol concentrations, the percentage of
deuterated a-tocopherol, and RRR-rac ratios at the time of
surgery; the RRR-rac ratios over the period beginning ≥ 5 d after
surgery; and g-tocopherol measured over the duration of the
study are shown in Table 3.
The trends already noted in the NRC plasma kinetics study
were also apparent in this study, although the changes were not
significant, unless noted otherwise. For example, plasma d 0-atocopherol concentrations of patients were consistently lower at
the end of supplementation, the decrease being larger the longer
the period of dosing. This finding was found to be significant,
not by a paired t test of the individual concentrations but by a
one-sample t test of the ratio of the d 0-a-tocopherol concentrations (before:after > 1, P = 0.002).
Plasma total and the percentage of deuterated a-tocopherol
increased with the duration of dosing. RRR-rac ratios generally
increased after the dosing ended (ie, after ≥ 5 d), attaining values
close to 2.0 for all but the first group. The rise in the RRR-rac
ratio after dosing ended was highly significant, as determined by
a one-sample t test [RRR:rac (≥ 5 d after surgery)/RRR:rac (end
of dosing) > 1, P < 0.0005].
Tissues obtained during elective surgery included adipose (n =
19), muscle (n = 8), skin (n = 6), vein (n = 3), and nerve (n = 3)
(Table 1). For each tissue examined, the mean length and range of
dosing time, the corresponding mean concentrations of labeled
and unlabeled a-tocopherols and g-tocopherol (where available),
and, for comparison, the corresponding values measured for
plasma obtained at the time of surgery are given in Table 4.
NATURAL AND SYNTHETIC VITAMIN E IN HUMAN TISSUES
677
TABLE 3
Percentage deuterated a-tocopherol (T), ratio of d3-RRR- to d6-all-rac-a-T (RRR:rac), and g-T in plasma and concentrations of d0- and total-a-T and g-T
obtained from six groups of elective surgery patients provided with deuterated vitamin E daily for increasing periods of time1
Group
1 (n = 2)
2 (n = 6)
3 (n = 5)
4 (n = 4)
5 (n = 4)
6 (n = 1)
Duration
of dosing2
Deuterated
a-T
Total
a-T
d
%
mmol/L
3 ± 06
8±1
14 ± 1
21 ± 1
29 ± 2
41
35 ± 6
42 ± 21
45 ± 19
50 ± 27
50 ± 19
59
28.2 ± 11.1
30.5 ± 10.5
36.1 ± 8.1
36.7 ± 22.6
41.3 ± 17.9
63.7
d0-a-T
RRR:rac
Before dosing End of dosing At surgery ≥ 5 d after surgery3
mmol/L
20.1 ± 10.4
18.3 ± 2.2
21.6 ± 7.8
22.0 ± 8.9
27.3 ± 11.4
43.7
19.1 ± 9.0
16.5 ± 4.3
19.0 ± 5.7
15.2 ± 4.3
18.2 ± 2.0
25.9
1.56 ± 0.02
1.79 ± 0.06
1.73 ± 0.10
1.74 ± 0.02
1.83 ± 0.13
1.77
1.61 ± 0.18 [5]
1.90 ± 0.13 [9]
1.93 ± 0.06 [10]8
2.08 ± 0.16 [5]
2.02 ± 0.07 [3]
—
g-T4
g-T4,5
mmol/L
%
—
2.2 ± 1.1 [1,6]7
6.7 ± 0.6 [2,13]9
5.5 ± 1.4 [2,11]9
3.5 ± 1.3 [4,19]
2.1± 1.3 [1,3]7
—
12 ± 4 [1,6] 7
22 ± 1 [2,13]9
20 ± 1 [2,11]9
14 ± 5 [4,19]
6 ± 2 [1,3]7
1
Values were determined from plasma obtained at the end of dosing unless noted otherwise.
Number of consecutive days of dosing with 75 mg d3 RRR-and 75 mg d6-all-rac-a-tocopheryl acetate/d.
3
Number of samples in brackets. Where the number of samples exceeds the number of subjects, the group mean was calculated as the average of the
individual subject means.
4
First number in brackets is the number of subjects; the second number is the number of samples. Plasma samples were obtained during the entire study
(ie, during and after dosing).
5
% g-T = 100 3 g-T/ (g-T + d0-a-T), ie, the percentage of total unlabeled tocopherol that is g-T.
6–
x ± SD.
7
The SD was calculated from the values obtained at different times for the one subject.
8
Significantly different from value at time of surgery, P < 0.005 (paired t test).
9
The SD was replaced by a value that is half the difference between the two numbers.
2
An unexpected result shown in Table 4 and Figure 6 is the substantially higher percentage of g-tocopherol (ie, percentage of gtocopherol, defined relative to total unlabeled tocopherols; see footnote 3 in Table 4) found in a significant portion of the tissue samples
compared with the corresponding value for plasma tocopherols at
either the time of surgery or before the start of dosing. The drop in
the percentage of plasma g-tocopherol during supplementation indicated, by the very definition of the term, that supplementation with
deuterated a-tocopherol produced a proportionately larger decrease
in the plasma concentration of g-tocopherol than of d0-a-tocopherol.
Study of terminally ill patients
A proper evaluation of the relative uptake and retention of natural compared with synthetic vitamin E requires measurements in
a broad range of tissues after a relatively long period of consumption of these two forms of vitamin E. To carry out such a
study, we enlisted two terminally ill subjects (Table 1). One subject (patient 1422) took 30 mg of the 1:1 mixture daily for 361 d;
the other subject (patient A6690) took 300 mg/d for 615 d. At
death, an autopsy was performed to obtain various tissues that
were analyzed for d0-, d3-, and d6-a-tocopherol in the usual way.
The concentration of deuterated and unlabeled a-tocopherol, the
RRR-rac ratios, and the percentage of deuterated a-tocopherol in
tissues, plasma, and erythrocytes at the time of autopsy for both
patients are given in Table 5. Although only one subject was studied for each dose, some remarkable observations were made.
Patient 1422, who received 30 mg vitamin E/d for almost 1 y,
had low tissue concentrations of deuterated a-tocopherols; the
percentage of deuterated a-tocopherol was in the range 2–11%,
with a mean value of 5.9 ± 2.2%. Thus, in this patient, supplementation with 30 mg/d apparently had little effect on either
plasma or tissue total a-tocopherol concentrations. RRR:rac
averaged 1.71 ± 0.24 for all tissues sampled, with the plasma
ratio being slightly higher (2.06).
Tissue concentrations of deuterated a-tocopherols were much
higher in patient A6690, who received 300 mg vitamin E/d for
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Tissues showed a wide range in the mean concentrations of
labeled and unlabeled a-tocopherols and, thus, the percentage of
deuterated a-tocopherol, whereas the corresponding values for
plasma showed much less variation, despite the substantially different lengths of time dosing was carried out (Figure 5B). Similarly, tissues showed more variability than plasma in their mean
RRR-rac ratios (Figure 5A).
The higher variability in the percentage of deuterated a-tocopherol and RRR:rac in tissues compared with plasma is not an
experimental artifact caused by, for example, adventitious oxidation after surgical removal of tissue samples or during sample
processing and extraction. This was confirmed by our experience
in handling many thousands of animal tissue samples in the NRC
laboratory over a period of almost 10 y, during which time samples were analyzed repeatedly after successive freeze-thaw cycles
with extended periods of storage in between. Excellent reproducibility of results was obtained. In the present study numerous
tissue results were confirmed by repeating the complete analysis
and even by analyzing duplicate samples stored for safekeeping
and backup purposes at the ETSU laboratory. Whereas there is no
doubt that oxidation would eventually destroy tissue tocopherol if
tissue were exposed to conditions such as prolonged standing at
room temperature, opportunities for this were minimized in both
the study of the elective surgery patients and the study of the terminally ill patients. Furthermore, even if oxidative degradation
were to occur, it is unlikely that it would differentially affect one
or the other forms of deuterated a-tocopherol, ie, RRR:rac should
not change.
Plots of the distributions of d0-a-tocopherol, the percentage of
deuterated a-tocopherol, RRR:rac, and the percentage of g-tocopherol values determined for all tissues and plasma obtained
from all subjects at the time of surgery are shown in Figure 6.
The magnitude and enormous variability of d 0-a-tocopherol in
adipose tissues, and to a lesser extent in other tissues, contrasts
with the small range of values seen in plasma. The highest atocopherol concentrations were seen in adipose tissues.
678
TABLE 4
d3-RRR-, d6-all-rac-, d0-a-tocopherol (T), and g-T concentrations in tissues and in plasma (after dosing or at or close to the time of surgery, ie, 0, 1, or 2 d before) of elective surgery patients, together with
percentage g-T in tissues and their corresponding plasma values
d3-RRR
Tissue
Tissue
Plasma
d6-all-rac
Tissue
Plasma
Tissue
Plasma
Tissue
g-T2
Percentage
tissue g-T2,3
d
nmol/g
mmol/L
nmol/g
mmol/L
nmol/g
mmol/L
nmol/g
%
12.1 ± 11.0
2.3 ± 2.3
4.0 ± 4.0
6.5 ± 5.5
1.2 ± 0.6
13.4 ± 3.3
19.7
12.1 ± 8.7
9.5 ± 5.5
12.2 ± 11.1
12.0 ± 3.4
8.0 ± 4.6
13.5 ± 2.8
16.3
7.7 ± 7.0
1.9 ± 1.7
2.6 ± 2.5
4.7 ± 4.0
0.8 ± 0.4
7.8 ± 2.2
21.6
7.0 ± 5.0
5.6 ± 3.2
7.3 ± 6.5
7.0 ± 1.9
5.0 ± 2.6
7.6 ± 1.4
7.1
440 ± 279
155 ± 163
127± 74
55 ± 40
254 ± 187
45 ± 30
27.6
18.3 ± 5.3
18.2 ± 6.3
22.6 ± 3.6
19.2 ± 5.5
20.4 ± 9.2
16.6 ± 4.5
15.4
19 ± 10 (3–41)
10 ± 5 (3–16)
13 ± 8 (3–23)
23 ± 10 (14–33)
4 ± 2 (3–7)
9 ± 3 (7–13)
7
5
d0-a-T
1
Number of consecutive days of dosing with 75 mg d3-RRR- and 75 mg d6-all-rac-a-tocopheryl acetate/d.
Samples taken at time of surgery; number of subjects in brackets.
3
% g-T = 100 3 g-T/(g-T + d0-a-T). The mean (± SD) plasma value was 13.5 ± 4.7% (range: 5–18%).
4–
x ± SD for all samples was 19.4 ± 7.1% (range: 8–28%).
5–
x ± SD; range in parentheses.
6
The SD was replaced by a value that is half the difference between the two numbers.
2
Plasma g-T
At surgery
Before dosing4
%
176 ± 79 [10] 31 ± 14 [10] 12.9 ± 5.3 [10]
107 [1]
38 [1]
15.7 [1]
180 ± 89 [2]6 53 ± 20 [2]6 14.6 ± 1.0 [2]6
23 ± 19 [2]6 32.7 ± 0.2 [2]6 17.4 ± 0.7 [2]6
3.7 [1]
21.6 [1]
8.0 [1]
17.7 ± 7.0 [10]
27.7 [1]
26.3 ± 1.4 [2]6
21.2 ± 3.5 [2]6
10.3 [1]
BURTON ET AL
Adipose (n = 19)
Muscle (n = 8)
Skin (n = 6)
Vein (n = 3)
Nerve (n = 3)
Gallbladder (n = 3)
Liver (n = 1)
Duration of dosing1
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NATURAL AND SYNTHETIC VITAMIN E IN HUMAN TISSUES
679
A6690 contrasts with the results obtained with the elective surgery
patients, who were dosed for much shorter periods of time.
DISCUSSION
almost 2 y. The percentage of deuterated a-tocopherol was in the
range 41–77%, with a mean value of 65 ± 10%. RRR:rac averaged 2.01 ± 0.17 for all tissue samples, a value that was similar to
the ratio in plasma, 2.11. In this patient, total a-tocopherol in
plasma and many of the tissues was more than double the concentration found in patient 1422. For example, the percentage of
deuterated a-tocopherol in plasma was 6.3% in patient 1422 and
68% in patient A6690, with the corresponding d0- and total atocopherol concentrations being 16 and 19 mmol/L and 17 and 60
mmol/L, respectively. Taking the comparison between the two
patients at face value, vitamin E supplementation with 300 mg/d
(ie, 354 IU containing 150 + 75 = 225 mg 2R-a-tocopherol
stereoisomers) apparently increased plasma concentrations of atocopherol by a factor of three and at least doubled the concentrations in most tissues.
Compared with patient 1422, patient A6690 showed less variability in both the tissue RRR-rac ratios and in the percentage of deuterated a-tocopherol, which were close to the corresponding plasma
values. This implies the existence of a greater degree of equilibration
between tissues and plasma in patient A6690 due, presumably, to the
250 additional days of dosing, the larger dose, or both. The convergence of the plasma and tissue values for these two indexes in patient
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FIGURE 5. Comparison of plasma (gray bars) and tissue (black bars)
ratios of d3-RRR-a-tocopherol to d6-all-rac-a-tocopherol (RRR:rac) and
the percentage of deuterated a-tocopherol determined on the day of
surgery in the elective surgery patients. The numbers of samples are in
brackets.
This study was undertaken to evaluate the response of human
plasma and tissues to vitamin E given in amounts and forms
commonly consumed as dietary supplements. The 30-mg dose is
similar to the amount found in multiple vitamin supplements and
the 300-mg (354-IU) dose is comparable with the amount commonly found in vitamin E capsules (eg, 400 IU).
Natural and synthetic forms of a-tocopherol were compared
in plasma via competitive uptake from 1:1 mixtures of RRR-aTAc and all-rac-a-TAc. Total a-tocopherol concentrations in
plasma increased only slightly when a 30-mg dose of the two
forms of vitamin E was given once or on 8 successive days.
However, it can be inferred from the decline to baseline concentrations shown in Figures 2E and 3E that the total a-tocopherol
concentration in plasma increased by <50% and <100% when a
300-mg dose was given once and on 8 successive days, respectively. These data confirm results obtained previously with unlabeled vitamin E (33–37).
One of the most obvious advantages that accrue with the use
of deuterated vitamin E is that the newly absorbed vitamin can
readily be distinguished from the preexisting, unlabeled a-tocopherol. For example, by the first morning after administration of
the labeled vitamin E with an evening meal, a 30-mg dose produced 11% deuterated a-tocopherol in the plasma (Figure 2C)
and 33% when the same dose was given for 8 consecutive days
(day 0; Figure 3C). The first morning after a 300-mg dose there
was 55% deuterated a-tocopherol in the plasma (Figure 2F),
and this rose to 80% after dosing for 8 consecutive days (day 0;
Figure 3F).
One of our most striking findings was the speed with which
the new (labeled) vitamin E became a large fraction of the vitamin E in plasma after a single 300-mg dose. It appears that there
is a rough homeostasis of vitamin E in plasma so that when new
vitamin E is absorbed it displaces old vitamin E. As a consequence, the 300-mg dose taken for 8 consecutive days produced
a relatively small increase in total a-tocopherol in plasma (Figure 3E). Because the body cannot distinguish between deuterated
and nondeuterated a-tocopherol per se, our results show that vitamin E homeostasis was achieved by the loss of old vitamin E
from the plasma lipoproteins when there was an abundance of
new vitamin E arriving in the chylomicrons. Exactly how this
homeostasis is controlled is unknown. However, it seems likely
that it is dependent on the hepatic a-tocopherol transfer protein,
which preferentially selects RRR-a-tocopherol for secretion into
nascent VLDL (12, 17–20, 38). A genetic defect in this protein
causes low plasma a-tocopherol concentrations (39), vitamin E
deficiency (26), and an impaired ability to discriminate between
stereoisomers of a-tocopherol (21).
A second advantage conferred by the deuterated vitamin E
technique is that the two distinctly labeled RRR- and all-racforms can be compared directly and simultaneously in the same
subject, which greatly increases the statistical power of the data
by eliminating much of the variations attributable to individual
differences and factors that change with time. The benefits are
twofold: 1) the relative availabilities of RRR-a-tocopherol and
all-rac-a-tocopherol are immediately evident in each and every
subject at any time, and 2) following the time dependence of the
680
BURTON ET AL
RRR-rac ratio provides insight into the mechanisms in the body
for absorbing, distributing, and eliminating vitamin E.
The behavior of the RRR-rac ratios also supports a stereoselective mechanism for the recycling of a-tocopherol through the liver.
The initial RRR-rac ratio in plasma was <1.5 the morning after a
single dose of either 30 mg or 300 mg of the two deuterated aTAcs (Figures 2C and 2F), and it was not much different after
eight doses of 30 mg and 300 mg (Figures 3C and 3F). However,
after dosing ceased, the RRR-rac ratio increased to <2. Because
the RRR-rac ratio is smaller during dosing than afterward, it is
likely that plasma and tissues (see below) acquire some 2S-a-tocopherol stereoisomers during the dosing period. These 2S stereoisomers, which, of course, constitute 50% of all-rac-a-TAc, are preferentially eliminated after dosing ceases, presumably by a
differential filtration in an overall and continuous recycling
process involving the liver and its a-tocopherol transfer protein.
After the dosing period, the percentage of deuterated a-tocopherol in plasma declined rapidly. For example, the percentage of
deuterated a-tocopherol in plasma decreased from 80% to < 20% in
2 wk after daily dosing with 300 mg of deuterated material for 8 successive days (Figure 3F). This must reflect the replacement and dilution of circulating a-tocopherol by newly ingested unlabeled vitamin
E and by unlabeled a-tocopherol stored in the tissues. These observations are consistent with a rapid recycling of RRR-a-tocopherol
and the conclusion that the liver secretes approximately one plasma
pool of a-tocopherol into the plasma daily (23).
There are relatively few data available regarding a-tocopherol
concentrations in human tissues (32, 40–44). The present results
add substantially to knowledge in this area. For the elective
surgery patients the tissue RRR-rac ratios were, except for the
liver, generally slightly lower than plasma RRR-rac ratios (Table
4 and Figure 5A). For tissues other than the liver, the RRR-rac
ratios cluster around 1.5 (compared with 1.7 for plasma), a value
that slightly exceeds the officially accepted relative biopotencies
of these two forms of vitamin E of 1.36 (7). The single sample
of liver examined had an RRR-rac ratio of 0.91 after 7 d of dosing (compared with 1.79 for plasma; Table 4). A low (< 1.0) ratio
for liver was expected on the basis of earlier continuous feeding,
competitive studies with an equimolar mixture of d 6-RRR-a-TAc
and d3-SRR-a-TAc with the rat as the experimental animal (12).
For example, in the study by Ingold et al (12), the RRR-SRR ratio
in the liver was 0.67 after 8 d.
The percentage of deuterated a-tocopherol in the tissues
declined through the following series: liver > gallbladder > vein
> skin > muscle > adipose tissue > nerve, with the percentage in
plasma being the same as in the liver (Figure 5B). These results
are consistent with those of earlier studies on animals continuously fed deuterated RRR-a-TAc (12, 16). The times required for
Downloaded from ajcn.nutrition.org by guest on May 29, 2014
FIGURE 6. Individual concentrations of d0-a-tocopherol, ratios of d3-RRR- to d6-all-rac-a-tocopherol (RRR:rac), the percentage of deuterated atocopherol, and the percentage of g-tocopherol in the tissues of the elective surgery patients as measured on the day of surgery. Patients consumed a
capsule containing 150 mg of a 1:1 mixture of deuterated vitamin E (d3-RRR-a-tocopheryl acetate and d6-all-rac-a-tocopheryl acetate) daily up to the
day of surgery. The percentage of deuterated a-tocopherol = 100 3 (d3-RRR-a-tocopherol + d6-all-rac-a-tocopherol)/(d0-a-tocopherol+ d3-RRR-atocopherol + d6-all-rac-a-tocopherol); percentage of g-tocopherol = 100 3 g-tocopherol/(g-tocopherol + d0-a-tocopherol). The numbers of samples
analyzed for a-tocopherol and g-tocopherol, respectively, were as follows: adipose, 19 and 10; muscle, 8 and 1; skin, 6 and 2; vein, 3 and 2; nerve, 3
and 0; gallbladder, 3 and 1; liver, 1 and 0; plasma, 22 and 10.
NATURAL AND SYNTHETIC VITAMIN E IN HUMAN TISSUES
681
TABLE 5
a-Tocopherol (T) concentrations, percentage deuterated a-tocopherol, and ratio of d3-RRR- to d6-all-rac-a-T (RRR:rac) in plasma, red cells, and tissues at
autopsy of two terminally ill patients who consumed 30 mg vitamin E (15 mg d3-RRR- and 15 mg d6-all-rac-a-tocopheryl acetate) for 361 d (patient
1422) and 300 mg vitamin E (150 mg d3-RRR- and 150 mg d6-all-rac-a-tocopheryl acetate) for 615 d (patient A6690)1
Tissue and site
Patient 1422
% Deut RRR:rac d3-RRR d6-rac
1
2.06
1.52
1.69
1.63
0.71
0.15
18.4
15.8
Patient A6690
d3-RRR d6-rac
d0-a-T
Total a-T
0.35
0.10
16
3
17
3
68
69
2.11
2.14
28
6
13
3
19
4
60
12
11.0
9.87
454
340
483
355
68
71
1.85
1.70
486
1369
268
816
347
891
1100
3076
% Deut
RRR:rac
1.62
1.59
1.24
2.96
0.78
1.90
23
67
25
71
72
70
1.93
1.90
26
10
14
5
15
7
54
22
2.06
1.77
1.75
1.29
1.45
2.26
0.64
0.83
1.31
20
37
56
22
39
59
73
71
63
2.02
1.90
1.87
22
124
221
11
67
120
12
80
196
44
271
586
1.89
1.86
0.66
0.90
0.36
0.49
19
55
20
56
1.84
1.56
1.65
2.16
1.71
1.76
0.87
1.48
0.43
0.68
2.73
11.2
10.8
0.39
0.82
0.28
0.42
1.28
6.67
6.22
0.46
76
37
29
47
208
355
50
78
38
30
52
226
372
51
60
50
58
70
55
45
48
41
2.21
2.14
2.15
1.99
2.21
2.19
2.20
2.09
25
35
32
53
45
19
20
17
11
17
15
27
21
9
9
8
24
51
35
34
54
34
32
36
60
102
83
114
121
61
61
61
61
1.89
73
39
73
185
1.81
0.62
0.14
0.35
15
1
16
1
72
74
1.89
2.18
45
17
24
8
26
9
95
34
1.51
1.59
2.22
4.10
1.49
2.63
51
100
54
107
77
72
1.55
1.74
71
422
46
247
35
260
153
929
1.75
1.69
0.22
0.19
0.13
0.12
6
5
6
5
71
70
2.06
2.01
10
16
5
8
6
10
20
34
1.86
1.63
1.77
7.89
0.45
0.64
4.32
0.28
0.37
170
20
14
183
21
15
69
69
71
2.10
2.06
2.13
201
11
20
97
6
10
131
8
12
430
25
42
% Deut, percentage deuterated a-tocopherol; d6-all-rac-a-tocopherol (d6-rac).
new deuterated RRR-a-tocopherol to become equal in concentration to old nondeuterated RRR-a-tocopherol in plasma, liver,
muscle, and brain were 3.7, 3.0, 24, and 107 d, respectively, for
guinea pigs and 6.2, 6.9, 23, and 40 d, respectively, for rats (16).
Insofar as comparison is possible, therefore, the kinetics of vitamin E in human tissues (ie, the relative rates of uptake of new
vitamin E) are similar to those in two other species of mammals.
Although the American diet contains 5–10 times as much gtocopherol as a-tocopherol, there have been few measurements
of g-tocopherol in human tissues and these have been largely
confined to adipose tissue (43). The data obtained from the elective surgery study show that g-tocopherol represents <31% of
adipose tissue vitamin E (unlabeled). More remarkable is the
38% contribution of g-tocopherol to muscle vitamin E and the
53% contribution to skin vitamin E (Table 4). The contribution
of g-tocopherol to plasma vitamin E was 19 ± 7% (range:
8–28%). This percentage refers to measurements before supplementation with deuterated a-tocopherol because supplementa-
tion is known to reduce plasma g-tocopherol concentrations
(Table 4) (45, 46). At the time of surgery the mean percentage
contribution of g-tocopherol to plasma vitamin E was 14 ± 5%
(range: 5–18%; Table 4). The unexpectedly high tissue concentrations of g-tocopherol invite reexamination of the potential
importance of this previously underestimated component of vitamin E, particularly in view of the recent report that g-tocopherol reacts with the pollutant, nitrogen dioxide, in a way fundamentally different to that of a-tocopherol (47).
In marked contrast with the elective surgery results, the terminally ill patients who took the deuterated a-tocopherols for
much longer periods of time attained an RRR-rac ratio close to
2:1 in plasma and in all tissues (particularly patient A6690, who
took the 300-mg supplement daily for almost 2 y; Table 5). Note
also that the tissue concentrations of a-tocopherol in subject
A6690 were much greater (generally by a factor of * 2) than
those in patient 1422, who took a 30-mg supplement daily for
nearly 1 y (Table 5). Moreover, the patient receiving the 300-mg
Downloaded from ajcn.nutrition.org by guest on May 29, 2014
Plasma (mmol/L)
6.3
Red cells (mmol/L)
7.6
Adipose tissue (nmol/g)
Abdominal
6.1
Perirenal
7.0
Muscle
Abdominal (nmol/g)
8.1
Skin (nmol/g)
6.9
Circulatory system (nmol/g)
Aorta above aortic valve
8.8
Apex of left ventricle
5.8
Right atrium
6.0
Nerve tissues (nmol/g)
Cerebellum
5.0
Substantia nigra
2.5
Cortex (frontal)
Pituitary gland
Pons
2.9
Spinal cord: cervical (C2-C4) 1.9
Spinal cord: thoracic (T2-T4) 3.6
Spinal cord: lumbar (L2-L4)
7.8
Vagus nerve
7.9
Femoral nerve
4.6
Optic nerve
1.7
Hepatobiliary system (nmol/g)
Liver
6.0
Bile
11.4
Pancreas (nmol/g)
Head of pancreas
6.8
Tail of pancreas
6.3
Kidney (nmol/g)
Cortex of left kidney
5.8
Medulla of right kidney
6.1
Miscellaneous (nmol/g)
Adrenal
6.7
Lung
3.5
Spleen
6.8
d0-a-T Total a-T
682
BURTON ET AL
surgery patients suggest that these tissues receive a particularly
large fraction of their vitamin E from chylomicrons. The relatively
low RRR:rac in muscle (1.40) also suggests that this tissue takes
up much of its vitamin E from chylomicrons.
After the delivery of chylomicron remnants to the liver, it
appears probable that the hepatic a-tocopherol transfer protein
preferentially enriches nascent VLDL with RRR- and probably
the other 2R-a-tocopherol stereoisomers (12, 18, 19). The 2R-atocopherol stereoisomers are then delivered to tissues by one or
more of the mechanisms outlined above. The net RRR:rac in
each tissue will be a composite that initially reflects the relative
contribution from chylomicron donors (all forms of vitamin E)
and the VLDL-derived vitamin E (mainly 2R-a-tocopherol
stereoisomers). Ultimately, RRR:rac becomes 2:1. The rate at
which this ultimate value is attained in a tissue will depend on
tissue clearance mechanisms, which are still unknown. This general model for a-tocopherol uptake and retention is consistent
with data from the literature (18, 19) and with the lipoprotein
results obtained during the dosing period (Table 2), which show
only slight enrichment of RRR-a-tocopherol in chylomicrons but
progressive enrichment in VLDL, LDL, and HDL.
In earlier competitive dosing studies with 1:1 mixtures of
deuterated RRR- and SRR-a-TAcs, we found a strong preferential retention of RRR-a-tocopherol in plasma, erythrocytes, and
tissues (except liver) of rats (12) and monkeys (19) and in plasma
and erythrocytes of humans (18, 20, 21). For example, in rats
continuously fed an equimolar mixture of deuterated RRR-(d6)
and SRR-(d3)-a-TAc, the RRR-SRR ratio increased with time,
reaching 2.4 in plasma and 5.3 in the brain after 22 wk (12). Similarly, an equimolar single dose of these same two labeled aTAcs given to humans produced an RRR-SRR ratio in plasma of
6.7 ± 2.8 4 d later (18). In the present study, the RRR-rac ratio
after a 300-mg daily dose for 8 d stabilized for months at <2.0,
some 7 d after the end of dosing (Figure 3I).
All our competitive uptake and retention studies indicate that
it is the stereochemistry at the 2-position that is the prime determinant of biodiscrimination between stereoisomers of a-tocopherol. That is, ultimately all four 2R stereoisomers will be
preferentially retained (included among these is RRR-a-tocopherol—natural vitamin E) and all four 2S stereoisomers will be
preferentially eliminated from the body. That is, eventually, just
half of a dose of synthetic vitamin E would be treated by animals
and humans as although it were the natural vitamin and the other
half would be eliminated from the liver via the bile, or from the
kidney via the urine, or both.
Attractive as this simple explanation is for a 2:1 RRR-rac
ratio, it may not fully reflect the real situation. For example, the
acetate esters of all eight a-tocopherol stereoisomers have been
reported to be active in the traditional rat fetal gestation-resorption assay (2). They not only were reported to have different
activities from each other but, also, the relative biopotencies
appeared not to be exclusively dependent on the stereochemistry
at the 2-position (RRR > RRS > RSS > SSS > RSR > SRS > SRR
> SSR), although the 2R stereoisomers generally appear to be
more active than 2S forms (2). It follows, then, that within the
organs and tissues critical to the bioassay, there will be a finite
amount of each stereoisomer present, at least for the duration of
the test. Indeed, a recently published bioavailability study carried out in rats dosed with all-rac-a-TAc for 90 d showed the
presence of 2S stereoisomers in tissues and plasma (54). However, there was a preferential accumulation of each of the four 2R
Downloaded from ajcn.nutrition.org by guest on May 29, 2014
dose had <65% deuterated a-tocopherol throughout his body,
whereas the patient receiving the 30-mg daily dose had only
<6% deuterated a-tocopherol in his body. Thus, the larger dose
significantly increased a-tocopherol concentrations in all tissues, including the brain.
Another striking result from these long-term dosing studies
was the general similarity of the RRR-rac ratios and the percentage of deuterated a-tocopherol between plasma and tissues in
both patients. It is clear, given sufficient time (1–2 y), that tissues eventually equilibrate with the various forms of a-tocopherol circulating in the plasma (as is expected).
There also are striking differences between the two patients in
the concentrations of a-tocopherol in some of the tissues. For
example, although plasma total a-tocopherol in patient A6690
was 3.5 times that in patient 1422, the relative difference in concentrations was sevenfold for the left ventricle, ninefold for the
right atrium, sixfold for liver, and ninefold for the tail of pancreas. (We have no explanation for the large difference between
the head and tail of the pancreas in patient A6690.) It is difficult
to draw conclusions from data obtained from just two patients.
The only other data we are aware of in which the effect of dosing on concentrations in human tissues (other than adipose) has
been examined was obtained in a limited study carried out by us
using patients undergoing elective heart surgery. In this study,
doses of 100–900 mg d 3-RRR-a-TAc given orally for 14 consecutive days caused a twofold increase in myocardial vitamin E
concentrations (48).
In patient A6690 the RRR-rac ratio in bile was as high as the
value determined in plasma. This finding is supported by results
we obtained in bile collected from rats and humans fed 1:1 mixtures of RRR- and SRR-a-TAc (ie, RRR > SRR; unpublished
observations). Evidently, the 2S stereoisomers were not preferentially eliminated from the body as unchanged tocopherol
excreted from the liver in bile.
The hydrophobic, water-insoluble nature of vitamin E and the
absence of a specific plasma protein carrier means that its delivery to tissues must, by default, occur via lipoprotein-mediated
mechanisms. Delivery to tissues has been suggested to take place
via lipoprotein lipase-mediated transfer (49), LDL receptormediated uptake of LDL (50), and aqueous phase-mediated
transfer from HDL (51). There is also evidence for a phospholipid transfer protein-mediated exchange between lipoproteins
and tissue (52). None of these mechanisms have as yet been
shown to involve preferential transfer of one particular stereoisomeric form of a-tocopherol. The differences in discrimination
between RRR-a-tocopherol and roughly half the stereoisomers
present in all-rac-a-tocopherol in plasma and the various tissues
can most simply be explained by invoking a dual pathway for
delivery of a-tocopherol to tissues, as was originally suggested
in the first kinetics study using two deuterated a-tocopherol
stereoisomers (RRR and SRR) (12).
After absorption of all the various forms of vitamin E, including RRR-a-tocopherol, from the intestine, apparently without discrimination (18, 20, 53), they may each be delivered without discrimination directly to tissues via the lipoprotein lipase-mediated
hydrolysis of chylomicrons (49). Thus, tissues to which vitamin E
is delivered mainly by chylomicrons and the lipoprotein lipase
route (49) are likely to become enriched with all stereochemical
and structural forms of vitamin E, including g-tocopherol, and not
enriched just with RRR-a-tocopherol. The high concentrations of
g-tocopherol in adipose tissue and skin obtained from the elective
NATURAL AND SYNTHETIC VITAMIN E IN HUMAN TISSUES
Excellent technical assistance was provided by Malgorzata Daroszewski,
Urszula Wronska, Nora Lagmay, and Sandy Thedford. The helpful support of
the Ottawa branch of the Canadian Red Cross Society is gratefully acknowledged. We thank the patients and their families at the Veterans’ Administration Medical Center Hospital, Johnson City, TN, for their enthusiasm and participation in this project.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
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