Free Radical Biology & Medicine, Vol. 29, No. 9, pp.

834 – 845, 2000

Copyright © 2000 Elsevier Science Inc.
Printed in the USA. All rights reserved
0891-5849/00/$–see front matter

PII S0891-5849(00)00371-3

Original Contribution
STUDIES OF LDL OXIDATION FOLLOWING ␣-, ␥-, OR ␦-TOCOTRIENYL
ACETATE SUPPLEMENTATION OF HYPERCHOLESTEROLEMIC HUMANS

D. O’BYRNE,*,† S. GRUNDY*‡ L. PACKER,§ S. DEVARAJ*,†,‡ K. BALDENIUS,㛳 P. P. HOPPE,㛳 K. KRAEMER,㛳

I. JIALAL,*,†,‡ and M. G. TRABER¶
*Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USA; †Division of Clinical
Biochemistry and Human Metabolism, University of Texas Southwestern Medical Center, Dallas, TX, USA; ‡Department of
Pathology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; §Department of Molecular
and Cell Biology, University of California, Berkeley, CA, USA; 㛳BASF Aktiengesellschaft, Ludwigshafen, Germany; ¶Linus
Pauling Institute, Oregon State University, Corvallis, OR, USA

(Received 5 April 2000; Revised 27 June 2000; Accepted 29 June 2000)

Abstract—In vitro tocotrienols (T3s) have potent vitamin E antioxidant activity, but unlike tocopherols can inhibit
cholesterol synthesis by suppressing 3-hydroxy-3-methyl-glutarylCoA (HMG-CoA) reductase. Because hypercholes-
terolemia is a major risk factor for coronary artery disease and oxidative modification of low-density lipoprotein (LDL)
may be involved in atherogenesis, we investigated whether daily supplements of placebo, or alpha-, gamma-, or delta-
(␣-, ␥-, or ␦-) tocotrienyl acetates would alter serum cholesterol or LDL oxidative resistance in hypercholesterolemics
in a double-blind placebo controlled study. Subjects were randomly assigned to receive placebo (n ⫽ 13), ␣- (n ⫽ 13),
␥- (n ⫽ 12), or ␦- (n ⫽ 13) tocotrienyl acetate supplements (250 mg/d). All subjects followed a low-fat diet for 4 weeks,
then took supplements with dinner for the following 8 weeks while still continuing diet restrictions. Plasma ␣- and
␥-tocopherols were unchanged by supplementation. Plasma T3s were undetectable initially and always in the placebo
group. Following supplementation in the respective groups plasma concentrations were: ␣-T3 0.98 ⫾ 0.80 ␮mol/l, ␥-T3
0.54 ⫾ 0.45 ␮mol/l, and ␦-T3 0.09 ⫾ 0.07 ␮mol/l. Alpha-T3 increased in vitro LDL oxidative resistance (⫹22%, p ⬍
.001) and decreased its rate of oxidation (p ⬍ .01). Neither serum or LDL cholesterol nor apolipoprotein B were
significantly decreased by tocotrienyl acetate supplements. This study demonstrates that: (i) tocotrienyl acetate
supplements are hydrolyzed, absorbed, and detectable in human plasma; (ii) tocotrienyl acetate supplements do not
lower cholesterol in hypercholesterolemic subjects on low-fat diets; and (iii) ␣-T3 may be potent in decreasing LDL
oxidizability. © 2000 Elsevier Science Inc.

Keywords—Vitamin E, Tocotrienol, Tocopherol, Cholesterol, HMG CoA reductase, Low-density lipoprotein oxida-
tion, Antioxidant, Free radicals

INTRODUCTION disease (CAD) [2,3]. There is in vitro evidence that

Hypercholesterolemia is a major contributing factor for
the risk of atherosclerosis [1] and clinical trials have
shown that lowering LDL cholesterol with 3-hydroxy-3-
methyl-glutarylCoA (HMG-CoA) reductase inhibitors
slows the progression of atherosclerosis and significantly
reduces morbidity and mortality from coronary artery
This work was presented in part at Experimental Biology ’99 in
Washington, DC, on April 19, 1999, and was published in abstract form
(FASEB J. 13:A536; 1999).
Address correspondence to: Dr. Maret G. Traber, Oregon State
University, Linus Pauling Institute, 561A Weniger Hall, Corvallis, OR
97333-6512, USA; Tel: (541) 737-7977; Fax: (541) 737-5077; E-Mail:
maret.traber@orst.edu.
tocotrienols (T3s), members of the vitamin E family, are
potent inhibitors of HMG-CoA reductase and decrease
synthesis of apolipoprotein B [4 –7]. In animal and hu-
man studies, supplementation with mixed T3s (Palmvi-
tee; Lane Labs, Malaysia) has resulted in significant
reductions in serum total cholesterol and low-density
lipoprotein cholesterol in chickens [8], hyperlipidemic
pigs [9], and hypercholesterolemic humans [10 –12].
However, two placebo-controlled clinical trials failed to
confirm the cholesterol-lowering effects of mixed T3s
(Palmvitee) in hyperlipidemic patients [13,14]. These
divergent findings may be due to the varying amounts
and proportions of T3s and tocopherols in the Palmvitee

834
 Tocotrienols and LDL oxidation 835

supplements used in the studies. In vitro evidence sug- Table 1. Composition of the T3 Preparations
gests there may be as much as a 30-fold difference in the Tocotrienol preparations (g/100 g)
ability of ␣-, ␥-, and ␦-T3 isomers to inhibit cholesterol
synthesis [4]. Furthermore, ␣-tocopherol present in the ␣-Tocotrienyl ␥-Tocotrienyl ␦-Tocotrienyl
tocol blends of Palmvitee may compete for binding with Analyzed compounds acetate acetate acetate
the ␣-tocopherol transfer protein [15], and thus interfere ␣-tocotrienyl acetate 83 0.7 1.9
with the transport of T3s in the circulation. In several ␣-T3 0.5 —
studies [14,16,17], fasting plasma T3s levels have been ␥-tocotrienyl acetate 0.3 94 10.6
␥-T3 — ⬍0.1 —
undetectable or considerably lower than plasma ␣-to- ␦-tocotrienyl acetate — — 81
copherol after subjects have been supplemented with ␣-T3 — —
Palmvitee. In addition, ␣-tocopherol has been reported to ␤-tocotrienyl ⫹ 0.2 3.4 —
␤-tocotrienyl acetates
attenuate the inhibitory effects of T3s on HMG CoA ␣-tocomonoenola acetate 2.5 — 0.6
reductase activity [8]. In contrast, when hyperlipidemic ␣-tocopherol ⫹ 4.2 0.3 3.6
subjects have been given ␣-tocopherol–free ␥-T3 sup- ␣-tocopheryl acetate
Other tocopherols and 1.4 — 0.2
plements (200 –220 mg) on a short-term basis, significant their acetates
cholesterol reductions have occurred [10,11]. Currently,
a
no clinical trials have been undertaken to determine the Acetic acid ester of 2,5,7,8-Tetramethyl-2-(4⬘,8⬘,12⬘-trimethyl-
tride-11-enyl)-chroman-6-ol[CAS 65291-81-8].
efficacy of purified ␣-, ␥-, or ␦- T3 on serum cholesterol
reduction in humans.
Epidemiological studies have shown that increased and decreasing LDL oxidative susceptibility, we inves-
vitamin E consumption is correlated with reduced risk of tigated the efficacy of supplementing hypercholester-
cardiovascular disease in men [18] and women [19]. The olemic men and women with 250 mg/d of purified ␣-, ␥-,
results of clinical intervention trials investigating the or ␦- tocotrienyl acetates for 8 weeks. In this communi-
benefits of ␣-tocopherol supplements have not been as cation we report the results of a placebo-controlled sup-
consistent [20 –22]. In one study, supplementation with plementation with different purified T3 isomers on lipid
400 – 800 IU ␣-tocopherol in patients with angiographi- and lipoprotein levels, and resistance of LDL to oxida-
cally proven coronary atherosclerosis significantly re- tion in these hypercholesterolemic patients.
duced the incidence of nonfatal myocardial infarction
[20]. In two other studies, vitamin E (doses of 300 mg or
MATERIALS AND METHODS
400 IU, respectively) did not have significant protective
benefits in high-risk cardiovascular patients [21,22]. We
Isolation of individual tocotrienols from palm oil
have shown that supplementation with at least 400 IU
␣-tocopherol is necessary to protect LDL from in vitro Kilogram quantities of individual T3s were isolated
oxidative modification [23–25]. Since oxidized LDL has from a food-grade palm oil T3 mixture (Tocomin 50;
been proposed as an initiating factor in atherosclerotic Carotech; Malaysia). Separation of the homologues was
lesion formation [26], it is hypothesized that antioxidants achieved using preparative HPLC [30] with a mobile
such as ␣-tocopherol may play a role in reducing the risk phase of ethyl acetate/heptane, the fractions collected,
of cardiovascular disease by protecting LDL from oxi- and the solvents removed in vacuo. Free T3s were pro-
dative modification. T3s are structurally similar to toco- tected at all stages of isolation by purging with nitrogen.
pherols and in some systems have even greater antioxi- The ␥-T3 and ␦-T3 isolates were further purified by
dant activities [6,27,28]. Suarna et al. [29] reported that selective absorption to a strongly basic ion-exchange
following acute supplementation of rats or humans with resin and subsequent elution with 5% acetic acid in
a mixed T3 and tocopherol preparation, T3s provided ethanol. Following removal of solvents, the tocotrienols
oxidative protection to plasma (rats) or LDL (human) were acetylated with acetic anhydride (vol/vol ⫽ 1) at
comparable to that of the corresponding tocopherols. The 140°C (3 h for ␥- and ␦-tocotrienol, 6 h for ␣-tocotrien-
antioxidant benefits of T3 supplementation have also ol). Excess acetic anhydride and acetic acid were re-
been reported in a group of patients with cerebrovascular moved in vacuo.
disease [13]. The supplements used in this clinical inter- The purity of the main component was determined by
vention study contained both tocopherols and 240 mg of gas chromatography (GC) and verified by 1H-NMR with
mixed T3s. To date, no clinical trials have been con- internal standard. The content of the other tocols was
ducted to determine the efficacy of supplementation with determined by GC and verified by HPLC. The amounts
purified T3 isomers on LDL oxidative resistance. of tocotrienyl acetates, as well as contaminating toco-
Since T3 supplementation may have potential thera- pherols and T3s, are given in Table 1. Acetic anhydride
peutic benefits through lowering LDL cholesterol levels and acetic acid were less than 1 g/100 g. Solvents were
836
Table 2. Actual Amount of Tocotrienyl Acetates (Estimated 125 mg)
in the Soft Gel Capsules
Tocotrienol preparation
␣-Tocotrienyl ␥-Tocotrienyl ␦-Tocotrienyl
acetate acetate acetate
Tocotrienyl acetate (mg) 148 140
Equivalent free T3 (mg) 134 127
a
Additional 17 mg of ␥-tocotrienyl acetate.
b
Additional 15 mg of ␥-tocotrienol.
verified to be below 50 mg/kg. Metals were not detect-
able (⬍ 10 mg/kg). Residual components were palm oil
lipids, mostly phytosterols and glycerides, none of which
exceeded 1 g/100 g.
The purified tocotrienyl acetates were diluted with
medium-chain triglycerides (MCT-Oil; Grünau; Illertis-
sen, Germany) to a final concentration of 35 g T3 per
100 g, then were encapsulated in soft gels. Each soft gel
capsule was formulated to contain a total of 125 mg of
free T3 equivalent; exact amounts are shown in Table 2.
In the case of the ␦-T3 acetate preparation, the sum of ␦-
and ␥-T3 was intended to be 125 mg per soft gel, because
the ␥-homologue contributed significantly to the sum of
␦- and ␥-tocotrienols (␦/␥ ⫽ 7:1). The placebo prepara-
tion contained MCT-Oil free of tocotrienols. All soft gels
were filled in encoded bottles allowing double-blind ap-
plication. Analysis of the capsules was repeated 5
months after production and shelving. No significant
losses were observed.
Study design
The study design was approved by the Institutional
Review Board, University of Texas Southwestern Med-
ical Center, Dallas, Texas. Hypercholesterolemic sub-
jects were recruited from the campus and clinics of
University of Texas Southwestern Medical Center and
gave informed consent to participate in the study. Sub-
jects were randomly assigned to the four treatment
groups (placebo, ␣-T3, ␥-T3, or ␦-T3) using an adaptive
randomization program that matched individuals on the
basis of age, gender, body mass index (BMI), LDL
cholesterol, and estrogen use in order to provide a bal-
anced distribution. All subjects were placed on an Amer-
ican Heart Association (AHA) Step 1 Diet for 12 weeks.
After stabilization on the diet for 4 weeks, subjects took
250 mg/d placebo, or ␣-, ␥-, or ␦- tocotrienyl acetate
supplement with their evening meal for 8 weeks. Sub-
jects and investigators were blinded to the identity of the
supplements. Two fasting blood samples were obtained
prior to initiation of the study, after 4 weeks of AHA
Step 1 diet, and after 8 weeks of dietary restrictions plus
D. O’BYRNE et al.
supplementation. Three-day diet records were completed
by subjects prior to entry, and during weeks 2, 4, 8, and
12. Compliance was monitored by pill counts and by
measurement of fatty acid and antioxidant levels.
119a Subject population
108b
Fifty-two healthy volunteers with fasting LDL cho-
lesterol greater than 130 mg/dL gave informed consent to
participate in this study. The exclusion criteria for the
study were: (i) smoking; (ii) ingestion of antioxidant
supplements, fish oil supplements, hypolipidemic medi-
cations, anticoagulant therapy, and thyroxin; (iii) alcohol
consumption in excess of 1 oz. per day; (iv) diabetes,
hypertension, or endocrine disorders; (v) abnormal blood
chemistry profile; (vi) fasting triglyceride greater than
300 mg/dL; or (vii) clinical evidence of coronary heart
disease.
Diet
In order to minimize the confounding effects of di-
etary cholesterol, fat, and saturated fat on HMG CoA
reductase activity, all subjects were assigned to follow an
AHA Step 1 diet. The subjects received instructions on
the diet by a registered dietitian. The instructions entailed
a 2–3 h class outlining practical means of reducing fat,
saturated fat, and cholesterol in the diet in order to meet
the diet recommendations (total dietary fat ⬍ 30% en-
ergy; saturated fat ⬍ 10% energy; ⬍ 300 mg cholesterol/
d). Literature from the AHA was given to each partici-
pant. Subjects received personalized information based
on evaluation of their initial dietary record. Participants
completed additional 3-d dietary records after following
the Step 1 diet for 2, 4, 8, and 12 weeks. Dietary records
were evaluated for compliance and feedback was pro-
vided by telephone, letters, and personal contact. Addi-
tional literature emphasizing various aspects of the Step
1 diet was provided at each blood drawing session in
order to encourage compliance and motivation. Overall
dietary compliance was determined by evaluating 7 d,
randomly selected from each subject’s dietary records
using the MEDFICTS questionnaire (NCEP). An evalu-
ation score of greater than 75 indicated lack of compli-
ance to the dietary recommendations; 40 –75 indicated
compliance with Step 1 guidelines and a score of less
than 40 indicated compliance with Step 2 guidelines (⬍
20% dietary fat, ⬍ 7% saturated fat, ⬍ 200 mg/d cho-
lesterol).
Subjects were advised to maintain their usual activi-
ties and weight during the study. Weight was measured
prior to initiation of the diet and during weeks 2, 4, 8, and
12. If the subject’s weight varied more than 1 to 2 kg
 Tocotrienols and LDL oxidation
from their initial weight, they were advised on how to
modify their diet in order to stabilize weight.
Blood collection and storage
Blood samples were obtained from each participant
after a 10 –12 h fast using 10 ml Vacutainer collection
sets (Vacutainer; Becton, Dickinson and Co., Franklin
Lakes, NJ, USA). When collecting blood samples for
LDL isolation, plasma tocopherols, plasma T3s, fatty
acids, and CBC profiles, Vacutainer tubes containing 1
mg/ml EDTA were used. Blood samples for lipid profile,
glucose, total protein, liver, and renal function profiles
were collected using Vacutainer tubes for serum separa-
tion. Two fasting blood samples were collected at each
phase of the study (0 week, 4 weeks, 12 weeks), with an
interval of at least 3 d between blood samples.
Plasma and serum were separated by low-speed cen-
trifugation at 4°C. Freshly separated serum was used for
determination of the lipoprotein profile and blood chem-
istries. Aliquots of plasma were stored at ⫺70°C for
subsequent determination of fatty acids, tocopherols, and
T3s; and at ⫺20°C for assessment of the oxidative re-
sistance of LDL at the conclusion of the study.
Analytical procedures
Serum lipid and lipoprotein levels were assayed en-
zymatically using the automated Paramax system from
Parkland Hospital’s Clinical Chemistry department. The
Clinical Chemistry laboratory participates in the Alert
Proficiency program and has a CV ⬍ 3% for total cho-
lesterol. Serum total protein, albumin, glucose, creati-
nine, alanine transaminase, and complete blood cell
count with differential were performed by Clinical Lab-
oratory, Parkland Hospital. Plasma fatty acids were de-
termined by gas liquid chromatography after extraction
and transmethylation [31], using C17:0 as the internal
standard (Nuchek-Prep; Elysian, MN, USA). Protein
concentration was determined by the method of Lowry et
al. [32]. Plasma tocopherols and T3s were measured by
high-pressure liquid chromatography (HPLC) and elec-
trochemical detection according to the method of Podda
et al. [33], with the exception that only the isocratic
mobile phase was used for the HPLC system. Plasma
tocopherol and T3 concentrations are expressed as
␮mol/l.
Lipoprotein isolation
LDL (d 1.019 –1.063 g/ml) was isolated by rapid
two-step method ultracentrifugation using a modification
of the method of Kleinveld et al. [34]. NaBr-EDTA salts
837
were used to adjust density, and ultracentrifugation was
performed at 100,000 rpm using a TI-100 tabletop ultra-
centrifuge (Beckman Coulter Inc.; Fullerton, CA, USA).
The EDTA and salts were removed by passage of the
LDL sample through a 10 ml Sephadex DG-10 column
(Amersham Pharmacia Biotech, Inc.; Piscataway, NJ,
USA). Samples were purged with nitrogen, refrigerated
until the protein concentration was measured, and used
for oxidation experiments within 24 h of isolation.
LDL oxidation indices
Samples of freshly isolated LDL were diluted to 200
␮g LDL protein/ml with phosphate-buffered saline (pH
7.4) and incubated at 37°C with 5 ␮mol/l Cu2⫹ for 5 h.
Formation of conjugated dienes were measured via con-
tinuous spectrophotometric monitoring at 234 nm [35].
Measurements were recorded every 10 min and used to
assess LDL oxidation. Conjugated dienes were deter-
mined using the molar extinction coefficient 2.95 ⫻ 104
M⫺1cm⫺1 and expressed relative to the amount of LDL
protein. Lag time, oxidative rate, and maximum conju-
gated diene concentration produced during the 5 h time
course were calculated and used to characterize the ox-
idative profile of each LDL sample.
Statistical analysis
Results are expressed as mean ⫾ standard deviation
(SD). Kruskal Wallis nonparametric test was used to
assess overall differences between the four groups on: (i)
presupplementation data (i.e., after 4 weeks of diet sta-
bilization), and (ii) response to supplementation (post-
supplementation/presupplementation data). Follow-up
comparisons between the placebo vs. ␣-, ␥-, or ␦-tocot-
rienyl acetate supplementation were determined by Wil-
coxon Rank Sums tests, if the Kruskal-Wallis test was
significant. When analyzing the plasma T3 data, fol-
low-up comparisons between the placebo vs. ␣-, ␥-, or
␦-tocotrienyl acetate supplementation were made by
2-tailed Fisher’s Exact Test. Pairwise correlations be-
tween variables of interest were determined a priori and
Spearman rank correlation coefficients were used to as-
sess these relationships. The level of significance was set
at p ⬍ .05. Analyses were performed using SAS (SAS
Institute; Cary, NC, USA) by the biostatistician at the
General Clinical Research Center.
RESULTS
Fifty-two subjects participated in the study, but one
participant receiving ␥-tocotrienyl acetate dropped out of
the study due to time constraints. Baseline descriptive
838 D. O’BYRNE et al.

Table 3. Baseline Descriptive Characteristics of Subjects Receiving Placebo, ␣-, ␥-, or ␦-Tocotrienyl Acetate Supplements

Placebo ␣-Tocotrienyl acetate ␥-Tocotrienyl acetate ␦-Tocotrienyl acetate

Characteristic (n ⫽ 13) (n ⫽ 13) (n ⫽ 12) (n ⫽ 13)

Age (years) 38.7 ⫾ 9.7 40.0 ⫾ 11.0 43.2 ⫾ 13.2 41.4 ⫾ 9.8
Gender (male, female) 6, 7 5, 8 6, 6 5, 8
Weight (kg) 83.7 ⫾ 21.9 82.2 ⫾ 17.0 79.7 ⫾ 23.7 79.7 ⫾ 19.3
BMI 30.0 ⫾ 9.0 29.6 ⫾ 6.9 28.8 ⫾ 7.7 28.3 ⫾ 5.8
Total cholesterol (mg/dL) 239.9 ⫾ 22.9 239.6 ⫾ 25.8 256.4 ⫾ 23.0 238.1 ⫾ 16.4
Triglycerides (mg/dL) 156.9 ⫾ 76.3 142.9 ⫾ 44.9 173.6 ⫾ 67.9 144.8 ⫾ 65.8
LDL cholesterol (mg/dL) 160.3 ⫾ 20.6 160.7 ⫾ 18.1 174.0 ⫾ 22.6 158.9 ⫾ 16.9
HDL cholesterol (mg/dL) 48.3 ⫾ 13.8 50.3 ⫾ 13.2 47.8 ⫾ 13.6 50.2 ⫾ 9.5

Values represent the mean ⫾ standard deviation. Two fasting blood samples were taken from subjects at baseline (before diet restrictions),
with at least a 3 d interval between blood samples. The mean concentration for blood lipids was determined from the two blood samples.

characteristics of the 51 participants completing the
study are given in Table 3. No significant differences
were observed between groups for any of the variables.
Subjects tolerated the T3 supplements, except four sub-
jects receiving ␥-tocotrienyl acetate supplements re-
ported transient abdominal distention, gastric upset, and
pain during the first week of supplementation. Two of
these subjects also reported increased flatulence that per-
sisted during the study. One subject, who had a hiatel
hernia and gastric reflux, reported nausea and vomiting
1 h after taking ␣-tocotrienyl supplements with the
evening meal. These symptoms stopped when supple-
ments were taken with the afternoon meal. No abnormal
blood chemistry profiles resulted from taking the supple-
ments.
8 weeks, a maximum of 32 capsules would remain when
subjects returned the unused pills. Most participants took
the capsules for approximately 60 d. The mean pill count
for each group was: placebo, 22.2 ⫾ 11.1; ␣-T3, 26.3 ⫾
12.8; ␥-T3, 25.1 ⫾ 12.5; and ␦-T3, 29.5 ⫾ 9.1. There
were no significant differences in compliance between
groups.
Plasma fatty acids
There were no significant differences in plasma fatty
acids between groups prior to supplementation or in
response to supplementation (Table 4).
Plasma tocopherols and tocotrienols

Dietary compliance Prior to supplementation, plasma ␣-tocopherol con-

The mean dietary compliance scores for the groups
receiving the placebo, ␣-, ␥-, or ␦-tocotrienyl acetate
supplements were: 62.9 ⫾ 16.3, 63.23 ⫾ 23.64, 60.8 ⫾
15.1, and 63.3 ⫾ 17.6, respectively. The mean scores
were within the compliance range (40 –75), but approx-
imately two to three subjects in each group had scores
exceeding 75. We found no significant difference in the
results when statistical analysis was performed using
data from subjects with dietary compliance scores of less
than 75, compared with data including all subjects. Since
dietary compliance was similar in all groups, all subjects’
data were included in the analysis.
There were no significant changes in weight within or
between groups during the supplementation phase
(weight change: placebo group, ⫺1.1 ⫾ 2.26 kg; ␣-T3
group, ⫺0.34 ⫾ 1.60 kg; ␥-T3 group, ⫺0.51 ⫾ 1.97 kg;
␦-T3 group, ⫺0.377 ⫾ 1.54 kg).
Pill compliance
Each subject was given 150 capsules. If subjects con-
sumed the recommended dose of 2 capsules per day for
centrations were 19.1 ⫾ 5.5, 16.3 ⫾ 5.0, 18.6 ⫾ 5.3, and
24.3 ⫾ 12.6 ␮mol/l for subjects assigned to receive the
placebo, ␣-, ␥-, or ␦-tocotrienyl acetate supplements,
respectively. No significant differences were detected
between groups before or after supplementation (Fig.
1A), even when standardized for plasma lipids (data not
shown). Plasma ␥-tocopherol levels were 3.1 ⫾ 1.7,
3.1 ⫾ 1.6, 3.4 ⫾ 1.8, and 3.3 ⫾ 1.7 ␮mol/l prior to
supplementation in groups assigned to receive the pla-
cebo, ␣-, ␥-, or ␦-tocotrienyl acetate supplements, re-
spectively (Fig. 1B). No significant differences were
observed between groups, and T3 supplementation did
not exert an appreciable effect on plasma ␥-tocopherol
levels.
Plasma T3s were undetectable in fasting plasma prior
to supplementation, therefore only postsupplementation
values are displayed in Fig. 2. Supplementation with ␣-,
␥-, or ␦-tocotrienyl acetates resulted in significantly
greater (p ⬍ .001) plasma concentrations of the respec-
tive T3 compared with the placebo group: ␣-T3, 0.97 ⫾
0.80 ␮mol/l; ␥-T3, 0.54 ⫾ 0.45; ␦-T3, 0.096 ⫾ 0.068
(Figs. 2A, 2B, and 2C, respectively). These data indicate
that the tocotrienyl acetate supplements were hydro-
 Tocotrienols and LDL oxidation 839

despite similarity in administered T3 doses. Interest-

ingly, in subjects who consumed ␦-tocotrienyl acetate,
compared to the placebo, there were significant, albeit
small, increases in both plasma ␣- and ␥-T3 concentra-
tions (Fig. 2, compare A, B, and C). There was also a
significant increase in plasma ␥-T3 concentrations after
subjects consumed ␣-tocotrienyl acetates, compared with
the placebo (Fig. 2B). These may be a result of contam-
inating ␣- and ␥-T3s and tocotrienyl acetates in the
supplements (Table 1).

Lipid and lipoprotein profile

Presupplementation lipid and lipoprotein profiles of

Fig. 1. Plasma tocopherol concentrations in subjects assigned to the
placebo (n ⫽ 13), ␣-tocotrienyl acetate (n ⫽ 13), ␥-tocotrienyl acetate
(n ⫽ 12), or ␦-tocotrienyl acetate (n ⫽ 13) supplementation groups.
The data presented represents the mean ⫾ standard deviation for the
presupplementation phase: after 4 weeks of AHA Step 1 diet, and the
postsupplementation phase: after subjects consumed 250 mg/d placebo
or individual tocotrienyl acetate supplements for 8 weeks while con-
tinuing to follow the AHA Step1 diet. (A) Plasma ␣-tocopherol con-
centrations (␮mol/l). (B) Plasma ␥-tocopherol concentrations (␮mol/l).
lyzed, absorbed, and still present in the circulation after
a 10 –12 h fast. Of interest was the difference in the
resulting plasma concentrations of the three T3 isomers,
subjects were not significantly different between groups
(Table 5). After 4 weeks of adherence to the AHA Step
1 diet, serum cholesterol decreased by approximately
4 –5%. The addition of tocotrienyl acetate supplements to
the diet failed to lower total or LDL cholesterol com-
pared to the placebo. In contrast, the ␦-tocotrienyl acetate
supplement resulted in a significant increase in total
cholesterol and LDL cholesterol levels (p ⬍ .05). No
significant changes in serum HDL cholesterol, LDL cho-
lesterol/HDL cholesterol ratio, or in triglycerides were
observed in any group.
Apolipoprotein B
The apolipoprotein B concentrations were not altered
by tocotrienyl acetate supplements (Table 6), indicating
that the number of circulating LDL particles was un-
changed. Furthermore, ␣-, ␥-, and ␦-tocotrienyl acetate
supplements did not modify the cholesterol content of
the LDL particles, as reflected by the LDL cholesterol/
apolipoprotein B ratios.

Table 4. Plasma Fatty Acid Profile of Subjects Receiving Placebo, ␣-, ␥-, or ␦-Tocotrienyl Acetate Supplements

Fatty Supplementation Placebo ␣-Tocotrienyl acetate ␥-Tocotrienyl acetate ␦-Tocotrienyl acetate

acid phase (n ⫽ 13) (n ⫽ 13) (n ⫽ 12) (n ⫽ 13)

16:0 Pre- 2.73 ⫾ 0.43 2.65 ⫾ 0.61 2.71 ⫾ 0.52 2.74 ⫾ 0.71
Post- 2.63 ⫾ 0.76 2.80 ⫾ 0.71 2.81 ⫾ 0.69 2.83 ⫾ 0.62
18:0 Pre- 0.72 ⫾ 0.07 0.68 ⫾ 0.14 0.78 ⫾ 0.17 0.78 ⫾ 0.14
Post- 0.75 ⫾ 0.14 0.72 ⫾ 0.14 0.80 ⫾ 0.19 0.79 ⫾ 0.12
18:1 Pre- 2.72 ⫾ 0.48 2.47 ⫾ 0.59 2.59 ⫾ 0.64 2.67 ⫾ 0.81
Post- 2.58 ⫾ 0.98 2.55 ⫾ 0.63 2.79 ⫾ 0.83 2.60 ⫾ 0.47
18:2 Pre- 3.47 ⫾ 0.58 3.26 ⫾ 0.64 3.83 ⫾ 0.77 3.69 ⫾ 0.61
Post- 3.49 ⫾ 0.64 3.45 ⫾ 0.71 3.84 ⫾ 0.76 3.85 ⫾ 0.61
18:3 Pre- 0.08 ⫾ 0.02 0.07 ⫾ 0.04 0.09 ⫾ 0.04 0.09 ⫾ 0.03
Post- 0.08 ⫾ 0.05 0.07 ⫾ 0.04 0.08 ⫾ 0.03 0.10 ⫾ 0.05
20:4 Pre- 0.81 ⫾ 0.14 0.83 ⫾ 0.29 0.81 ⫾ 0.14 0.72 ⫾ 0.15
Post- 0.81 ⫾ 0.17 0.87 ⫾ 0.32 0.85 ⫾ 0.18 0.75 ⫾ 0.19

Values represent the mean ⫾ standard deviation of fasting plasma fatty acids mmol/l. Presupplementation concentrations were determined from
samples taken after subjects followed an AHA Step 1 diet for 4 weeks. Postsupplementation concentrations were determined from samples taken
after subjects consumed 250 mg/day placebo or tocotrienyl acetate supplements for 8 weeks while continuing to follow the AHA Step 1 diet.
840 D. O’BYRNE et al.
Fig. 2. Plasma T3 concentrations in subjects assigned to the placebo
(n ⫽ 13), ␣-tocotrienyl acetate (n ⫽ 13), ␥-tocotrienyl acetate (n ⫽ 12),
or ␦-tocotrienyl acetate (n ⫽ 13) supplementation groups. The data
presented represents the mean ⫾ standard deviation for the postsupple-
mentation phase: after subjects consumed 250 mg/d placebo or tocot-
rienyl acetate supplements for 8 weeks while continuing to follow the
AHA Step 1 diet. Presupplementation plasma T3 concentrations were
below the limits of detection. (A) Plasma ␣-T3 concentration (␮mol/l).
(B) Plasma ␥-T3 concentration (␮mol/l). (C) Plasma ␦-T3 concentra-
tion (␮mol/l). *p ⬍ .05, ***p ⬍ .001 indicates statistically significant
differences in postsupplementation plasma T3 concentrations com-
pared with placebo at 8 weeks.
LDL oxidative profile
The ability of T3 supplements to protect LDL against
Cu2⫹-induced oxidation was measured ex vivo, and as-
sessed by the length of time prior to onset of conjugated
diene formation (lag time), rate of oxidation, and the
maximum concentration of conjugated dienes formed
during the 5 h incubation (Table 7). No statistically
significant differences were detected in the LDL oxida-
tion profile between groups during the presupplementa-
tion phase, although it should be noted that the group
receiving ␣-tocotrienyl acetate supplements had slightly
shorter lag times and greater oxidation rates than the
other groups. The change in LDL oxidation profile was
calculated for each of the parameters from the post-
supplementation minus presupplementation values. Sup-
plementation with placebo, ␣-, ␥-, and ␦-tocotrienyl ac-
etates resulted in 4.7, 22, 8.8, and 11.6% increases in lag
time, respectively, however, only ␣-tocotrienyl acetate
supplements resulted in significantly greater increases in
lag time compared to those observed in the placebo
group (p ⬍ .001). Alpha-tocotrienyl acetate supplements
also significantly reduced the rate of LDL oxidation (p ⬍
.01). The effect of ␥-tocotrienyl acetate on the oxidative
indices of LDL supplements did not significantly differ
from the placebo. Delta-tocotrienyl acetate supplements
resulted in significantly greater reductions in the rate of
LDL oxidation and the amount of conjugated dienes
formed, compared to the placebo group (p ⬍ .05).
DISCUSSION
Our study is the first clinical trial to determine the
hypocholesterolemic and antioxidant effects following
supplementation with individual purified ␣-, ␥-, or ␦-to-
cotrienyl acetates. Consistent with other investigators’
observations, we did not detect appreciable amounts of
T3s in fasting plasma prior to supplementation [14,16,
28,29,36]. Subsequent to supplementation with the indi-
vidual tocotrienyl acetates, fasting plasma levels of the
respective isomers increased to detectable levels. This
indicates that the individual tocotrienyl acetates were
hydrolyzed, absorbed, and retained in the circulation.
Fasting plasma concentrations of T3 isomers were higher
after supplementation with the purified isomers than re-
ported in the literature for mixed T3/tocopherol supple-
ments [14,16,17]. We believe that competition with ␣-to-
copherol in the supplement used by others [15] is likely
to decrease the ability for the tocotrienols to be recog-
nized by the hepatic ␣-tocopherol transfer protein (␣-
TTP).
Of interest is the observation that plasma concentra-
tions of ␣-T3 were twice those of ␥-T3 and almost ten
times higher than ␦-T3 after subjects were supplemented
with the respective T3 isomers. The supplement dosage
was the same for all groups, which indicates that T3
isomers may have different rates of absorption, affinities
with ␣-TTP, and/or are retained in tissues to different
extents. Alpha-TTP has some affinity for ␣-T3 [37], but
it is not clear if this is significantly greater than for the
other T3 isomers. Alpha-T3 may be better absorbed than
the other tocotrienols; Ikeda et al. [38] have shown
preferential absorption of ␣-T3 compared to ␥- or ␦-T3
in rats. It is, however, unknown if the same is true in
humans.
 Tocotrienols and LDL oxidation 841

Table 5. Serum Lipid Profile of Subjects Receiving Placebo, ␣-, ␥-, or ␦-Tocotrienyl Acetate Supplements

Placebo ␣-Tocotrienyl acetate ␥-Tocotrienyl acetate ␦-Tocotrienyl acetate

(n ⫽ 13) (n ⫽ 13) (n ⫽ 12) (n ⫽ 13)

Total cholesterol (mg/dL)

Presupplementation 228.1 ⫾ 23.6 230.1 ⫾ 25.0 243.7 ⫾ 33.5 234.1 ⫾ 18.4
Postsupplementation 232.6 ⫾ 29.1 234.9 ⫾ 30.1 237.7 ⫾ 32.2 245.5 ⫾ 26.9*
LDL cholesterol (mg/dL)
Presupplementation 149.7 ⫾ 20.1 148.7 ⫾ 20.9 163.3 ⫾ 28.9 149.6 ⫾ 23.2
Postsupplementation 151.9 ⫾ 24.5 156.6 ⫾ 21.1 160.1 ⫾ 25.8 163.8 ⫾ 27.2*
HDL cholesterol (mg/dL)
Presupplementation 46.7 ⫾ 13.7 49.1 ⫾ 14.1 46.7 ⫾ 15.4 48.8 ⫾ 9.4
Postsupplementation 46.6 ⫾ 14.1 46.4 ⫾ 13.0 44.4 ⫾ 12.9 50.6 ⫾ 11.3
LDL:HDL cholesterol ratio
Presupplementation 3.42 ⫾ 0.93 3.24 ⫾ 0.93 3.84 ⫾ 1.36 3.15 ⫾ 0.67
Postsupplementation 3.45 ⫾ 0.86 3.61 ⫾ 1.02 3.85 ⫾ 1.13 3.37 ⫾ 0.82
Triglycerides (mg/dL)
Presupplementation 166.8 ⫾ 75.2 160.9 ⫾ 64.5 173.6 ⫾ 73.7 176.0 ⫾ 100.7
Postsupplementation 175.8 ⫾ 98.7 158.8 ⫾ 66.9 166.0 ⫾ 61.6 155.8 ⫾ 58.7

Values represent the mean ⫾ standard deviation of two fasting blood samples, taken with at least a 3 d interval between blood samples.
Presupplementation concentrations were determined from samples taken after subjects followed an AHA Step 1 diet for 4 weeks.
Postsupplementation concentrations were determined from samples taken after subjects consumed 250 mg/d placebo or tocotrienyl acetate
supplements for 8 weeks while continuing to follow the AHA Step 1 diet. *p ⬍ .05 indicates a statistically significant difference in the
response (postsupplementation minus presupplementation value for a given variable) to T3 supplementation compared to the reponse to
placebo.

In vitro experiments in mammalian cells have dem-
onstrated that T3s influence cholesterol synthesis by
post-transcriptional suppression of HMG-CoA reductase
and stimulation of apolipoprotein B degradation [4 – 6].
However, we found that compared to placebo, supple-
mentation with 250 mg/d of purified ␣-, ␥-, or ␦- toco-
trienyl acetate failed to lower total cholesterol, LDL
cholesterol, and apolipoprotein B concentrations in hy-
percholesterolemic subjects. Qureshi et al. [10,39] re-
ported that T3s have a dose-dependent effect on serum
cholesterol and LDL cholesterol; therefore, it is possible
that the dose of 250 mg/d of purified T3s was insufficient
to inhibit hepatic HMG-CoA reductase activity in hu-
mans. However, this explanation seems unlikely since
studies documenting the hypocholesterolemic effects of
T3 mixtures generally used a total T3 dose of ⬍ 220
mg/d [10 –12]. Furthermore, Qureshi et al [10,11] also
reported as much as a 31% reduction in serum total
cholesterol after hyperlipidemic individuals took 200
mg/d ␥-T3 for 4 weeks. We observed a nonstatistically
significant decline in serum total cholesterol and LDL
cholesterol levels after subjects took 250 mg/d ␥-tocot-
rienyl acetate supplements for 8 weeks. Surprisingly, a
significant increase in serum total cholesterol and LDL
cholesterol was observed after subjects consumed 250
mg/d ␦-tocotrienyl acetate supplements. These undesir-
able changes in the ␦-T3 group were not attributed to
factors such as weight or compliance with the diet and
supplement regimens. Since no other clinical trial has
investigated the effects of purified ␦-T3, it is difficult to
determine if the results observed are spurious or not.
It should be noted that only clinical trials with a
duration of less than 30 d have provided evidence that
␥-T3 or mixed T3/tocopherol preparations lower serum
cholesterol [10 –12], suggesting that T3s only have short-
term inhibitory effects on cholesterol synthesis. Other

Table 6. Serum Apolipoprotein B Profiles of Subjects Receiving Placebo, ␣-, ␥-, or ␦-Tocotrienyl Acetate Supplements

Placebo ␣-Tocotrienyl acetate ␥-Tocotrienyl acetate ␦-Tocotrienyl acetate

(n ⫽ 13) (n ⫽ 13) (n ⫽ 12) (n ⫽ 13)

Serum apolipoprotein B (mg/dL)

Presupplementation 113.7 ⫾ 12.7 111.5 ⫾ 13.2 122.7 ⫾ 15.6 113.8 ⫾ 14.7
Postsupplementation 113.6 ⫾ 13.3 116.1 ⫾ 16.1 121.9 ⫾ 19.5 118.5 ⫾ 19.2
LDL cholesterol/Apo B ratio
Presupplementation 1.32 ⫾ 0.12 1.33 ⫾ 0.14 1.33 ⫾ 0.12 1.32 ⫾ 0.19
Postsupplementation 1.35 ⫾ 0.22 1.35 ⫾ 0.10 1.32 ⫾ 0.15 1.39 ⫾ 0.18

Values represent the mean ⫾ standard deviation of fasting blood samples. Presupplementation concentrations were determined from
samples taken after subjects followed an AHA Step 1 diet for 4 weeks. Postsupplementation concentrations were determined from samples
taken after subjects consumed 250 mg/d placebo or tocotrienyl acetate supplement for 8 weeks while continuing to follow the AHA Step
1 diet.
842 D. O’BYRNE et al.

Table 7. LDL-Conjugated Diene Lag Time, Oxidation Rate, and LDL-Conjugated Diene Maximum Concentration of Subjects Receiving Placebo,
␣-, ␥-, or ␦-Tocotrienyl Acetate Supplements

Placebo ␣-Tocotrienyl acetate ␥-Tocotrienyl acetate ␦-Tocotrienyl acetate

(n ⫽ 13) (n ⫽ 13) (n ⫽ 12) (n ⫽ 13)

Presupplementation lag time (min) 55.2 ⫾ 12.8 51.6 ⫾ 8.7 57.3 ⫾ 10.2 58.8 ⫾ 11.7
Postsupplementation lag time (min) 57.8 ⫾ 11.8 62.9 ⫾ 8.2 62.3 ⫾ 9.4 65.6 ⫾ 9.2
Change in lag time (min) 2.6 ⫾ 4.5 11.3 ⫾ 4.6*** 5.1 ⫾ 4.7 6.8 ⫾ 13.9
Presupplementation oxidation rate (nmol/mg/min) 6.0 ⫾ 1.2 6.9 ⫾ 1.2 6.4 ⫾ 0.9 6.4 ⫾ 1.6
Postsupplementation oxidation rate (nmol/mg/min) 6.3 ⫾ 1.0 6.3 ⫾ 0.8 6.7 ⫾ 0.9 6.3 ⫾ 1.2
Change in oxidation rate (nmol/mg/min) 0.3 ⫾ 0.4 ⫺0.6 ⫾ 0.9** 0.3 ⫾ 1.2 ⫺0.1 ⫾ 1.1*
Presupplementation maximum concentration (nmol/mg) 394.8 ⫾ 37.8 427.4 ⫾ 55.7 438.8 ⫾ 49.9 447.7 ⫾ 62.4
Postsupplementation maximum concentration (nmol/mg) 406.5 ⫾ 25.9 430.1 ⫾ 45.4 446.1 ⫾ 49.5 410.7 ⫾ 61.9
Change in maximum concentration (nmol/mg) 11.7 ⫾ 20.1 2.7 ⫾ 22.0 7.3 ⫾ 26.0 ⫺37.0 ⫾ 65.6*

Values represent the mean ⫾ standard deviation. LDL (200 ␮g protein/ml) was incubated with 5 ␮M Cu2⫹ for 5 h at 37°C and monitored
continuously at 234 nm for the formation of conjugated dienes. Presupplementation values were determined from samples taken after subjects
followed an AHA Step 1 diet for 4 weeks. Postsupplementation values were determined from samples taken after subjects consumed 250 mg of
placebo or tocotrienyl acetate supplements for 8 weeks while continuing to follow the diet. *p ⬍ .05, **p ⬍ .01, ***p ⬍ .001 indicate a statistically
significant difference in the response (postsupplementation minus presupplementation value for a given variable) to T3 supplementation compared to
the response to placebo.

studies in which mixed T3/tocopherol supplements were
given for 6 weeks, 20 weeks, or 18 months failed to show
significant reductions in serum cholesterol or LDL cho-
lesterol [13,14,17]. However, these later studies may be
somewhat confounded by the relatively high content of
␣-tocopherol (29 –56% of the vitamin E content) in the
mixed T3 supplements, as well as the fact that subjects
did not specifically consume low-fat diets in all studies.
According to Qureshi et al. [9,10], the cholesterol-low-
ering effects of T3s are prevented by supplements con-
taining more than 15–20% ␣-tocopherol and by con-
sumption of diets high in fat, saturated fat, and
cholesterol.
The hyperlipidemic subjects in our study consumed
low-fat diets and took 250 mg/d of individual T3 isomer
supplements, free of ␣-tocopherol; yet, after 8 weeks of
this regimen, serum cholesterol levels had not decreased.
It must be emphasized that subjects in each group expe-
rienced a reduction in serum cholesterol during the equil-
ibration phase of the study after 4 weeks of following the
AHA Step 1 diet. The addition of the T3 supplements to
the dietary regimen did not provide further benefits.
Thus, we can not confirm the findings of short-term
clinical trials in which T3 supplements lowered total
cholesterol and LDL cholesterol. The design of our study
was such that we can not comment on the efficacy of T3
mixtures, and the possibility of a synergistic effect. How-
ever, our results indicate that when the diet is carefully
controlled and is typical of AHA Step 1 diet, supplemen-
tation with 250 mg/d of purified ␣-, ␥-, or ␦-tocotrienyl
acetate does not significantly lower serum cholesterol in
hypercholesterolemic patients.
In our study, subjects received supplements of 250 mg
of either ␣-, ␥-, or ␦-tocotrienyl acetates, yet there were
a striking differences in the resultant plasma T3 isomer
concentrations. Plasma ␣-T3 concentrations were twice
that of ␥-T3 and almost ten times that of ␦-T3. Suarna et
al. [29] found that when human volunteers were supple-
mented with Palmvitee capsules containing approxi-
mately the equal amount of ␣-T3 and ␥-T3, the resulting
LDL concentrations of ␣-T3 were 1.4- to 3.0-fold higher
than those of ␥-T3. In that study, vitamin E concentra-
tions were reported in as a ratio to cholesterol, not per ml
plasma. To our knowledge, no studies have reported
lipoprotein T3 concentrations. Although we did not mea-
sure LDL T3 concentrations due to limited sample size,
based on the observation that ␣- and ␥-tocopherols are
similarly distributed into plasma lipoproteins [40], it is
likely that LDL carries roughly 40% of the plasma T3.
An important finding in this study, is that supplemen-
tation with 250 mg/d ␣-tocotrienyl acetate provided sig-
nificant protection of LDL against copper-catalyzed ox-
idation. Compared to the changes observed in the
placebo group, supplementation with ␣-tocotrienyl ace-
tate resulted in significantly greater increases in LDL lag
time and greater reductions in the rate of LDL oxidation.
Correlations using pooled data from all groups revealed
a significant association between the change in plasma
␣-T3 concentration and the change in LDL lag time (r ⫽
0.273, p ⬍ .05) and LDL oxidation rate (r ⫽ ⫺0.340,
p ⬍ .02). No significant correlations were found between
other plasma T3 levels and indices of LDL oxidative
resistance. This suggests that in vivo ␣-T3 supplemen-
tation provides significant antioxidant protection com-
pared with supplementation with other T3s.
Based on the chemical structure of the T3s, the anti-
oxidant capacity was anticipated to be in the order of
␣-T3 ⬎ ␥-T3 ⬎ ␦-T3 [28]. Alpha-tocotrienyl acetate
supplements provided the greatest protection against
LDL oxidation, but the antioxidant protection of the LDL
from subjects taking other T3 supplements did not follow
the expected order. Gamma-tocotrienyl acetate supple-
 Tocotrienols and LDL oxidation
ments appeared to provide no significant antioxidant
protection to LDL. Suarna et al. [29] also found that
enrichment of LDL with ␣-T3 provided significantly
greater antioxidant protection than did ␥-T3. Unexpect-
edly, supplementation with ␦-tocotrienyl acetate pro-
vided a measure of antioxidant protection to LDL as
demonstrated by the lower rate of LDL oxidation and
decreased level of oxidation products. The clinical sig-
nificance of ␦-T3 antioxidant activity is unclear, since lag
time was not prolonged. Of the three LDL oxidation
indices, lag time is thought to be the most important
clinical marker because it is correlated with coronary
artery disease in several studies [41– 43]. It is also un-
clear whether the direct enrichment of the lipoproteins
with the T3 isomers, or a decrease in the potential oxi-
dizablility as a result of T3 supplementation was the
cause of the protective effect. Measurements of the LDL
fatty acid composition showed that there were no obvi-
ous changes in this parameter.
Alpha-T3 has similar if not greater antioxidant activ-
ity than ␣-tocopherol [27–29]. It is possible that supple-
mentation with ␣-tocotrienyl acetate may protect against
coronary artery disease in a similar manner as ␣-tocoph-
erol [18 –20], but clinical and epidemiological data are
lacking. Tomeo et al. [13] have provided promising
preliminary evidence that supplementation with ␣-T3,
␥-T3 (total T3 of 240 mg/d), and ␣-tocopherol (93 mg/
d), led to regression in carotid atherosclerosis in hyper-
lipidemic patients. The exact mechanism eliciting regres-
sion in atherosclerotic plaque is unclear, since serum
cholesterol levels were not altered, although significant
decreases in serum oxidation products were noted. The
antioxidant effects of ␣-T3 and ␣-tocopherol in the sup-
plements may have played a role in the observed carotid
atherosclerotic regression.
Previously, our laboratory has shown that 8 –12
weeks of supplementation with 400 – 800 IU/d ␣-to-
copherol results in an approximate 24 –37 ␮mol/l in-
crease in plasma ␣-tocopherol and a corresponding
27–79% increase in LDL oxidative resistance as man-
ifested by prolonged lag time [23,25,44]. In compari-
son, the findings of the present study indicate that
supplementation with 250 mg/d of ␣-tocotrienyl ace-
tate increases plasma concentrations by about 1
␮mol/l, yet results in a significant 22% increase in
LDL resistance to oxidation. The in vivo antioxidant
potency of ␣-T3 appears to be quite impressive. The
superior antioxidant activity of ␣-T3 is thought to be
related to (i) its higher recycling efficiency from chro-
manoxyl radicals; (ii) its more uniform distribution in
the membrane bilayer; and (iii) its disordering effect
on the membrane lipids, which enhances the interac-
tion between the chromanols and lipid radicals [28,29,
45]. However, Suarna et al. [29] failed to observe the
843
superior antioxidant activity of ␣-T3 in LDL, but
found the antioxidant protection of ␣-T3 to be com-
parable to ␣-tocopherol. It must be emphasized that
the supplementation experiments we have conducted
do not directly compare the antioxidant activities of
tocopherols and T3s in LDL. Although it appears that
␣-T3 is a potent antioxidant in vivo, it is uncertain
whether supplementation with ␣-tocotrienyl acetate
actually confers superior protection against LDL oxi-
dation compared to doses of 400 – 800 IU ␣-tocoph-
erol, since there appears to be a limited capacity to
retain ␣-T3 in the circulation.
In conclusion, our results indicate that supplements
of purified ␣-, ␥-, or ␦-tocotrienyl acetates, when
given in the dosage of 250 mg/d for 8 weeks, do not
provide the dual benefits of cholesterol reduction and
antioxidant protection in hyperlipidemic patients, as
suggested by other investigators. Contrary to the find-
ings of in vitro studies or short-term clinical trials
using mixed T3/tocopherols or ␥-T3 supplements, we
found that supplementation with purified tocotrienyl
acetates did not result in significant reductions in
serum cholesterol, LDL cholesterol, or apolipoprotein
B levels. At the dosage given, only ␣-tocotrienyl ac-
etate supplements provided significant protection to
LDL against Cu2⫹-induced oxidation. Tomeo et al.
[13] has shown that supplementation with T3s pro-
vides some degree of cardiovascular protection. Our
study suggests that these therapeutic benefits may be
derived from ␣-T3 in the T3 supplement, although the
presence of ␣-tocopherol in the supplement used by
Tomeo et al. is also an important contributing factor.
In order to determine if ␣-T3 or ␣-tocopherol is the
clinically relevant factor, placebo-controlled long-
term trials comparing the antioxidant activity, athero-
sclerotic regression, and clinical outcomes of the two
supplements in cardiac patients are required.
Acknowledgements — The authors wish to thank Eleanor Goodwin
MT, Helen Mancuso MT, Steven Lee MT, the phlebotomy staff of
Pathology Client Services, and the staff of Clinical Chemistry at
Parkland Hospital in Dallas, Texas; Suben Naidu MD, Mita Patel BS,
Runna Awil BS, and Anh Nguyen MS for technical assistance; and
Beverly Adams-Huet for statistical expertise. This research was sup-
ported by a grant from BASF (Germany) and National Institutes of
Health grants MO1-RR 00633, RO1-AT00005 and K24 AT00596.
REFERENCES
[1] Ross, R. Atherosclerosis—an inflammatory disease. New Engl.
J. Med. 340:115–126; 1999.
[2] Pederson, T. R. Statin trials and goals of cholesterol-lowering
therapy after AMI. Am. Heart J. 138:177–182; 1999.
[3] Rifkind, B. M. Clinical trials of reducing low-density lipoprotein
concentrations. Endocrinol. Metab. Clin. North. Am. 27:585–595;
1998.
[4] Pearce, B. C.; Parker, R. A.; Deason, M. E.; Qureshi, A. A.;
Wright, J. J. Hypocholesterolemic activity of synthetic and natural
tocotrienols. J. Med. Chem. 35:3595–3606; 1992.
844 D. O’BYRNE et al.
[5] Parker, R. A.; Pearce, B. C.; Clark, R. W.; Gordon, D. A.; Wright,
J. J. Tocotrienols regulate cholesterol production in mammalian
cells by post-transcriptional suppression of 3-hydroxy-3-methyl-
glutaryl-coenzyme A reductase. J. Biol. Chem.
268:11230 –11238; 1993.
[6] Pearce, B. C.; Parker, R. A.; Deason, M. E.; Dischino, D. D.;
Gillespie, E.; Qureshi, A. A.; Volk, K.; Wright, J. J. Inhibitors of
cholesterol biosynthesis. 2. Hypocholesterolemic and antioxidant
activities of benzopyran and tetrahydronaphthalene analogues of
the tocotrienols. J. Med. Chem. 37:526 –541; 1994.
[7] Theriault, A.; Wang, Q.; Gapor, A.; Adeli, K. Effects of ␥-toco-
trienol on apoB synthesis, degradation, and secretion in HepG2
cells. Arterioscler. Thromb. Vasc. Biol. 19:704 –712; 1999.
[8] Qureshi, A. A.; Pearce, B. C.; Nor, R. M.; Gapor, A.; Peterson,
D. M.; Elson, C. E. Dietary ␣-tocopherol attenuates the impact of
␥-tocotrienol on hepatic 3-hydroxy-3-methylglutaryl coenzyme A
reductase activity in chickens. J. Nutr. 126:389 –394; 1996.
[9] Qureshi, A. A.; Qureshi, N.; Hasler-Rapacz, J. O.; Weber, F. E.;
Chaudhary, V.; Crenshaw, T. D.; Gapor, A.; Ong, A. S.; Chong,
Y. H.; Peterson, D.; Rapacz, J. Dietary tocotrienols reduce con-
centrations of plasma cholesterol, apolipoprotein B, thromboxane
B2, and platelet factor 4 in pigs with inherited hyperlipidemias.
Am. J. Clin. Nutr. 53:1042S–1046S; 1991.
[10] Qureshi, A. A.; Qureshi, N.; Wright, J. J.; Shen, Z.; Kramer, G.;
Gapor, A.; Chong, Y. H.; DeWitt, G.; Ong, A.; Peterson, D. M.;
Bradlow, B. A. Lowering of serum cholesterol in hypercholester-
olemic humans by tocotrienols (palmvitee). Am. J. Clin. Nutr.
53:1021S–1026S; 1991.
[11] Qureshi, A. A.; Bradlow, B. A.; Brace, L.; Manganello, J.; Peter-
son, D. M.; Pearce, B. C.; Wright, J. J.; Gapor, A.; Elson, C. E.
Response of hypercholesterolemic subjects to administration of
tocotrienols. Lipids 30:1171–1177; 1995.
[12] Tan, D. T.; Khor, H. T.; Low, W. H.; Ali, A.; Gapor, A. Effect of
a palm-oil-vitamin E concentrate on the serum and lipoprotein
lipids in humans. Am. J. Clin. Nutr. 53:1027S–1030S; 1991.
[13] Tomeo, A. C.; Geller, M.; Watkins, T. R.; Gapor, A.; Bieren-
baum, M. L. Antioxidant effects of tocotrienols in patients with
hyperlipidemia and carotid stenosis. Lipids 30:1179 –1183; 1995.
[14] Mensink, R. P.; van Houwelingen, A. C.; Kromhout, D.; Hornstra,
G. A vitamin E concentrate rich in tocotrienols had no effect on
serum lipids, lipoproteins, or platelet function in men with mildly
elevated serum lipid concentrations. Am. J. Clin. Nutr. 69:213–
219; 1999.
[15] Hosomi, A.; Arita, M.; Sato, Y.; Kiyose, C.; Ueda, T.; Igarashi,
O.; Arai, H.; Inoue, K. Affinity for ␣-tocopherol transfer protein
as a determinant of the biological activities of vitamin E analogs.
FEBS Lett. 409:105–108; 1997.
[16] Hayes, K. C.; Pronczuk, A.; Liang, J. S. Differences in the plasma
transport and tissue concentrations of tocopherols and tocotrien-
ols: observations in humans and hamsters. Proc. Soc. Exp. Biol.
Med. 202:353–359; 1993.
[17] Wahlqvist, M. L.; Krivokuca-Bogetic, Z.; Lo, C. S.; Hage, B.;
Smith, R.; Lukito, W. Differential serum responses of tocopherols
and tocotrienols during vitamin supplementation in hypercholes-
terolemic individuals without change in coronary risk factors.
Nutr. Res. 12:S181–S201; 1992.
[18] Rimm, E. R.; Stampfer, M. J.; Ascherio, A.; Giovannucci, E.;
Colditz, G. A.; Willett, W. C. Vitamin E consumption and the risk
of coronary heart disease in men. New Engl. J. Med. 328:1450 –
1456; 1993.
[19] Stampfer, M.; Hennekens, C.; Manson, J.; Colditz, G.; Rosner, B.;
Willett, W. Vitamin E consumption and the risk of coronary
disease in women. New Engl. J. Med. 328:1444 –1449; 1993.
[20] Stephens, N. G.; Parsons, A.; Schofield, P. M.; Kelly, F.; Cheese-
man, K.; Mitchinson, M. J. Randomized controlled trial of vita-
min E in patients with coronary disease: Cambridge Heart Anti-
oxidant Study (CHAOS). Lancet 347:781–786; 1996.
[21] Dietary supplementation with n-3 polyunsaturated fatty acids and
vitamin E after myocardial infarction: results of the GISSI-Pre-
venzione trial. Lancet 354:447– 455; 1999.
[22] Yusuf, S.; Dagenais, G.; Pogue, J.; Bosch, J.; Sleight, P. Vitamin
E supplementation and cardiovascular events in high-risk pa-
tients. The Heart Outcomes Prevention Evaluation Study Inves-
tigators. New Engl. J. Med. 342:154 –160; 2000.
[23] Jialal, I.; Fuller, C. J.; Huet, B. A. The effect of ␣-tocopherol
supplementation on LDL oxidation. A dose-response study. Ar-
terioscler. Thromb. Vasc. Biol. 15:190 –198; 1995.
[24] Fuller, C. J.; Chandalia, M.; Garg, A.; Grundy, S. M.; Jialal, I.
RRR-alpha-tocopheryl acetate supplementation at pharmacologic
doses decreases low-density lipoprotein oxidative susceptibility
but not protein glycation in patients with diabetes mellitus. Am. J.
Clin. Nutr. 63:753–759; 1996.
[25] Devaraj, S.; Adams-Huet, B.; Fuller, C. J.; Jialal, I. Dose-response
comparison of RRR-␣-tocopherol and all-racemic ␣-tocopherol
on LDL oxidation. Arterioscler. Thromb. Vasc. Biol. 17:2273–
2279; 1997.
[26] Witztum, J. L.; Steinberg, D. Role of oxidized low-density li-
poprotein in atherogenesis. J. Clin. Invest. 88:1785–1792; 1991.
[27] Serbinova, E.; Kagan, V.; Han, D.; Packer, L. Free radical recy-
cling and intramembrane mobility in the antioxidant properties of
␣-tocopherol and ␣-tocotrienol. Free Radic. Biol. Med. 10:263–
275; 1991.
[28] Kamal-Eldin, A.; Appelqvist, L. A. The chemistry and antioxidant
properties of tocopherols and tocotrienols. Lipids 31:671–701;
1996.
[29] Suarna, C.; Hood, R. L.; Dean, R. T.; Stocker, R. Comparative
antioxidant activity of tocotrienols and other natural lipid-soluble
antioxidants in a homogeneous system, and in rat and human
lipoproteins. Biochim. Biophys. Acta 1166:163–170; 1993.
[30] Schuep, W.; Rettenmaier, R. Analysis of vitamin E homologs in
plasma and tissue: high-performance liquid chromatography.
Methods Enzymol. 234:294 –302; 1994.
[31] Lepage, G.; Roy, C. C. Direct transesterification of all classes of
lipids in a one-step reaction. J. Lipid Res. 27:114 –120; 1986.
[32] Lowry, O. H.; Rosebrough, J. J.; Farr, A. L.; Randall, R. J. Protein
measurement with the folin phenol reagent. J. Biol. Chem. 193:
265–275; 1951.
[33] Podda, M.; Weber, C.; Traber, M. G.; Packer, L. Simultaneous
determination of tissue tocopherols, tocotrienols, ubiquinols, and
ubiquinones. J. Lipid Res. 37:893–901; 1996.
[34] Kleinveld, H. A.; Hak-Lemmers, H. L.; Stalenhoef, A. F.;
Demacker, P. N. Improved measurement of low-density lipopro-
tein susceptibility to copper-induced oxidation: application of a
short procedure for isolating low-density lipoprotein. Clin. Chem.
38:2066 –2072; 1992.
[35] Esterbauer, H.; Gebicki, J.; Puhl, H.; Jurgens, G. The role of lipid
peroxidation and antioxidants in oxidative modification of LDL.
Free Radic. Biol. Med. 13:341–390; 1992.
[36] Chow, C. K. Distribution of tocopherols in human plasma and red
blood cells. Am. J. Clin. Nutr. 28:756 –760; 1975.
[37] Sato, Y.; Hagiwara, K.; Arai, H.; Inoue, K. Purification and
characterization of the ␣-tocopherol transfer protein from rat
liver. FEBS Lett. 288:41– 45; 1991.
[38] Ikeda, I.; Imasato, Y.; Sasaki, E.; Sugano, M. Lymphatic transport
of ␣-, ␥-, and ␦-tocotrienols and ␣-tocopherol in rats. Int. J. Vit.
Nutr. Res. 66:217–221; 1996.
[39] Qureshi, A. A.; Burger, W. C.; Peterson, D. M.; Elson, C. E. The
structure of an inhibitor of cholesterol biosynthesis isolated from
barley. J. Biol. Chem. 261:10544 –10550; 1986.
[40] Traber, M. G.; Cohn, W.; Muller, D. P. R. Absorption, transport,
and distribution to tissues. In: Packer, L.; Fuchs, J., eds. Vitamin
E in health and disease. New York: Marcel Dekker, Inc.; 1993:
35–52.
[41] Halevy, D.; Thiery, J.; Nagel, D.; Arnold, S.; Erdmann, E.;
Hofling, B.; Cremer, P.; Seidel, D. Increased oxidation of LDL in
patients with coronary artery disease is independent from dietary
vitamins E and C. Arterioscler. Thromb. Vasc. Biol. 17:1432–
1437; 1997.
[42] Regnstrom, J.; Nilsson, J.; Tornvall, P.; Landou, C.; Hamsten, A.
Susceptibility to low-density lipoprotein oxidation and coronary
atherosclerosis in man. Lancet 339:1183–1186; 1992.
[43] Cominacini, L.; Garbin, U.; Pastorino, A. M.; Davoli, A.; Campag-
 Tocotrienols and LDL oxidation 845

nola, M.; De Santis, A.; Pasini, C.; Faccini, G. B.; Trevisan, M. T.;
Bertozzo, B. L.; Pasini, F.; LoCascio, V. Predisposition to LDL
oxidation in patients with and without angiographically established
coronary artery disease. Atherosclerosis 99:63–70; 1993.
[44] Islam, K. N.; O’Byrne, D.; Devaraj, S.; Palmer, B.; Grundy,
S. M.; Jialal, I. Alpha-tocopherol supplementation decreases the
oxidative susceptibility of LDL in renal failure patients on dial-
ysis therapy. Atherosclerosis 150:217–224; 2000.
[45] Serbinova, E.; Kagan, V.; Han, D.; Packer, L. Free radical recy-
cling and intramembrane mobility in the antioxidant properties of
␣-tocopherol and ␣-tocotrienol. Free Radic. Biol. Med. 10:263–
275; 1991.