![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
2
Departments of
*
Medicine,
**
Pathology and
Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
2To whom correspondence should be addressed.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tocopherol
(mixed isomers); 0.5 g/100 g palm tocopherols (palm-E; 33%
-tocopherol, 16.1% -tocotrienol, 2.3% ß-tocotrienol, 32.2%
-tocotrienol, 16.1% 
-tocotrienol); 1.5 g/100 g palm-E; or 0.01
g/100 g palm-carotenoids (58% ß-carotene, 33% -carotene, 9%
other carotenoids). Compared with mice fed the control diet, plasma
cholesterol was fourfold greater in mice fed the atherogenic diet. Mice
fed the 1.5 g/100 g palm-E supplement had 60% lower plasma
cholesterol than groups fed the other atherogenic diets. Mice fed the
atherogenic diet had markedly higher VLDL, intermediate density
lipoprotein (IDL) and LDL cholesterol and markedly lower HDL
cholesterol than the controls. Lipoprotein patterns in mice
supplemented with -tocopherol or palm carotenoids were similar to
those of the mice fed the atherogenic diet alone, but the pattern in
mice supplemented with 1.5 g/100 g palm-E was similar to that of
mice fed the control diet. In mice fed the atherogenic diet, the
hepatic cholesterol plus cholesterol ester concentration was 4.4-fold
greater than in mice fed the control diet. Supplementing with 1.5 g/100
g palm-E lowered hepatic cholesterol plus cholesterol ester
concentration 66% compared with the atherogenic diet alone. Mice fed
the atherogenic diet had large atherosclerotic lesions at the level of
the aortic valve. With supplements of 0.5 g/100 g palm-E or 1.5
g/100 g palm-E, the size of the lesions was 92 or 98% smaller,
respectively. The 0.5 g/100 g -tocopherol and palm carotenoid
supplements had no effect. Supplements did not alter mRNA abundance for
apolipoproteins A1, E, and C3. The beneficial effect of tocotrienols on
atherogenesis, the plasma lipoprotein profile and accumulation of
hepatic cholesterol esters cannot be attributed to their antioxidant
properties.
KEY WORDS: • antioxidants • ß-carotene • vitamin E • atherosclerosis • apoE gene knockout mice
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Ross 1993
, Witztum 1994
).
The oxidation of lipoproteins is believed to play a major role in
stimulating the processes that lead to plaque formation; however, it
has not been determined directly whether preventing lipoprotein
oxidation will decrease atherosclerotic plaque formation. Further, it
has not been determined whether the proposed effects of vitamin E or
ß-carotene on atherosclerosis are due to antioxidant properties of
these vitamins. Epidemiologic studies and studies in animal models of
atherosclerosis have shown variable responses to vitamin E and
ß-carotene. Vitamin E supplements are positively associated with a
lower risk of atherosclerotic disease (Rimm et al. 1993
,
Stampfer et al. 1993
), but intervention studies with
antioxidants have provided conflicting results. In human studies,
supplementation with -tocopherol resulted in fewer myocardial events
or deaths (Stampfer et al. 1993
, Stephens et al. 1996
), but benefits from ß-carotene have not been shown
(Hennekens et al. 1996
, Omenn et al. 1996
). Animal studies with -tocopherol have been similarly
inconclusive (Upston et al. 1999
). The antioxidant
Probucol decreases atherosclerotic lesion formation in Wantanabe
heritable hyperlipidemic rabbits (Carew et al. 1987
),
but paradoxically increases lesion formation in apolipoprotein E
(apoE)3
knockout mice (Zhang et al. 1997
), whereas the
antioxidant, N,N'-diphenyl-1,4-phenylenediamine,
decreases aortic plaque formation in these mice (Tangirala et al. 1995
). In other studies of homozygous apoE knockout mice,
0.05 g/100 g -tocopherol or 0.05 g/100 g ß-carotene had no effect
on the spontaneous formation of atherosclerotic lesions (Shaish et al. 1999
), whereas 2000 IU/kg vitamin E (
0.2
g/100 g) reduced the progression of atherosclerotic lesions
(Pratico et al. 1998
). Of the carotenoids,
all-trans-ß-carotene but not 9-cis-ß-carotene
decreased atherogenic plaques in New Zealand White rabbits fed an
atherogenic diet (Shaish et al. 1995
).
To test the effects of vitamin E and ß-carotene on the development of
atherosclerotic lesions in a systematic manner, we used adult female
apoE +/- mice, which are deficient in apoE, the high affinity ligand
for the LDL receptor (Weisgraber 1994
). ApoE is the
major ligand for removing cholesterol-rich chylomicra and VLDL
remnants from the blood. Unlike homozygous apoE -/- mice, which
develop atheromas when fed a rodent nonpurified diet, heterozygous apoE
+/- mice develop atherosclerotic lesions only when they are fed a diet
that is high in saturated fat and cholesterol (Zhang et al. 1994
).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animal protocols were approved by the University of North Carolina at
Chapel Hill Institutional Animal Care and Use Committee. ApoE +/-
female mice were bred on site from adult female C57BL/6J (B6) mice
(Charles River Laboratory, Wilmington, MA) and male homozygous apoE
-/- mice. The male apoE -/- mice were the result of backcrossing
B6:129 F1 heterozygotes(Piedrahita et al. 1992
) to B6
for six generations before intercrossing to obtain the apoE +/-
heterozygotes. The mice were maintained on a 12-h light:dark cycle.
After weaning, the pups had free access to a nonpurified diet until
they were 8–12 wk old (see below). Females, in groups of 4, were then
fed one of six diets (see below) for 3 mo. The mice were weighed at the
start (d 0) and the completion (d 90) of the experimental diets. At the
beginning of the study, each group contained 16 mice except the 1.5
g/100 g palm-E group which contained 14. Deaths in each group
occurred in association with anesthesia used during blood drawing.
There were 3 deaths in the atherogenic group, 1 in the 0.5 g/100 g
palm-E group, 2 in the 1.5 g/100 g palm-E group and 1 in the
-tocopherol group.
Diets.
The nonpurified diet (Prolab RMH 3000, Purina Mills, St Louis, MO)
contained 5.3 g/100 g triglyceride and 0.2 g/100 g cholesterol. The
atherogenic diets were prepared by Dyets (Bethlehem, PA) to contain
18.5 g/100 g triglyceride, 1.25 g/100 g cholesterol and 0.5 g/100 g
sodium cholate and supplemented with either 0.5 g/100 g
(+)--tocopherol (mixed isomers; Sigma Chemical, St. Louis, MO); 0.5
g/100 g mixed palm-tocopherols (palm-E) (33% -tocopherol,
16.1% -tocotrienol, 2.3% ß-tocotrienol, 32.2% -tocotrienol,
16.1% -tocotrienol); 1.5 g/100 g palm-E; or 0.01g/100 g mixed palm
carotenoids (58% ß-carotene, 33% -carotene, 9% other
carotenoids). The palm-E supplements were added at 0.5 g/100 g to
be equivalent as vitamin E to the -tocopherol supplement, and at 1.5
g/100 g, which contains 0.5 g/100 g -tocopherol. The
palm-derived supplements were kindly supplied by Dr. Kalyana
Sundram of the Palm Oil Research Institute of Malaysia. The mice had
free access to food and water during the study period.
Lipid analyses.
Plasma for total cholesterol and triglyceride was obtained at d 0 and 90 of the study. The mice were deprived of food overnight, then anesthetized with 0.2–0.4 mL of 20 g/L tribromoethanol (Avertin, Aldrich Chemical, Milwaukee, WI). Heparinized whole blood was placed in 1.5-mL Eppendorf tubes containing 10 µL of 10 mmol/L EDTA. The blood was then centrifuged for 10 min at 12,000 x g at 4°C. Plasma was assayed for triglyceride (GPO-Trinder kit, #339–20, Sigma Chemical) and cholesterol (Cholesterol CII kit, #276–64909, Wako Pure Chemical, Osaka, Japan) with colorimetric enzyme methods.
Lipoprotein separation.
Plasma lipoproteins were separated by size using fast-performance liquid chromatography (FPLC) (Amersham Pharmacia Biotech, Piscataway, NJ). The system fitted with a Superose-6 gel column (Pharmacia) was equilibrated with PBS containing 20 g sodium azide/L. Then 100 µL of mouse serum combined from four mice in an experimental group was loaded onto the column. The sample was run at 0.4 mL/min, and 0.5-mL fractions were collected. After the first 9 mL were discarded, the remainder of the fractions were assayed for total cholesterol as described above. Briefly, the samples were dried, dissolved in 100 µL of the cholesterol reagent as above and read at 500 nm. Absorbance at 500 nm provides a measure of relative amounts of cholesterol in each fraction.
Tissue preparation.
At the end of each treatment, mice were anesthetized with an
intraperitoneal injection of 2–2-2-tribromoethanol. After blood was
obtained, the heart and arterial tree were perfused with 4% phosphate
buffered paraformaldehyde (pH 7.4) under physiologic pressure
(Zhang et al. 1994
). The perfused hearts were stored in
fresh paraformaldehyde until sectioned. The proximal aorta and aortic
sinus were cryosectioned serially and stained with Sudan IVB (Fisher
Scientific, Pittsburgh, PA).
Livers from control, atherogenic and 1.5 g/100 g palm-E diet groups were removed after the above perfusion and stored in fresh paraformaldehyde for 24 h. The paraformaldehyde solution was changed and the liver was soaked for another 24 h. The storage solution was then replaced with 300 g/L sucrose in 0.1 mol/L phosphate buffer (pH 2.7) solution; the tissue was frozen, sectioned and stained with oil red O and counterstained with hematoxylin.
Morphometric analysis.
Lesion size represents an average of the lesions of four sections
spanning the proximal aorta to the aortic sinus at the appearance of
three complete valve leaflets (Reddick et al. 1994
).
Individual lesion sizes were determined by quantifying the
lesion-covered areas on the aortic vessel walls (NIH image 1.57
program; National Institutes of Health, Bethesda, MD).
mRNA analyses.
RNA was prepared from liver that had been flash-frozen in liquid
nitrogen. RNA was extracted into 4 mol/L guanidinium isothiocyanate
containing 0.5% ß-mercaptoethanol, isolated (Chirgwin et al. 1979
), subjected to formaldehyde gel electrophoresis
(Lehrach et al. 1977
) and transferred to nitrocellulose.
The mRNA abundance was determined by Northern blot analysis
(Pullinger et al. 1989
). RNA blots containing 20
µg of total RNA were electrophoresed on a 1.2%
agarose formaldehyde gel and blotted onto a nylon transfer membrane
(Hybound-H+, Amersham Pharmacia Biotech) in the presence of high salt.
After transfer, RNA was fixed to the membrane under UV light for 5 min.
Probes were labeled with [32P]dCTP (Amersham) by a
random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN)
and then purified on a G-25 Sephadex Column (Boehringer Mannheim). The
blot was prehybridized in a buffer containing 0.5 mol/L sodium
phosphate and 7% SDS at 65°C for 30 min and hybridized with a
32P-labeled probe in 5 mL of the same buffer at 65°C
overnight. The membrane was washed with a primary buffer (2X SSC, 0.5%
SDS) at 65°C for 30 min, washed with a secondary buffer (0.5X SSC,
0.5% SDS) at 65°C for 2–10 min, then exposed to X-ray film
overnight at -80°C. Filters were hybridized sequentially after
washing. Probes for apoA1 and apoC3 (Maeda et al. 1994
)
and for apoE and GAPDH (Sullivan et al. 1997
) were
described previously. Relative amounts of mRNA were assessed by using
an imaging program (NIH Image) and normalizing to the amounts of mRNA
for glyceraldehyde phosphate dehydrogenase.
Liver lipids.
Unperfused liver was removed and flash-frozen in liquid nitrogen.
Lipids in weighed portions of liver were extracted (Bligh and Dyer 1959
). To measure total cholesterol (free plus cholesterol
esters), lipid aliquots were resuspended in 0.2 mL 10% Triton X-100 in
isopropanol before measurement as described by the manufacturer
(Cholesterol CII kit, Wako Pure Chemical). Triacylglycerol and
phospholipid were measured by colorimetric analysis of glycerol
(Fletcher 1968
) and phosphate (Bartlett 1959
), respectively.
Statistical analysis.
Treatment comparisons were performed for mice fed the atherogenic
diets. For atherogenic lesions, weight, plasma cholesterol and plasma
triglycerides, comparisons were performed after log transformation
using ANOVA methods with Scheffé’s test. Log transformation
was used because of the variability of the standard deviations among
the groups. Data for liver cholesterol, triglyceride and phospholipid
were analyzed using the General Linear Models procedure of SAS,
appropriate for a completely randomized design (SAS 1989
). Treatment differences were assessed using a protected
Least Significant Difference test (Steel et al. 1997
) at
P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mouse weights did not differ among groups at 90 d (P
= 0.28) (Table 1
). Although plasma triglyceride concentrations were lower in most of the
atherogenic diet groups compared with controls, the differences were
not significant. In mice fed the atherogenic diet, plasma cholesterol
concentration was fourfold greater than in mice fed the control diet.
When the atherogenic diet was supplemented with -tocopherol, palm
carotenoids or 0.5 g/100 g palm-E, plasma cholesterol tended to be
an additional 20–33% greater. In contrast, compared with mice fed the
atherogenic diet alone, plasma cholesterol was 58% lower when mice
were fed the atherogenic diet supplemented with 1.5 g/100 g
palm-E (Table 1)
.
|
When apoE +/- mice were fed the nonpurified control diet, the major
lipoprotein peak was HDL (fractions 17–24) (Fig. 1
). The atherogenic diet changed this pattern radically, i.e., the major
cholesterol-containing fractions were VLDL (fractions 3–6),
intermediate density lipoprotein (IDL) (fractions 6–8), and LDL
(fractions 8–16), and the HDL peak decreased. Supplementation with
palm carotenoids had no effect on this pattern but 0.5 g/100 g
-tocopherol appeared to decrease the LDL peak and the 0.5 g/100 g
palm-E supplements appeared to decrease both IDL and LDL peaks,
although sufficient samples were not available for statistical
analysis. In mice supplemented with 1.5 g/100 g palm-E, however,
the VLDL, IDL and LDL peaks were markedly diminished and the HDL peak
was restored. The changes in the areas under the curve suggest that the
numbers of VLDL, IDL and LDL particles decreased, consistent with the
decrease in serum cholesterol.
|
The apoE +/- mice fed the nonpurified control diet had no arterial
atherosclerotic lesions. As was reported previously (Zhang et al. 1997
), the atherogenic diet resulted in large lesions in
the proximal aorta (Fig. 2
). The size of the diet-induced atheromas was larger than reported
previously in heterozygous mice that had not been backcrossed
(Zhang et al. 1994
). The average size of the
diet-induced lesions in the eight mice studied was 166,391 ± 14,086 µm2 (mean ± SEM). Palm carotenoid supplementation had no effect on
lesion size (144,452 ± 28,086
µm2). Although supplementation with
-tocopherol appeared to decrease lesion size 55% (75,375 ± 11,411 µm2), this decrease was not
significant. In mice that were fed the atherogenic diet plus the
palm-E supplements, however, lesion sizes were diminished
substantially compared with mice fed the atherogenic diet alone.
Palm-E at 0.5 g/100 g and 1.5 g/100 g reduced lesion sizes 92%
(13,573 ± 5,161 µm2) and 97%
(3,835 ± 613 µm2),
respectively. (Some groups contained fewer samples because of technical
problems with the staining that became apparent too late in the
analysis to be rectified.) The atherogenic diet induced multilayered
early fatty streaks with both superficial and intimal foam cells
(Fig. 3
), as reported previously (Zhang et al. 1994
). Similar
lesions were observed in the mice fed the atherogenic diet supplemented
with palm carotenoids. Lesion size and thickness were diminished in the
mice supplemented with 0.5 g/100 g palm-E, and the aortic histology
in mice supplemented with 1.5 g/100 g palm-E was virtually
indistinguishable from that of heterozygote mice fed the nonpurified
control diet.
|
|
Because the livers of the mice fed the atherogenic diet alone appeared
pale and fatty, whereas those of mice consuming the atherogenic diet
plus 1.5 g/100 g palm-E appeared normal, we examined liver
histology and analyzed liver lipids. Oil red O, which interacts with
neutral lipids such as triglyceride and cholesteryl esters, stained
numerous lipid droplets in livers from mice fed the atherogenic diet
and fewer droplets from the mice supplemented with 1.5 g/100 g
palm-E (Fig. 4
). In the 1.5 g/100 g palm-E group, lipid droplets were well formed,
discrete and smaller. The unsupplemented atherogenic group had large
lakes of lipid accumulation with clefts suggestive of cholesterol
crystals. The cholesterol content of the droplets was confirmed by
chemical analysis. In mice fed all of the atherogenic diets, hepatic
triglyceride contcentration was 30% lower than in mice fed the
nonpurified control diet. The phospholipid concentration in liver did
not differ among the groups (Table 2
). In contrast, the atherogenic diet caused the concentration of liver
free cholesterol plus cholesteryl ester to increase 4.4-fold.
Supplemented palm carotenoids, -tocopherol, and 0.5 g/100 g
palm-E had no effect on the high liver cholesterol concentration
produced by the atherogenic diet; however, the 1.5 g/100 g palm-E
supplement reduced the hepatic concentration of cholesterol plus
cholesterol ester 66%.
|
|
No significant differences were observed in liver mRNA abundance of apolipoproteins A1, E and C3 (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tocopherol or
palm-carotenoids had no effect. Of the diets that contained
palm-E, the more striking effect occurred in the 1.5 g/100 g
palm-E group. This effect was probably due to the content of
tocotrienols in the supplement, which contained as much -tocopherol
(33%) as was present in the 0.5 g/100 g -tocopherol supplement.
Moreover, the 0.5 g/100 g palm-E supplement was more effective than
the 0.5 g/100 g -tocopherol supplement. Tocotrienols are similar
in structure to tocopherols, but contain three double bonds in the
isoprenoid chain. In addition to their antioxidant properties
(Kamal-Eldin and Appelqvist 1996
), tocotrienols lower
total and LDL cholesterol in hamsters (Khor and Chieng 1996
) and rats (Watkins et al. 1993
).
Tocotrienols have been shown to decrease serum cholesterol in some
(Qureshi et al. 1991
, 1995
and 1997
), but not all
(Mensink et al. 1999
, Tan et al. 1991
)
human studies. These reported effects on serum cholesterol may have
occurred because tocotrienols suppress 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase activity and decrease the secretion of apoB.
Tocotrienols both decrease translation of HMG-CoA reductase mRNA
(Parker et al. 1993
) and increase degradation of the
reductase protein (Pearce et al. 1992
). They also
increase proteosome-mediated degradation of apolipoprotein B so
that less is secreted in VLDL (Wang et al. 1998
).
However, the effects on HMG-CoA reductase that would lead to
decreased synthesis and secretion of VLDL cholesterol are unlikely to
be relevant to this study. The apoE +/- mice were fed atherogenic
diets that contained a high amount of cholesterol (1.25 g/100 g), and
the mice had a large excess of cholesterol stored in their livers.
Excess hepatic cholesterol would be expected to suppress the
proteolytic release of the nuclear transcription factor SREBP
from the endoplasmic reticulum and minimize its entry into the nucleus
and its action on gene transcription (Goldstein and Brown 1990
). Thus, it is likely that de novo synthesis of cholesterol
was already severely down-regulated in mice fed the atherogenic
diets, and we would expect that little endogenous cholesterol synthesis
would occur.
If tocotrienol effects on the already low HMG-CoA reductase do not
provide a mechanism for attenuating either hepatic or serum
hypercholesterolemia, what then accounts for the ability of the 1.5
g/100 g palm-E supplement to decrease hepatic cholesterol
concentration and to normalize plasma lipoproteins? Possibilities
include increased hepatic bile synthesis and secretion as well as
reduced intestinal cholesterol absorption. Although not previously
investigated, either of these possibilities might occur via
tocotrienol-mediated effects that increase gene transcription of
the 7--hydroxylase or that decrease bile acid and cholesterol
transporters in the intestinal mucosa. Another possibility is that the
pharmacologic amounts of tocotrienols present in the supplements might
be preventing cholesterol absorption. Alternatively, increased
degradation of apoB (Wang et al. 1998
) might decrease
plasma VLDL, LDL and cholesterol, although changes in apoB would not
explain the lower hepatic cholesterol concentration.
The second remarkable finding of this study was the dramatic prevention
of atheroma formation induced by the palm-E supplements. These
results cannot be attributed to antioxidant effects alone. Vitamin E
compounds are effective antioxidants because they can donate phenolic
hydrogens to quench lipid free radicals. Although relative antioxidant
potency has not been established, tocotrienols with their unsaturated
side chain may be more mobile in membranes than tocopherols and thus
better able to interact with lipid free radicals (Serbinova and Packer 1994
). Others, however, have reported little difference
between antioxidant potencies of tocopherols and tocotrienols
(Kamal-Eldin and Appelqvist 1996
). The mean lesion size
for the 0.5 g/100 g -tocopherol group was 3.7 times larger than that
for the 0.5 g/100 g palm-E group. Because the diet of the 0.5 g/100
g palm-E group contained less -tocopherol and gave a better
response than did -tocopherol alone, it appears that the tocotrienol
content of the palm-E supplement had an important independent
effect. Further, because the 0.5 g/100 g palm-E–mediated decreases in
atheroma formation occurred in mice whose plasma cholesterol
concentration and lipoprotein pattern had changed little, if at all,
attenuation of lesion formation in these diets must have occurred by
mechanisms other than cholesterol control alone.
Thus, it appears that one or more of the tocotrienols reduced lesion formation in atherosclerosis-prone apoE +/- mice by at least two mechanisms. One may have been an antioxidant effect with no alteration in hepatic or serum cholesterol or in serum lipoproteins; the second is largely independent of antioxidant action and may relate to effects of the tocotrienols on foam cell formation and on either hepatic cholesterol secretion or intestinal absorption.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: apoE, apolipoprotein E; B6, C57BL/6J; FPLC, fast-performance liquid chromatography; IDL, intermediate density lipoprotein; HMG, 3-hydroxy-3-methylglutaryl; palm-E, palm-tocopherols.
Manuscript received February 7, 2000. Initial review completed March 15, 2000. Revision accepted May 30, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1.
Bartlett G. R. Phosphorus assay in column chromatography. J. Biol. Chem. 1959;234:466-468
2.
Berliner J. A. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation 1995;91:2488-2496
3. Bligh E. G., Dyer W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37:911-917
4.
Carew T., Schwenke D., Steinberg D. Antiatherogenic effect of Probucol unrelated to its hypercholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow progression of atherosclerosis in Wantanabe heritable hyperlipidemic rabbits. Proc. Natl. Acad. Sci. U.S.A. 1987;84:7725-7729
5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979;18:5294-5299[Medline]
6. Fletcher M. J. A colorimetric method for estimating serum triglycerides. Clin. Chim. Acta 1968;22:393-397[Medline]
7. Goldstein M. S., Brown J. L. Regulation of the mevalonate pathway. Nature (Lond.) 1990;343:425-430[Medline]
8.
Hennekens C. H., Buring J. E., Manson J. E. Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. N. Engl. J. Med. 1996;334:1145-1149
9. Kamal-Eldin A., Appelqvist L.-A. The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 1996;31:671-693[Medline]
10. Khor H., Chieng D. Effect of dietary supplementation of tocotrienols and tocopherols on serum lipids in the hamster. Nutr. Res. 1996;16:1393-1401
11. Lehrach H., Diamond D., Woznay J. M., Boedtler H. RNA molecule weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 1977;16:4743-4751[Medline]
12.
Maeda N., Li H., Lee D., Oliver P., Quarfordt S. H., Osada J. Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia. J. Biol. Chem. 1994;269:23610-23616
13.
Mensink R., van Houwelingen A., 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. 1999;69:213-219
14.
Omenn G., Goodman G., Thornquist M., Blames J., Cullen M. R., Glass A., Keogh J. P., Meyskens F. L., Valanis B., Williams J. H. Effects of combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N. Engl. J. Med. 1996;334:1150-1155
15.
Parker R., Pearce B., Clark R., Gordon D., Wright J. Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 2-hydroxy-3-methylglutaryl-coenzyme A reductase. J. Biol. Chem. 1993;268:11230-11238
16. Pearce B., Parker R., Deason M., Qureshi A., Wright J. Hypocholesterolemic activity of synthetic and natural tocotrienols. J. Med. Chem. 1992;35:3595-3606[Medline]
17.
Piedrahita J. A., Zhang S. H., Hagaman J. R., Oliver P. M., Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 1992;89:4471-4475
18. Pratico D., Tangirala R. K., Rader D. J., Rokach J., FitzGerald G. A. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in apoE-deficient mice. Nature Med 1998;4:1189-1192[Medline]
19. Pullinger C. R., North J. D., Teng B., Rifici V. A., Ronhild de Brito A. E., Scott J. The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life. J. Lipid Res. 1989;30:1065-1077[Abstract]
20. Qureshi A., Bradlow B., Brace L., Manganello J., Peterson D., Pearce B., Wright J., Gapor A., Elson C. Response of hypercholesterolemic subjects to administration of tocotrienols. Lipids 1995;30:1171-1177[Medline]
21. Qureshi A., Bradlow B., Salser W., Brace L. Novel tocotrienols of rice bran modulate cardiovascular risk parameters of hypercholesterolemic humans. Nutr. Biochem. 1997;8:290-298
22.
Qureshi 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 hypercholesterolemic humans by tocotrienols (palmvitee). Am. J. Clin. Nutr. 1991;53:1021S-1026S
23.
Reddick R. L., Zhang S. H., Maeda N. Atherosclerosis in mice lacking apo E: evaluation of lesional development and progression. Arterioscler Thromb 1994;14:141-147
24.
Rimm E. B., Stampfer M. J., Ascherio A., Giovannucci E., Colditz G. A., Willett C. Vitamin E consumption and risk of coronary artery disease in men. N. Engl. J. Med. 1993;328:1444-1449
25. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990’s. Nature (Lond.) 1993;362:801-142[Medline]
26. SAS Institute Inc SAS/STAT User’s Guide 6th ed. 1989 SAS Institute Cary, NC.
27.
Serbinova E. A., Packer L. Antioxidant properties of -tocopherol and -tocotrienol. Methods Enzymol 1994;234:354-366[Medline]
28. Shaish A., Daugherty A., O’Sullivan F., Schonfeld G., Heinecke J. W. Beta-carotene inhibits atherosclerosis in hypercholesterolemic rabbits. J. Clin. Investig. 1995;96:2075-2082
29.
Shaish A., George J., Gilburd B., Keren P., Levkovitz H., Harats D. Dietary beta-carotene and alpha-tocopherol combination does not inhibit atherogenesis in an apoE-deficient mouse model. Arterioscler. Thromb. Vasc. Biol. 1999;19:1470-1475
30. Stampfer M. J., Hennekens C. H., Manson J. E., Colditz G. A., Rosner B., Willett W. C. Vitamin E consumption and the risk of coronary heart disease in women. N. Engl. J. Med. 1993;328:1444-1449
31. Steel R.G.D., Torrie J. H., Dickey D. A. Principles and Procedures of Statistics: A Biometrical Approach 1997 McGraw-Hill New York, NY.
32. Stephens N. G., Parsons A., Schofield P. M., Kelly F., Cheeseman K., Mitchinson M. J. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 1996;347:781-786[Medline]
33.
Sullivan P. M., Mezdour H., Aratani Y., Knouff C., Najib J., Reddick R. L., Quarfordt S. H., Maeda N. Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis. J. Biol. Chem. 1997;272:17972-17980
34.
Tan D.T.S., Khor H. T., Low W.H.S., Ali A., Gapor A. Effect of a palm-oil-vitamin E concentrate on the serum and lipoprotein lipids in humans. Am. J. Clin. Nutr. 1991;53:1027S-1030S
35.
Tangirala R. K., Casanada F., Miller E., Witztum J. L., Steinberg D., Palinski W. Effect of the antioxidant N,N'-diphenyl 1,4-phenylenediamine (DPPD) on atherosclerosis in apoE-deficient mice. Arterioscler. Thromb. Vasc. Biol. 1995;15:1625-1630
36.
Upston J. M., Terentis A. C., Stocker R. Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J 1999;13:977-994
37. Wang Q., Theriault A., Gapor A., Adeli K. Effects of tocotrienol on the intracellular translocation and degradation of apolipoprotein B: possible involvement of a proteasome independent pathway. Biochem. Biophys. Res. Commun. 1998;246:640-643[Medline]
38.
Watkins T., Lenz P., Gapor A., Struck M., Tomeo A., Bierenbaum M. -Tocotrienol as a hypocholesterolemic and antioxidant agent in rats fed atherogenic diets. Lipids 1993;28:1113-1118[Medline]
39. Weisgraber K. H. Apolipoprotein E: structure-function relationships. Adv. Protein Chem. 1994;45:249-302[Medline]
40. Witztum J. L. The oxidation hypothesis of atherosclerosis. Lancet 1994;344:793-795[Medline]
41. Zhang S. H., Reddick R. L., Avdievich E., Surles L. K., Jones R. G., Reynolds J. B., Quarfordt S. H., Maeda N. Paradoxical enhancement of atherosclerosis by Probucol treatment in apolipoprotein E-deficient mice. J. Clin. Investig 1997;99:2858-2866[Medline]
42. Zhang S. H., Reddick R. L., Burkey B., Maeda N. Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption. J. Clin. Investig. 1994;94:937-945
This article has been cited by other articles:
|
|
 |
|
  K. S. Ahn, G. Sethi, K. Krishnan, and B. B. Aggarwal {gamma}-Tocotrienol Inhibits Nuclear Factor-{kappa}B Signaling Pathway through Inhibition of Receptor-interacting Protein and TAK1 Leading to Suppression of Antiapoptotic Gene Products and Potentiation of Apoptosis J. Biol. Chem., January 5, 2007; 282(1): 809 - 820. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Schaffer, W. E. Muller, and G. P. Eckert Tocotrienols: Constitutional Effects in Aging and Disease J. Nutr., February 1, 2005; 135(2): 151 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ikeda, T. Tohyama, H. Yoshimura, K. Hamamura, K. Abe, and K. Yamashita Dietary {alpha}-Tocopherol Decreases {alpha}-Tocotrienol but Not {gamma}-Tocotrienol Concentration in Rats J. Nutr., February 1, 2003; 133(2): 428 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Schwenke, L. L. Rudel, M. G. Sorci-Thomas, and M. J. Thomas {alpha}-Tocopherol protects against diet induced atherosclerosis in New Zealand white rabbits J. Lipid Res., November 1, 2002; 43(11): 1927 - 1938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Y. Fan, K. S. Ramos, and R. S. Chapkin Dietary {{gamma}}-Linolenic Acid Suppresses Aortic Smooth Muscle Cell Proliferation and Modifies Atherosclerotic Lesions in Apolipoprotein E Knockout Mice J. Nutr., June 1, 2001; 131(6): 1675 - 1681. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
