![[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}
|
|
|
|
|
||
,**
*
Advanced Medical Research, Madison, Wisconsin 53719;
U.S. Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Madison, Wisconsin 53705;
**
Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706 and
Departments of Genetics and Animal Sciences, University of Wisconsin, Madison, Wisconsin
2To whom correspondence should be addressed at Advanced Medical Research, 8251 Raymond Road, Madison, WI 53719. E-mail: nqureshi{at}mhub.facstaff.wisc.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tocotrienol, d-P21-T3
or d-P25-T3 per g for 6 wk. Group 5 was fed
the control diet for 6 wk and served as a control. After 6 wk, serum
total cholesterol was reduced 32–38%, low density lipoprotein
cholesterol was reduced 35–43%, apolipoprotein B was reduced
20–28%, platelet factor 4 was reduced 12–24%, thromboxane
B2 was reduced 11–18%, glucose was reduced 22–25%
(P < 0.01), triglycerides were reduced 15–19%
and glucagon was reduced 11–17% (P < 0.05) in
the treatment groups relative to the control. Insulin was 100% greater
(P < 0.01) in the treatment groups than in the
control group. Preliminary data (n = 1) indicated
that hepatic activity of the 3-hydroxy-3-methylglutaryl–coenzyme A
reductase was lower in the treatment groups, and cholesterol
7
-hydroxylase activity was unaffected. Cholesterol and fatty acid
levels in various tissues were lower in the treatment groups than in
control. After being fed the tocotrienol-supplemented diets, two
swine in each group were transferred to the control diet for 10 wk. The
lower concentrations of serum lipids in these four treatment groups
persisted for 10 wk. This persistent effect may have resulted from the
high tocotrienol levels in blood of the treatment groups, suggesting
that the conversion of tocotrienols to tocopherols may not be as rapid
as was reported in chickens and humans.
KEY WORDS: • hereditary hypercholesterolemic (HH) swine • rice bran • novel tocotrienols • lipid metabolism • cholesterol metabolism
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
,
Hood and Sidhu 1992
, Khor et al.1995
,
Parker et al. 1993
, Qureshi et al. 1997
,
Qureshi and Qureshi 1993
, Watkins et al. 1993
). Furthermore, the hypocholesterolemic effects of rice
bran have been demonstrated in experimental animals (Kahlon et al. 1991
, Seetharamaiah and Chandrashekhara 1989
, Sharma and Rukmini 1987
). This lowering of
serum total cholesterol (total-chol) levels by rice bran was
initially attributed to constituents of the unsaponifiable fractions of
the oil, such as cycloartenol (triterpene alcohols) or ferulic acid
esters (-oryzanols), which are present in large amounts
(Kahlon et al. 1991
, Seetharamaiah and Chandrashekhara 1989
, Sharma and Rukmini 1987
).
However, we have demonstrated that tocotrienols, minor constituents
present in various cultivars of rice brans, exert powerful
hypocholesterolemic effects in humans and chickens (Qureshi et al. 1997
and 2000
). The lipid variables and lipoprotein
profiles of swine are more similar to those of humans than to those of
rats (Hasler-Rapacz et al. 1995
, Huang and Kummerow 1976
, Leveille et al. 1975
,
Shrago et al. 1971
).
A strain of swine that expresses spontaneous hyperlipidemia and
hypercholesterolemia (hereditary/genetically) is derived from a unique
gene pool population that has resulted from a worldwide survey of 37
breeds and subsequent experimental breeding of uncommon lipid
phenotypes and lipoprotein genotypes (Rapacz 1978
). The
serum total-chol concentrations in these swine are 7.76–12.93
mmol/L compared with 3.10–4.14 mmol/L in normal adult swine. The
phenotype in these swine shows twofold to fourfold elevated
concentrations of serum or plasma lipids [total-chol, LDL-chol,
triglycerides (TG)] and apolipoprotein (apo)B, apoC3 and apoE and
reduced levels of HDL cholesterol (HDL-chol), apoA1 and apoA4
(Hasler-Rapacz et al. 1995
). Plasma from these swine is
characterized by cholesterol ester–enriched buoyant LDL (Lee et al. 1990
) as well as dense LDL particles (Rapacz and Hasler-Rapacz 1984
). These hereditary hypercholesterolemic
(HH) swine develop complicated atherosclerotic plaques that closely
resemble advanced atherosclerotic lesions found in humans
(Hasler-Rapacz et al. 1995
, Prescott et al. 1995
). Therefore, these swine are a good model for studying the
effects of tocotrienols from rice brans on LDL-chol and other lipid
parameters. High concentrations of serum LDL-chol or apoB and low
concentrations of HDL-chol or apoA1 are found to be better
predictors for the severity of coronary artery stenosis than are
total-chol and TG concentrations. Therefore, the ratios of
HDL-chol to LDL-chol and apoA1 to apoB are used as indicators
for assessment of the risk for coronary heart disease (Naito 1986
, Vega and Grundy 1990
).
The present study was carried out to evaluate the effects of
tocotrienol-rich fraction (TRF25) and its
individual components, -tocotrienol, desmethyl
(d-P21-T3) and didesmethyl
(d-P25-T3) tocotrienols, in HH swine.
The TRF25 is mainly a mixture of -, - and
-tocotrienols plus two novel tocotrienols,
d-P21-T3 and
d-P25-T3, isolated and purified from
stabilized and heated rice brans by flash chromatography
(Qureshi et al. 2000
).
It has been established that the relative contributions of various
tissues to overall fatty acids and cholesterol synthesis vary among
animal species (Dietschy et al. 1993
). All of the fatty
acid synthesis takes place in the liver of chickens and humans, whereas
in rats and swine, adipose tissue is a major contributor (Huang and Kummerow 1976
, Leveille et al. 1975
,
Shrago et al. 1971
). In swine, cholesterol biosynthesis
supported by acetate or glucose occurs mainly in the liver and to some
extent in adipose tissue (Huang and Kummerow 1976
);
however, the lipid parameters and profiles in swine are similar to
those of humans (Hasler-Rapacz et al. 1995
, Huang and Kummerow 1976
, Leveille et al. 1975
,
Shrago et al. 1971
). The present study describes the
effects of TRF25, -T3,
d-P21-T3 and
d-P25-T3 fed to HH swine on the levels
of serum total-chol, LDL-chol, HDL-chol, apoA1, apoB,
thromboxane B2 (TXB2),
platelet factor 4 (PF4), glucose, insulin and glucagon, as well as on
the hepatic enzymatic activities of HMG-CoA reductase and
cholesterol 7-hydroxylase. The concentrations of cholesterol and the
fatty acid compositions were also determined in liver, intestines,
lungs, heart, loin muscles, adipose tissues and ham muscles in the
experimental and control HH swine.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sources of chemicals, substrates, labeled substrates, enzymes and
diagnostic kits have been identified previously (Qureshi et al. 1997
and 2000
). Chemicals and solvents were of analytical
grade. Sigma Diagnostic Kits (Sigma Chemical Co., St. Louis, MO) were
used to estimate serum total-chol (kit 352; 500 nm), LDL-chol
(kit 352; 500 nm), HDL-chol (kit 352; 500 nm), TG (kit 336; 500
nm), apoAI (kit 356-A; 340 nm), apoB (kit 357; 340 nm) and glucose (kit
315; 505 nm). PF4 was determined by using Elisa Kit for PF4 (kit 601;
American Bioproduct, Parsippany, NJ), and a radioimmunoassay (RIA) kit
was used to determine TXB2 levels (Chemicon International,
El Segundo, CA). Serum insulin concentrations were estimated by using a
Double Antibody RIA Kit (Ventrex Laboratories, Portland, OR), and
glucagon was estimated by using an RSL Glucagon Kit (ICN Biomedicals,
Costa Mesa, CA).
Purification of -oryzanol–free TRF25 by flash
chromatography.
The stabilized and heated rice brans of L-487 rice variety were
supplied by M. Wells (Riviana Foods, Abbeville, LA). Large quantities
of tocotrienol-rich fraction (TRF25) and its individual
tocols (tocopherols and tocotrienols) were prepared by extracting 10
batches (10 kg of each) of stabilized and processed rice bran (L-487
variety; tocols 476 µg/g) twice with methanol (20 L) in a stainless
steel pan (50 L). The mixture was stirred six times during the day with
a stainless steel spoon for 3 d. The methanol (selective extracts
polyphenols, tocols, sterols, -oryzanols, fatty acids esters and
some phospholipids)-soluble fraction was separated by filtering over a
pad of four layers of cheese cloth. The combined methanol fractions
from all 10 batches were dried under vacuum (50°C). The semiviscous
oily residue (4600 g) of the methanol-soluble fraction was
extracted with hexane (5 L), filtered and left at 0°C overnight to
remove some of the sterols and triacylglycerides as precipitates. The
hexane-soluble fraction was filtered and evaporated under vacuum at
40°C, yielding 2412 g of a mixture of TRF25,
-oryzanols, some low-molecular-weight polyphenols, some free fatty
acids, fatty acid esters and triacylglycerides. HPLC of viscous oil for
tocols revealed the presence of -tocopherol, -tocotrienol,
ß-tocotrienol, -tocotrienol, -tocopherol, -tocotrienol and
two novel tocotrienols (d-P21-T3 and
d-P25-T3). The molecular structures of
d-P21-T3 and
d-P25-T3 have been established as
3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E),7'(E),11'-trienyl)-2H-1-benzopyran-6-ol
and
3,4-dihydro-2-(4,8,12-trimethyltrideca-3'(E),7',(E),11'-trienyl)-2H-1-benzopyran-6-ol,
respectively (Qureshi et al. 1999
and 2000
). The
purification of large quantities of TRF25 free from
-oryzanols and most of -tocopherol was carried out by flash
chromatography, using silica gel as reported recently (Qureshi et al. 2000
).
The tocols of TRF25 mixture were identified according to
the retention time and absorption profiles against standards of tocols
described earlier (Qureshi et al. 2000
). The composition
of various tocols in TRF25 was 4.5% -tocopherol, 15.7%
-tocotrienol, 2.3% ß-tocotrienol, 34.6% -tocotrienol, 4.8%
-tocopherol, 7.9% -tocotrienol, 13.7%
d-P21-T3, 14.4%
d-P25-T3 and 2.1% unidentified
tocotrienols.
Purification of -tocotrienol,
d-P21-T3 and
d-P25-T3 by flash chromatography.
The purification of -tocotrienol,
d-P21-T3 and
d-P25-T3 was carried out with the remaining
viscous oil (1912 g) by using the same procedure as described
previously (Qureshi et al. 2000
), except various tocols
were eluted with the sequential application of 1 L diethyl ether (DE)
(5, 10, 12, 14, 16, 18, 20, 22, 25 and 30% v/v) in hexane. The
composition of the combined fractions obtained from 19 pools (1912 g
starting material) with each elution was 5% DE; fatty acids esters and
sterols (1266 g); 10% DE, -tocopherol (3.3 g); 12% DE, a mixture
of -tocopherol and -tocotrienol (2.7 g); 14% DE, -tocotrienol
(3.7 g); 16% DE, ß-tocotrienol (0.4 g); 18% DE, -tocotrienol
(11.6 g); 20% DE, a mixture of -tocopherol and -tocotrienol
contaminated with d-P21-T3 (3.9 g); 22% DE,
d-P21-T3 (4.7 g); 25% DE,
d-P25-T3 (2.1 g); and 30% DE,
d-P25-T3 (4.1 g). Washing with 100% DE or
methanol gave -oryzanols (29.4 g). Each of these fractions contained
> 95% of its major component of the respective tocol.
Animals.
The protocol was reviewed and approved by the University of Wisconsin-Madison College of Agriculture and Life Sciences Animal Care Committee.
The HH swine used in this study were from the Immunogenetic Project
Herd, University of Wisconsin, Madison. The swine were derived from the
gene pool population and were selected based on the cholesterol
phenotypes (total-chol, HDL-chol) determined at 5 mo of age and
immunogenetically defined lipoprotein polymorphisms
(Hasler-Rapacz et al. 1995
). They represented both sexes
and were fed a standard 20% protein, corn-soybean meal swine
(control, tocotrienols 0.0%) diet as reported previously
(Qureshi et al. 1991a
), formulated at the University of
Wisconsin Experiment Station. The control diet contains 717.7 g corn
(9.3%), 210 g soybean meal (44%), 30 g lard, 11.3 g
dicalcium phosphate, 8 g calcium carbonate, 3 g NaCl and
10 g mineral-vitamin premix (Qureshi et al. 1991a
). The National Research Council guide for the care and
use of laboratory animals was followed. Fifteen 20-wk-old HH and
hereditary hyperlipidemic swine were divided into five groups
(n = 3, two males and one female), and each group
of three was placed into an individual 2.5 x 2.5-m pen. Four of
these five groups represented the treatment groups, and the fifth
served as the control group. The treatment diets were prepared by
supplementing the standard 20% protein, corn-soybean meal
(control) diet as described here (Qureshi et al. 1991a
)
with 50 µg of either TRF25, -T3,
d-P21-T3 or
d-P25-T3 per d and fed for 42 d. A dose
of 50 µg/g for each treatment was used in the present study, which
was very effective in inhibiting the activity of HMG-CoA reductase
in chickens (Qureshi et al. 2000
). Swine consumed feed
and water ad libitum. Feed intake by pen and individual body weights
was recorded weekly. Six normolipemic swine (20-wk-old; four males and
two females), derived from the Nutrition Project, University of
Wisconsin-Madison, were included to compare the weight gain and
other variables at 21, 42 and 70 d of feeding with that of the
HH/hereditary hyperlipidemic swine.
Blood samples, drawn by a syringe from the jugular vein, were taken on d 0, 21 and 42 of feeding after overnight food deprivation to facilitate chylomicron and very low density lipoprotein clearance. The blood was collected (5 mL each time), processed for serum or plasma and kept at -20°C for blood chemistry analyses. After being fed the diets for 42 d and having blood drawn after overnight food deprivation, the swine were further starved for 36 h. Then the swine were refed for 48 h, and one swine (male, randomly selected) from each group and two males and one female from the normolipemic group were killed according to procedures approved by the Research Animal Resources Center (RARC), University of Wisconsin-Madison. Samples of liver, intestine, lung, heart, loin muscle, adipose tissue and ham muscles were collected at the Muscle Biology Laboratory, University of Wisconsin, and stored at -20°C for subsequent analyses.
In addition, livers (15–20 g) were washed with saline solution, placed
on ice and processed to prepare cytosolic and microsomal fractions to
carry out cholesterol biosynthesis assays (Qureshi et al. 1991a
and 1982
). The food deprivation and refeeding segments of this
treatment were performed to induce hepatic HMG-CoA reductase (EC
1.1.1.34) and cholesterol 7-hydroxylase (EC 1.14.13.17) activities
as described here (Leveille et al. 1975
). The remaining
two swine (one male and one female of the hypercholesterolemic group
and two males and one female of the normolipemic group) were
transferred to a corn-soybean control diet for 10 wk, and serum and
plasma samples prepared from blood were collected after overnight food
deprivation for lipid analyses. The microsomal fractions of liver,
serum and plasma samples were stored at -20°C for subsequent
analyses. Feed consumption and feed efficiencies were calculated from
body and feed weights taken at the start and the end of the experiment.
Serum lipid analyses and apolipoprotein assays.
Serum total-chol and TG were assayed using kits 352 and 336, respectively. LDL-chol and very low density lipoprotein cholesterol were precipitated using 400-µL aliquots of serum with 50 µL of a mixture of 9.7 mmol phosphotungstic acid and 0.4 mol MgCl2 per L with gentle shaking for 10 min at room temperature, followed by centrifugation at 12,000 x g for 10 min. The supernatant, containing HDL-chol, was analyzed with Sigma reagent (kit 352). LDL-chol was calculated according to the formula LDL-chol = (total chol) - (HDL-chol) - (TG/5). Serum apoA1 and apoB were determined by RIA using antibodies to humans apoA1 and apoB (Sigma kits). Similarly, plasma PF4 and TXB2 concentrations were determined with RIA kits. Triplicates of each sample were run analyzed in each assay, and the average was used for the final calculation.
Estimation of tissue cholesterol levels and fatty acid composition.
The estimation of cholesterol in various tissues was carried out by homogenizing each tissue (0.2 g) in sodium citrate buffer (0.1 mol/L; 1 mL) with a microprobe from Omni GLH International Homogenizer (Warrenton, VA) for 30–60 s; 10 µL of the emulsion was used to estimate the cholesterol as described here with Sigma diagnostic reagent kit 352.
The fatty acid compositions were determined as described by
Hirai et al. (1984
). Each tissue (1 g) was homogenized
for 30 s with a Polytron homogenizer in 2 mL saline solution.
After homogenization, the sample was extracted with 8 mL hexane by
shaking for 20 min and then centrifuging for 10 min at 12,000 x g. The hexane layer was dried at 40°C under vacuum,
and the residue was treated with 0.5 mL diazomethane to yield the fatty
acid esters. The levels of fatty acids were measured as their esters by
using a Hewlett Packard model 5710A gas chromatograph equipped with
automatic sampler 7672A (Hewlett Packard, Boise, ID). The
identification of each fatty acid ester was established by comparison
against a standard mixture of fatty acid esters (Sigma Chemical Co.).
Triplicates of each sample were included in each assay, and the average
was used for the final calculation.
Assays for cholesterol biosynthesis.
Hepatic enzyme activities were assayed in the microsomal fraction of
liver homogenates. Fresh livers were homogenized and the microsomes
were obtained through centrifugation as described previously
(Qureshi et al. 1982
and 1991a
) and stored at -20°C
until used for assays. The liver homogenates were prepared in 0.1 mol
potassium phosphate/L buffer, pH 7.4, containing 4 mmol
MgCl2, 1 mmol EDTA and 2 mmol dithiothreitol per L
(Qureshi et al. 1991a
and 1982
). Microsomal protein was
measured by the Biuret method with bovine serum albumin used as a
standard. HMG-CoA reductase activity was assayed from the
microsomal fractions by the production of radioactive mevalonic acid
from
dl-3-hydroxymethyl-[3-14C]glutaryl-CoA.
Likewise, cholesterol 7-hydroxylase activity was assayed from
microsomal fractions by the production of
[14C]7-hydroxycholesterol from
[14C]cholesterol (Qureshi et al. 1991a
and 1982
). Aquasol scintillation solution,
dl-3-hydroxy-3-methyl-[3-14C]glutaryl-CoA
and [14C]cholesterol were purchased from New England
Nuclear (Boston, MA). An average of triplicate analyses of each sample
was used for these two enzymatic assays. Enzyme data are presented as
specific activities.
Expression of data and statistical methods.
StatView software (1992; Abacus Concepts, Berkeley, CA) was used for
the analysis of treatment-mediated effects. Treatment-mediated
differences in serum lipid variables were identified with ANOVA, and
when the F test indicated a significant effect, the
differences between the means were analyzed by a Fisher’s Protected
Least Significant Difference (LSD) test (1992; Abacus Concepts). The
statistical significance level was set at 5% (P < 0.05). Data are reported as means ± SD in the text.
Time-mediated differences in serum lipid variables in Table 2
were
calculated using ANOVA as described earlier (P < 0.05). Similarly, the strain differences (Table 2
; normolipemic versus
hypercholesterolemic) for each lipid variables were analyzed as one
block using ANOVA and by Fisher’s LSD, and differences were considered
significant at P < 0.001.
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The gain in body weight in the normolipemic swine did not differ from
the HH swine groups (Table 1)
.
|
). In normolipemic swine, these values were 10, 19 and 24%
(P < 0.05) at 21, 42 and 70 d, respectively,
compared with zero time (Table 2)
. The serum LDL-chol levels for HH
swine and normolipemic swine were 76 and 25% (P < 0.05), respectively, at 70 d compared with zero time (Table 2)
.
Feeding of TRF25, -T3,
d-P21-T3 or
d-P25-T3 to HH swine for 42 d
significantly decreased serum total-chol concentrations by 32, 34,
36 and 38% (P < 0.01), respectively, compared with
the concentration in the HH swine control group (Table 3
). The levels of LDL-chol were decreased by 35, 38, 40 and 43%
(P < 0.01), respectively, compared with the HH swine
control group, whereas HDL-chol levels did not differ among groups
(Table 3)
.
|
-T3, d-P21-T3 and
d-P25-T3. However, the ratios of
HDL-chol to total chol and HDL-chol to LDL-chol in the
treatment groups generally differed from the control group. The
HDL-chol/total chol ratios were greater in swine fed
TRF25, -T3,
d-P21-T3 and
d-P25-T3 by 39, 59, 68 and 81%
(P < 0.01), whereas the HDL-chol/LDL-chol ratios were
increased by 44, 70, 80 and 95% (P < 0.05),
respectively (Table 3)
. Similarly, the total chol/HDL-chol and
LDL-chol/HDL-chol ratios in the four HH treatment groups were
significantly lower than the HH swine control group. The ratios of
total chol/HDL-chol compared with control HH swine group were lowered
with TRF25 (30%), -T3 (39%),
d-P21-T3 (42%) and
d-P25-T3 (46%) (P < 0.01), whereas the LDL-chol/HDL-chol ratios were lowered to 31, 41, 44
and 49% (P < 0.05), respectively, compared with the
control group (Table 3)
.
The reduction in total-chol and LDL-chol concentrations in HH
swine fed TRF25, -T3,
d-P21-T3 and
d-P25-T3 were more pronounced after
42 d of feeding experimental diets than after 21 d of
feeding. Serum total-chol at 21 d was lower in swine fed
TRF25, -T3,
d-P21-T3 and
d-P25-T3 by 12, 10, 14 and 17%
(P < 0.05), and LDL-chol was lower by 13, 14, 17
and 19% (P < 0.05), respectively, compared with the
control group (data not shown).
Levels of apoA1 did not differ among groups (Table 4
); apoB was 20–28% lower (P < 0.01), TG were 15–19%
lower (P < 0.05), PF4 was 12–24% lower and
TXB2 was 11–18% lower (P < 0.01) in treated HH swine than in control HH swine (Table 4)
. The
maximum effects on these variables were observed in the group fed
d-P25-T3. The four treatment groups
were significantly different (P < 0.05) from the
control group, although they were not different from one another.
|
). Decreases of 11–17% (P < 0.05) were observed in
the levels of glucagon. Insulin was doubled (P < 0.01)
in treated groups relative to the controls (Table 5)
.
|
-hydroxylase and the levels of cholesterol and total
fatty acid compositions of various tissues are considered preliminary
because analyses were conducted on samples obtained from a single pig
fed each of the diets (n = 1). Normolipidemic swine
(n = 3, an average of nine analyses) fed the control
diet were included for comparison. Dietary supplementation of
tocotrienols significantly reduced the induced (by food deprivation and
refeeding regimen; Leveille et al. 1975
) enzymatic
activity of HMG-CoA reductase compared with HH swine controls
(Table 6
). However, the activity of cholesterol 7-hydroxylase did not appear
to be affected by any of these treatments compared with HH swine
controls (Table 6)
. The hepatic activity of HMG-CoA reductase in
the normolipemic swine controls was higher than the in HH swine
controls (Table 6)
.
|
-T3,
d-P21-T3 or
d-P25-T3 compared with HH swine
controls (data not shown). The levels of cholesterol in all of these
tissues of the normolipemic swine control group (n = 3)
were lower than those of the HH swine controls (data not shown). Total
fatty acids in liver and loin muscle were lower in the treated HH swine
than in the HH swine controls. Myristic, palmitic, palmitoleic,
palmitelaidic, oleic, linoleic and arachidonic acids were lower in the
liver of the treated HH swine, whereas stearic and linolenic acids were
greater than controls. In the loin muscle, only palmitic, stearic and
oleic acids were found, and they were lower in treated swine than in
control swine (data not shown).
Two pigs (one male and one female) in each group were transferred to
the corn-soybean control diet for 10 wk after being fed the
experimental diets for 6 wk. The reduced levels of serum total-chol
and LDL-chol persisted even after the control diet was fed for 10
wk in all of the treatment groups compared with control group. The
serum total-chol concentrations (in mmol/L) in HH swine after being
fed control or treatment diets for 42 d were 9.23 for the control
group, 6.29 for TRF25, 6.05 for -T3, 5.95 for
d-P21-T3 and 5.74 for
d-P25-T3 (n = 3)
(Table 3)
. The serum total-chol concentration (in mmol/L) after
transferring all of the groups to the corn-soybean control diet for
10 wk (70 d) were 9.69 for the control group, 7.19 for
TRF25, 7.39 for -T3, 7.14 for
d-P21-T3 and 6.95 for
d-P25-T3 (n = 2). The
reductions were 74, 76, 74 and 72% in the treatment groups compared
with the control group. These reductions were slightly lower than those
reported in Table 3
. Similarly reduced serum LDL-chol levels
persisted in the HH swine treatment groups (data not shown). These
results suggest that the tocotrienols may have remained in the
bloodstream over extended periods of time as reported earlier
(Qureshi et al. 1991a
, 1991b
and 1997
).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Hypercholesterolemia in these swine results from a
hereditary defect in catabolism that leads to an elevation of LDL. The
increase in cholesterol-rich LDL particles causes atherosclerotic
arteries similar to those observed in humans (Checovich et al. 1988
). The life-span of HH swine is only one third that of
normolipemic swine (Prescott et al. 1995
).
Although body weight gains were similar in HH and normolipemic swine
fed corn-soybean control diet from 20 to 36 wk (65–115 kg in all
the groups), serum total-chol (or LDL-chol) concentrations
increased far more in HH swine (63%) compared with normolipemic swine
(24%) during the 16-wk growth period (Table 2)
. The plasma lipids and
lipoproteins in HH swine differed significantly from those of
normolipemic swine (Qureshi et al. 1991a
). Tocotrienols
are more effective in hypercholesterolemic individuals, which explains
the large magnitude of their effects in the HH swine (Qureshi et al. 1991a
, 1991b
, 1997
and 1999
).
The significant decline in the concentrations of plasma
TXB2 and PF4 in swine fed
TRF25 and its novel tocotrienols
(d-P21-T3 and
d-P25-T3) is of great interest,
because these results show that TRF25 and its
components not only produce favorable lipid profiles but also can have
a protective effect on the endothelium and/or platelets. Thromboxanes
induce platelet aggregation and vasoconstriction, whereas prostacyclins
inhibit platelet aggregation and produce vasodilation. These results
may be mediated in part through reduced prostaglandin synthesis from
fatty acids. The preliminary results of the effects of
TRF25, -T3,
d-P21-T3 and
d-P25-T3 isolated from rice bran on
the fatty acid compositions of various tissues of swine showed a
significant decrease in levels of arachidonic acid in liver and loin
muscles, which might explain the dramatic reduction in the serum
TXB2 and PF4 levels (Qureshi et al. 1999
). The lowering of arachidonic acid by these novel
tocotrienols in various tissues of HH swine may result in the reduction
of oxygen metabolites in blood. Thus, there is an overall reduction in
prostaglandins and leukotrienes, both of which are synthesized from
arachidonic acid (Qureshi et al. 1999
). Some
investigators attribute these tocotrienol-mediated actions to their
unique in situ antioxidant activity, which is at least
threefold to fivefold better than that of -tocopherol
(Serbinova et al. 1991
). The decrease in concentrations
of arachidonic acid by these novel tocotrienols may be due to the
inhibition of phospholipase A2 activity.
The changes in levels of insulin and glucagon may have mediated the
decrease in glucose levels observed in this study. We found a greater
insulin-to-glucagon ratio (>100%) in all the treatment groups
(0.533–0.563) compared with controls (0.245). Such an effect may
decrease glucose intolerance in patients with diabetes mellitus and
restore the acute glucose-induced insulin response in
non–insulin-dependent diabetes mellitus patients. The 100% increase
in the serum insulin levels with novel tocotrienols in HH swine is
probably due to their antioxidant property. The positive effects of
vitamin E (tocopherol) due to its antioxidant activity on the
complications of diabetes have been reported (Karpen et al. 1982
and 1985
). The novel tocotrienols isolated from rice bran are
8- to 33-fold better antioxidants than tocopherols (Qureshi et al. 2000
).
Hepatic HMG-CoA reductase activity was 26–31% in HH swine fed
TRF25, -T3,
d-P21-T3 or
d-P25-T3. The activity of cholesterol
7-hydroxylase was unaffected. The reductions in hepatic HMG-CoA
reductase are consistent with the results reported for chickens fed
TRF25, -T3,
d-P21-T3 or
d-P25-T3 (16–36%), in which eight
chickens per group were studied (Qureshi et al. 2000
).
Moreover, the hepatic HMG-CoA reductase of adipose tissue in the
normolipemic swine (n = 3) control group was 27%
higher, as expected, compared with the HH swine control group
(Qureshi et al. 1991a
). The activities of HMG-CoA
reductase and cholesterol 7-hydroxylase enzymes are generally
considered to be rate-limiting steps for cholesterol and bile acids
synthesis in liver, respectively. The activities of HMG-CoA
reductase and cholesterol 7-hydroxylase are regulated coordinately,
but in opposite directions, by phosphorylation and dephosphorylation
(Carlson et al. 1978
).
Novel tocotrienols are potent cholesterol inhibitors, acting most
likely through feedback inhibition. The suppression of HMG-CoA
reductase requires two regulators: cholesterol and a nonsterol. The
cholesterol regulator is expressed predominantly through changes in the
rate of transcription of the HMG-CoA reductase gene, and the
nonsterol product regulator modulates the efficiency of the translation
of HMG-CoA reductase mRNA or by degradation of the enzyme itself
(Brown and Goldstein 1986
, Goldstein et al. 1985
). As reported earlier, both the mass and activity of
HMG-CoA reductase are decreased in the liver after the
administration of tocotrienols, and inhibit cholesterol biosynthesis by
suppressing HMG-CoA reductase through a novel
post-transcriptional mechanism involving protein degradation
(Parker et al. 1993
).
The efficacies of the individual components -T3,
d-P21-T3 and
d-P25-T3 compared with TRF25 in
these experiments are very similar, which may be attributed to either
the 50 µg/g concentration being at the saturation level or the fact
that swine consume large amounts of feed daily compared with other
experimental models (rats and chickens), resulting in a high
accumulation of tocotrienols in the body, of which only small amounts
might be converted to tocopherols in this model. These findings are in
sharp contrast to those for chickens (Qureshi et al. 1999
). Recently, Khor et al. (1995)
reported a
dose-dependent inhibition (17–50%) of HMG-CoA reductase
activity resulted from the administration of -tocotrienol for 6 d to guinea pigs. The authors reported that low doses of pure
-tocotrienol are much more effective than the higher doses and
confirmed our earlier observation that -tocopherol increases
HMG-CoA reductase activity (Khor et al. 1995
,
Khor and Ng 1999
, Qureshi et al. 1996
,
Qureshi and Qureshi 1993
).
The lower inhibitory effect on HMG-CoA reductase activity by
tocotrienols at higher doses is due to its conversion to tocopherols in
the tissue, which may counteract the effect of tocotrienols. In our
earlier studies, we reported high levels of -tocopherol in serum
after feeding of TRF from palm oil or -tocotrienol in humans
(Qureshi et al. 1991b
). To confirm this result, we fed
radioactive synthetic [3H]-tocotrienol to chickens for
4 wk (2 x 10 6 dpm). The serum, analyzed by HPLC
(Qureshi et al. 1997
), showed radioactivity in
-tocopherol (3265 dpm), -tocotrienol (5815 dpm), -tocopherol
(4650 dpm) and -tocotrienol (12,926 dpm) but none in
-tocotrienol, indicating a similar metabolic biosynthetic pathway
for tocotrienols in animals and plants (Qureshi and Qureshi 1993
). The enzymes of the gut bacteria in the chickens may be
responsible for these conversions (Hood 1995
). The
conversion of [3H]-tocotrienol into various tocols was
only 1.3%; -tocopherol accounted for 
10% of the total counts
recovered after 1 mo of feeding. Although this conversion into
-tocopherol is small, it could exert a negative effect on the
reduction in lipid concentrations produced by tocotrienols.
However, the results of the present study demonstrate that the
reductions in serum total-chol and LDL-chol levels by
tocotrienols were time dependent and that the effects were more
pronounced after 42 d compared with after 21 d of feeding.
Moreover, the reductions in total-chol and LDL-chol observed
after 42 d persisted over 10 wk after the termination of
tocotrienol feeding. This result suggests that the tocotrienols may
have remained in the bloodstream and that the conversion of
tocotrienols to tocopherols in this HH swine model is not as rapid as
that in humans (Qureshi et al. 1997
). In humans, higher
levels of tocotrienols were found in the HDL moiety compared with the
LDL moiety after feeding of tocotrienols for 4 wk, as reported earlier
(Qureshi et al. 1997
), and these tocotrienols are
transported nonspecifically like other lipid-soluble compounds to
different tissues by lipoproteins (Qureshi et al. 1997
).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: apo, apolipoprotein; HH, hereditary hypercholesterolemic; HMG-CoA, 3-hydroxy 3-methylglutaryl–coenzyme A; LDL-chol, low density lipoprotein cholesterol; PF 4, platelet factor 4; d-P21-T3, desmethyl tocotrienol from stabilized and processed rice bran;
d-P25-T3, didesmethyl tocotrienol from stabilized and heated rice bran; total-chol, total cholesterol; RIA, radioimmunoassay; d-TRF25, tocotrienol-rich fraction from stabilized and heated rice bran; TXB2, thromboxane B2.
Manuscript received July 14, 2000. Initial review completed August 23, 2000. Revision accepted November 13, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1.
Brown M. S., Goldstein J. L. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34-47
2. Carlson S. E., Mitchell A. D., Goldfarb S. Sex-related differences in diurnal activities and development of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7a-hydroxylase. Biochem. Biophys. Acta 1978;531:115-124[Medline]
3. Checovich W. J., Fitch W. L., Krauss R. M., Smith M. P., Rapacz J., Smith C. L., Attie A. D. Defective catabolism and abnormal composition of low density lipoprotein from mutant pigs with hypercholesterolemia. Biochemistry 1988;27:1934-1941[Medline]
4. Dietschy J. M., Turley S. D., Spady D. K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J. Lipid Res. 1993;34:1637-1659[Medline]
5. Goldstein J. L., Brown M. S., Anderson G. W., Russell D. W., Schneider W. J. Receptor mediated endocytosis: Concepts emerging from the LDL receptor system. Annu. Rev. Cell Biol. 1985;1:1-39
6.
Hasler-Rapacz J. O., Prescott M. F., Von Linden-Reed J., Rapacz J. M., Jr, Hu Z., Rapacz J. Elevated concentrations of plasma lipids and apolipoprotein B, C-III, and E are associated with the progression of coronary disease in familial hypercholesterolemic swine. Arterioscler. Thromb. Vasc. Biol. 1995;15:583-592
7. Hirai K., Ohno Y., Nakano T., Izutani K. Effects of dietary fats and phytosterols on serum fatty acid composition and lipoprotein cholesterol in rats. J. Nutr. Sci. Vitaminol. 1984;30:101-112
8. Hood R. L., Sidhu G. S. Effects of guar gum and tocotrienols on cholesterol metabolism on the Japanese quail. Nutr. Res. 1992;12:S117-S127
9. Hood R. L. Tocotrienols and cholesterol metabolism. Ong A.S.H. Niki E. Packer L. eds. Nutrition, Lipids, Health, and Diseases 1995:96-103 AOCS Press Champaign, IL.
10. Huang W. Y., Kummerow F. A. Cholesterol and fatty acid synthesis in swine. Lipids 1976;11:34-41[Medline]
11. Kahlon T. S., Sayre R. N., Chow F. I., Chiu M.C.A.A. The relative effects of rice bran at various levels, defatted rice bran and rice bran oil on plasma and liver cholesterol in hypercholesterolemic hamsters. FASEB J 1991;5:A1287
12. Karpen C. W., Pritchard K. A., Arnold J. H., Cornwell D. G., Panganamala R. V. Restoration of prostacyclin/thromboxane A2 balance in the diabetic rat: Influence of dietary vitamin E. Diabetes 1982;31:947-951
13. Karpen C. W., Cataland S., Odorisio T. M., Panganamala R. V. Production of 12-hydroxyeicosatetraenoic acid and vitamin E status in platelets from type I human diabetic subjects. Diabetes 1985;34:526-531[Abstract]
14. Khor H. T., Chieng D. Y., Ong K. K. Tocotrienols inhibit HMG-CoA reductase activity in the guinea pig. Nutr. Res. 1995;15:537-544
15.
Khor H. T., Ng T. T. Effects of administration of -tocopherol and tocotrienols on serum lipid and liver HMG-CoA reductase activity. Proceeding of the PORIM International Palm Oil Congress (Nutrition), February 1–6, pp. 177–186 1999
16. Lee D. M., Mok T., Hasler-Rapacz J., Rapacz J. Concentrations and compositions of plasma lipoprotein subfractions of Lpb5-Lpu1 homozygous and heterozygous swine with hypercholesterolemia. J. Lipid Res. 1990;31:839-847[Abstract]
17. Leveille G. A., Romsos D. R., Yeh Y. Y., O’Hea E. K. Lipid biosynthesis in the chick: A consideration of site of synthesis, influence of diet and possible regulatory mechanisms. Poultry Sci 1975;54:1075-1093[Medline]
18. Naito N. K. The clinical significance of apolipoprotein measurements. J. Clin. Immunoassay 1986;9:11-20
19.
Parker R. A., Pearce B. C., Clark R. W., Gordon D. A., Wright J.J.K. Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase. J. Biol. Chem. 1993;268:11230-11238
20. Prescott M. F., Hasler-Rapacz J. O., Von Linden-Reed J., Rapacz J. Familial hypercholesterolemia associated with coronary atherosclerosis in swine bearing different alleles for apolipoprotein B. Ann. N.Y. Acad. Sci. 1995;748:283-293[Abstract]
21. Qureshi A. A., Bradlow B. A., Salser W. A., Brace L. D. Novel tocotrienols of rice bran modulate cardiovascular disease risk parameters of hypercholesterolemic human. J. Nutr. Biochem. 1997;8:290-298
22. Qureshi A. A., Burger W. C., Elson C. E., Benevenga N. J. Effects of cereals and culture filtrate of Trichoderma viride on lipid metabolism of swine. Lipids 1982;17:924-934[Medline]
23. Qureshi, A. A., Lane, R. H. & Salser, A. W. (1999) Tocotrienols and tocotrienol-like compounds and method of their use. US Patent 5,919,818, July 6.
24. Qureshi A. A., Mo H., Packer L., Peterson D. M. Isolation and identification of novel tocotrienols from rice bran with hypocholesterolemic, antioxidant, and antitumor properties. J. Agric. Food Chem. 2000;48:3130-3140[Medline]
25.
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-methyl glutaryl coenzyme A reductase activity in chickens. J. Nutr. 1996;126:389-394
26. Qureshi N., Qureshi A. A. Tocotrienols: novel hypocholesterolemic agents with antioxidant properties. Packer L. Fuchs J. eds. Vitamin E in Health and Disease 1993:247-267 Marcel Dekker, Inc New York, NY.
27.
Qureshi A. A., Qureshi N., Hasler-Rapacz J. O., Weber F. E., Chaudhary V., Crenshaw T. D., Gapor A., Chong Y. H., Ong A.S.H., Peterson D. M., Rapacz J. Dietary tocotrienols reduce concentrations of plasma cholesterol, apolipoprotein B, thromboxane B2 and platelet factor 4 in pigs with inherited hyperlipidemias. Am. J. Clin. Nutr. 1991a;53:1042S-1046S
28.
Qureshi A. A., Qureshi N., Wright J.J.K., Shen Z., Kramer G., Gapor A., Chong Y. H., DeWitt G., Ong A.S.H., Peterson D. M., Bradlow B. A. Lowering of serum cholesterol in hypercholesterolemic humans by tocotrienols (Palmvitee). Am. J. Clin. Nutr. 1991b;53:1021S-1026S
29. Rapacz J. Lipoprotein immunogenetics and atherosclerosis. Am. J. Med. Genet. 1978;1:377-405[Medline]
30. Rapacz J., Hasler-Rapacz J. O. Investigations on the relationship between immunogenetic polymorphism of ß-lipoproteins and cholesterol levels in swine. Lenzi S. Descovich G. D. eds. Atherosclerosis and Cardiovascular Diseases 1984:99-108 Editrice Composition Bologna, Italy.
31. Seetharamaiah G. S., Chandrashekhara N. Studies on the hypocholesterolemic activity of rice bran oil. Atherosclerosis 1989;78:219-223[Medline]
32. Serbinova E., Kagan V., Han D., Packer L. Free radical recycling and intermembrane mobility in the antioxidant properties of alpha-tocopherol, and alpha-tocotrienol. Free Radic. Biol. Med. 1991;10:263-275[Medline]
33. Sharma R. D., Rukmini C. Hypocholesterolemic activity of unsaponifiable matter of rice bran oil. Indian J. Med. Res. 1987;85:278-281[Medline]
34. Shrago E., Glennon J. A., Gordon E. S. Comparative aspects of lipogenesis in mammalian tissues. Metabolism 1971;20:54-62[Medline]
35. Vega G. L., Grundy S. M. Does measurement of apolipoprotein B have a place in cholesterol management. Atherosclerosis 1990;10:668-671
36.
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]
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
 |
|
 V. A Mustad, C. A Smith, P. P Ruey, N. K Edens, and S. J DeMichele Supplementation with 3 compositionally different tocotrienol supplements does not improve cardiovascular disease risk factors in men and women with hypercholesterolemia Am. J. Clinical Nutrition, December 1, 2002; 76(6): 1237 - 1243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Qureshi, W. A. Salser, R. Parmar, and E. E. Emeson Novel Tocotrienols of Rice Bran Inhibit Atherosclerotic Lesions in C57BL/6 ApoE-Deficient Mice J. Nutr., October 1, 2001; 131(10): 2606 - 2618. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
