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Department of Nutritional Sciences, Oklahoma State University, Stillwater, Oklahoma 74078
2To whom correspondence should be addressed at Department of Nutritional Sciences, 416 Human Environmental Sciences; Oklahoma State University, Stillwater, OK 74078. E-mail: arjmand{at}okstate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • lipids • triglycerides • casein • ovariectomy • isoflavones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Fukami et al. 1995
, Goldstein and Stampfer 1995
, Sullivan 1996
). These changes are
a consequence of reductions in the levels of circulating estrogens,
which is the basis for estrogen replacement therapy (ERT). Estrogen
replacement therapy reduces the risk of CHD in part through the
modulation of serum cholesterol (Godsland et al. 1987
,
Knight and Eden 1996
). However, ERT and other
cholesterol-lowering pharmacological agents may be accompanied by
side effects (U.S. Department of Health and Human Services 1993
) and therefore are recommended only for women without
known contraindications. Moreover, many women chose not to comply with
a recommendation for ERT because of safety concerns (Kessel 1998
). Therefore, other means that present no known side
effects for the treatment of postmenopausal hypercholesterolemia are
preferred.
Recently, the Food and Drug Administration approved a CHD risk
reduction claim for soy protein (U.S. Food and Drug Administration 1999
); the health claim does not specify a
requirement for the presence or quantity of the nonprotein constituents
such as saponins, phytic acid, trypsin inhibitors and isoflavones known
to influence cholesterol. The amount of these compounds, in a given soy
protein preparation, can be affected by processing. For instance,
isolates prepared through extraction with water and ethanol can be
substantially depleted in their nonprotein components (Anderson and Wolf 1995
). Hence, variable results can be obtained from
the use of different soy protein preparations.
Another issue of importance is to investigate whether the
cholesterol-lowering efficacy of nonprotein constituents of soy,
such as isoflavones, differ when given with soy or other protein
sources. The findings from some human (Crouse et al. 1999
, Wong et al. 1998
) and animal
(Anthony et al. 1996
and 1997
, Arjmandi et al. 1997
, Kirk et al. 1998
) studies suggest the
importance of isoflavones in reducing total and LDL cholesterol in the
context of soy protein. However, when isoflavone-rich extract of
soy has been fed to cynomolgus monkeys in the absence of soy protein,
it has not produced any cholesterol-lowering effects
(Greaves et al. 1999
). The question remains whether it
is the isoflavone or other components of soy, soy protein itself or
their combination that lower cholesterol.
A previous hamster study in this laboratory has shown
hypercholesterolemic changes after ovariectomy (Sohn et al. 1999a
). Hence, in the present study, we used this animal model
to address the question of whether soy protein depleted of its
nonprotein constituents such as isoflavones would be able to suppress
the ovariectomy-induced rise in serum cholesterol.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Forty-eight 6-mo-old female Golden Syrian hamsters (Harlan
Sprague-Dawley, Indianapolis, IN) were individually housed and kept
in an environmentally controlled laboratory. Guidelines for the ethical
care and treatment of animals from the Animal Care and Use Committee at
Oklahoma State University were strictly followed. After 3 d of
acclimation, hamsters were either sham-operated (sham) or
ovariectomized (ovx) and divided into four groups of 12 hamsters each
as follows: sham + casein, ovx + casein, sham + SPe and ovx + SPe.
Animals were fed for 70 d a semipurified cholesterol-free
powdered diet that was either casein or SPe based (Table 1
). Diet composition was a modification of the formulation by
Terpstra et al (1991
). Ovariectomized hamsters were
pair-fed to the sham group; deionized water access was
unrestricted. Food intake was determined daily.
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Serum triglycerides (TG); phospholipids; total, free, esterified and high density lipoprotein (HDL) cholesterol and 17ß-estradiol.
Serum TG and total cholesterol concentrations were determined
enzymatically with Sigma Diagnostics kits (St. Louis, MO). Serum HDL
cholesterol was determined with a precipitation technique (Sohn et al. 1999a
, Sjoblom and Elklund 1989
). Serum
phospholipids and free cholesterol concentrations were determined
through colorimetric methods with commercially available kits (Wako,
Richmond, VA). These tests were performed with a Cobas-Fara II
Clinical Analyzer (Montclair, NJ). Non-HDL cholesterol was
calculated by subtracting HDL cholesterol from total cholesterol.
Esterified cholesterol was calculated by subtracting free cholesterol
from total cholesterol. Serum 17ß-estradiol was determined with a
radioimmunoassay kit (ICN Biomedical, Costa Mesa, CA).
Liver total lipids and cholesterol.
Portions of the liver were homogenized and then extracted with a 2:1
(v/v) chloroform/methanol mixture. After the addition of 0.12 NaCl
mol/L solution to the extraction solution and the separation of phases,
aliquots of the organic phase were analyzed for liver total
cholesterol. Liver total cholesterol was determined with a color
reagent of glacial acetic
acid/FeSO4/H2SO4 (Searcy and Bergquist 1960
). Total liver lipids were determined with the
Folch gravimetric method (Folch et al. 1957
).
Statistical analyses.
The data were analyzed as a 2 x 2 factorial arrangement in a completely randomized design with SAS software, Version 6.11 (SAS Institute, Cary, NC). Analysis of variance and least squares means were calculated using the general linear model procedure. Data are reported as least squares means ± SEM; unless otherwise indicated, P < 0.05 is regarded as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Daily food consumption did not differ among the groups (Table 2
). Hamsters in all groups had similar initial and final mean body
weights. Ovariectomy caused atrophy of uterine tissue, indicating the
success of the surgical procedure. There was no difference in relative
liver weight among the treatments. The small intestine relative weight
was significantly (P < 0.05) lower in the ovx groups
than in the sham groups.
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As expected, ovariectomy significantly (P < 0.001)
reduced circulating serum levels of 17ß-estradiol (Table 3
), confirming our earlier observations in this model (Sohn et al. 1999a
)
ovariectomy significantly increased serum total- and non-HDL cholesterol
concentrations regardless of dietary protein source. Serum phospholipid
and free and esterified cholesterol concentrations were also
significantly elevated in ovx hamsters compared with sham hamsters
regardless of whether they were fed SPe- or casein-based diets.
Regardless of ovarian hormone status, hamsters that were fed the
casein-based diet had significantly higher TG concentrations than
did those fed SPe-based diet.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Our previous study in this model (Sohn et al. 1999a
) reported a significant rise in total plasma cholesterol
and a trend (P = 0.073) toward higher concentrations of
non-HDL cholesterol. In the present study, however, the increase in
the non-HDL cholesterol was significant.
In agreement with our earlier observations (Arjmandi et al. 1997
, Sohn et al. 1999a
), ovariectomy did not
alter liver weight, lipids or cholesterol concentrations. Although
ovariectomy has been reported to reduce the activity of hepatic
hydroxymethylglutaryl (HMG)-coenzyme A (CoA) reductase in rats
(Abul-Hajj 1981
, Carlson et al. 1999
),
this may not be the case in ovx hamsters. We previously observed no
change in hepatic rates of sterol synthesis due to ovariectomy in this
animal model (Sohn et al. 1999a
). Hence, altered de novo
cholesterol synthesis cannot be a mechanism by which ovariectomy causes
hypercholesterolemia in hamsters. A reduction in the conversion of
cholesterol to bile acids may in part explain the
ovariectomy-induced rise in serum cholesterol, as ovarian hormone
deficiency has been shown to reduce both hepatic 7
-hydroxylase mRNA
expression and activity (Colvin et al. 1998,
Kushawa and Born 1991
, Sohn et al. 1999b
).
Similar to our earlier observations (Sohn et al. 1999a
),
ovariectomy reduced the weight of the small intestine compared with
that of intact hamsters. The atrophy of the intestine, a tissue that
expresses estrogen receptors (Arjmandi et al. 1993
),
suggests that the intestine may be a target tissue for estrogen, and
hence the impact of estrogen deficiency on the function of this
important organ is meritorious of further study.
The inability of the SPe to prevent the ovx-induced rise in
circulating total, non-HDL, esterified and free cholesterol
concentrations may further indicate the importance of soy isoflavones
or other nonprotein components in modulating lipid metabolism. A study
by Balmir et al. (1996
) of male hamsters confirmed the
importance of isoflavones in reducing cholesterol. Their results showed
that hamsters that were fed soy protein with normal isoflavone content
or casein with added isoflavones had lower serum total cholesterol
concentrations than those fed casein alone.
A study by Greaves et al. (1999
) concluded that soy
components other than isoflavones are responsible for its
cholesterol-lowering properties in ovx monkeys; however, their
findings neither confirmed nor rejected the observations made in the
present study. Their results suggested that isoflavone-rich extract
of soy alone fed in conjunction with protein other than soy does not
produce the desirable hypocholesterolemic effects in ovarian hormone
deficiency. Our findings in this and earlier work (Arjmandi et al. 1997
) and the majority of the studies by other
investigators (Anthony et al. 1996 and 1997, Crouse et al. 1999
, Kirk et al. 1998
, Wong et al. 1998
) imply that the combination of soy protein and its
isoflavones is necessary for their effectiveness in lowering serum
cholesterol in ovarian hormone deficiency.
In the present study, SPe significantly reduced serum TG levels,
regardless of ovarian hormone status, compared with casein. The effects
of soy on circulating TG concentrations in humans (Ashton and Ball 2000
, Crouse et al. 1999
) and in rats
(Iritani et al. 1996
) have been reported previously;
however, the data are not conclusive and are sometimes contradictory.
Therefore, the effects of soy protein on serum TG, an important risk
factor for cardiovascular disease, as well as possible modes of action
warrant investigation.
Our results suggest that SPe alone is not sufficient to prevent the ovarian hormone deficiency–induced hypercholesterolemia. Ethanol extractable components, either alone or in conjunction with the soy protein, may be needed to lower circulating cholesterol concentrations and therefore reduced the risk of CHD in ovarian hormone deficiency.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CHD, coronary heart disease; ERT, estrogen replacement therapy; HDL, high density lipoprotein(s); LDL, low density lipoprotein(s); ovx, ovariectomy; SPe, ethanol-extracted soy protein isolate; TG, triglycerides.
Manuscript received June 27, 2000. Initial review completed August 23, 2000. Revision accepted October 27, 2000.
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REFERENCES |
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