Nutrient Metabolism

Hamsters and Guinea Pigs Differ in Their Plasma Lipoprotein Cholesterol

Distribution when Fed Diets Varying in Animal Protein, Soluble Fiber, or
Cholesterol Content1
Maria Luz Fernandez,* Thomas A. Wilson, Karin Conde,* Marcela Vergara-Jimenez* and
Robert J. Nicolosi2
*Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269 and Department of Health
and Clinical Sciences, University of Massachusetts, Lowell, Lowell, MA 01854

KEY WORDS:

hamsters

guinea pigs

dietary cholesterol

The interest in finding appropriate animal models to study

diet or drug effects on plasma lipid levels and atherosclerosis
has increased in recent years (Krause and Newton, 1991,
Sullivan et al. 1993). However, the use of animal models is
associated with a number of disadvantages, including the lack
of similarity to humans in plasma lipoprotein profiles or in the
activity of regulatory enzymes of hepatic cholesterol and lipoprotein metabolism.
Pigs are well accepted models to study hypercholesterolemia and atherosclerosis because of their similarities to humans in both of these areas (Van Tol et al. 1991). Yet, there
is a need for the utilization of smaller animals for more rapid
studies on the effects of diet on hyperlipidemia and atherosclerosis. The cholesterol-fed rabbit has been used as a model
of human atherosclerosis for the rapid development of aortic
lesions (Daley et al. 1994); nevertheless, rabbits carry most of
the cholesterol in the VLDL fraction, a situation different

1
2

casein

pectin

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ABSTRACT There were two objectives to these studies: 1) to compare the lipoprotein cholesterol distribution in
two animal models in response to different dietary treatments and 2) to assess whether the hypercholesterolemia
induced by high cholesterol intake could be reversed by consumption of vegetable-protein and/or dietary fiber.
Guinea pigs, which carry the majority of plasma cholesterol in LDL, and hamsters, with a higher distribution of
cholesterol in HDL, were evaluated in three different studies. In Study 1, animals were fed semi-purified diets for
4 wk with proportions of 60:40, 20:80 or 0:100 (w/w) of casein/ soybean protein. Hamsters and guinea pigs that
consumed 100% soybean protein had lower plasma total cholesterol (TC) than those fed diets containing casein
(P , 0.01). In Study 2, three doses of dietary pectin (2.7, 5.4, or 10.7 g/100g) added in place of cellulose were
tested. Intake of 10.7 g/100 g pectin resulted in the lowest plasma TC concentrations for both species (P , 0.01).
Although the TC lowering was similar in studies 1 and 2, the lipoprotein cholesterol distribution differed. Whereas
the differences in plasma cholesterol were in LDL in guinea pigs, hamsters exhibited differences in both non-HDL
and HDL cholesterol. In study 3, animals were fed 100% soybean protein, 10.7 g/100 g pectin, and three doses of
dietary cholesterol: 0.04, 0.08, or 0.16 g/100 g, which is equivalent to 300, 600, or 1,200 mg/d in humans. Guinea
pigs and hamsters had the highest plasma LDL and hepatic cholesterol concentrations when they consumed 0.16
g/100 g of cholesterol (P , 0.01). However, intake of 0.08 g/100 g of cholesterol resulted in lower plasma LDL
cholesterol concentrations than did consuming high animal protein (60:40 casein/ soy) or low soluble fiber (2.7
g/100 g). Relatively high levels of dietary cholesterol combined with vegetable protein and soluble fiber resulted in
desirable lipoprotein profiles in animal models that significantly differ in their lipoprotein cholesterol distribution. J.
Nutr. 129: 13231332, 1999.

from humans (Badimon et al. 1990). The use of the rat has
been in decline because of the major differences between this
animal model and humans, including responses to diet, HDL
being the major plasma cholesterol carrier in the rat, and its
resistance to atherosclerosis (Shefer et al. 1992).
Hamsters and guinea pigs have been extensively used for
studying the effects of diet on plasma lipid levels and the
mechanisms involved because these two animal models
present characteristics that might resemble the human situation (Fernandez and McNamara 1991, Fernandez et al. 1993,
1994, 1995, and 1997, Fernandez 1995, Nicolosi and Wilson
1997, Spady and Dietschy 1988, Terpstra et al. 1991, Woollett
et al. 1992). Guinea pigs carry the majority of cholesterol in
the LDL fraction (Fernandez and McNamara 1991), and hamsters develop atherosclerosis when challenged with a hypercholesterolemic diet (Otto et al. 1995).
The response to different hypercholesterolemic diets in
general, and to dietary cholesterol in particular, is an important area of research that continues to be under debate. The
heterogeneity of the human response to dietary cholesterol was
documented (McNamara et al. 1987), and studies have dem-

Supported by an award from the American Egg Board.

To whom reprint requests should be addressed.

0022-3166/99 $3.00 1999 American Society for Nutritional Sciences.

Manuscript received 12 November 1998. Initial review completed 22 December 1998. Revision accepted 10 March 1999.
1323

FERNANDEZ ET AL.

1324

MATERIALS AND METHODS

Materials. Reagents were obtained from the following sources:
phosphotungstate reagent, glucose 6-phosphate, NADP1, glucose-6phosphate dehydrogenase, Tween 80, triton-100, 7a-hydroxycholesterol, unlabeled cholesterol, [4-14C] cholesterol, and triacylglycerol
enzymatic kits from Sigma (St. Louis, MO). Cholesterol enzymatic
assay kit, cholesterol oxidase, and cholesterol esterase were purchased
from Boehringer-Mannheim (Indianapolis, IN); free cholesterol and
phospholipid enzymatic assay kits were obtained from Waco Pure
Chemical Industries (Richmond, VA); halothane from Halocarbon
(Hackensack, NJ); [3-14C] HMG-CoA, [5-3H] mevalonolactone,
[1-14C ] oleoyl CoA, [4, 14C] cholesterol, and [1,2,6,7 3H] cholesteryl
oleate from Du Pont New England Nuclear (Boston, MA). Silica gel
TLC plates were from Eastman Kodak (Rochester, NY); silica gel G
TLC plates were from Analtech (Newark, DE), 7 b-hydroxycholesterol was from Steraloids (Wilton, NH); quickseal ultracentrifugation
tubes from Beckman Instruments (Palo Alto, CA).
Diets. Diets were prepared by Research Diets (New Brunswick,
NJ). Diets for the three experiments had the same composition
except for the nutrient under evaluation as indicated in Table 1. For
study 1, the difference was the proportion of casein/soybean protein
used, which was 60/40, 20/80, or 0/100 (Table 1). For study 2, the
protein used was 0/100 casein/soybean protein, and the proportion of
pectin/cellulose varied: 2.7/9.8, 5.4/7.1, 10.7/1.8 g/100 g for a total of
12.5 g/100 g dietary fiber (Table 1). For study 3, the diet contained
0/100 casein/soybean protein and 10.7 g/100g pectin, while the
amount of dietary cholesterol varied 0.04, 0.08, or 0.16 g/100g (Table
1), which is equivalent to 300, 600, or 1,200 mg/d in humans (Lin et
al. 1992).
Animals. Male Hartley guinea pigs (Harlan Sprague Dawley,
Indianapolis, IN) weighing 300 400 g were randomly assigned to
each one of dietary treatments for 4 wk to achieve a metabolic steady
state prior to analysis. Guinea pigs (6 8/dietary treatment) were
slowly weaned onto the experimental diets by feeding them increasing amounts of the test diet over a period of 1 wk. They were housed
in a room of controlled light (light 0700 1900 h) and had free access
to diets and water. At the end of the experiment (4 wk), guinea pigs
were anesthetized with halothane and killed by exsanguination after
cardiac puncture.
Male, 10-wk-old Golden Syrian hamsters were obtained from
Charles River Laboratories (Wilmington, MA). Hamsters were fed a
nonpurified diet (Purina, St. Louis, MO) for 1 wk prior to the
treatment period. Following this acclimation period, hamsters were
randomly assigned to three groups of 15 for each study and fed the
respective treatment diets for 4 wk. They were housed in individual,

TABLE 1
Composition of experimental diets for studies 1, 2 and 3
Diet composition

Components
Protein1
L-Methionine
Sucrose
Corn starch
Fat mix2
Fiber3
Mineral mix4
Vitamin mix4
Cholesterol5

g/100g

J/100J

22.5
0.5
25.0
15.0
15.1
12.5
8.2
1.1
0.04

23.3

26.0
15.6
35.1

1 Protein for study 1 was 100% soybean protein, 80% soybean/

20% casein, or 40% soybean/60% casein. For Studies 2 and 3, 100%
soybean protein was used.
2 Fat was a mixture of 5 g beef tallow/100 g, 23 g olive oil/100 g,
13 g palm kernel oil/100 g, 22 g palm oil/100 g, and 37 g safflower
oil/100 g yielding a polyunsaturated:mononsaturated:saturated fatty
acid ratio of 1:1:1 for all diets.
3 Fiber was 10 g cellulose/100 g and 2.5 g guar gum/100 g for Study
1; 2.7, 5.4, or 10.7 g pectin/100 g adjusted to 12.5 g fiber/100 g with
cellulose for Study 2; and 10.7 g pectin/100 g and 1.8 g cellulose/100
g for study 3.
4 Mineral and vitamin mixes were adjusted to meet National Research Council requirements for guinea pigs and hamsters. Detailed
composition of the vitamin and mineral mix has been reported elsewhere (Fernandez & McNamara 1991).
5 Dietary cholesterol was 0.04 g/100 g for studies 1 and 2 and 0.04,
0.08, or 0.16 g/100 g for study 3.

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onstrated that saturated fat or trans fatty acids appear to have

a more pronounced effect on plasma lipid levels than does
dietary cholesterol (Hu et al. 1997). Consumption of fruits,
vegetables, and grains was postulated as a healthy diet, which
results in lowering of plasma cholesterol (Jenkins et al. 1997).
Further, studies have reported that lacto-ovo-vegetarians have
low plasma cholesterol concentrations and desirable lipoprotein profiles (Masarei et al. 1984), which suggests that consuming relatively high amounts of dietary cholesterol with
high amounts of vegetable protein and adequate amounts of
dietary fiber will not result in elevated plasma cholesterol
concentrations.
The present studies were designed to compare the response
to different dietary factors, including animal protein, dietary
fiber, and dietary cholesterol, in two animals models that differ
in terms of cholesterol-carrying lipoproteins in plasma (Fernandez and McNamara 1991, Woollett et al. 1992). We hypothesized 1) that dietary challenges would alter lipoprotein
distribution differently in hamsters and guinea pigs and 2)
that, independent of the animal model used, the response to
dietary cholesterol would be lower with diets high in vegetable
protein and soluble fiber.

stainless steel hanging cages at room temperature with a 12-h light:

dark cycle and had free access to food and water.
All animal experiments were conducted in accordance with US
Public Health Service/US Department of Agriculture guidelines, and
experimental procedures were approved by the University of Connecticut and the University of Massachusetts Institutional Animal
Care and Use Committee.
Plasma lipid concentrations in hamsters. Blood samples were
taken after 4 wk from food-deprived hamsters (12 h) and collected,
via the retro-orbital sinus, into heparinized capillary tubes under
ultra-pure CO2/O2 (50:50) gas (Northeast Airgas, Salem, NH) anesthesia. Plasma was harvested after centrifugation at 1,500 x g at room
temperature for 20 min, and plasma total cholesterol (TC)4 (Allain
et al. 1974) and triacylglycerol (TAG) concentrations (Bucolo and
David 1973) were measured enzymatically. Plasma VLDL and LDL
cholesterol, non-HDL cholesterol (non-HDL-C), were precipitated
with phosphotungstate reagent (Weingand and Daggy 1990), and
HDL cholesterol (HDL-C) was measured in the supernatant. The
concentration of non-HDL-C was calculated as the difference between plasma TC and HDL-C.
Isolation and characterization of VLDL and LDL for guinea
pigs. For the determination of plasma lipids and isolation of lipoproteins, blood samples were taken by cardiac puncture after guinea pigs
were anesthetized with halothane. Plasma total and lipoprotein cholesterol (Allain et al. 1974) and TAG (Bucolo and David 1973) were
determined by enzymatic methods. Guinea pig VLDL (d 5 1.006
kg/L) and LDL (d 5 1.021.09 kg/L) were isolated by ultracentrifugation at 120,000 x g with the use of a Ti50 rotor. Plasma HDL
cholesterol was measured according to Warnick et al. (1982), with
the modification of using 2 mol/L MgCl2 to precipitate the apo

4
Abbreviations used: ACAT, acyl coenzyme A cholesterol acyltransferase;
HDL-C, HDL-cholesterol; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A
reductase; non-HDL-C, non-HDL cholesterol; TAG, triacylglycerol; TC, total cholesterol.

HAMSTERS, GUINEA PIGS, DIET AND LIPOPROTEIN DISTRIBUTION

1325

TABLE 2
Plasma total cholesterol (TC) and triacylglycerol (TAG) concentrations in hamsters and guinea pigs fed different levels of casein/
soybean protein for 4 wk (Study 1)1
Hamsters
Diet, casein/soybean
protein

TC

Guinea pigs
TAG

TC

TAG

2.94 6 0.59a
2.56 6 0.57ab
2.17 6 0.47b

1.22 6 0.57
1.00 6 0.43
0.70 6 0.26

mmol/L
60/40
20/80
0/100

4.53 6 0.29a
4.11 6 0.19b
3.71 6 0.32c

1.97 6 0.43a
1.99 6 0.60a
1.84 6 0.44b

1 Values represent mean 6 SD, n 5 15 hamsters or 6 8 guinea pigs. Values in a column with different superscripts differ, P , 0.05.

RESULTS
Study 1. There were no differences in weight gain or food
consumption in hamsters and guinea pigs fed the different
diets (data not shown). In addition, both species exhibited
significantly higher plasma TC concentrations when fed 40%
compared to 80 or 100% soybean protein (Table 2). Plasma
cholesterol was 9% lower in hamsters fed 80% soybean protein
and 18% lower in those fed 100%; in guinea pigs, values were
13% (P 5 0.08) and 26% lower than in those fed 40% soybean
protein respectively. In addition, there was 7% lower (P
5 0.14) plasma TAG in hamsters fed the 100% soybean diet
than in those fed 80 or 40% soybean protein (Table 2).
Although guinea pigs fed 100% soybean protein had 43%
lower plasma TAG than those fed 40% soybean protein, the
differences were not significant because of the large standard
deviations (P 5 0.17; Table 2). In this and subsequent experiments, hamsters had higher plasma TC concentrations than
guinea pigs (Tables 2 4).
Guinea pigs and hamsters had substantially different lipoprotein profiles. Hamsters had 34% higher concentration of
HDL cholesterol compared to non-HDL cholesterol (Fig. 1).
The concentrations of LDL were not determined in hamsters,
and the results are expressed as non-HDL cholesterol.
In guinea pigs, the majority of the cholesterol was carried in
the LDL fraction and only small percentages were carried in
VLDL and HDL (data not shown). In the rest of the experiments, hamsters and guinea pigs presented lipoprotein distributions similar to those found in study 1. The percentage
carried by VLDL in guinea pigs was added to LDL and expressed as non-HDL cholesterol for purpose of comparisons
between hamsters and guinea pigs.

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B-containing lipoproteins. The accuracy of the procedures used for

the measurements of plasma TC, HDL-C, and TAG concentrations
for hamsters and guinea pigs were checked by participation in the
Lipid Standardization Program of the Centers for Disease Control and
the National Heart, Blood and Lung Institute (Atlanta, GA).
Lipoprotein composition was determined by measuring protein
(Markwell et al. 1978), TAG (Bucolo and David 1973), phospholipids, and free and esterified cholesterol (Carr et al. 1993). The number
of component molecules of LDL was calculated assuming one apo B
per particle with a molecular weight of 412,000, as reported for guinea
pigs (Chapman et al. 1975). The molecular weight of TAG, cholesterol, cholesteryl ester, and phospholipids were calculated as 885.4,
386.6, 664, and 734, respectively, as previously reported (Fernandez
et al. 1994).
Hepatic cholesterol and triacylglycerol determination. Hepatic
lipids were determined as described by Carr et al. (1993). Briefly, for
lipid extraction 1 g of liver was homogenized in chloroform:methanol
overnight. After mixing with acidified water, phases were separated
and aliquots evaporated and resuspended in ethanol to measure total
and free cholesterol and TAG by enzymatic methods. Cholesteryl
ester was calculated by subtracting free from total hepatic cholesterol.
Hepatic HMG-CoA Reductase assay. Microsomal 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34)
activity was measured as previously described (Shapiro et al. 1969).
HMG-CoA reductase activity was expressed as picomoles of [14C]
mevalonate produced per min per mg microsomal protein. Recoveries
of [3H] mevalonate were between 70 and 80%.
Hepatic ACAT assay. Acyl coenzyme A cholesterol acyltransferase (ACAT) (EC 2.3.2.26) activity was determined by pre-incubating microsomal protein 0.8 1.0 mg per assay with 84 g albumin/L,
an amount of albumin equivalent to the molar ratio of the substrate
(1:1 albumin:14C-oleoyl CoA) (Smith et al. 1986), and buffer for
ACAT isolation (Fernandez et al. 1993). Recoveries of [3H] cholesteryl oleate were between 75 and 90%.
Hepatic cholesterol 7a-hydroxylase assay. Cholesterol 7a-hydroxylase (EC 1.14.13.7) activity was assayed by the method of
Jelinek et al. (1990) using [14C] cholesterol as substrate, except that
cholesterol was delivered as cholesterol:phosphotidylcholine liposomes (1:8 by weight) prepared by sonication, and an NADPHregenerating system (glucose-6-phosphate dehydrogenase, NADP,
and glucose 6-phosphate) was included in the assay as a source of
NADPH (Fernandez 1995).
Statistical analysis. One-way ANOVA was used to determine
differences in plasma TC, TAG, lipoprotein composition, hepatic
lipids, and hepatic enzyme activity in hamsters or guinea pigs fed the
diets in experiments 1, 2, or 3. Newman-Keuls was used for post-hoc
analysis. P , 0.05 was considered significant. Two-way ANOVA was
used to compare the specific responses in plasma TC between hamsters and guinea pigs consuming 60% casein, 2.7g/100 g pectin, or
0.08 g/100 g dietary cholesterol. Linear regressions were calculated
between TC for hamsters and guinea pigs and increasing doses of diet
and for ACAT and cholesterol 7a-hydroxylase activities and pectin
doses in guinea pigs.

TABLE 3
Plasma total cholesterol (TC) and triacylglycerol (TAG)
concentrations between hamsters and guinea pigs fed
different levels of pectin for 4 wk (Study 2)1
Hamsters
Diet, pectin,
g/100g

TC

Guinea Pigs
TAG

TC

TAG

2.69 6 0.46a
2.02 6 0.41b
1.66 6 0.31b

1.44 6 0.32
1.28 6 0.43
0.81 6 0.28

mmol/L
2.7
5.4
10.7

4.29 6 0.51a
3.41 6 0.27b
2.94 6 0.26c

1.82 6 0.46a
0.88 6 0.53b
1.30 6 0.30b

1 Values represent mean 6 SD, n 5 15 hamsters or 6 guinea pigs.

Values in a column with different superscripts differ, P , 0.05.

FERNANDEZ ET AL.

1326

TABLE 4
Plasma total cholesterol (TC) and triacylglycerol (TAG) concentrations in hamsters and guinea pigs fed different levels of dietary
cholesterol for 4 wk (Study 3)1
Hamsters
Diet, dietary cholesterol, g/100g

TC

Guinea pigs
TAG

TC

TAG

2.25 6 0.41a
2.72 6 1.22a
4.37 6 1.60b

1.40 6 0.90
1.52 6 0.50
0.91 6 0.25

mmol/L
0.04
0.08
0.16

3.08 6 0.27a
3.35 6 0.34a
4.61 6 0.66b

1.16 6 0.12a
1.13 6 0.22a
1.43 6 0.28b

1 Values represent mean 6 SD, n 5 15 hamsters or 6 guinea pigs. Values in a column with different superscripts differ, P , 0.01.

FIGURE 1 Lipoprotein cholesterol concentrations in hamsters or

guinea pigs fed diets containing 60/40, 20/80 or 0/100 casein/soybean
protein ratios for 4 wk (Study 1). Values are mean 6 SD for 15 hamsters
or 6 guinea pigs. Comparisons are made between non-HDL (upper
panel) or HDL cholesterol (lower panel) in hamsters or guinea pigs by
one-way ANOVA and the Newman-Keuls as post hoc test. Different
letters indicate significant differences (P ,0.01). In guinea pigs VLDL
cholesterol represents ,5% of the non-HDL fraction.

served in the relative percentages of cholesteryl ester, free

cholesterol, protein, and phospholipid for these two lipoproteins for guinea pigs fed the three protein diets (data not
shown).
Hepatic HMG-CoA reductase, cholesterol 7a-hydroxylase,
and ACAT activities did not differ among the three dietary
treatments. Values were 17 6 12, 16 6 9 and 11 6 5
pmol/(minzmg protein) for HMG-CoA reductase activity; 1.70
6 0.33, 1.33 6 0.44, and 1.72 6 0.50 pmol/(minzmg protein)
for cholesterol 7a-hydroxylase activity; and 48 6 18, 50 6 19,
and 44 6 5 pmol/(minzmg protein) for ACAT activity in
guinea pigs fed the 60/40, 20/80, or 0/100 casein:soybean
protein diets, respectively.
Study 2. Plasma TC was 21 and 32% lower in hamsters
and 25 and 39% lower in guinea pigs fed the 5.4 and 10.7
g/100 g pectin diets, respectively, compared to those fed 2.7
g/100 g pectin (Table 3; P , 0.01). Plasma TAG were lower
by an average of 40% (P , 0.05) in the hamsters that consumed higher amounts of pectin. Increasing the amount of
soluble fiber in the diet resulted in a dose response in the
plasma cholesterol lowering in both hamsters and guinea pigs
(r 5 -0.985, P , 0.05).
Hamsters fed the two higher levels of pectin had lower
plasma non-HDL and HDL cholesterol concentrations than
the 2.7 g/100 g pectin group, whereas guinea pigs had similar
responses only in non-HDL cholesterol (Fig. 2; P , 0.01).
The plasma TC and non-HDL cholesterol lowering ranged
from 27 to 40% for hamsters and guinea pigs (Fig. 2).
No significant differences in the VLDL components were
found. However, differences in the number of cholesteryl ester
and free cholesterol molecules in LDL were observed among
groups. Guinea pigs fed the highest dose of pectin (10.7 g/100
g) had fewer free cholesterol and cholesteryl ester molecules
than those fed 2.7 or 5.4 g/100 g pectin, respectively (P
, 0.05). The values were 249 6 97, 204 6 48, and 135 6 22
for free cholesterol and 607 6 127, 615 6 159, and 409 6 134
cholesteryl ester molecules for the 2.5, 5.4, and 10.7 g/100 g
pectin groups, respectively. The other components of LDL,
TAG, and phospholipids were not different among dietary
treatments. These results indicate that the LDL from guinea
pigs fed 10.7 g/100 g pectin were cholesterol-depleted and
smaller in size than the LDL derived from guinea pigs fed 2.7
or 5.4 g/100 g pectin.
In both of these studies, 1 and 2, guinea pigs presented a
proportionally greater reduction in plasma non-HDL cholesterol concentrations than hamsters when given 100% soybean
protein (Fig. 1) or 10.7 g/100 g pectin (Fig. 2).
Hepatic total and free cholesterol were lower in guinea pigs
fed 5.4 or 10.7 g/100g pectin compared to those fed the lower

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Increasing soybean protein resulted in significantly lower

concentrations of non-HDL and HDL cholesterol in hamsters.
Non-HDL cholesterol was 24% lower in hamsters fed the
100% soybean protein diet compared to those fed 40% soybean protein. There was 34% lower plasma non-HDL cholesterol in guinea pigs fed 100% than in those fed 40% soybean
protein (Fig. 1). No other significant differences in plasma
lipoprotein cholesterol were observed in guinea pigs.
Guinea pigs fed the 100% soybean diet had lower hepatic
total cholesterol concentrations than those fed lower ratios of
soybean protein:casein; differences were associated with free
cholesterol concentrations (Table 5). No significant differences among treatments were observed for esterified cholesterol or hepatic TAG.
No differences in VLDL and LDL composition were ob-

HAMSTERS, GUINEA PIGS, DIET AND LIPOPROTEIN DISTRIBUTION

1327

TABLE 5
Hepatic total cholesterol (TC), free cholesterol (FC), esterified cholesterol (EC), and triacylglycerol (TAG) of guinea pigs fed
different levels of casein/soybean protein for 4 wk (Study 1)1
Hepatic lipids
Diet, casein/soybean protein

TC

FC

EC

TAG

0.49 6 0.13
0.59 6 0.13
0.52 6 0.10

7.6 6 2.0
7.9 6 2.2
8.6 6 2.0

mmol/g
60/40
20/80
0/100

4.91 6 0.28a
5.10 6 0.59a
4.27 6 0.44b

4.40 6 0.39a
4.50 6 0.54a
3.36 6 1.32b

1 Values represent mean 6 SD, n 5 6. Values in a column with different superscripts differ, P , 0.05.

FIGURE 2 Lipoprotein cholesterol concentrations in hamsters or

guinea pigs fed diets containing 2.7, 5.4, or 10.7 g/100 g pectin for 4 wk
(Study 2). Values are mean 6 SD for 15 hamsters or 6 guinea pigs.
Comparisons are made between non-HDL (upper panel) or HDL cholesterol (lower panel) in hamsters or guinea pigs by one-way ANOVA
and the Newman-Keuls as post hoc test. Different letters indicate
significant differences (P , 0.01). In guinea pigs VLDL cholesterol
represents ,5% of the non-HDL fraction.

were 9% (P 5 0.27) and 50% higher in hamsters and 20% (P

5 0.09) and 94% higher in guinea pigs fed 0.08 and 0.16 g/100
g dietary cholesterol, respectively, compared to those fed 0.04
g/100 g dietary cholesterol (P , 0.01). In hamsters fed 0.16
g/100 g of cholesterol, plasma TAG concentrations were
greater than in those fed the two lower levels. However, no
differences in plasma TAG were observed in guinea pigs fed
the three levels of dietary cholesterol, indicating differences
between animal models.
In hamsters, both non-HDL and HDL cholesterol concentrations increased because of the greater intake of dietary
cholesterol. However, the proportion of non-HDL cholesterol/
HDL cholesterol changed as the concentration of dietary
cholesterol increased. The proportions were 0.64, 0.76, and
0.99, respectively, indicating that the non-HDL-C increased
more than the HDL-C. In guinea pigs, there was a slightly
greater plasma non-HDL-C concentration in those fed 0.08
g/100 g cholesterol and a 100% greater concentration in those
fed the 0.16 g/100 g cholesterol diet. No differences were
observed in HDL-C (Fig. 4).
Differences were found in both VLDL and LDL compositions of guinea pigs fed different levels of dietary cholesterol.
The concentration of cholesteryl ester in VLDL was seven
times higher in guinea pigs fed the 0.16 g/100 g cholesterol
diet compared to the other two groups (data not shown). In
contrast, the concentration of VLDL TAG was lowest in
guinea pigs fed the highest level of dietary cholesterol (P
, 0.01). In LDL, a significantly greater number of free cholesterol molecules was observed in guinea pigs fed the highest
dose of dietary cholesterol than in the other two groups.
Values were 261 6 33, 324 6 55, and 465 6 75 free cholesterol molecules for guinea pigs fed 0.04, 0.08, and 0.16 g/100
dietary cholesterol, respectively.
Hepatic total free, and esterified cholesterol concentrations
were higher for both hamsters and guinea pigs fed 0.16 g/100
g dietary cholesterol compared to the other dietary cholesterol
doses (Table 7). Guinea pigs had higher concentrations of free
versus the esterified cholesterol, even in the case of high
dietary cholesterol, whereas hamsters had higher concentrations of esterified cholesterol at the highest dose of dietary
cholesterol (Table 7).
Hamsters fed 0.16 g/100 g dietary cholesterol had significantly lower HMG-CoA reductase activity than the other two
groups (Table 8). In contrast, guinea pigs fed 0.08 g/100 g had;
70% lower HMG-CoA reductase activity than those fed 0.04
g/100 g, indicating that the first compensatory mechanism in
guinea pigs to maintain plasma cholesterol homeostasis is the
suppression of the activity of the regulatory enzyme of cholesterol synthesis. In contrast, cholesterol 7a-hydroxylase activity

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dose (Table 6). No other differences were observed in hepatic

lipids. As the amount of pectin in the diet increased, there was
a dose response for some of the regulatory enzymes of hepatic
cholesterol metabolism. Hepatic cholesterol 7 a-hydroxylase
activity increased as the amount or pectin in the diet increased. In contrast, ACAT activity decreased as the amount
of pectin in the diet increased (Fig. 3). HMG-CoA reductase
activity was higher in guinea pigs fed pectin levels of 5.4 and
10.7 g/100 g, although a dose response was not observed.
Values were 8.1 6 3.2, 15.3 6 8.4, and 14.1 6 4.1 pmol/
(minzmg protein) for guinea pigs fed 2.7, 5.4, and 10.7 g/100 g
pectin, respectively.
Study 3. Plasma TC concentrations increased in both
hamsters and guinea pigs as the amount of cholesterol in the
diet increased (Table 4). Plasma cholesterol concentrations

FERNANDEZ ET AL.

1328

TABLE 6
Hepatic total cholesterol (TC), free cholesterol (FC), esterified
cholesterol (EC), and triacylglycerol (TAG) of guinea pigs fed
different levels of dietary pectin for 4 wk1
Hepatic lipids
Diet, pectin,
g/100g

TC

FC

EC

TAG

0.57 6 0.21
0.57 6 0.44
0.67 6 0.33

8.5 6 1.8
8.5 6 1.8
6.7 6 1.2

mmol/g
2.7
5.4
10.7

8.70 6 2.11a
4.30 6 1.54b
4.70 6 1.36b

8.13 6 2.05a
3.76 6 1.42b
4.01 6 1.34b

1 Values represent the mean 6 SD, n 5 6. Values in a column with

different superscripts differ, (P , 0.01).

DISCUSSION
A main question addressed in the current investigation was
whether cholesterolemic responses to diet would be different

Downloaded from jn.nutrition.org by guest on May 6, 2015

was not altered by increasing the amount of dietary cholesterol

fed to hamsters or guinea pigs (Table 8). ACAT activity was
not altered by diet in hamsters, whereas in guinea pigs ACAT
activity and the amount of free cholesterol (Table 8) increased
in parallel with higher intakes of dietary cholesterol. These
results indicate different mechanisms to maintain cholesterol
homeostasis when challenged with high levels of dietary cholesterol exist in hamsters and guinea pigs.
Summary of the three studies. There was a dose response
in plasma TC for both hamsters and guinea pigs in studies 1,
2, and 3. Plasma total cholesterol decreased as the amount of
soybean protein increased in the diet (Fig. 5 upper panel).
Similarly, as the amount of pectin increased in the diet, TC
decreased (Fig. 5 middle panel). Both correlations were linear
(P ,0.05). Although there was a dose response in which
plasma TC increased as the amount of dietary cholesterol
increased, the best fit was obtained using a power correlation
(r 5 0.95 for hamsters and r 5 0.98 for guinea pigs; Fig. 5 lower
panel). Plasma TAG concentrations were not consistently
correlated with the amount of each nutrient in diet (data not
shown).
Hamsters and guinea pigs fed the intermediate level of
dietary cholesterol (0.08 g/100g) had lower plasma non-HDL
cholesterol concentrations than those fed either the high
animal protein diet (60% casein, 40% soybean protein) or the
low soluble fiber diet (2.7 g/100 g pectin; Fig. 6). Although
hamsters had higher plasma TC concentrations than guinea
pigs in all three studies, guinea pigs had higher plasma nonHDL cholesterol concentrations (Fig. 6). This was because
guinea pigs carry the majority of cholesterol in the LDL
fraction, whereas hamsters have different proportions of LDL
and HDL cholesterol , depending on the diet. When data were
analyzed by two-way ANOVA, diet and species effects were
present. Guinea pigs had higher plasma non-HDL cholesterol
concentrations when compared to hamsters in all three studies
(P , 0.01). In addition, intake of 0.08g/100 g dietary cholesterol with 10.7 g/100 pectin and 100% soybean protein resulted in significantly lower plasma non-HDL cholesterol concentrations for both hamsters and guinea pigs than did intake
of 60% casein or 2.7g/100 g pectin with 0.04 g/100 g dietary
cholesterol (P , 0.05).

in guinea pigs (predominantly an LDL species) and hamsters

(high HDL/LDL ratio). In this study, we demonstrated that,
although hamsters and guinea pigs have different plasma lipoprotein cholesterol distributions, they have similar responses to dietary factors that are known to alter plasma
cholesterol levels in humans.
Hamsters and guinea pigs responses to casein/soybean and
soluble fiber. Soybean protein lowers plasma cholesterol levels in experimental animals (Nicolosi and Wilson 1997, Terpstra et al. 1991) and humans (Nagata et al. 1998). In rats and
hamsters, the hypocholesterolemic effect was associated with
increases in fecal steroid excretion (Hayashi et al. 1994).
Rabbits are very susceptible to dietary casein because they
develop endogenous hypercholesterolemia and develop atherosclerosis with diets high in casein (Daley et al. 1994). In
this study, we have shown that hamsters had similar hypocholesterolemic responses to soy relative to casein as previously
reported (Terpstra et al. 1991) and that guinea pigs also
exhibit higher plasma LDL cholesterol concentrations as the
amount of dietary casein is increased. All these studies demonstrate that there is a consistent response to casein intake
across animal species, although with different degrees of hypercholesterolemia.
Although increases of fecal steroid secretion were proposed
as a possible mechanism for the action of soybean protein
(Huff and Carroll 1980), there are many sites of cholesterol
and lipoprotein metabolism that could be affected and that
have not been studied. We found no effect from the intake of
different casein/soybean protein ratios in any of the regulatory

FIGURE 3 Hepatic cholesterol 7a-hydroxylase (upper panel) and

acyl-CoA:choelsterol acyltransferase (ACAT) (lower panel) activities of
guinea pigs fed increasing doses of pectin for 4 wk (Study 2). Values are
mean 6 SD n 5 6 . There was a significant, positive, linear relationship
between cholesterol 7a-hydroxylase activity and pectin dose (r
5 0.993, P , 0.05) and a negative relationship between ACAT activity
and pectin dose (r 5 0.994, P , 0.05).

HAMSTERS, GUINEA PIGS, DIET AND LIPOPROTEIN DISTRIBUTION

1329

TABLE 7
Hepatic total cholesterol (TC), free cholesterol (FC), esterified cholesterol (EC), concentrations in hamsters and guinea pigs fed
different levels of dietary cholesterol for 4 wk (Study 3)1
Hamsters
Diet, dietary
cholesterol, g/100g

TC

FC

Guinea pigs
EC

TC

FC

EC

3.9 6 0.4b
6.0 6 1.2b
13.1 6 3.0a

3.4 6 0.4b
5.3 6 0.8b
8.9 6 1.6a

0.5 6 0.1b
0.8 6 0.5b
4.1 6 1.9a

mmol/g
0.04
0.08
0.16

7.8 6 0.7b
8.7 6 1.9b
21.4 6 6.3a

6.8 6 0.7b
6.5 6 0.9b
8.3 6 1.9a

1.9 6 0.4b
2.9 6 2.4b
13.1 6 1.9a

1 Values represent mean 6 SD, n 5 15 hamsters or 6 guinea pigs. Values in a column with different superscripts differ, P , 0.01.

circulation of bile acids and increasing the demand for bile

acid synthesis. In addition, hepatic ACAT activity decreased
in guinea pigs fed the higher doses of pectin in a dosedependent manner, indicating that ACAT activity is related
to the availability of free cholesterol in the liver, which was
altered by the intake of pectin as was observed in guinea pigs
(Fernandez et al. 1994).
These data suggest that the alterations in hepatic cholesterol pools by dietary factors are associated with different
mechanisms, which warrant further investigation. Although
plasma cholesterol was decreased both by soybean protein
intake and higher amounts of pectin, the hypocholesterolemic
mechanisms are likely to be different.
The intake of fiber decreases the delivery of cholesterol to
the liver through the chylomicron remnants and decreases

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enzymes of hepatic cholesterol homeostasis in guinea pigs.

These data suggest that other mechanisms, not related to the
synthesis or catabolism of cholesterol, are being affected by the
intake of different levels of casein and soybean protein in this
species. We did find lower hepatic cholesterol concentrations
in guinea pigs fed the 100% soybean protein diet, but these
differences were not associated with modifications in hepatic
enzyme activity.
The mechanisms associated with the hypocholesterolemic
effects of soybean are not clear. For example, because soybean
protein is deficient in some of the essential amino acids,
studies in which soybean protein is supplemented with seven
essential amino acids to make it similar to animal protein,
were conducted, and no hypercholesterolemic responses were
observed (Carroll 1982). The most consistently postulated
mechanism in animal studies appears to be related to increases
in fecal steroid secretion caused by the reduced availability of
bile acids (Huff and Carroll 1980). What is clear from studies
conducted in humans and animals is that soybean protein has
a hypocholesterolemic effect that we were able to reproduce in
the present study of hamsters and guinea pigs.
In the case of dietary soluble fiber, there were dose responses to pectin intake in total and LDL cholesterol in guinea
pigs and in total, HDL, and non-HDL cholesterol in hamsters.
Another interesting observation was that LDL composition
was altered by dietary fiber in guinea pigs. The highest intake
of pectin resulted in particles that contained less cholesteryl
ester. Higher concentrations of cholesteryl ester induced by
diet were associated with an increased risk for atherosclerosis
in African green monkeys (Carr et al. 1992). We have also
shown that smaller LDL particles resulting from dietary interventions are removed by plasma more rapidly (Fernandez et al.
1993).
In guinea pigs, the plasma cholesterol lowering induced by
pectin is proportional to the dose of dietary pectin and dependent on the depletion of hepatic cholesterol induced by the
action of fiber in the intestinal lumen (Fernandez et al. 1994).
This indicates that pectin is possibly delaying the delivery of
cholesterol to the liver by affecting cholesterol absorption or
by interrupting the enterohepatic circulation of bile acids
(Fernandez 1995). In addition, when hepatic enzyme activities
were determined in guinea pigs fed the higher doses of dietary
fiber and compared to the lower dose, we found increases in
HMG-CoA reductase and a dose response in cholesterol 7ahydroxylase activity similar to our previous reports (Fernandez
et al. 1994, Fernandez 1995). These effects of fiber on the
regulatory enzymes of cholesterol synthesis and catabolism
indicate a compensatory response to produce more cholesterol
and bile acids because of the pectin altering the enterohepatic

FIGURE 4 Lipoprotein cholesterol concentrations in hamsters or

guinea pigs fed 0.04, 0.08, or 0.16 g/100 g dietary cholesterol for 4 wk
(Study 3). Values are mean 6 SD for 15 hamsters or 6 guinea pigs.
Comparisons are made between non-HDL (upper panel) or HDL cholesterol (lower panel) in hamsters or guinea pigs by one-way ANOVA
and the Newman-Keuls as post hoc test. Different letters indicate
significant differences (P , 0.01). In guinea pigs VLDL cholesterol
represents ,5% of the non-HDL fraction.

FERNANDEZ ET AL.

1330

TABLE 8
Hepatic 3hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-R), cholesterol 7a-hydroxylase (C7H) and acyl-CoA:cholesterol
acyltransferase (ACAT) activities in hamsters and guinea pigs fed different levels of dietary cholesterol for 4 wk (Study 3)1
Hamsters
Diet, dietary
cholesterol, g/100 g

HMG-R

C7H

Guinea pigs
ACAT

HMG-R

C7H

ACAT

2.2 6 0.2
2.3 6 0.1
2.1 6 0.3

42.5 6 12.2c
100.6 6 53.1b
162.3 6 66.7a

pmol/(min z mg protein)
0.04
0.08
0.16

2.3 6 0.2a
2.0 6 0.1a
1.4 6 0.1b

8.6 6 18.4
5.4 6 7.4
3.9 6 6.8

17.2 6 5.8
16.4 6 6.3
20.1 6 7.3

13.9 6 5.7a
4.2 6 3.1b
3.0 6 0.6b

1 Values represent mean 6 SD, n 5 15 hamsters or 6 guinea pigs. Values in a column with different superscripts differ, P , 0.01.

Other important differences between guinea pigs and hamsters are the responses of regulatory enzymes of cholesterol
metabolism to high dietary cholesterol. In guinea pigs, HMGCoA reductase activity was suppressed, even after concentraDownloaded from jn.nutrition.org by guest on May 6, 2015

hepatic cholesterol via increases in its catabolic pathway in

hamsters and guinea pigs (Fernandez 1995, Horton et al.
1994). In guinea pigs, alterations in lipoprotein secretion,
remodeling, and catabolism by soluble fiber were also demonstrated (Fernandez et al. 1997). Other than increases in fecal
steroid secretion (Hayashi et al. 1994, Huff and Carroll 1980),
it is not clear how the source of dietary protein exerts its
hypocholesterolemic action.
Hamsters and guinea pig responses to dietary cholesterol.
The responses in plasma TC to dietary cholesterol were
similar in hamsters and guinea pigs. However, if we analyze
the effects on the lipoproteins, species differences were
evident. In guinea pigs, there were increases in plasma LDL
cholesterol concentrations caused by intake of increasing
doses of dietary cholesterol. In hamsters, the plasma HDL
cholesterol also increased so that it accounted for 50% of
the total cholesterol at the highest dose tested (0.16
g/100g), where the highest plasma LDL cholesterol concentrations also occurred. In addition, in hamsters, there was a
significant increase in plasma TAG caused by increasing
dietary cholesterol. In hamsters, high plasma TAG and
VLDL cholesterol caused by dietary cholesterol were observed by other investigators (Sessions et al. 1993, Woollett
et al. 1992). The elevated plasma non-HDL cholesterol
fraction observed in the present study may also be caused by
elevations in VLDL cholesterol.
The observed changes in VLDL and LDL composition
caused by increasing levels of dietary cholesterol were previously reported in guinea pigs (Fernandez et al. 1997, Lin et al.
1994). Cholesteryl ester-enriched VLDL from hyperlipidemic
subjects was demonstrated to be more easily converted to
intermediate-density lipoprotein and then to LDL through the
delipidation cascade (Nestel et al. 1983). In addition, studies
in African green monkeys have demonstrated that high-cholesterol diets lead to the formation of cholesteryl ester-enriched VLDL that are readily converted to LDL (Marzetta et
al. 1989). Our present observations of guinea pigs agree with
these reports because those guinea pigs with the highest proportion of cholesteryl esters in VLDL also had the highest
plasma LDL cholesterol concentrations.
Hepatic cholesterol concentrations were increased proportionately to dietary cholesterol; however, significant increases
were observed only with the highest dose. Hamsters and
guinea pigs have different distributions of hepatic cholesterol
in response to a cholesterol challenge. Whereas in guinea pigs
the majority of the hepatic cholesterol exists in the free form,
in hamsters most of it exists as esterified cholesterol. In humans, the majority of the cholesterol in liver is in the free form
(Angelin et al. 1992).

FIGURE 5 Correlation between plasma total cholesterol (TC) in

hamsters or guinea pigs and decreasing casein/soybean protein ratios
(Study 1) (upper panel) (r 0.999, P , 0.01), increasing pectin doses
(Study 2) (middle panel) (r 5 20.985, P , 0.05), and increasing concentrations of dietary cholesterol (Study 3) (lower panel) (r 5 0.95 for
hamsters and 0.99 for guinea pigs, P , 0.05 using a power correlation).

HAMSTERS, GUINEA PIGS, DIET AND LIPOPROTEIN DISTRIBUTION

1331

sible for the elevated plasma LDL cholesterol concentrations

(Horton et al. 1993, Lin et al. 1994).
From these studies we conclude that hamsters and guinea
pigs respond to dietary treatment by lowering plasma LDL
cholesterol, which is similar to humans. Based on the data
presented here, we speculate that the increases in plasma
cholesterol levels in response to dietary cholesterol are affected
in the presence of other dietary components, such as dietary
soluble fiber and the source of protein. Thus, diets containing
vegetable protein or relatively high amounts of dietary fiber
lessen the elevations in plasma cholesterol concentrations
induced by high amounts of dietary cholesterol.

tions of 0.08 g/100g dietary cholesterol, which is equivalent to

600 mg of dietary cholesterol per day. But, in hamsters the
enzyme activity was suppressed only after feeding pharmacological doses of dietary cholesterol.
Interestingly, hepatic cholesterol 7a-hydroxylase activity
was not up-regulated by high cholesterol diets in guinea pigs,
and hamsters as was reported for rats (Horton et al. 1995). Rats
have the ability to convert excess dietary cholesterol to bile
acids. The activity of this bile acid synthesis regulatory enzyme
is extremely high, even in the absence of dietary cholesterol
(Horton et al. 1995). The inability of hamsters and guinea pigs
to respond to excess dietary cholesterol by increasing hepatic
cholesterol catabolism may be one of the reasons why these
two animal models are more responsive than rats to dietary
cholesterol. Thus in hamsters and guinea pigs, the responses to
dietary cholesterol are more consistent with that in humans.
In guinea pigs, ACAT activity was regulated by the increases of free cholesterol concentrations in the liver. However, in hamsters, no effect on this enzyme activity was observed, even in those hamsters in which the concentration of
free cholesterol in the liver was increased. Guinea pigs presented higher ACAT activity with higher levels of dietary
cholesterol possibly caused by the increased substrate (free
hepatic cholesterol) for this enzyme, which occurs when they
were fed high cholesterol diets.
We have demonstrated previously that elevations in hepatic cholesterol concentrations caused by high intake of
dietary cholesterol result in the suppression of hepatic apo B/E
receptors in guinea pigs (Lin et al. 1994). Suppression of LDL
receptors is the main cause of elevated plasma LDL cholesterol
in this animal model. In hamsters, decreases in mRNA levels
for the apo B/E receptor that are associated with decreases in
LDL uptake were also reported with high intakes of dietary
cholesterol (Horton et al. 1993). These reports and our present
data suggest that dietary cholesterol increases the concentrations of hepatic cholesterol. As a compensatory mechanism, in
guinea pigs these higher concentrations of cholesterol in the
liver suppress HMG-CoA reductase activity and increase
ACAT activity, possibly because of increases in substrate
availability. However, the suppression of LDL receptors by
dietary cholesterol appears to be the major mechanism respon-

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FIGURE 6 Comparisons of plasma non-HDL cholesterol concentrations in guinea pigs or hamsters from studies 1, 2, and 3. Values are
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