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Department of Food Science, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan and * Tsukuba Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Ibaraki 305-0841, Japan
1To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • serum cholesterol • soy protein • phospholipid • Caco-2 cells • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Nagaoka et al. 1991 and 1992
,
Potter 1995
, Sirtori et al. 1993
,
Zhang and Beynen 1993
). Soy protein, a vegetable
protein, reduces serum cholesterol relative to casein, an animal
protein.
In addition, the cholesterol-lowering effect of purified
phospholipid also has been reported (Imaizumi et al. 1989
, O'Mullane and Hawthorne 1982
).
Sirtori et al. (1985)
studied the effects of textured
soy protein2
containing 6% lecithin in type-II hyperlipidemic patients, among
whom they noted an increase in HDL cholesterol concentration. However,
there is little information about textured soy protein containing 6%
lecithin, and no studies have yet been reported on the effects of
proteins that bind phospholipids in large quantities and, in
particular, their hydrolysates.
A soy protein peptic hydrolysate
(SPH)3
has been reported to have a stronger lowering effect on serum
cholesterol than that of intact soy protein (Sugano et al. 1990
). These authors showed that SPH both decreased the blood
cholesterol level and promoted fecal excretion of steroids, compared
with casein. From the fecal steroid excretion data, they suggested that
SPH might have inhibited cholesterol absorption. Although the soy
protein effect on cholesterol absorption has been examined previously
in comparison with that by casein (Nagata et al. 1982
),
the in vivo experimental system did not allow determination of its
direct effect on cholesterol absorption. Saeki et al. (1987)
suggested that the inhibition of cholesterol absorption
was not the major factor involved in the differential effects of
dietary proteins on serum cholesterol. Moreover, Lovati et al. (1992)
suggested that the activation of LDL receptor activity
in liver cells induced by soybean globulin may be related to the serum
cholesterol–lowering action of soy protein. Therefore, to elucidate
the molecular mechanism of the inhibitory effect of soy protein on
cholesterol absorption, we evaluated this effect with the use of a cell
strain cultured in vitro. In an earlier paper (Nagaoka et al. 1997
), we used cultured Caco-2 cells and found that SPH
directly inhibited the absorption of micellar cholesterol. However, no
direct in vivo or in vitro studies of the effects of SPHP on
cholesterol absorption from the intestine have been reported.
Interestingly, no work has been reported to date evaluating the effects
of proteins or their hydrolysates on the micellar solubility of
cholesterol.
We postulate that SPHP-induced hypocholesterolemia may have resulted in the inhibition of both cholesterol absorption in the intestinal epithelial cells (accompanying the lowering of micellar cholesterol solubility) and ileal reabsorption of bile acids. Thus, we used Caco-2 cells, rats or in vitro assays related to both the micellar solubility of cholesterol and the binding capacity of taurocholate to investigate the mechanisms of the serum cholesterol–lowering action of SPHP.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Soy protein (New Fujipro-E; Fuji Oil, Osaka, Japan) was hydrolyzed by porcine pepsin (activity; 1:10,000, Nacalai Tesque, Kyoto, Japan) at pH 2.0 and 37°C for 24 h. Porcine pepsin (1 g/100 g) was added to the protein. The digest was heated at 90°C for 30 min and neutralized with 2 mol/L NaOH. The digest was centrifuged at 4500 x g for 20 min. The sediment was washed with water three times and centrifuged at 4500 x g for 20 min. The sediment was freeze-dried and identified as SPH.
Preparation of soy protein with bound phospholipids (SP).
Enzyme-modified soy phospholipids (Elmizer AC; T & K Lecithin, Mie,
Japan) were used as a phospholipid source. Enzyme-modified soy
phospholipids were prepared from soy phospholipids (SLP; True Lecithin,
Mie, Japan) by phospholipase A2 (Novo Industry, Bagsvaerd,
Denmark) hydrolysis as described previously (Pryde 1985
).
The components of enzyme-modified soy phospholipids were analyzed
as described previously (Erdahl et al. 1973
) and were as
follows (g/100 g): phosphatidylcholine, 8.1; phosphatidylethanolamine,
5.4; phosphatidylinositol, 13.2; phosphatidic acid, 4.9;
lysophosphatidylcholine, 25.8; lysophosphatidic acid, 8.4; phytic acid,
7.0; glycerophosphorylethanolamine, 15.4; glycerophosphoinositol, 3.5;
triglyceride, 3.0; water, 0.5; lysophosphatidylethanolamine, 0.1;
lysophosphatidylinositol, 0.1.
Soy protein (New Fujipro-E; Fuji Oil, Osaka, Japan) was dispersed in water and stirred with the use of a homogenizer (Ace homogenizer AM-7, Nihonseiki, Tokyo, Japan) at 2800 x g for 5 min. Enzyme-modified soy phospholipids were then added to the solution and stirred as described above. Soy protein and enzyme-modified soy phospholipids were added in the ratio of 4:1 (wt/wt). The resulting mixture was freeze-dried and identified as SP.
Preparation of soy protein peptic hydrolysate with bound phospholipids (SPHP).
SP was hydrolyzed by the method described in the preparation of SPH. The digest was treated with the same procedures as those for SPH. Finally, the sediment was freeze-dried and identified as SPHP.
Chemical analyses.
Protein content was determined by the Kjeldahl method, with a
N-to-protein conversion factor of 6.25. Lipids were extracted using
chloroform:methanol (2:1, v/v) and weighed. Sugar content was
determined by the phenol/sulfonic acid method (Dubois et al. 1956
). Moisture was determined as the loss in weight after
drying at 105°C for 24 h. Ash content was determined by the
direct ignition method (550°C overnight). The chemical compositions
are shown in Table 1
.
|
, model L-8500, Hitachi, Tokyo, Japan). Tryptophan content was
determined by the p-dimethyl-aminobenzaldehyde method
(Spies and Chambers 1948 and 1949
).
|
Various lipid concentrations were determined using commercially
available kits as follows: serum and liver cholesterol with Monotest
cholesterol (Boehringer Mannheim Yamanouchi, Tokyo, Japan); HDL
cholesterol with HDL-cholestase (Nissui, Tokyo, Japan); serum and
liver triglyceride with Triglycolor III (Boehringer Mannheim
Yamanouchi); serum phospholipid with Phospholipid C-Test Wako (Wako
Pure Chemical, Osaka, Japan). Liver lipids were extracted by the method
of Folch et al. (1957)
, and total lipids were determined
gravimetrically as described previously (Nagaoka et al. 1990
). Fecal acidic steroids were measured according to the
method of Bruusgaard et al. (1977)
and
Malchow-Moller et al. (1982)
, and fecal neutral steroids
were assayed with trimethylsilyl ether by using 1.5% OV-17 with a
GC-14A instrument (Shimadzu, Kyoto, Japan) and 5 
-cholestane as the
internal standard (Miettinen et al. 1965
).
Animals and diets.
Male rats of the Wistar strain (Japan SLC, Hamamatsu, Japan) were used
in two experiments. Room temperature was maintained at 22 ± 2°C
with a 12-h light:dark cycle (lights on 0800–2000 h). The approval of
Gifu University Animal Care and Use Committee was given for our animal
experiments. All of the rats were housed individually in metal cages
and were allowed free access to food and water. After acclimation to a
commercial nonpurified MF
diet4
(Oriental Yeast, Osaka, Japan) for 3 d, rats were divided into
groups on the basis of body weight in Experiments 4 and 5. The basal
diet was that recommended by the AIN (1977)
. All diet
compositions for Experiment 5 are given in Table 3
.
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The binding capacities of cholestyramine, CTH, SPH or SPHP with
taurocholate were measured by the method of Kritchevsky and Story (1974)
with some modifications as described previously
(Hirose et al. 1991
). The mixtures containing 1.85 kBq
of tauro [carbonyl-14C] cholic acid (sodium salt) (1.89
Gbq/mmol, Amersham International, Buckinghamshire, UK), 0.1 mol/L
sodium taurocholate in 5 mL of 0.1 mol/L Tris-HCl buffer (pH 7.4),
and 1–500 mg binding substances (cholestyramine, CTH, SPH or SPHP)
were incubated at 37°C for 2 h; the radioactivity in the
supernatant (15,000 x g for 15 min) was measured
by liquid scintillation counting.
Experiment 2: Micellar solubility of cholesterol and taurocholate.
Micellar solubility of cholesterol with various proteins in vitro was
measured by the method of Ikeda et al. (1988)
with some
modifications. Micellar solutions (1 mL) containing 6.6 mmol/L sodium
taurocholate, 0.5 mmol/L cholesterol, 1 mmol/L oleic acid, 0.5 mmol/L
monoolein, 0.6 mmol/L phosphatidylcholine, 132 mmol/L NaCl, 15 mmol/L
sodium phosphate (pH 7.4), CTH, SPH or SPHP (5.0 g/L) were prepared by
sonication. Then the mixture was incubated at 37° for 24 h and
ultracentrifuged at 100,000 x g for 60 min at
37°C. The supernatant was collected for the determination of
cholesterol and bile acids as described previously (Nagaoka et al. 1997
).
Experiment 3: Cholesterol absorption in Caco-2 cells in vitro.
Caco-2 cells were generously provided by the Central Research Institute
of Meiji Milk Products, (Tokyo, Japan). The cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum (FCS), 4 mmol/L L-glutamine, 50,000
IU/L of penicillin and 50 mg/L streptomycin. The cells were
incubated at 37°C in a humidified atmosphere of 5% CO2
in air. The monolayers became confluent 3–4 d after seeding at between
7 x 105 and 1.2 x 106 cells per
100-mm diameter dish; the cells were passaged at a split ratio of 4–8
by trypsinizing with 0.25% trypsin and 0.8 mmol/L EDTA in PBS.
Monolayers were grown in 24-well plastic dishes containing 1 mL FCS
supplemented with DMEM as described previously (Nagaoka et al. 1997
); fresh medium was added every 2 d. The experiments
described usually used cultures 12–15 d after plating and were
performed in medium-199/Earle's (Gibco, Grand Island, NY) containing 1
mmol/L HEPES. Cell viability, as ascertained by trypan-blue
exclusion, was unaffected by any of the experimental procedures. The
number of passages of the cell line ranged from 60 to 77.
[14C]-Labeled micellar cholesterol uptake in Caco-2 cells
was measured by the method described previously (Nagaoka et al. 1997
). The final concentration of each
[14C]-labeled micellar solution (0.5 mL) was as follows:
3.7 kBq [4-14C]-cholesterol (2.1 Gbq/mmol, NEN, Boston,
MA), 0.1 mmol/L cholesterol, 1 mmol/L oleic acid, 0.5 mmol/L monoolein,
6.6 mmol/L sodium taurocholate, 0.6 mmol/L phosphatidylcholine, and
CTH, SPH or SPHP (2.5 mg/0.5 mL). The micellar solution was mixed by
ultrasonic vibration.
After 14 d, the cells were rinsed two times with 1 mL of PBS. A [14C]-labeled micellar solution (0.5 mL) containing CTH, SPH or SPHP was then added to the dishes, which were incubated at 37°C for 20 min in a CO2 incubator. After this incubation, the cells were rinsed two times with 1 mL of PBS. The cells were finally lysed in 0.1% SDS solution; then 7.5 mL of Aquasol-2 (NEN) was added, and the radioactivity in the cellular debris was counted to determine the amount of cholesterol associated with the cells.
Experiment 4: Cholesterol absorption in vivo.
After acclimation to a commercial nonpurified diet (MF, Oriental Yeast,
Osaka, Japan) for 3 d, 8-wk-old rats weighing 176–205 g were
deprived of food for 48 h with free access to water. Casein
tryptic hydrolysate (CTH) was provided by Meiji Milk Products. The
chemical composition of CTH was as follows (g/kg): protein, 862; ash,
47; moisture, 91; lipid, 0; sugar, 0. Rats received the test solutions
by intragastric intubation with the use of a polyethylene catheter.
Rats were anesthetized with diethyl ether and killed 1 h after the
administration of the test solutions. Blood was collected by cardiac
puncture for the separation of serum. The liver and intestine were
excised quickly. The liver was rinsed with ice-cold saline, and the
luminal contents of the small intestine were removed by flushing with
ice-cold saline. The test solutions consisted of 1 mmol/L monoolein
(Sigma, St. Louis, MO), 5 mmol/L taurocholic acid (Sigma), 37 kBq
[1,2-3H]-cholesterol (1972.1 GBq/mmol, NEN) and CTH, soy
protein, SPH or SPHP (62.5 mg) in 1 mL of 15 mmol/L phosphate buffer
(pH 7.4). These solutions were emulsified by sonication (Ultrasonic
Homogenizer, Model VP-5, Taitec, Saitama, Japan). [3
H]-Cholesterol incorporated into the serum, liver and intestine was
extracted with hexane after saponification of KOH-ethanol as
described previously (Borel et al. 1990
). Aliquots of
the organic extract were used for scintillation counting.
Experiment 5: Lipid metabolism in rats fed casein, soy protein, SP, SPH or SPHP.
After acclimation to a commercial nonpurified diet (MF, Oriental
Yeast), 5-wk-old rats weighing 115–130 g were divided into five groups
of six rats each on the basis of body weight. Each group had free
access to one of the test diets (Table 3)
containing casein, soy
protein, SP, SPH or SPHP as the protein source for 10 d. After
24 h without food, the rats were anesthetized with diethyl ether.
Blood was collected by cardiac puncture and the liver removed. Fecal
collections (d 7–9) were completed before the 24-h food restriction
and blood sampling. Feces were used for determining fecal steroids.
Statistical analyses.
Results are expressed as means and pooled SEM. The
statistical significance of differences was evaluated by Duncan's
multiple range test (Duncan 1957
) after one-way
ANOVA. Differences were considered significant at P
< 0.05.
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RESULTS |
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From 25 to 300 mg, the bile acid–binding capacity of SPHP was
significantly higher than that of CTH or SPH, indicating that SPHP has
the highest bile acid–binding capacity among the protein hydrolysates
tested (Fig. 1
).
|
We used cholestyramine as a standard for the micellar solubility of
cholesterol in preliminary test (in mmol/L: cholestyramine, 0.02
± 0.001; SPH, 0.18 ± 0.02). The micellar solubility of
cholesterol was significantly less in the presence of cholestyramine
compared with SPH. As shown in Table 4
, the micellar solubility of cholesterol was significantly lower in the
presence of SPH or SPHP than CTH or soy protein. In contrast, no
significant effects of protein hydrolysates on bile acids solubility
were observed.
|
Cholesterol uptake from micelles containing SPH (5.70 ± 0.34 pmol/well) or SPHP (4.87 ± 0.21 pmol/well) was significantly lower than that from cholesterol micelles containing casein, CTH or soy protein (9.85 ± 0.97, 9.50 ± 0.87, 8.12 ± 0.82 pmol/well, respectively).
Experiment 4: Cholesterol absorption in rats infused with SPHP, SPH, soy protein or CTH.
Final body weights (CTH, 170.0 ± 2.0 g; soy protein, 170.7
± 2.1 g; SPH, 170.5 ± 2.5 g; SPHP, 170.3 ± 2.0 g) and relative liver weights (in g/100 g body weight: CTH,
2.88 ± 0.03; soy protein, 2.85 ±0.03; SPH, 2.87 ± 0.02;
SPHP, 2.85 ± 0.02) were unaffected by treatments. The
incorporation of [3 H]-cholesterol into the
serum, liver and intestine was significantly lower in the SPH and SPHP
groups than in the CTH and soy protein groups (Table 5
).
|
Food intake and body weight gains were unaffected by dietary treatment,
but the relative liver weight was significantly greater in the
casein-fed group than in all other groups (Table 6
). Serum total cholesterol levels in the SP, SPH and SPHP groups were
significantly lower than in the casein group. Serum HDL cholesterol
concentration and the ratio of HDL cholesterol to total cholesterol in
groups fed soy protein or its hydrolysates were significantly higher
than in the casein-fed group. Serum triglyceride and phospholipid
concentrations were unaffected by dietary treatments. Liver total lipid
and cholesterol concentrations were significantly lower in all soy
protein–fed groups than in the casein-fed group. Fecal dry weight
was significantly higher in the SPH- and SPHP-fed groups than in
the casein-fed group. The fecal outputs of acidic and total
steroids were significantly higher in the SPHP group than in all other
groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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reported that textured soybean protein containing 6% lecithin
increased HDL cholesterol levels in type-II hyperlipidemic
patients. We found that SP and SPHP had higher phospholipid
concentrations (~20%) than those reported by Sirtori et al. (1985)
. In particular, SPHP clearly demonstrated serum
cholesterol–lowering effects. In these studies, soy protein and soy
phospholipids were added in the ratio of 4:1 (wt/wt). By increasing the
content of soy phospholipids in SPHP, the level of unbound
phospholipids was increased. SPHP, rich in unbound phospholipids, is
oxidized more readily than SPHP that are rich in bound phospholipids.
Thus, this ratio (4:1) is appropriate for the quality control of SPHP.
We are trying to identify the active component or components related to
the hypocholesterolemic action of SPHP. We hypothesized that the active
components of SPHP may relate to the combined effects of peptides
derived from soy protein and soy phospholipids. Studies on the active
components of SPHP are currently underway.
It has been postulated that the degree of serum cholesterol lowering
depends on the extent of fecal steroid excretion (Nagata et al. 1982
). We found a significant correlation between fecal total
steroid excretion and serum total cholesterol in rats (r
= 0.60, P < 0.001). Fecal excretion of total
steroids (acidic steroids + neutral steroids) was higher in rats fed
SPHP, indicating that the effect is due, at least in part, to an
enhancement of fecal steroid excretion.
We previously found that serum total cholesterol was significantly
lower in rats fed SPH than in those fed CTH (Nagaoka et al. 1997
). The cholesterol micelles containing SPH significantly
suppressed cholesterol uptake by Caco-2 cells compared with the
cholesterol micelles containing CTH (Nagaoka et al. 1997
). Because intact proteins (casein, soy protein) are
hydrolyzed by digestive enzymes in the body, CTH or SPH is more
appropriate than the intact proteins to identify the active components
affecting the cholesterol absorption. Also, the extent of the effect of
casein on serum cholesterol level (Nagaoka et al. 1997
)
or on cholesterol uptake in Caco-2 cells in this study is almost the
same as that of CTH. Thus, we used the CTH group instead of the casein
group as a control group in Experiments 1, 2 and 4.
In recent studies of lipid metabolism, monolayers of Caco-2 cell
cultures have been used as a model system (Field et al. 1987
, Hughes et al. 1987
, Ranheim et al. 1992
). For example, Field et al. (1987)
reported
that Caco-2 cells, like the small intestine, have the ability to absorb
micellar cholesterol and to express marker enzymes like alkaline
phosphatase, as in small intestinal epithelial cells. However, there
have been few experimental studies to date to evaluate any effects of
peptides on cholesterol uptake by using cultured intestinal cells. In
this study, we found that SPHP also directly inhibited the uptake of
micellar cholesterol in Caco-2 cells in vitro. These results suggest
that the suppression of cholesterol absorption by direct interaction
between cholesterol-mixed micelles and SPHP in the intestinal
epithelia is part of the mechanism of the hypocholesterolemic action of
SPHP.
Cholesterol is rendered soluble in bile salt–mixed micelles and then
absorbed (Wilson and Rudel 1994
). We have found for the
first time that micellar solubility of cholesterol in the presence of
SPH or SPHP was significantly lower than with CTH. Sitosterol
(Ikeda et al. 1988
), sesamine (Hirose et al. 1991
) or catechin (Ikeda et al. 1992)
also
lowered the micellar solubility of cholesterol, in conjunction with the
serum cholesterol–lowering effects in rats. These findings suggest
that suppression of the micellar solubility of cholesterol induces the
inhibition of cholesterol absorption in the jejunum, and this may be
closely related to the lowering action of serum cholesterol. As shown
in the cases of SPH and SPHP, some other dietary proteins or peptides
may also affect such solubility. Although the micellar solubility of
cholesterol in the presence of SPH and SPHP was almost the same, fecal
excretion of acidic steroids was significantly greater in rats fed the
SPHP diet compared with those fed the SPH diet. Iwami et al. (1986)
showed a correlation between the hydrophobicity of a
protein hydrolysate and its binding capacity to bile acids and
suggested that a peptide with a high bile acid–binding capacity could
inhibit the reabsorption of bile acids in the ileum and decrease the
blood cholesterol level. In our feeding study, SPHP had a significantly
greater bile acid–binding capacity than did SPH, concomitant with a
greater increase in fecal bile acid excretion. Thus, the difference in
the degree of hypocholesterolemic action between SPH and SPHP may
result from the differences in their bile acid–binding capacities.
In summary, there have been many studies on the hypocholesterolemic
effects of proteins, most of which emphasized the hypothesis that a
peptide with high bile acid–binding capacity could inhibit the
reabsorption of bile acid in the ileum and decrease the blood
cholesterol level. These possibilities may be applicable to the case of
SPHP on the basis of the evidence of fecal bile acid excretion and bile
acid–binding capacity in this study. However, our earlier study
(Nagaoka et al. 1997
) and this study clearly suggest
that the inhibition of micellar solubility of cholesterol, which causes
the suppression of cholesterol absorption by direct interaction between
cholesterol-mixed micelles and SPHP in the jejunal epithelia, is
part of the mechanism of hypocholesterolemic action induced by SPHP.
Thus, the hypocholesterolemic action of SPHP may involve both jejunal
and ileal effects, as shown in this study.
Our experimental system to evaluate cholesterol uptake in Caco-2 cells is useful for clarifying both the molecular mechanism and active components underlying the inhibitory effect of soy protein or other proteins on cholesterol absorption from the small intestine; it can also be expected to facilitate greatly an elucidation of the effects of various food constituents on cholesterol absorption in the future.
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FOOTNOTES |
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). 
3 Abbreviations used: CTH, casein tryptic
hydrolysate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal
calf serum; SP, soy protein with bound phospholipids; SPH, soy protein
peptic hydrolysate; SPHP, soy protein peptic hydrolysate with bound
phospholipids.
4 Commercial nonpurified MF diet contains the
following nutrients (g/kg): water, 76; protein, 246; lipid, 56;
carbohydrate, 552; minerals, 63; vitamins, 7.
Manuscript received September 10, 1998. Initial review completed December 8, 1998. Revision accepted May 28, 1999.
|
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