Biosci. Biotechnol. Biochem.

, 72 (6), 1506–1512, 2008

Mechanism for the Cholesterol-Lowering Action

of Egg White Protein in Rats
Ryosuke M ATSUOKA,1;2; y Mamoru K IMURA,1 Ayano M UTO,1 Yasunobu M ASUDA,1
Masao S ATO,2 and Katsumi IMAIZUMI2
1
R&D Division, Q.P. Corporation, 403-2 Motokurihashi, Goka-machi, Sashima-gun, Ibaraki 306-0313, Japan
2
Laboratory of Nutrition Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate
School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received January 9, 2008; Accepted March 3, 2008; Online Publication, June 7, 2008
[doi:10.1271/bbb.80016]

Eggs are a popular source of dietary cholesterol, but
their consumption does not necessarily result in an
increased serum cholesterol concentration. We inves-
tigated the cholesterol-lowering activity of egg white
protein (EWP) and its potential mechanism in rats. The
consumption of EWP resulted in a decreased concen-
tration of cholesterol in the serum, liver and intestinal
mucosa. The excretion of fecal neutral sterols and bile
acids was greater by rats fed with EWP than by those
fed with casein. The ratio of cholesterol and bile acids in
the micellar phase to those in the solid phase was lower
in the intestinal contents from rats fed with EWP than
from those fed with casein.
These results suggest that the cholesterol-lowering
activity of EWP can be attributed to lowering the
cholesterol absorption by intervening in the micellar
formation in the intestines.
Key words: egg white protein; cholesterol; intestine;
micelle; rat
Hypercholesterolemia is one of the most prevalent
risk factors for arteriosclerosis.1) Eggs are cholesterol-
containing food; therefore, their consumption is thought
to result in an increased serum cholesterol concentra-
tion.2) Avoidance of eggs is the usual dietary recom-
mendation to reduce hypercholesterolemia. However,
the results of many clinical trials have shown no
correlation between egg consumption and the serum
cholesterol concentration.3–6) An epidemiological survey
has fact in shown a negative correlation between the
intake of eggs and cardiovascular diseases.7) Thus, it is
thought that a component in egg may help in controlling
the serum cholesterol concentration.
It has previously been reported that egg yolk
phospholipids lowered the concentration of serum
cholesterol by inhibiting cholesterol absorption.8) How-
ever, there have been a few studies reporting the effect
of egg white on the cholesterol concentration.9–11) Egg
white consists mainly of water and protein, but no
cholesterol. If egg white protein (EWP) has a choles-
terol-lowering effect, it may be of benefit during dietary
therapy for hypercholesterolemia. It has been reported
that such vegetable proteins as soy protein decreased
the serum cholesterol concentration,12–17) whereas such
animal proteins as casein increased it.12) Asato et al.
have reported that consumption of egg white compared
with cheese (milk protein) reduced the serum total- and
low-density lipoprotein (LDL)-cholesterol concentra-
tions in humans. The cholesterol-lowering activity of
egg white was similar to that of tofu (a processed food
made from soy protein).9) Therefore, it is considered
that EWP may also reduce the serum cholesterol
concentration.
Nagaoka et al. have reported that ovomucin, which
is an EWP, inhibited the incorporation of cholesterol
into Caco-2 cells by binding to bile acids.11) However,
the ovomucin content in EWP is only at the level of 1.5–
3.5%.18) Therefore, it is unlikely that the cholesterol-
lowering activity of EWP is due to ovomucin alone.
EWP is rich in cystine compared with casein. Some
workers have reported that cystine supplementation or
cystine-enriched protein reduced the serum total choles-
terol concentration in rats fed on a high-cholesterol
diet.19,20) Others, however, did not confirm the hypo-
cholesterolemic action of cystine, but instead reported
the hypercholesterolemic action in rats.21,22) According-
ly, the role of EWP in cholesterol metabolism after
egg consumption remains unclear. We showed in this
study the hypocholesterolemic action of EWP in rats
and propose a possible mechanism that includes the
inhibition of cholesterol absorption in the intestines.

y
To whom correspondence should be addressed. Fax: +81-280-84-3141; E-mail: ryosuke matsuoka@kewpie.co.jp
Abbreviations: EWP, egg white protein; SD, Sprague Dawley; LDL, low-density lipoprotein; HDL, high-density lipoprotein; AIN, American
Institute of Nutrition; ANOVA, analysis of variance
 Cholesterol-Lowering Action of Egg White Protein
Material and Methods
Material. EWP was prepared from un-sterilized egg
white (Q.P. Egg Corporation, Tokyo). The un-sterilized
egg white was freeze-dried and crushed uniformly.
Casein was purchased from Oriental Yeast Co., Ltd.
(Tokyo, Japan). The protein contents, which were
determined by the Kjeldahl method, were calculated as
the nitrogen content
6.25. The measured protein
content was 84.8% casein and 80.2% EWP. Their
cystine content, which was measured by high-perform-
ance liquid chromatography, was 2.1% for EWP and
0.4% for casein.
Animals and diet. Male SD rats (Japan SLC Inc.,
Shizuoka) weighing 250–300 g were used in this study.
The rats were bred in a temperature (23  1  C) and
humidity (50  2%)-controlled room with a 12-h light
cycle (light from 08.00 to 20.00). The control diet
was formulated according to the AIN-93G formula23)
and contained the following ingredients [in weight %:
protein (casein or EWP), 20;
-cornstarch (Oriental
Yeast Co., Ltd.), 13.2; sucrose (Mitsui Sugar Co., Ltd.,
Tokyo, Japan), 10; cellulose (Oriental Yeast Co., Ltd.),
5; mineral mixture (AIN-93G, Oriental Yeast Co., Ltd.),
3.5; vitamin mixture (AIN-93, Oriental Yeast Co., Ltd.),
1; soybean oil (The Nisshin OilliO Group Ltd., Tokyo,
Japan), 7; choline bitartrate (Wako Pure Chemical
Industries Ltd., Osaka, Japan), 0.25; tert-butyl hydro-
quinone (Junsei Chemical Co., Ltd., Tokyo, Japan),
0.0014; and -cornstarch (Oriental Yeast Co., Ltd.),
40.05]. The hypocholesterolemic action of soy protein is
well-known. The cholesterol-lowering action of soy
protein has been investigated for a diet containing 20%
soy protein in animal experiments.13–16) Therefore, in all
experiment in this series, EWP was added at the level
of 20%. To the high-cholesterol diet, 0.5% cholesterol
(Wako Pure Chemical Industries Ltd.) and 0.125%
cholic acid (Wako Pure Chemical Industries Ltd.) were
added at the expense of -cornstarch to the control
diet. To the casein+cystine diet, 0.3% L-cystine (Wako
Pure Chemical Industries Ltd.) was added at the expense
of -cornstarch to the high-cholesterol diet containing
casein. The cystine content of a casein+cystine diet
was equal to that of the high-cholesterol diet containing
EWP.
In Experiment 1, the rats were fed daily on the high-
cholesterol diet for 3 weeks ad libitum. In Experiment 2,
the rats were meal-fed for 2 h (09.00 to 11.00) with the
high-cholesterol diet or control diet containing casein or
EWP for 3 weeks. In this experiment, the tail vain
plasma was collected each week to analyze the changes
in cholesterol concentration. The plasma total choles-
terol concentration of the rats was influenced by the
time when the rats were fed. Therefore, the rats in this
experiment were meal-fed to unify the feeding and
fasting times. In Experiment 3, the rats were fed with
the high-cholesterol diet containing casein or EWP, or
1507
with the casein+cystine diet for 3 weeks. EWP contains
about 0.5% avidin,18) which inhibits biotin absorption,
thereby causing biotin deficiency.24) Avidin consists of a
tetramer; thus, 4 mol of biotin could be bound to 1 mol
of avidin. The molecular weight of avidin is about
68,000, and of biotin is 244. We calculated the biotin
content that could be bound to avidin in the diet
containing 20% EWP as 0.0002%. Thus, the all diets in
Experiment 3 were supplemented with 0.0002% biotin
(Wako Pure Chemical Industries Ltd.) at the expense
of -cornstarch.25) Feces were collected for 3 d just
before the end of the feeding period. In Experiment 4,
the rats were meal-fed for 1 h (10.00 to 11.00) with
the high-cholesterol diet for 1 week, and the small
intestine was removed. The intestinal contents were
allowed to drain as much as possible into centrifuge
tubes by gentle massage. The tubes were placed in a
water bath at 70  C for 15 min to deactivate lipase and
cholesterol esterase and then centrifuged at 100,000 g
for 15 h to obtain two phases: a solid (pellet) phase and
a micellar (clear yellowish supernatant) phase.26) The
small intestine was washed with ice-cold saline, divided
into four equal lengths, and the intestinal mucosa was
scraped off.
All rats were freely provided with distilled water
throughout the experiments. On the last day, after 8 h of
fasting (in Experiment 4, two hours of fasting), the rats
were anesthetized with sodium pentobarbital (Dainippon
Sumitomo Pharma Co., Ltd., Osaka, Japan), blood
was collected from the abdominal aorta, and the liver
was removed. The blood was centrifuged at 3,000 g for
10 min to collect the serum. The serum and liver were
then stored at 20  C until needed for analysis.
This study was conducted in accordance with the
guidelines set for the care and use of laboratory animals
by the Ministry of the Environment, Government of
Japan.
Chemical analysis. The total cholesterol concentra-
tions in the serum from the abdominal aorta and plasma
from the tail vain were analyzed with a Cholesterol
C-test Wako kit (Wako Pure Chemical Industries
Ltd.).27) The serum triglyceride, phospholipid, and
high-density lipoprotein (HDL)-cholesterol concentra-
tions were respectively analyzed with Triglyceride
C-test Wako, Phospholipid E-test Wako, and HDL-
cholesterol C-test Wako kits (Wako Pure Chemical
Industries Ltd.).28–30) Hepatic lipids were extracted and
analyzed for their cholesterol, triglyceride, and phos-
pholipid concentrations.31–34) The collected feces and
solid phase of the intestinal contents were freeze-dried
and extracted with ethanol.35) The neutral sterols and
bile acid contents of the feces and solid phase were
analyzed by gas chromatography, using 5
-cholestane
(Sigma-Aldrich Inc., St. Louis, USA) and nor-desoxy-
cholic acid (Toronto Research Chemicals Inc., North
York, Canada) as respective internal standards.36,37) The
lipids of the intestinal mucosa were extracted, and the
1508 R. M ATSUOKA et al.

cholesterol content was analyzed by gas chromatogra- Table 1. Serum and Hepatic Lipid Concentrations in Rats Fed with a
phy.31,36) The cholesterol content of the micellar phase Casein- or EWP-Containing Diet (Experiment 1)
of the intestinal contents was analyzed with a Choles- Casein EWP
terol C-test Wako kit (Wako Pure Chemical Industries
Growth parameter (g/d)
Ltd.).27) The bile acid content of the micellar phase was Body weight gain 5:22  0:35 5:22  0:47
analyzed enzymatically.38) Food intake 23:0  0:8 21:9  0:7

Statistical analysis. Each result is expressed as the Serum lipids (mg/100 ml)
Total cholesterol 122  5 101  7
mean  standard error (SE). The statistical difference
HDL-cholesterol 41:5  3:6 45:9  4:6
between casein and EWP was determined by Student’s Triglyceride 194  23 144  19
t-test, and that between the feeding period and dietary Phospholipid 144  11 142  14
protein was analyzed with a two-way analysis of
variance (ANOVA) followed by Dunnett’s test and Hepatic lipids (mg/g of liver)
Cholesterol 36:7  2:0 23:3  2:4
Student’s t-test. The statistical difference among casein,
Triglyceride 109:8  4:8 70:4  5:1
casein+cystine, and EWP was analyzed with one-way Phospholipid 29:1  0:9 31:0  0:6
ANOVA followed by Tukey’s test. A p value < 0:05
Mean  SE of 6 rats.
indicates significant difference. Statistical analyses were 
, p < 0:05 vs. casein group by Student’s t-test.
conducted with Dr. SPSS for Windows (SPSS Japan
Inc., Tokyo, Japan).
Effects of dietary cholesterol on the cholesterol-
Results lowering activity of EWP (Experiment 2)
SD rats were meal-fed on a high-cholesterol diet or a
Effect of EWP on the serum and hepatic lipid cholesterol-free diet containing casein or EWP for 3
concentrations in rats (Experiment 1) weeks (09.00 to 11.00). With the high-cholesterol diet,
SD rats were fed on a high-cholesterol diet containing no significant difference in body weight gain (3:4 
casein or EWP for 3 weeks ad libitum. No significant 0:4 g/d for EWP and 3:2  0:2 g/d for casein consump-
differences in body weight gain and food intake were tion) or food intake (15:6  0:5 g/d for EWP and 15:4 
apparent throughout the experiment (Table 1). The 0:6 g/d for casein consumption) was apparent. With
serum and hepatic cholesterol concentrations had de- the cholesterol-free diet, no significant difference in the
creased more after the consumption of EWP than of body weight gain (3:1  0:3 g/d for EWP and 2:6 
casein. EWP consumption resulted in a decrease in the 0:2 g/d for casein consumption) or food intake (14:7 
hepatic triglyceride concentration. There were no sig- 0:8 g/d for EWP and 15:0  0:3 g/d for casein con-
nificant differences between the casein and EWP diets in sumption) was apparent. Changes in the plasma choles-
the serum HDL-cholesterol, triglyceride, phospholipid, terol concentration after EWP and casein consumption
and hepatic phospholipid concentrations. with the cholesterol-containing or cholesterol-free diets
are shown in Fig. 1. Casein consumption with the high-

A B
Total cholesterol Total cholesterol
(mg/100 ml) (mg/100 ml)
120 120

100 100
80 80
60 60
40 40
20 20
0 0
0 1 2 3 0 1 2 3
Feeding period (weeks) Feeding period (weeks)

Fig. 1. Changes in the Plasma Cholesterol Concentrations of Rats Fed with a Casein- or EWP-Containing Diet Supplemented with Cholesterol (A)
or without Supplementation (B) (Experiment 2).
, Casein group; , EWP group. Mean  SE of 6 rats.  , p < 0:05 vs. 0 weeks by Dunnett’s test; # , p < 0:05 vs. casein group by Student’s
t-test.
 Cholesterol-Lowering Action of Egg White Protein 1509
Table 2. Serum and Hepatic Lipid Concentrations, and Fecal Steroid Table 3. Amounts of Cholesterol and Bile Acids in the Micellar
Excretion in Rats Fed with a Casein-, EWP-, or Casein+Cystine- Phase and Solid Phase Prepared from the Intestinal Contents of Rats
Containing Diet (Experiment 3) Fed with a Casein- or EWP-Containing Diet (Experiment 4)

Casein EWP Casein+cystine Casein EWP

Growth parameter (g/d) Intestinal contents
Body weight gain 4:89  0:24 5:15  0:26 5:21  0:15 Total weight (g/rat) 1:94  0:19 3:34  0:38
Food intake 20:5  0:2 19:8  0:4 20:5  0:1
Micellar phase
Serum lipids (mg/100 ml) Total volume (ml/rat) 1:33  0:16 2:42  0:28
Total cholesterol 152  11a 115  5b 136  9ab Cholesterol (mg/rat) 1:27  0:13 0:89  0:09
HDL-cholesterol 28:6  1:9a 40:6  2:9b 35:3  3:6ab Bile acid (mmol/rat) 109  14 131  10
Triglyceride 130  14 108  15 163  20
Phospholipid 150  4 156  8 164  9 Solid phase
Total weight (mg/rat) 499  32 705  61
Hepatic lipids (mg/g of liver) Cholesterol (mg/rat) 2:64  0:37 4:71  0:46
Cholesterol 37:8  1:6a 26:2  1:1b 30:0  2:4b Bile acid (mmol/rat) 33:2  6:2 87:5  12:2
Triglyceride 81:3  2:5 82:1  5:3 91:2  6:9
Phospholipid 30:6  1:4 31:2  1:7 29:8  1:7 Distribution (micellar/solid)
Cholesterol 0:52  0:06 0:19  0:02
Fecal steroids (mg/d) Bile acid 4:04  0:92 1:67  0:32
Neutral sterols 43:0  1:4a 49:4  1:0b 39:8  1:3a
Mean  SE of 6 rats.
Bile acids 13:9  0:7a 21:4  0:7b 13:7  0:7a 
, p < 0:05 vs. casein group by Student’s t-test.
Mean  SE of 6 rats.
ab
, Different letters show a significant difference at p < 0:05 by Tukey’s test.

of fecal neutral sterols and bile acids, but there was no

cholesterol diet resulted in a significant time-dependent
increase in the plasma total cholesterol concentration
from 68:2  3:2 mg/100 ml at 0 weeks to 101:4 
8:1 mg/100 ml at 3 weeks (Fig. 1A). However, EWP
consumption did not result in such a change over 3
weeks (65:3  4:5 mg/100 ml at 0 weeks and 75:4 
7:0 mg/100 ml at 3 weeks). Hence, the levels of plasma
cholesterol after 2 and 3 weeks were lower in the EWP
group than in the casein group. With the cholesterol-free
diet (Fig. 1B), no change was apparent in the plasma
total cholesterol concentration over 3 weeks after the
consumption of casein or EWP.
Effect of cystine and biotin on the serum, hepatic, and
fecal steroids in rats (Experiment 3)
SD rats were fed on a high-cholesterol diet containing
casein, EWP, or casein+cystine for 3 weeks. These
three diets were supplemented with 0.0002% biotin.
No significant difference in body weight gain or
food intake was apparent throughout the experiment
(Table 2). The serum cholesterol concentrations were
significantly lower after EWP consumption than after
casein consumption, but there was no significant effect
of dietary cystine. The hepatic cholesterol concentra-
tions were lower after EWP or casein+cystine con-
sumption than after casein consumption. Although the
rats were fed with a diet containing 0.0002% biotin in
this experiment, EWP still resulted in reduced serum and
hepatic cholesterol concentrations. EWP enhanced the
serum HDL-cholesterol concentration when compared
with the effect from casein. No significant differences
were observed in the serum triglyceride, phospholipids,
hepatic triglyceride, and phospholipids concentrations.
EWP consumption resulted in an increased excretion
significant effect from dietary cystine.
Effect of EWP on the steroid distribution between the
micellar and solid phases of the intestinal contents
(Experiment 4)
SD rats were meal-fed a high-cholesterol diet con-
taining casein or EWP for 1 week. No significant
difference in body weight gain (2:8  0:5 g/d for EWP
and 2:4  0:4 g/d for casein consumption) or food
intake (13:8  0:5 g/d for EWP and 14:3  0:9 g/d for
casein consumption) was apparent. The serum total
cholesterol concentration was significantly lower in the
EWP group than casein group (75:6  5:5 mg/100 ml
and 102:4  5:8 mg/100 ml serum, p < 0:05). The
hepatic cholesterol concentration was also significantly
lower in the EWP group than casein group (4:9 
0:7 mg/g and 9:5  1:5 mg/g of liver, p < 0:05). No
significant difference was apparent in the cholesterol
amount in the stomach contents (46:5  1:5 mg for
the EWP and 47:7  2:3 mg for the casein group,
respectively).
The amount of intestinal contents was significantly
higher in the EWP group than in the casein group
(Table 3). The micellar volume and solid phase weight
were significantly higher in the EWP group than in the
casein group (Table 3). In the micellar phase, the
cholesterol content was significantly lower in the EWP
group than in the casein group. No significant difference
was apparent in the bile acid contents between the
groups. In the solid phase, the cholesterol and bile acid
contents were significantly higher in the EWP group
than in the casein group. The ratio of cholesterol or
bile acid in the micellar phase to that in the solid phase
was significantly lower in the EWP group than in the
casein group.
1510 R. M ATSUOKA et al.
Cholesterol
(mg/g of intestinal mucosa)
4 Accordingly, the ratio of cholesterol in the micellar
3
2 experiment, the amount of intestinal contents was 1.7-
1 intestinal segment no. 2 and the greater excretion of
0
1 2 3 4
(Upper) (Lower)
Intestinal segment no.
Fig. 2. Cholesterol Content of the Intestinal Mucosa in Rats Fed with
a Casein- or EWP-Containing Diet (Experiment 4).
, Casein group; , EWP group. Upper, stomach side. Lower,
cecum side. Mean  SE of 6 rats. # , p < 0:05 vs. casein group by
Student’s t-test.
No significant difference was apparent for the
mucosal weight in the intestine (3:43  0:18 g for
EWP and 3:34  0:15 g for casein consumption). The
cholesterol content in the intestinal mucosa is shown in
Fig. 2. The cholesterol content of intestinal segment
no. 2 was significantly lower in the EWP group than in
the casein group. The cholesterol content of intestinal
segment no. 2 was higher than in any another segment.
No significant difference was apparent in other intestinal
segments between the groups.
Discussion
The hypocholesterolemic action of EWP has been
reported in clinical trials and animal experiments.9,10)
We confirmed the cholesterol-lowering effect of EWP
compared with casein in rats. The cholesterol-lowering
action of EWP was observed only for the diet containing
cholesterol and bile acid, but not for the cholesterol-free
diet. Furthermore, the excretion of fecal neutral sterols
and bile acids was significantly higher in the rats fed
with the high-cholesterol diet containing EWP than with
casein. EWP lowered the hepatic cholesterol concen-
tration. It is therefore likely that EWP reduced the serum
cholesterol concentration by inhibiting cholesterol ab-
sorption.
A critical step in cholesterol absorption is the efficient
incorporation of cholesterol into bile acid micelles from
the solid phase in the intestines.39) In order to address
this issue, the intestinal contents were collected 2 h after
the diet had been consume, and the steroid content in
the micellar and non-micellar phases (solid phase) was
determined. The cholesterol content in the micellar
phase was significantly lower in the rats fed with the
EWP diet than in those fed with the casein diet. The
cholesterol content in the solid phase was significantly
higher in the EWP group than in the casein group.
phase to that in the solid phase was significantly lower in
the EWP group than in the casein group. These results
indicate that EWP inhibited the micellar solubility of
cholesterol from the solid phase. Furthermore, in this
fold higher in the EWP group than in the casein group.
It is also considered that the lower cholesterol content in
fecal neutral sterol by the EWP group compared with the
casein group is attributable to the lower cholesterol
absorption in the former group. In the present study, the
bile acid content of the solid phase was significantly
higher in the EWP group than in the casein group. These
results suggest that bile acids derived from bile in the
EWP group was not effectively used for the formation
of micelles. Furthermore, it remains to be determined if
an increased proportion of bile acids in the solid phase
might have caused the decreased absorption from the
ileum, hence increasing the fecal excretion of bile acids.
A question is here how the EWP diet influenced
the partition of cholesterol and bile acids between the
micellar and solid phases. It has been reported that
ovalbumin constituted about 54% of EWP and was
denatured by acidic pH and pepsin.40,41) Denatured
ovalbumin causes the gelation and formation of hydro-
phobic particles.42) Therefore, it is likely that the EWP-
derived substances formed during the passage through
the digestive tract resulted in the higher amount of
intestinal contents in the EWP group than in the casein
group, and lowered the proportion of cholesterol and
bile acids in the micellar phase.
Furthermore, our preliminary experiment showed that
pepsin hydrolysates of ovalbumin (the most abundant
component of EWP) and ovotransferrin (the second
most abundant component of EWP, about 13%) inhib-
ited the micellar solubility of cholesterol in vitro
(Matsuoka, R., un published results). Therefore, it is
thought that the protein component of EWP was
responsible for the lower serum cholesterol concentra-
tion by inhibiting the micellar solubility of cholesterol in
the intestines. The main component of EWP are 54%
ovalbumin, 13% ovotransferrin, 11% ovomucoid, and
3.5% lysozyme.18) We confirmed that the water-soluble
fraction of EWP containing ovalbumin and ovotransfer-
rin pepsin hydrolysates inhibited the micellar solubility
of cholesterol, whereas, in soy protein, it has been
reported that insoluble fractions such as 7S and 11S
globulin had a cholesterol-lowering action.13) Therefore,
it is possible that the cholesterol-lowering mechanism
of EWP is different from that of soy protein. In this
context, it is interesting to note that ovomucin, which is
a less abundant EWP insoluble component (about 1.5–
3.5% of EWP), reduced the incorporation of cholesterol
into Caco-2 cells by binding to bile acids.11) In any case,
 Cholesterol-Lowering Action of Egg White Protein
it is thought that further studies are required to identify
the cholesterol-lowering component, including the ami-
no acid composition and the peptides of EWP.
It has been reported that an increased amount of
dietary cystine resulted in a reduced serum cholesterol
concentration.20) Egg white contains a larger amount of
cystine than does casein. In the present study, dietary
cystine resulted in a reduced hepatic cholesterol con-
centration, but had no effect on the serum cholesterol
concentration. EWP contains about 0.5% avidin,18)
which can inhibit biotin absorption,24) hence leading
to biotin deficiency. It has been reported that biotin
deficiency decreased the serum cholesterol concentra-
tion.25,43) For this reason, it is possible that biotin
deficiency is related to the cholesterol-lowering activity
of EWP. However, the present results indicate that there
was no correlation between biotin deficiency and the
cholesterol-lowering action of EWP.
In all experiments in this series, rats were fed with a
diet containing 20% casein or EWP. Therefore, we did
not investigate whether the cholesterol-lowering action
of EWP was dose dependent; however, our preliminary
experiments showed that an EWP pepsin hydrolysate
inhibited the micellar solubility of cholesterol in vitro in
a dose-dependent manner (Matsuoka, R., un published
results). The cholesterol-lowering action of EWP may
also be a dose-dependent function.
In summary, the present results indicate the choles-
terol-lowering activity of EWP and that the possible
mechanism is the reduction of cholesterol absorption by
inhibition of the micellar solubility of cholesterol in the
intestines.
References
1) Kannel, W. B., Neaton, J. D., Wentworth, D., Thomas,
H. E., Stamlar, J., Hulley, S. B., and Kjelsberg, M. O.,
Overall and coronary heart disease mortality rates in
relation to major risk factors in 325,348 men screened
for the MRFIT multiple risk factor intervention trial. Am.
Heart J., 112, 825–836 (1986).
2) Ingr, I., Simenova, J., Stavkova, J., Petrovsky, E., and
Dostal, F., Cholesterol content in market hen eggs.
Nahrung, 31, 933–940 (1987).
3) Morgen, J. M., Tavani, D. M., Garwin, J. L., Stowell,
R. L., Stowell, R. L., Richardson, M. P., and Capuzzi,
M., Effect of dietary (egg) cholesterol on serum
cholesterol in free-living adults. J. Appl. Nutr., 45, 74–
84 (1993).
4) Schnohr, P., Thomsen, O., Hansen, P. R., Boberg-ans,
G., Lawaetz, H., and Weeke, T., Egg consumption and
high-density-lipoprotein cholesterol. J. Int. Med., 235,
249–251 (1994).
5) Knopp, R. H., Retzlaff, B. M., Walden, C. E., Dowdy,
A. A., Tsunehara, C. H., Austin, M. A., and Nguyen,
T., A double-blind, randomized, controlled trial of the
effects of two eggs per day in moderately hypercholes-
terolemic and combined hyperlipidemic subjects taught
the NCEP Step I diet. J. Am. Coll. Nutr., 16, 551–561
(1997).
1511
6) Blanco-Molina, A., Castro, G., Mortı́n-Escalante, D.,
Bravo, D., López-Miranda, J., Castro, P., López-Segura,
F., Fruchart, J. C., Odovás, J. M., and Pérez-Jiménez,
F., Effects of different dietary cholesterol concentrations
on lipoprotein plasma concentrations and on cholesterol
efflux from Fu5AH cells. Am. J. Clin. Nutr., 68, 1028–
1033 (1998).
7) McNamara, D. J., Dietary cholesterol and atheroscle-
rosis. Biochim. Biophys. Acta, 1529, 310–320 (2000).
8) Ikeda, I., Matsuoka, R., Hamada, T., Mitsui, K.,
Imabayashi, S., Uchino, A., Sato, M., Kuwano, E.,
Itamura, T., Yamada, K., Tanaka, K., and Imaizumi, K.,
Cholesterol esterase accelerates intestinal cholesterol
absorption. Biochim. Biophys. Acta, 1571, 34–44 (2001).
9) Asato, L., Wang, M. F., Chan, Y. C., Yeh, S. H., Chung,
H. M., Chung, S. Y., Chida, S., Uezato, T., Suzuki, I.,
Yamagata, N., Kokubu, T., and Yamamoto, S., Effect of
egg white on serum cholesterol concentration in young
women. J. Nutr. Sci. Vitaminol., 42, 87–96 (1996).
10) Yamamoto, S., Kita, T., Yamagata, N., Kokubu, T.,
Shinjo, S., and Asato, L., Favorable effects of EWP on
lipid metabolism in rats and mice. Nutr. Res., 13, 1453–
1457 (1993).
11) Nagaoka, S., Masaoka, M., Zhang, Q., Hasegawa, M.,
and Watanabe, K., Egg ovomucin attenuates hyper-
cholesterolemia in rats and inhibits cholesterol absorp-
tion in Caco-2 cells. Lipids, 37, 267–272 (2002).
12) Carrol, K. K., and Hamilton, R. M. G., Effects of dietary
protein and carbohydrate on plasma cholesterol levels in
relation to atherosclerosis. J. Food Sci., 40, 18–23
(1975).
13) Okita, T., and Sugano, M., Effect of dietary soybean
globulins on plasma lipid and fecal excretion of neutral
sterols in rats. J. Nutr. Sci. Vitaminol., 27, 379–388
(1981).
14) Nagata, Y., Ishikawa, N., and Sugano, M., Studies on
the mechanism of antihypercholesterolemic action of
soy protein and soy protein-type amino acid mixture in
relation to the casein counterparts in rats. J. Nutr., 112,
1614–1625 (1982).
15) Sugano, M., Yamada, Y., Yoshida, K., Hashimoto, Y.,
Matsuo, T., and Kimoto, M., The hypocholesterolemic
action of undigested fraction of soybean protein in rats.
Atherosclerosis, 72, 115–122 (1988).
16) Choi, Y. S., Goto, S., Ikeda, I., and Sugano, M.,
Interaction of dietary cholesterol and age on lipid
metabolism in rat. Br. J. Nutr., 61, 531–543 (1989).
17) van Raaij, J. M., Katan, M. B., West, C. E., and
Hautvast, J. G., Influence of diets containing casein, soy
isolate, and soy concentrate on serum cholesterol and
lipoprotein in middle-aged volunteers. Am. J. Clin. Nutr.,
35, 925–934 (1982).
18) Nakamura, R., Takeyama, M., Nakamura, K., and
Umemura, O., Constituent proteins of globulin fraction
obtained from egg white. Agric. Biol. Chem., 44, 2357–
2363 (1980).
19) Sugiyama, K., Mizuno, M., and Muramatsu, K., Effect
of individual amino acids on plasma cholesterol in
rats fed a high cholesterol diet. J. Nutr. Sci. Vitaminol.,
32, 623–633 (1986).
20) Oda, H., Functions of sulfur-containing amino acids in
lipid metabolism. J. Nutr., 136, 1666S–1669S (2006).
21) Serougne, C., Felginess, G., Ferezou, J., Hajri, T., Bertin,
1512 R. M ATSUOKA et al.
C., and Mazur, A., Hypercholesterolemia induced by
cholesterol- or cystine-enriched diets is characterized by
different plasma lipoprotein and apolipoprotein concen-
trations in rats. J. Nutr., 125, 35–41 (1995).
22) Aoyama, Y., Amano, N., and Yoshida, A., Cholesterol
synthesis and degradation in normal rats fed a choles-
terol-free diet with excess cystine. Lipids, 34, 583–589
(1999).
23) Reeves, P. G., Nielsen, F. H., and Fahey Jr., G. C., AIN-
93G purified diets for laboratory rodents: final report of
the American Institute of Nutrition ad hoc Writing
Committee on the reformulation of the AIN-76A rodent
diet. J. Nutr., 123, 101–109 (1993).
24) Rasmussen, M. V., Barker, T. T., and Silbart, L. K., High
affinity binding site-mediated prevention of chemical
absorption across the gastrointestinal tract. Toxicol. Lett.,
125, 51–59 (2001).
25) Klevay, L. M., The biotin requirement of rats fed 20%
egg white. J. Nutr., 106, 1643–1646 (1976).
26) Ikeda, I., and Sugano, M., Some aspects of mechanism
of inhibition of cholesterol absorption by -sitosterol.
Biochim. Biophys. Acta, 732, 651–658 (1983).
27) Allain, C. C., Poon, L. S., Chan, C. S., Richmond, W.,
and Fu, P. C., Enzymatic determination of total serum
cholesterol. Clin. Chem., 20, 470–475 (1974).
28) Burstein, M., Scholnick, H. R., and Morfin, R., Rapid
method for the isolation of lipoproteins from human
serum by precipitation with polyanions. J. Lipid Res., 11,
583–595 (1970).
29) Spayd, R. W., Bruschi, B., Burdick, B. A., Dappen,
G. M., Eikenberry, J. N., Esders, T. W., Figueras, J.,
Goodhue, C. T., LaRossa, D. D., Nelson, R. W., Rand,
R. N., and Wu, T. W., Multilayer film elements for
clinical analysis: applications to representative chemical
determinations. Clin. Chem., 24, 1343–1350 (1978).
30) Takayama, M., Itoh, S., Nagasaki, T., and Tanimizu,
I., A new enzymatic method for determination of serum
choline-containing phospholipids. Clin. Chem. Acta, 79,
93–98 (1977).
31) Folch, J., Lee, M., and Stanley, G. H. S., A simple
method for the isolation and purification of total lipids
for animal tissue. J. Biol. Chem., 226, 497–509 (1957).
32) Sperry, W. M., and Webb, M., A revision of the
Schoenheimer-Sperry method for cholesterol determina-
tion. J. Biol. Chem., 187, 97–106 (1948).
33) Fletcher, M. J., A colorimetric determining serum
triglyceride. Clin. Chim. Acta, 22, 393–397 (1968).
34) Gomori, G., A modification of the colorimetric phos-
phorus determination for use the photoelectric color-
imeter. J. Lab. Clin. Med., 27, 955–960 (1942).
35) Mate, J. D., and Schneider, D. L., Extraction of bile
acids from rat feces containing cholestylamine. J. Lipid
Res., 12, 376–377 (1971).
36) Miettinen, T. A., Ahrens, E. H., and Grundy, S. M.,
Quantitative isolation and gas-liquid chromatographic
analysis of total dietary and fecal neutral steroids. J.
Lipid Res., 6, 411–424 (1965).
37) Uchida, K., Nomura, Y., Kadowaki, M., Takeuchi, N.,
and Yamamura, Y., Effect of dietary cholesterol on
cholesterol and bile acid metabolism in rats. Jpn. J.
Pharmacol., 27, 193–204 (1977).
38) Eaton, D. L., and Klaassen, C. D., Effects of acute
administration of taurocholic and taurochenodeoxychol-
ic acid on biliary lipid excretion in the rats. Proc. Soc.
Exp. Biol. Med., 151, 198–202 (1976).
39) Ikeda, I., and Sugano, M., Inhibition of cholesterol
absorption by plant sterols for mass intervention. Curr.
Opin. Lipidol., 9, 527–531 (1998).
40) Tatsumi, E., Yoshimatsu, D., and Hirose, M., Confor-
mation state of disulfide-reduced ovalbumin at acidic
pH. Biosci. Biotechnol. Biochem., 63, 1285–1290
(1999).
41) Walker, V., and Taylor, W. H., Ovalbumin digestion by
human pepsins 1, 3 and 5. Biochem. J., 176, 429–432
(1978).
42) Tomimatsu, Y., A light-scattering study of the aggrega-
tion of ovalbumin near its isoelectric pH. Biochim.
Biophys. Acta, 29, 525–534 (1965).
43) Suchy, S. F., and Wolf, B., Effect of biotin deficiency
and supplementation on lipid metabolism in rats:
cholesterol and lipoproteins. Am. J. Clin. Nutr., 43,
831–838 (1986).