A comparison of the potential unfavorable effects of oxycholesterol and oxyphytosterol in mice: Different effects, on cerebral 24S-hydroxychoelsterol and serum triacylglycerols levels

Article abstract of DOI:10.1271/bbb.80256

Sterol oxidation products derived from cholesterol and phytosterol are formed during the processing and storage of foods. The objective of the present study was to assess the potential unfavorable effects of oxysterols in mice. C57BL/6J mice were fed an AIN-93G-based diet containing 0.2 g/kg of oxycholesterol or oxyphytosterol for 4 weeks. The most abundant oxysterol in the diet was 7-ketosterol, but α-epoxycholesterol, β-epoxycholesterol, or 7α-hydroxyphytosterol, and 7β-hydroxyphytosterol were more prominent than 7-ketosterol in the serum and liver respectively. Consumption of both oxysterols resulted in an increased in 4β-hydroxycholesterol and total oxycholesterol in the liver, but the oxycholesterol-fed mice had a lower level of cerebral 24S-hydroxycholesterol and a higher level of the serum triacylglycerols than the control and oxyphytosterol groups. These results indicate that both oxysterols in the diet are accumulated in the body, but that the biological effect of oxycholesterol is different from that of oxyphytosterol.

Full text of DOI:10.1271/bbb.80256

Biosci. Biotechnol. Biochem., 72 (12), 3128–3133, 2008
A Comparison of the Potential Unfavorable Effects
of Oxycholesterol and Oxyphytosterol in Mice: Different Effects,
on Cerebral 24S-Hydroxychoelsterol and Serum Triacylglycerols Levels
Hyun-jung BANG,y Chiyo ARAKAWA, Michihiro TAKADA, Masao SATO, and Katsumi IMAIZUMI
Laboratory of Nutrition Chemistry, Division of Bioresource and Bioenvironmental Sciences,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan
Received April 16, 2008; Accepted August 9, 2008; Online Publication, December 7, 2008
[doi:10.1271/bbb.80256]
Sterol oxidation products derived from cholesterol
and phytosterol are formed during the processing and
storage of foods. The objective of the present study was
to assess the potential unfavorable effects of oxysterols
in mice. C57BL/6J mice were fed an AIN-93G-based
diet containing 0.2 g/kg of oxycholesterol or oxyphyto-
sterol for 4 weeks. The most abundant oxysterol in the
diet was 7-ketosterol, but ꢀ-epoxycholesterol, ꢁ-epox-
ycholesterol, or 7ꢀ-hydroxyphytosterol, and 7ꢁ-hydro-
xyphytosterol were more prominent than 7-ketosterol in
the serum and liver respectively. Consumption of both
oxysterols resulted in an increased in 4ꢁ-hydroxycho-
lesterol and total oxycholesterol in the liver, but the
oxycholesterol-fed mice had a lower level of cerebral
24S-hydroxycholesterol and a higher level of the serum
triacylglycerols than the control and oxyphytosterol
groups. These results indicate that both oxysterols in the
diet are accumulated in the body, but that the biological
effect of oxycholesterol is different from that of oxy-
phytosterol.
Our previous study indicated that oxidation products
derived from cholesterol and plant sterols accumulated
to a considerable extent in the tissues of apolipopro-
tein E deficient mice, but the atherosclerotic severity in
the aortic value of the mice fed the oxysterols was not
different from that in the oxysterol-free group.7,8) Our
findings are different from those showing a role of
dietary oxycholesterols in the development of athero-
sclerosis in experimental animals.10,11) Very few data
except those from Tomoyori et al.8) are available on the
atherosclerotic potential of oxyphytosterols. Although
the biological effects of oxycholesterol have been
extensively studied,12) similar research on oxyphytoster-
ols is scarce.13)
As reviewed by Hovenkamp et al.,13) recent data
suggest that oxysterols have beneficial biological prop-
erties through the modulation of cholesterol metabolism,
lipid-lowering, and anti-diabetic properties, modulation
of inflammation and immunity, estrogenic and/or
androgenic activity, and so on. Alternatively, the usual
perception of oxysterols is that they present a concern in
terms of food quality and health. This perception
originates from the parallel drawn between oxycholes-
terol and oxyphytosterol. Hence, the object of the
present study was to examine how dietary oxysterols
influence the accumulation of individual oxysterols
in the serum, liver, and brain, and to compare the
biological action of oxyphytosterols to that of oxy-
cholesterols. The results indicates that those oxysterols
differently affect the levels of cerebral 24S-hydroxy-
cholesterol and serum triacylglycerol.
Key words: oxycholesterol; oxyphytosterol; 24S-hy-
droxycholesterol; triacylglycerols
Recently there has been an unprecedent fortification
of phytosterols worldwide in various foods, since they
are claimed to be efficacious cholesterol-lowering
agents. Cholesterol and phytosterol oxidation products
are formed during the processing and storage of
foods.1–5) Among oxysterols prepared by heating cho-
lesterol or phytosterols, 7-ketosterols is the predominant
kind, but various oxysterols are simultaneously pro-
duced to an extent lower than the 7-keto types.6–8) These
sterol oxidation products are transported to the circu-
lation via the lymphatic system after being absorbed by
the small intestine.6,8,9) It is expected that each oxysterol
has a different biological effect in the body, but this
remains to be elucidated.
Materials and Methods
Materials. Cholesterol (Daiichi Pure Chemicals,
Tokyo) and a phytosterol mixture (Merck-Japan, Tokyo)
composed (on a weight basic) of 1.6% brassicasterol,
44.8% campesterol, 2.0% stigmasterol, 50.8% ꢀ-sitos-
y
To whom correspondence should be addressed. Fax: +81-92-642-3005; E-mail: misroom@hotmail.com
Abbreviations: LXR, liver X receptor; CONT, control group; OP, oxyphytosterol-fed group; OC, oxycholesterol-fed group

Comparison of Effects of Oxycholesterol and Oxyphytosterol in Mice
3129
controlled room at 21–23 ꢀC with a light-dark cycle
(light on 8:00–20:00).
Table 1. Compositions of Cholesterol Oxidation Products
Oxycholesterols
Weight %
Experimental diets were packed in a pouch containing
O2 absorbent (Ageless S-200, Mitsubishi Gas Chemical,
Tokyo), flushed with N2, and stored at 4 ꢀC. The diet
was freshly prepared every week and was changed every
2 d. Any remaining food was discarded. At the end of the
4-week feeding period, the mice were deprived of food
for 6 h (7:00–13:00), anesthetized with diethyl ether, and
killed by withdrawal of blood from the left ventricle.
The blood was transferred to microcentrifuge tubes
containing 50 mg, BHT. The serum was separated,
bubbled with Ar, and stored at ꢂ85 ꢀC. The livers, and
brains, were immediately removed from the carcasses
and frozen in liquid nitrogen.
7ꢁ-Hydroxycholesterol
7ꢀ-Hydroxycholesterol
ꢀ-Epoxycholesterol
ꢁ-Epoxycholesterol
ꢀ-Cholestanetriol
ꢁ-Cholestanetriol
7-Ketocholesterol
25-Hydroxycholesterol
Unknown
3:08 ꢁ 0:05
2:60 ꢁ 0:03
12:1 ꢁ 0:3
10:4 ꢁ 0:3
2:91 ꢁ 0:10
1:00 ꢁ 0:02
27:7 ꢁ 1:2
1:25 ꢁ 0:05
39:0 ꢁ 1:8
Values are means ꢁ SEM of triplicate analyses.
Table 2. Compositions of Phytosterol Oxidation Products
Oxyphytosterols
Weight %
Experiments were carried out under the Guidelines
for Animal Experiments of the Faculty of Agriculture
and the Graduate Course, Kyushu University, Fukuoka,
and Law no. 105 and Notification no. 6 of the Govern-
ment of Japan.
Oxidized sitosterol
7ꢁ-Hydroxysitosterol
7ꢀ-Hydroxysitosterol
ꢀ-Epoxysitosterol
ꢁ-Epoxysitosterol
Dihydroxysitosterol
7-Ketositosterol
2:55 ꢁ 0:25
1:67 ꢁ 0:20
5:89 ꢁ 0:40
6:03 ꢁ 0:13
3:65 ꢁ 0:41
14:8 ꢁ 1:0
Analysis of sterol and oxysterols. Oxysterols were
measured as described previously.7,8) Briefly, to 0.25 g
of liver and 100 ml of serum was added 50 mg, 5ꢁ-
cholestane (Sigma, St. Louis, MO), and 1 mg, 19-
hydroxycholesterol (Steraloids, Newport, RI) as an
internal standard. The lipids in the sample were
extracted with 20 vol of chloroform/methanol (2:1,
v/v) containing 0.01% (w/v) BHT (Nacalai Tesque,
Kyoto). The extracted lipids were applied to a Sep-Pak
Vac silica cartridge (Nihon Waters, Tokyo) to separate
the oxysterols from the sterols. The cartridge was
sequentially eluted with 1 ml of hexane, 8 ml of a
solvent mixture composed of hexane and 2-propanol
(1:200, v/v), and 5 ml of one composed of hexane and
2-propanol (3:10, v/v), which allowed for sequential
elution of 5ꢁ-cholestane, cholesterol plus phytosterols,
and 19-hydroxycholesterol plus oxysterol respectively.
The samples were allowed to saponify at room temper-
ature overnight in the dark, and unsaponifyed lipids
were converted into trimethlysilylethers. The GC of
sterols was performed as described previously.15)
GC-MS was performed on a Shimazu GC-17A ver. 3
coupled with SPB-1 fused silica capillary column
connected to a Shimadzu QP5050A series mass-selec-
tive detector. Variables ions monitored, relative reten-
tion times, correlation confficient for calibration curves,
response factors for the monitored ions, detection limits,
and CV for repeated injection, were determined as
previously described.7,8)
Oxidized campesterol
7ꢁ-Hydroxycampesterol
7ꢀ-Hydroxycampesterol
ꢀ-Epoxycampesterol
ꢁ-Epoxycampesterol
Dihydroxycampesterol
7-Ketocampesterol
Unkown
0:95 ꢁ 0:05
1:14 ꢁ 0:05
5:89 ꢁ 0:39
6:07 ꢁ 0:12
3:45 ꢁ 0:55
13:9 ꢁ 0:9
34:0 ꢁ 3:7
Values are means ꢁ SEM of triplicate analyses.
terol, and 0.9% ꢀ-sitostanol were used as starting
materials, in the preparation of oxycholesterols and
oxyphytosterols respectively.7,8) They were heated at
150 ꢀC for 12 h. Then the heated products were applied
to a silicic acid column, and the resulting oxysterols
were eluted with acetone after being washed with
diethyl ether as described previously.7,8) The composi-
tion of heat-prepared oxysterols is shown in Tables 1
and 2.
Animals and diets. Male C57BL/6J mice, 6 weeks
old, with an initial body weight of 23:6 ꢁ 0:2 g, were
divided into three groups. The control group mice were
fed a basal diet containing 100 g/kg, lard and 0.2 g/kg,
phytosterol mixture. The other mice were fed the basal
diet containing 0.2 g/kg, oxycholesterol or 0.2 g/kg,
oxyphytosterol. The phytosterol mixture was added to
the three dietary groups in order to determine whether
dietary oxycholesterol or oxyphytosterol would modu-
late the absorption and transport of dietary phytosterol.
This amount of oxysterols was used previously to
evaluate their effects, on atherosclerosis in apolipopro-
tein E deficient mice.7,8) The diet was based on the
AIN-93G diet formulation,14) as previously described.9)
The mice were individually housed in a temperature-
Serum and liver lipids were determined as previously
described.16)
Statistical analysis. Results were expressed as
means ꢁ SEM. Statistical analysis was carried out with
Statcel (Excel 2000). Variance among the groups was
first checked by Bartlett’s test. The Turkey-Kramer test
was used to compare the three groups, following

3130
H. BANG et al.
Table 3. Oxycholesterol Levels in Serum
Table 4. Oxycholesterol Levels in Liver
Groups
Groups
Oxysterols
Oxysterols
CONT
OP
OC
CONT
OP
OC
mg/dl
mg/g
Autoxidation products
7ꢀ-Hydroxycholesterol
ꢀ-Epoxycholesterol
ꢁ-Epoxycholesterol
ꢀ-Cholestanetriol
Autoxidation products
7ꢀ-Hydroxycholesterol
ꢀ-Epoxycholesterol
ꢁ-Epoxycholesterol
ꢀ-Cholestanetriol
11:9 ꢁ 3:0a 18:9 ꢁ 2:6a 28:3 ꢁ 1:8b
0:28 ꢁ 0:01a 0:29 ꢁ 0:05a 0:65 ꢁ 0:04b
1:62 ꢁ 0:05 1:48 ꢁ 0:29 1:86 ꢁ 0:29
1:15 ꢁ 0:09 1:96 ꢁ 0:35 1:97 ꢁ 0:26
0:18 ꢁ 0:04a 0:14 ꢁ 0:02a 0:47 ꢁ 0:05b
75:1 ꢁ 24:7a 136 ꢁ 18b
136 ꢁ 20b
252 ꢁ 38b
25:1 ꢁ 7:5
34:4 ꢁ 7:8
16:6 ꢁ 3:1
128 ꢁ 26a
29:6 ꢁ 6:6
29:3 ꢁ 0:1
21:2 ꢁ 2:6
123 ꢁ 42a
26:4 ꢁ 4:2
28:7 ꢁ 8:9
23:5 ꢁ 5:2
ꢁ-Cholestanetriol
7-Ketocholesterol
ꢁ-Cholestanetriol
7-Ketocholesterol
Auto and enzymatic oxidation products
n.d.
n.d.
0:09 ꢁ 0:01
0:27 ꢁ 0:04 0:26 ꢁ 0:03 0:38 ꢁ 0:03
Auto and enzymatic oxidation products
7ꢁ-Hydroxycholesterol
25-Hydroxycholesterol
Enzymatic oxidation products
6:13 ꢁ 0:58a 10:1 ꢁ 0:9b 10:1 ꢁ 1:0b
7ꢁ-Hydroxycholesterol
25-Hydroxycholesterol
Enzymatic oxidation products
4ꢀ-Hydroxycholesterol
27-Hydroxycholesterol
0:15 ꢁ 0:03 0:12 ꢁ 0:01 0:16 ꢁ 0:03
1:93 ꢁ 0:43 2:81 ꢁ 0:40 2:34 ꢁ 0:71
0:02 ꢁ 0:01 0:04 ꢁ 0:01 0:03 ꢁ 0:01
24(S)-Hydroxycholesterol 13:9 ꢁ 2:6
10:9 ꢁ 3:8
25:8 ꢁ 4:9
10:4 ꢁ 2:1
35:6 ꢁ 8:5
0:87 ꢁ 0:15a 1:65 ꢁ 0:16b 1:88 ꢁ 0:30b
0:20 ꢁ 0:02 0:21 ꢁ 0:04 0:27 ꢁ 0:02
ng/g
27-Hydroxycholesterol
25:5 ꢁ 0:6
343 ꢁ 11a
Total oxycholesterols
407 ꢁ 63ab 544 ꢁ 50b
22(R)-Hydroxycholesterol 5:61 ꢁ 1:99 4:58 ꢁ 1:06 6:42 ꢁ 2:14
Total oxycholesterols
4:75 ꢁ 0:17a 6:14 ꢁ 0:57b 7:75 ꢁ 0:59b
a,bDifferent letters show signicant differences at P < 0:05.
Values are means ꢁ SEM for five mice per group.
a,bDifferent letters show signicant differences at P < 0:05.
Values are means ꢁ SEM for five mice per group. n.d., not detected.
detection of effects, by one-way ANOVA. Differences
were considered significant at P < 0:05.
Table 5. Oxyphytosterol Levels in Serum
Serum
Oxysterols
Results
CONT
OP
OC
mg/dl
Growth parameters
Oxidized sitosterols
7ꢁ-Hydroxysitosterol
7ꢀ-Hydroxysitosterol
ꢀ-Epoxysitosterol
The oxysterols diets did not affect final body weights,
(g: 27:4 ꢁ 0:5, n ¼ 5 for the control, 27:9 ꢁ 0:5, n ¼ 5
for the oxycholesterol and 26:6 ꢁ 0:6, n ¼ 5 for the
oxyphytosterol group), food intake (g/d: 3:8 ꢁ 0:2,
3:6 ꢁ 0:3, and 3:8 ꢁ 0:1 for the control, oxycholesterol
and oxyphytosterol groups respectively), liver weight
(g: 1:7 ꢁ 0:2, 1:8 ꢁ 0:1 and 1:8 ꢁ 0:1 for the control,
oxycholesterol, and oxyphytosterol groups respectively),
and cerebrum weight (g: 0:22 ꢁ 0:00, 0:23 ꢁ 0:01 and
0:23 ꢁ 0:02 for the control, oxycholesterol and oxy-
phytosterol groups respectively).
138 ꢁ 29a
355 ꢁ 90:0b 131 ꢁ 25:7a
3:20 ꢁ 1:05a 97:8 ꢁ 13:0b 6:38 ꢁ 1:46a
34:8 ꢁ 13:4ab 52:5 ꢁ 13:6a 17:2 ꢁ 2:7b
1:73 ꢁ 0:64ab 2:88 ꢁ 0:47a 0:80 ꢁ 0:20b
1:84 ꢁ 0:52 2:40 ꢁ 0:75 4:40 ꢁ 0:93
16:1 ꢁ 2:5a 41:1 ꢁ 3:0b 21:7 ꢁ 5:7a
176 ꢁ 32:7a 532 ꢁ 79:4b 154 ꢁ 28a
ꢁ-Epoxysitosterol
Dihydroxysitosterol
7-Ketositosterol
Total oxysitosterols
Oxidized campesterols
7ꢁ-Hydroxycampesterol 57:7 ꢁ 13:3a 164 ꢁ 36:6b 67:4 ꢁ 12:7a
7ꢀ-Hydroxycampesterol 2:07 ꢁ 0:36a 153 ꢁ 21:3b 6:90 ꢁ 0:8a
ꢀ-Epoxycampesterol
ꢁ-Epoxycampesterol
Dihydroxycampesterol
7-Ketocampesterol
24:7 ꢁ 7:3
48:3 ꢁ 21:2 42:0 ꢁ 9:1
5:74 ꢁ 2:37 1:13 ꢁ 0:24 2:63 ꢁ 0:75
0:90 ꢁ 0:03a 1:60 ꢁ 0:60ab 3:40 ꢁ 0:58b
1:95 ꢁ 0:58a 8:75 ꢁ 1:65b 3:70 ꢁ 0:80a
94:8 ꢁ 22:8a 365 ꢁ 30:3b 126 ꢁ 18a
Oxycholesterol levels in serum and liver
Total oxycampesterols
The concentrations of serum 7ꢁ-hydroxycholesterol,
7ꢀ-hydroxycholesterol, ꢁ-epoxycholesterol, ꢀ-epoxy-
cholesterol, and total oxycholesterol were higher in
the oxycholesterol-fed mice than the control group
(Table 3). The oxyphytosterol-fed mice had in increased
concentrations of 7ꢁ-epoxycholesterol and ꢀ-epoxycho-
lesterol as compared with the control group. The total
oxycholesterol level in the oxyphytosterol group was not
different from that in the oxycholesterol group. The liver
of the oxycholesterol group showed increased concen-
trations of 7ꢀ-hydroxycholesterol, ꢀ-cholestenetriol, 4ꢀ-
hydroxycholesterol, and total oxycholesterol as com-
pared with the control group (Table 4). The oxyphytos-
terol group also had increased concentration of 4ꢀ-
hydroxycholesterol and total oxycholesterol as com-
pared with the control group. There was no difference in
the total oxycholesterol level between the oxycholester-
ol and the oxyphytosterol group.
Total oxyphytosterols
271 ꢁ 53a
897 ꢁ 102b 279 ꢁ 37a
a,bDifferent letters show significant differences at P < 0:05.
Values are means ꢁ SEM for five mice per group.
Oxyphytosterol levels in serum and liver
The oxyphytosterol group had a increased concentra-
tions of serum 7ꢁ-hydroxysitosterol, 7ꢀ-hydroxysito-
sterol, 7-ketositosterol, total oxysitosterols, 7ꢁ-hydroxy-
campesterol, 7ꢀ-hydroxycampesterol, 7-ketocampesterol
total oxycampesterols, and total oxyphytosterols as
compared with the control group (Table 5). The oxy-
cholesterol group had increased concentration of the
serum dihydroxycampesterol as compared with the
control group. The oxyphytosterol group had increased
concentrations of the liver 7ꢀ-hydroxysitosterol, total
oxysitosterols, 7ꢀ-hydroxycampesterol, ꢀ-epoxycam-
pesterol, total oxycampesterols, and total oxyphytoster-

Comparison of Effects of Oxycholesterol and Oxyphytosterol in Mice
3131
Table 6. Oxyphytosterol Levels in Liver
Liver
Oxysterols
CONT
OP
OC
mg/g
Oxidized sitosterols
7ꢁ-Hydroxysitosterol
7ꢀ-Hydroxysitosterol
ꢀ-Epoxysitosterol
ꢁ-Epoxysitosterol
Dihydroxysitosterol
7-Ketositosterol
6:35 ꢁ 0:93
0:815 ꢁ 0:551a
0:181 ꢁ 0:086
0:037 ꢁ 0:021
0:037 ꢁ 0:014a
0:413 ꢁ 0:160
7:83 ꢁ 1:33a
7:16 ꢁ 1:43
6:85 ꢁ 1:99b
0:208 ꢁ 0:081
0:072 ꢁ 0:025
0:109 ꢁ 0:037ab
0:710 ꢁ 0:172
13:5 ꢁ 1:5b
4:24 ꢁ 0:06
0:160 ꢁ 0:035a
1:53 ꢁ 0:35
0:229 ꢁ 0:081
0:154 ꢁ 0:008b
0:266 ꢁ 0:077
6:58 ꢁ 0:47a
Total oxysitosterols
Oxidized campesterols
7ꢁ-Hydroxycampesterol
7ꢀ-Hydroxycampesterol
ꢀ-Epoxycampesterol
ꢁ-Epoxycampesterol
Dihydroxycampesterol
7-Ketocampesterol
Total oxycampesterols
2:58 ꢁ 0:37ab
0:199 ꢁ 0:055a
0:207 ꢁ 0:060a
0:026 ꢁ 0:01
0:026 ꢁ 0:01
0:064 ꢁ 0:04
3:04 ꢁ 0:39a
4:04 ꢁ 0:76a
6:03 ꢁ 1:04b
2:60 ꢁ 0:97b
0:044 ꢁ 0:028
0:083 ꢁ 0:041
0:479 ꢁ 0:192
10:7 ꢁ 2:89b
1:40 ꢁ 0:13b
0:144 ꢁ 0:018a
0:570 ꢁ 0:091c
0:048 ꢁ 0:01
0:043 ꢁ 0:01
0:080 ꢁ 0:03
2:28 ꢁ 0:08a
Total oxyphytosterols
10:9 ꢁ 1:7a
24:3 ꢁ 4:3b
8:86 ꢁ 0:53a
a,b,cDifferent letters show significant differences at P < 0:05.
Values are means ꢁ SEM for five mice per group.
Table 7. Levels of Cholesterol and Enzymatically-Produced Oxy-
cholesterols in Cerebrum
A
B
150
100
50
20
15
10
5
b
Groups
OP
14:9 ꢁ 0:8 14:6 ꢁ 0:6 15:0 ꢁ 0:5
Sterols
CONT
OC
a
a
Cholesterol (mg/g)
Oxycholesterols (mg/g)
24(S)-Hydroxycholesterol 5:31 ꢁ 0:44a 5:16 ꢁ 0:25a 2:85 ꢁ 0:46b
0
0
25-Hydroxycholesterol
27-Hydroxycholesterol
0:11 ꢁ 0:04 0:22 ꢁ 0:01 0:24 ꢁ 0:05
CONT
OP
OC
CONT OP
OC
1:23 ꢁ 0:98 0:85 ꢁ 0:43 0:34 ꢁ 0:05
a,bDifferent letters show signicant differences at P < 0:05.
Values are means ꢁ SEM for five mice per group.
Fig. 1. Triacylglycerols Levels of Serum (A) and Liver (B) in Mice.
Data represent the means ꢁ SEM for five rats in each group.
Different letters show significant differences at P < 0:05. Abbrevia-
tions: CONT, control; OP, oxyphytosterol; OC, oxycholesterol
ols as compared with the control group (Table 6). The
oxycholesterol group had an increased concentration of
dihydroxysitosterol and ꢀ-epoxycampesterol as com-
pared with the control group.
concentration of liver triacylglycerol among the groups
(Fig. 1B).
Cholesterol and enzymatically-produced oxycholes-
terol levels in the cerebrum
Discussion
There were no differences in cerebral cholesterol level
among the groups (Table 7). The oxycholesterol group
had a lower level of 24S-hydroxycholesterol than the
control or the oxyphytosterol group.
The present study indicates that the most abundant
oxysterol in the diet was 7-ketooxysterols, but they were
a relatively minor component in the serum and liver.
Instead, ꢁ- and ꢀ-epoxycholesterol or 7ꢁ- and 7ꢀ-
hydroxyphytosterol were the most abundant in the body.
These results are in accordance with ones for apolipo-
protein E deficient mice fed oxycholesterol or oxy-
phytosterol.7,8) When rats were fed a diet containing
oxycholesterol or oxyphytosterol,6,8) the amount of
individual oxysterol transported into the thoracic lymph
closely reflected the amount consumed. Therefore, it is
likely that each oxysterol absorbed was differently
metabolized in the body, and that this was reflected in
the concentrations in the serum and liver.
Cholesterol, phytosterol and triacylglycerol levels in
the serum and liver
There were no significant differences in the concen-
trations of cholesterol or plant sterols (campesterol and
ꢀ-sitosterol) in the serum and liver among the groups
(data not shown). The oxycholesterol group had an
increased concentration of the serum triacylglycerol as
compared with the control and the oxyphytosterol group
(Fig. 1A). There was no significant difference in the

3132
H. BANG et al.
Furthermore, the present study indicates that oxy-
When rodents were treated with a synthetic ligand
(TO901317) or natural ligands to bind to liver X
receptor (LXR),22–25) the levels of serum triacylglycerols
were higher than non-treated groups. These reports
support our results that dietary oxycholesterol may act
as a LXR ligand to induce hypertriglyceridemia. In the
present study, however, the oxycholesterol as well as
the oxyphytosterol diet resulted in elevations in liver
4ꢀ-hydroxycholesterol which is known to be one of the
strongest ligands for LXR.26) Accordingly, it remains to
the elucidated whether the oxycholesterol diet led to
hypertriglyceridemia via a LXR-related or an unknown
mechanism.
We have evaluated the roles of dietary oxycholesterol
and oxyphytosterol in the development of arterioscle-
rosis in apolipoprotein E-deficient mice.7,8) Neither
oxycholesterol nor oxyphytosterol in the diet accelerated
atherosclerosis in our apolipoprotein E-deficient mice.
In contrast to our results, Stapran et al.11) reported the
acceleration of atherosclerosis in apolipoprotein E defi-
cient mice fed an oxycholesterol diet. According to a
recent review by Havenkamp et al.,13) oxycholesterol as
well as oxyphytosterol has an action inducing similar
cell toxicity and apoptosis in cultured cells and in vivo,
but the former oxysterol exerted an unfavorable effect at
smaller amount than the latter oxysterol did. Taken
together with previous results, as described above,13) and
the present findings indicate that for oxycholesterol
lowers cerebral 24S-hydroxycholesterol and increases
serum triacylglycerol, and hence it is likely that the
action of the oxycholesterol diet on the body is stronger
than that of the oxyphytosterol diet.
cholesterol as well as oxyphytosterol in the diet resulted
in increases in the concentrations of 7ꢁ-hydroxycholes-
terol and ꢀ-epoxycholesterol in the serum and of 4ꢀ-
hydroxycholesterol in the liver. These results are in
agreement with our previous ones when apolipoprotein E
deficient mice were fed an oxyphytosterol-containing
diet.8) The apolipoprotein E deficient mice fed the
oxyphytosterol diet showed increased concentrations of
7ꢁ-hydroxycholesterol, 7ꢀ-hydroxycholesterol, choles-
tanetriol, 7-ketochoelsterol, and 25-hydroxycholesterol
in the serum. In addition to an increase in the
concentrations of the diet-derived oxycholesterols, the
present study indicates that dietary sterol oxides resulted
in prominent increases in the concentration of enzymati-
cally-produced 4ꢀ-hyrodyxcholesterol as compared with
the control diet. Moreover, the oxycholesterol diet
resulted in increases in the concentrations of dihydro-
xycampesterol in the serum and of dihydroxysitosterol
and ꢀ-epoxycampesterol in the liver. These results
suggest that dietary oxycholesterol and oxyphytosterol
modulated enzymatical and/or non-enzymatical sterol
metabolism.
Alternatively, the present study indicates that there
were no significant differences in the concentrations of
cholesterol and plant sterols (campesterol and ꢀ-sitos-
terol) in the serum and liver among the groups (data not
shown). The oxycholesterol diet lowered the concen-
tration of 24S-hydroxycholesterol, a metabolite specific
to the brain,17) but this was not the case for the
oxyphytosterol diet. Cholesterol in the cerebrum is
hydroxylated to 24S-hydroxycholesterol and is then
transported to the circulation through the blood-brain
barrier.18–20) It is therefore conceivable that a decrease
in cerebral 24S-hydroxycholesterol leads to a defect of
cholesterol-removal in the brain, but, the concentration
of cerebral cholesterol was not affected by oxysterol
consumption. Accordingly, the oxycholesterol diet
might lower cholesterol synthesis in the cerebrum.21)
These results strongly suggest that the oxycholesterol
diet had a stronger effect on cerebral cholesterol
metabolism than the oxyphytosterol diet did. It remains
to be elucidated how dietary oxycholesterol affects
cerebral cholesterol metabolism through the blood-brain
barrier.
In summary, the present study indicates that the
dietary oxycholesterol and oxyphytosterol accumulated
and stimulated the enzymatic and non-enzymatic for-
mation of oxysterols in the mice. However, the effect
on metabolism of cerebral cholesterol and serum
triacylglycerols was more prominent in mice fed the
oxycholesterol than the oxyphytosterol diet. Because
phytosterol consumption is expected to increase, further
study is necessary to evaluate the unfavorable effects of
dietary oxyphytosterol.
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