A Comparison of the Potential Unfavorable Effects Of

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

y Hyun-jung BANG, 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 Our previous study indicated that oxidation products and phytosterol are formed during the processing and derived from cholesterol and plant sterols accumulated storage of foods. The objective of the present study was to a considerable extent in the tissues of apolipopro- to assess the potential unfavorable effects of oxysterols tein E deficient mice, but the atherosclerotic severity in in mice. C57BL/6J mice were fed an AIN-93G-based the aortic value of the mice fed the oxysterols was not diet containing 0.2 g/kg of oxycholesterol or oxyphyto- different from that in the oxysterol-free group.7,8) Our sterol for 4 weeks. The most abundant oxysterol in the findings are different from those showing a role of diet was 7-ketosterol, but -epoxycholesterol, -epox- dietary oxycholesterols in the development of athero- ycholesterol, or 7 -hydroxyphytosterol, and 7 -hydro- sclerosis in experimental animals.10,11) Very few data xyphytosterol were more prominent than 7-ketosterol in except those from Tomoyori et al.8) are available on the the serum and liver respectively. Consumption of both atherosclerotic potential of oxyphytosterols. Although oxysterols resulted in an increased in 4 -hydroxycho- the biological effects of oxycholesterol have been lesterol and total oxycholesterol in the liver, but the extensively studied,12) similar research on oxyphytoster- oxycholesterol-fed mice had a lower level of cerebral ols is scarce.13) 24S-hydroxycholesterol and a higher level of the serum As reviewed by Hovenkamp et al.,13) recent data triacylglycerols than the control and oxyphytosterol suggest that oxysterols have beneficial biological prop- groups. These results indicate that both oxysterols in the erties through the modulation of cholesterol metabolism, diet are accumulated in the body, but that the biological lipid-lowering, and anti-diabetic properties, modulation effect of oxycholesterol is different from that of oxy- of inflammation and immunity, estrogenic and/or phytosterol. androgenic activity, and so on. Alternatively, the usual perception of oxysterols is that they present a concern in Key words: oxycholesterol; oxyphytosterol; 24S-hy- terms of food quality and health. This perception droxycholesterol; triacylglycerols originates from the parallel drawn between oxycholesterol and oxyphytosterol. Hence, the object of the Recently there has been an unprecedent fortification present study was to examine how dietary oxysterols of phytosterols worldwide in various foods, since they influence the accumulation of individual oxysterols are claimed to be efficacious cholesterol-lowering in the serum, liver, and brain, and to compare the agents. Cholesterol and phytosterol oxidation products biological action of oxyphytosterols to that of oxy- are formed during the processing and storage of cholesterols. The results indicates that those oxysterols foods.1–5) Among oxysterols prepared by heating cho- differently affect the levels of cerebral 24S-hydroxy- lesterol or phytosterols, 7-ketosterols is the predominant cholesterol and serum triacylglycerol. kind, but various oxysterols are simultaneously produced to an extent lower than the 7-keto types.6–8) These Materials and Methods sterol oxidation products are transported to the circulation via the lymphatic system after being absorbed by Materials. Cholesterol (Daiichi Pure Chemicals, the small intestine.6,8,9) It is expected that each oxysterol Tokyo) and a phytosterol mixture (Merck-Japan, Tokyo) has a different biological effect in the body, but this composed (on a weight basic) of 1.6% brassicasterol, remains to be elucidated. 44.8% campesterol, 2.0% stigmasterol, 50.8% -sitos-

y To whom correspondence should be addressed. Fax: +81-92-642-3005; E-mail: [email protected] 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 Table 1. Compositions of Cholesterol Oxidation Products controlled room at 21–23 C with a light-dark cycle (light on 8:00–20:00). Oxycholesterols Weight % Experimental diets were packed in a pouch containing 7-Hydroxycholesterol 3:08 0:05 O2 absorbent (Ageless S-200, Mitsubishi Gas Chemical, 7-Hydroxycholesterol 2:60 0:03 -Epoxycholesterol 12:1 0:3 Tokyo), flushed with N2, and stored at 4 C. The diet -Epoxycholesterol 10:4 0:3 was freshly prepared every week and was changed every -Cholestanetriol 2:91 0:10 2 d. Any remaining food was discarded. At the end of the -Cholestanetriol 1:00 0:02 4-week feeding period, the mice were deprived of food 7-Ketocholesterol 27:7 1:2 for 6 h (7:00–13:00), anesthetized with diethyl ether, and 25-Hydroxycholesterol 1:25 0:05 Unknown 39:0 1:8 killed by withdrawal of blood from the left ventricle. The blood was transferred to microcentrifuge tubes Values are means SEM of triplicate analyses. containing 50 mg, BHT. The serum was separated, bubbled with Ar, and stored at 85 C. The livers, and Table 2. Compositions of Phytosterol Oxidation Products brains, were immediately removed from the carcasses and frozen in liquid nitrogen. Oxyphytosterols Weight % Experiments were carried out under the Guidelines Oxidized sitosterol for Animal Experiments of the Faculty of Agriculture 7-Hydroxysitosterol 2:55 0:25 and the Graduate Course, Kyushu University, Fukuoka, 7-Hydroxysitosterol 1:67 0:20 and Law no. 105 and Notification no. 6 of the Govern- -Epoxysitosterol 5:89 0:40 ment of Japan. -Epoxysitosterol 6:03 0:13 Dihydroxysitosterol 3:65 0:41 7-Ketositosterol 14:8 1:0 Analysis of sterol and oxysterols. Oxysterols were measured as described previously.7,8) Briefly, to 0.25 g Oxidized campesterol of liver and 100 ml of serum was added 50 mg, 5- 7-Hydroxycampesterol 0:95 0:05 cholestane (Sigma, St. Louis, MO), and 1 mg, 19- 7-Hydroxycampesterol 1:14 0:05 -Epoxycampesterol 5:89 0:39 hydroxycholesterol (Steraloids, Newport, RI) as an -Epoxycampesterol 6:07 0:12 internal standard. The lipids in the sample were Dihydroxycampesterol 3:45 0:55 extracted with 20 vol of chloroform/methanol (2:1, 7-Ketocampesterol 13:9 0:9 v/v) containing 0.01% (w/v) BHT (Nacalai Tesque, Unkown 34:0 3:7 Kyoto). The extracted lipids were applied to a Sep-Pak Values are means SEM of triplicate analyses. 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 terol, and 0.9% -sitostanol were used as starting solvent mixture composed of hexane and 2-propanol materials, in the preparation of oxycholesterols and (1:200, v/v), and 5 ml of one composed of hexane and oxyphytosterols respectively.7,8) They were heated at 2-propanol (3:10, v/v), which allowed for sequential 150 C for 12 h. Then the heated products were applied elution of 5-cholestane, cholesterol plus phytosterols, to a silicic acid column, and the resulting oxysterols and 19-hydroxycholesterol plus oxysterol respectively. were eluted with acetone after being washed with The samples were allowed to saponify at room temper- diethyl ether as described previously.7,8) The composi- ature overnight in the dark, and unsaponifyed lipids tion of heat-prepared oxysterols is shown in Tables 1 were converted into trimethlysilylethers. The GC of and 2. sterols was performed as described previously.15) GC-MS was performed on a Shimazu GC-17A ver. 3 Animals and diets. Male C57BL/6J mice, 6 weeks coupled with SPB-1 fused silica capillary column old, with an initial body weight of 23:6 0:2 g, were connected to a Shimadzu QP5050A series mass-selec- divided into three groups. The control group mice were tive detector. Variables ions monitored, relative reten- fed a basal diet containing 100 g/kg, lard and 0.2 g/kg, tion times, correlation confficient for calibration curves, phytosterol mixture. The other mice were fed the basal response factors for the monitored ions, detection limits, diet containing 0.2 g/kg, oxycholesterol or 0.2 g/kg, and CV for repeated injection, were determined as oxyphytosterol. The phytosterol mixture was added to previously described.7,8) the three dietary groups in order to determine whether Serum and liver lipids were determined as previously dietary oxycholesterol or oxyphytosterol would modu- described.16) late the absorption and transport of dietary phytosterol. This amount of oxysterols was used previously to Statistical analysis. Results were expressed as evaluate their effects, on atherosclerosis in apolipopro- means SEM. Statistical analysis was carried out with tein E deficient mice.7,8) The diet was based on the Statcel (Excel 2000). Variance among the groups was AIN-93G diet formulation,14) as previously described.9) first checked by Bartlett’s test. The Turkey-Kramer test The mice were individually housed in a temperature- 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 Autoxidation products 7-Hydroxycholesterol 11:9 3:0a 18:9 2:6a 28:3 1:8b 7-Hydroxycholesterol 0:28 0:01a 0:29 0:05a 0:65 0:04b -Epoxycholesterol 75:1 24:7a 136 18b 136 20b -Epoxycholesterol 1:62 0:05 1:48 0:29 1:86 0:29 -Epoxycholesterol 128 26a 123 42a 252 38b -Epoxycholesterol 1:15 0:09 1:96 0:35 1:97 0:26 -Cholestanetriol 29:6 6:626:4 4:225:1 7:5 -Cholestanetriol 0:18 0:04a 0:14 0:02a 0:47 0:05b -Cholestanetriol 29:3 0:128:7 8:934:4 7:8 -Cholestanetriol n.d. n.d. 0:09 0:01 7-Ketocholesterol 21:2 2:623:5 5:216:6 3:1 7-Ketocholesterol 0:27 0:04 0:26 0:03 0:38 0:03 Auto and enzymatic oxidation products Auto and enzymatic oxidation products 7-Hydroxycholesterol 6:13 0:58a 10:1 0:9b 10:1 1:0b 7-Hydroxycholesterol 0:15 0:03 0:12 0:01 0:16 0:03 25-Hydroxycholesterol 1:93 0:43 2:81 0:40 2:34 0:71 25-Hydroxycholesterol 0:02 0:01 0:04 0:01 0:03 0:01 Enzymatic oxidation products Enzymatic oxidation products 24(S)-Hydroxycholesterol 13:9 2:610:9 3:810:4 2:1 4-Hydroxycholesterol 0:87 0:15a 1:65 0:16b 1:88 0:30b 27-Hydroxycholesterol 25:5 0:625:8 4:935:6 8:5 27-Hydroxycholesterol 0:20 0:02 0:21 0:04 0:27 0:02 ng/g Total oxycholesterols 343 11a 407 63ab 544 50b 22(R)-Hydroxycholesterol 5:61 1:99 4:58 1:06 6:42 2:14 a,b P < : Different letters show signicant differences at 0 05. Total oxycholesterols 4:75 0:17a 6:14 0:57b 7:75 0:59b 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 Results Oxysterols CONT OP OC Growth parameters mg/dl The oxysterols diets did not affect final body weights, Oxidized sitosterols a b a : : ¼ : : ¼ 7-Hydroxysitosterol 138 29 355 90:0 131 25:7 (g: 27 4 0 5, n 5 for the control, 27 9 0 5, n 5 7-Hydroxysitosterol 3:20 1:05a 97:8 13:0b 6:38 1:46a for the oxycholesterol and 26:6 0:6, n ¼ 5 for the -Epoxysitosterol 34:8 13:4ab 52:5 13:6a 17:2 2:7b oxyphytosterol group), food intake (g/d: 3:8 0:2, -Epoxysitosterol 1:73 0:64ab 2:88 0:47a 0:80 0:20b 3:6 0:3, and 3:8 0:1 for the control, oxycholesterol Dihydroxysitosterol 1:84 0:52 2:40 0:75 4:40 0:93 a b a and oxyphytosterol groups respectively), liver weight 7-Ketositosterol 16:1 2:5 41:1 3:0 21:7 5:7 Total oxysitosterols 176 32:7a 532 79:4b 154 28a (g: 1:7 0:2, 1:8 0:1 and 1:8 0:1 for the control, Oxidized campesterols oxycholesterol, and oxyphytosterol groups respectively), 7-Hydroxycampesterol 57:7 13:3a 164 36:6b 67:4 12:7a and cerebrum weight (g: 0:22 0:00, 0:23 0:01 and 7-Hydroxycampesterol 2:07 0:36a 153 21:3b 6:90 0:8a 0:23 0:02 for the control, oxycholesterol and oxy- -Epoxycampesterol 24:7 7:348:3 21:242:0 9:1 phytosterol groups respectively). -Epoxycampesterol 5:74 2:37 1:13 0:24 2:63 0:75 Dihydroxycampesterol 0:90 0:03a 1:60 0:60ab 3:40 0:58b 7-Ketocampesterol 1:95 0:58a 8:75 1:65b 3:70 0:80a Oxycholesterol levels in serum and liver Total oxycampesterols 94:8 22:8a 365 30:3b 126 18a The concentrations of serum 7-hydroxycholesterol, Total oxyphytosterols 271 53a 897 102b 279 37a 7-hydroxycholesterol, -epoxycholesterol, -epoxycholesterol, and total oxycholesterol were higher in a,bDifferent letters show significant differences at P < 0:05. Values are means SEM for five mice per group. the oxycholesterol-fed mice than the control group (Table 3). The oxyphytosterol-fed mice had in increased concentrations of 7-epoxycholesterol and -epoxycho- Oxyphytosterol levels in serum and liver lesterol as compared with the control group. The total The oxyphytosterol group had a increased concentra- oxycholesterol level in the oxyphytosterol group was not tions of serum 7-hydroxysitosterol, 7-hydroxysito- different from that in the oxycholesterol group. The liver sterol, 7-ketositosterol, total oxysitosterols, 7-hydroxy- of the oxycholesterol group showed increased concen- campesterol, 7-hydroxycampesterol, 7-ketocampesterol trations of 7-hydroxycholesterol, -cholestenetriol, 4- total oxycampesterols, and total oxyphytosterols as hydroxycholesterol, and total oxycholesterol as com- compared with the control group (Table 5). The oxy- pared with the control group (Table 4). The oxyphytos- cholesterol group had increased concentration of the terol group also had increased concentration of 4- serum dihydroxycampesterol as compared with the hydroxycholesterol and total oxycholesterol as com- control group. The oxyphytosterol group had increased pared with the control group. There was no difference in concentrations of the liver 7-hydroxysitosterol, total the total oxycholesterol level between the oxycholester- oxysitosterols, 7-hydroxycampesterol, -epoxycam- ol and the oxyphytosterol group. 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 7-Hydroxysitosterol 6:35 0:93 7:16 1:43 4:24 0:06 7-Hydroxysitosterol 0:815 0:551a 6:85 1:99b 0:160 0:035a -Epoxysitosterol 0:181 0:086 0:208 0:081 1:53 0:35 -Epoxysitosterol 0:037 0:021 0:072 0:025 0:229 0:081 Dihydroxysitosterol 0:037 0:014a 0:109 0:037ab 0:154 0:008b 7-Ketositosterol 0:413 0:160 0:710 0:172 0:266 0:077 Total oxysitosterols 7:83 1:33a 13:5 1:5b 6:58 0:47a Oxidized campesterols 7-Hydroxycampesterol 2:58 0:37ab 4:04 0:76a 1:40 0:13b 7-Hydroxycampesterol 0:199 0:055a 6:03 1:04b 0:144 0:018a -Epoxycampesterol 0:207 0:060a 2:60 0:97b 0:570 0:091c -Epoxycampesterol 0:026 0:01 0:044 0:028 0:048 0:01 Dihydroxycampesterol 0:026 0:01 0:083 0:041 0:043 0:01 7-Ketocampesterol 0:064 0:04 0:479 0:192 0:080 0:03 Total oxycampesterols 3:04 0:39a 10:7 2:89b 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 AB 150 20 Groups b Sterols 15 CONT OP OC 100 a a 10 Cholesterol (mg/g) 14:9 0:814:6 0:615:0 0:5 50 5 Oxycholesterols (mg/g)

a a b (mg/g) Triacylglycerols (mg/dl) Triacylglycerols 24(S)-Hydroxycholesterol 5:31 0:44 5:16 0:25 2:85 0:46 0 0 25-Hydroxycholesterol 0:11 0:04 0:22 0:01 0:24 0:05 CONT OP OC CONT OP OC 27-Hydroxycholesterol 1:23 0:98 0:85 0:43 0:34 0:05

a,bDifferent letters show signicant differences at P < 0:05. Fig. 1. Triacylglycerols Levels of Serum (A) and Liver (B) in Mice. Values are means SEM for five mice per group. 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- concentration of liver triacylglycerol among the groups pared with the control group. (Fig. 1B).

Cholesterol and enzymatically-produced oxycholes- Discussion terol levels in the cerebrum There were no differences in cerebral cholesterol level The present study indicates that the most abundant among the groups (Table 7). The oxycholesterol group oxysterol in the diet was 7-ketooxysterols, but they were had a lower level of 24S-hydroxycholesterol than the a relatively minor component in the serum and liver. control or the oxyphytosterol group. Instead, - and -epoxycholesterol or 7- and 7- hydroxyphytosterol were the most abundant in the body. Cholesterol, phytosterol and triacylglycerol levels in These results are in accordance with ones for apolipo- the serum and liver protein E deficient mice fed oxycholesterol or oxy- There were no significant differences in the concen- phytosterol.7,8) When rats were fed a diet containing trations of cholesterol or plant sterols (campesterol and oxycholesterol or oxyphytosterol,6,8) the amount of -sitosterol) in the serum and liver among the groups individual oxysterol transported into the thoracic lymph (data not shown). The oxycholesterol group had an closely reflected the amount consumed. Therefore, it is increased concentration of the serum triacylglycerol as likely that each oxysterol absorbed was differently compared with the control and the oxyphytosterol group metabolized in the body, and that this was reflected in (Fig. 1A). There was no significant difference in the the concentrations in the serum and liver. 3132 H. BANG et al. Furthermore, the present study indicates that oxy- When rodents were treated with a synthetic ligand cholesterol as well as oxyphytosterol in the diet resulted (TO901317) or natural ligands to bind to liver X in increases in the concentrations of 7-hydroxycholes- receptor (LXR),22–25) the levels of serum triacylglycerols terol and -epoxycholesterol in the serum and of 4- were higher than non-treated groups. These reports hydroxycholesterol in the liver. These results are in support our results that dietary oxycholesterol may act agreement with our previous ones when apolipoprotein E as a LXR ligand to induce hypertriglyceridemia. In the deficient mice were fed an oxyphytosterol-containing present study, however, the oxycholesterol as well as diet.8) The apolipoprotein E deficient mice fed the the oxyphytosterol diet resulted in elevations in liver oxyphytosterol diet showed increased concentrations of 4-hydroxycholesterol which is known to be one of the 7-hydroxycholesterol, 7-hydroxycholesterol, choles- strongest ligands for LXR.26) Accordingly, it remains to tanetriol, 7-ketochoelsterol, and 25-hydroxycholesterol the elucidated whether the oxycholesterol diet led to in the serum. In addition to an increase in the hypertriglyceridemia via a LXR-related or an unknown concentrations of the diet-derived oxycholesterols, the mechanism. present study indicates that dietary sterol oxides resulted We have evaluated the roles of dietary oxycholesterol in prominent increases in the concentration of enzymati- and oxyphytosterol in the development of arterioscle- cally-produced 4-hyrodyxcholesterol as compared with rosis in apolipoprotein E-deficient mice.7,8) Neither the control diet. Moreover, the oxycholesterol diet oxycholesterol nor oxyphytosterol in the diet accelerated resulted in increases in the concentrations of dihydro- atherosclerosis in our apolipoprotein E-deficient mice. xycampesterol in the serum and of dihydroxysitosterol In contrast to our results, Stapran et al.11) reported the and -epoxycampesterol in the liver. These results acceleration of atherosclerosis in apolipoprotein E defi- suggest that dietary oxycholesterol and oxyphytosterol cient mice fed an oxycholesterol diet. According to a modulated enzymatical and/or non-enzymatical sterol recent review by Havenkamp et al.,13) oxycholesterol as metabolism. well as oxyphytosterol has an action inducing similar Alternatively, the present study indicates that there cell toxicity and apoptosis in cultured cells and in vivo, were no significant differences in the concentrations of but the former oxysterol exerted an unfavorable effect at cholesterol and plant sterols (campesterol and -sitos- smaller amount than the latter oxysterol did. Taken terol) in the serum and liver among the groups (data not together with previous results, as described above,13) and shown). The oxycholesterol diet lowered the concen- the present findings indicate that for oxycholesterol tration of 24S-hydroxycholesterol, a metabolite specific lowers cerebral 24S-hydroxycholesterol and increases to the brain,17) but this was not the case for the serum triacylglycerol, and hence it is likely that the oxyphytosterol diet. Cholesterol in the cerebrum is action of the oxycholesterol diet on the body is stronger hydroxylated to 24S-hydroxycholesterol and is then than that of the oxyphytosterol diet. transported to the circulation through the blood-brain In summary, the present study indicates that the barrier.18–20) It is therefore conceivable that a decrease dietary oxycholesterol and oxyphytosterol accumulated in cerebral 24S-hydroxycholesterol leads to a defect of and stimulated the enzymatic and non-enzymatic for- cholesterol-removal in the brain, but, the concentration mation of oxysterols in the mice. However, the effect of cerebral cholesterol was not affected by oxysterol on metabolism of cerebral cholesterol and serum consumption. Accordingly, the oxycholesterol diet triacylglycerols was more prominent in mice fed the might lower cholesterol synthesis in the cerebrum.21) oxycholesterol than the oxyphytosterol diet. Because These results strongly suggest that the oxycholesterol phytosterol consumption is expected to increase, further diet had a stronger effect on cerebral cholesterol study is necessary to evaluate the unfavorable effects of metabolism than the oxyphytosterol diet did. It remains dietary oxyphytosterol. to be elucidated how dietary oxycholesterol affects cerebral cholesterol metabolism through the blood-brain References barrier. The present study also indicates the different effect of 1) Paresh, C. D., and Geofferey, P. S., ‘‘Cholesterol and oxycholesterol on serum triacylglycerols metabolism Phytosterols Oxidation Products: Analysis, Occurrence, from that of dietary oxyphytosterol. The oxycholesterol and Biological Effects’’ Chapter 15, AOCS Press, IL, diet resulted in increased concentrations of serum pp. 319–334 (2002). triacylglycerols, whereas no such effect was found for 2) Lampi, A. M., Juntunen, L., Toivo, J., and Piironen, V., the oxyphytosterol diet. Our previous results indicate Determination of thermo-oxidation products of plant that rats fed the oxycholesterol diet showed increased sterols. J. Chromatogr., B777, 83–92 (2002). 3) Bortolomeazzi, R., Cordaro, F., Pizzale, L., and Conte, transport of lymphatic triaclyglycerols,8) whereas no L. S., Presence of phytosterol oxides in crude vegetable such increase was observed for the rats when fed the oils and their fate during refining. J. Agric. Food Chem., 9) oxyphytosterol diet. Accordingly, these results indicate 51, 2394–2401 (2003). that triacylglycerol metabolism is influenced to a greater 4) Dutta, P. C., Studies on phytosterol oxides. II. Content in extent in animals fed oxycholesterol than in those fed some vegetable oils and in French fries prepared in these oxyphytosterol. oils. J. Am. Oil Chem. Soc., 74, 659–666 (1997). Comparison of Effects of Oxycholesterol and Oxyphytosterol in Mice 3133 5) Oehrl, L. L., Hansen, A. P., Rohrer, C. A., Fenner, G. P., 17) Lutjohann, D., Breuer, O., Ahlborg, G., Nennesmo, I., and Boyd, L. C., Oxidation of phytosterols in a test Siden, A., Diczfalusy, U., and Bjorkhem, I., Cholesterol food system. J. Am. Oil Chem. Soc., 78, 1073–1078 homeostasis in the human brain: evidence for an age- (2001). dependent flux of 24S-hydroxycholesterol from the brain 6) Osada, K., Sasaki, E., and Sugano, M., Lymphatic into the circulation. Proc. Natl. Acad. Sci., 93, 9799– absorption of oxidized cholesterol in rats. Lipids, 29, 9804 (1996). 555–559 (1994). 18) Bjorkhem, I., Lutjohann, D., Breuer, O., Sakinis, A., and 7) Ando, M., Tomoyori, H., and Imaizumi, K., Dietary Wennmalm, A., Importance of a novel oxidative cholesterol-oxidation products accumulate in serum and mechanism for elimination of brain cholesterol: turnover liver in apolipoprotein E-deficient mice, but do not of cholesterol and 24(S)-hydroxycholesterol in the rat 18 accelerate atherosclerosis. Br. J. Nutr., 88, 339–345 brain as measured with O2 techniques in vivo and vitro. (2002). J. Biol. Chem., 272, 30178–30184 (1997). 8) Tomoyori, H., Kawata, Y., Higuchi, T., Ichi, I., Sato, H., 19) Bjorkhem, I., Lutjohann, D., Diczfalusy, U., Stahle, L., Sato, M., Ikeda, I., and Imaizumi, K., Phytosterol Ahlborg, G., and Wahren, J., Cholesterol homeostasis in oxidation products are absorbed in the intestinal lym- the human brain: turnover of 24S-hydroxycholesterol phatics in rats but do not accelerate atherosclerosis in and evidence for a cerebral origin of most of this apolipoprotein E-deficient mice. J. Nutr., 134, 1690– oxysterol in the circulation. J. Lipid Res., 39, 1594–1600 1696 (2004). (1998). 9) Tomoyori, H., Carvajal, O., Nakayama, M., Kishi, T., 20) Lund, E. G., Guileyardo, J. M., and Russell, D. W., Sato, M., Ikeda, I., and Imaizumi, K., Lymphatic cDNA cloning of cholesterol 24-hydroxylase, a mediator transport of dietary cholesterol oxidation products, of cholesterol homeostasis in the brain. Proc. Natl. Acad. cholesterol and triacylglycerols in rats. Biosci. Biotech- Sci., 96, 7238–7243 (1999). nol. Biochem., 66, 828–834 (2002). 21) Lund, E. G., Xie, C., Kotti, T., Turley, S. D., Dietschy, 10) Brown, A. J., and Jessup, W., Oxysterols and athero- J. M., and Russell, D. W., Knockout of the cholesterol sclerosis. Atherosclerosis, 142, 1–28 (1999). 24-hydroxylase gene in mice reveals a brain-specific 11) Staprans, I., Pan, X. M., Rapp, J. H., and Feingold, K. R., mechanism of cholesterol turnover. J. Biol. Chem., 278, The role of dietary oxidized cholesterol and oxidized 22980–22988 (2003). fatty acids in the development of atherosclerosis. Mol. 22) Schultz, J. R., Tu, H., Luk, A., Repa, J. J., Medina, J. C., Nutr. Food Res., 49, 1075–1082 (2005). Li, L., Schwendner, S., Wang, S., Thoolen, M., 12) Bjo¨rkhem, I., Meaney, S., and Diczfalusy, U., Oxysterols Mangelsdorf, D. J., Lustig, K. D., and Shan, B., Role in human circulation: which role do they have? Curr. of LXRs in the control of lipogenesis. Genes Dev., 14, Opin. Lipidol., 13, 247–253 (2002). 2831–2838 (2000). 13) Hovenkamp, E., Demonty, I., Plat, J., Lu¨tjohann, D., 23) Fu, X., Menke, J. G., Chen, Y., Zhou, G., MacNaul, Mensink, R. P., and Trautwein, E. A., Biological effects K. L., Wright, S. D., Sparrow, C. P., and Lund, E. G., of oxidized phytosterols: a review of current knowledge. 27-hydroxycholesterol is an endogenous ligand for Prog. Lipid Res., 47, 37–49 (2008). liver X receptor in cholesterol-loaded cells. J. Biol. 14) Reeves, P. G., Nielsen, F. H., and Fahey, G. C., Jr., Chem., 276, 38378–38387 (2001). AIN-93 purified diets for laboratory rodents: final report 24) DeBose-Boyd, R. A., Ou, J., Goldstein, J. L., and Brown, of the American Institute of Nutrition ad hoc Writing M. S., Expression of sterol regulatory element-binding Committee on recommendation of the AIN-76A rodent protein 1c (SREBP-1c) mRNA in rat hepatoma cells diet. J. Nutr., 123, 1939–1951 (1993). requires endogenous LXR ligands. Proc. Natl. Acad. Sci. 15) Ikeda, I., Nakagiri, H., Sugano, M., Ohara, S., Hamada, USA, 98, 1477–1482 (2001). T., Nonaka, M., and Imaizumi, K., Mechanism of 25) Inaba, T., Matsuda, M., Shimamura, M., Takei, N., phytosterolemia in stroke-prone spontaneously hyper- Terasaka, N., Ando, Y., Yasumo, H., Koishi, R., tensive and WKY rats. Metabolism, 50, 1361–1368 Makishima, M., and Shimomura, I., Angiopoietin-like (2001). protein 3 mediates hypertriglyceridemia induced by the 16) Nagao, K., Sato, M., Takenaka, M., Ando, M., Iwamoto, liver X receptor. J. Biol. Chem., 278, 21344–21351 M., and Imaizumi, K., Feeding unsaponifiable com- (2003). pounds from rice bran oil does not alter hepatic mRNA 26) Janowski, B. A., Willy, P. J., Devi, T. R., Falck, J. R., abundance for cholesterol metabolism-related proteins in and Mangelsdorf, D. J., An oxysterol signalling pathway hypercholesterolemic rats. Biosci. Biotechnol. Biochem., mediated by the nuclear receptor LXR alpha. Nature, 65, 371–377 (2001). 383, 728–731 (1996).