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 oxycholes- terol 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 pro- duced to an extent lower than the 7-keto types.6–8) These Materials and Methods sterol oxidation products are transported to the circu- lation 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 Æ 0:05 O2 absorbent (Ageless S-200, Mitsubishi Gas Chemical, 7 -Hydroxycholesterol 2:60 Æ 0:03 -Epoxycholesterol 12:1 Æ 0:3 Tokyo), flushed with N2, and stored at 4 C. The diet -Epoxycholesterol 10:4 Æ 0:3 was freshly prepared every week and was changed every -Cholestanetriol 2:91 Æ 0:10 2 d. Any remaining food was discarded. At the end of the -Cholestanetriol 1:00 Æ 0:02 4-week feeding period, the mice were deprived of food 7-Ketocholesterol 27:7 Æ 1:2 for 6 h (7:00–13:00), anesthetized with diethyl ether, and 25-Hydroxycholesterol 1:25 Æ 0:05 Unknown 39:0 Æ 1:8 killed by withdrawal of blood from the left ventricle. The blood was transferred to microcentrifuge tubes Values are means Æ SEM of triplicate analyses. containing 50 mg, BHT. The serum was separated, bubbled with Ar, and stored at À85 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 Æ 0:25 and the Graduate Course, Kyushu University, Fukuoka, 7 -Hydroxysitosterol 1:67 Æ 0:20 and Law no. 105 and Notification no. 6 of the Govern- -Epoxysitosterol 5:89 Æ 0:40 ment of Japan. -Epoxysitosterol 6:03 Æ 0:13 Dihydroxysitosterol 3:65 Æ 0:41 7-Ketositosterol 14:8 Æ 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 Æ 0:05 cholestane (Sigma, St. Louis, MO), and 1 mg, 19- 7 -Hydroxycampesterol 1:14 Æ 0:05 -Epoxycampesterol 5:89 Æ 0:39 hydroxycholesterol (Steraloids, Newport, RI) as an -Epoxycampesterol 6:07 Æ 0:12 internal standard. The lipids in the sample were Dihydroxycampesterol 3:45 Æ 0:55 extracted with 20 vol of chloroform/methanol (2:1, 7-Ketocampesterol 13:9 Æ 0:9 v/v) containing 0.01% (w/v) BHT (Nacalai Tesque, Unkown 34:0 Æ 3:7 Kyoto). The extracted lipids were applied to a Sep-Pak Values are means Æ 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 Æ 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 Æ 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 Æ 3:0a 18:9 Æ 2:6a 28:3 Æ 1:8b 7 -Hydroxycholesterol 0:28 Æ 0:01a 0:29 Æ 0:05a 0:65 Æ 0:04b -Epoxycholesterol 75:1 Æ 24:7a 136 Æ 18b 136 Æ 20b -Epoxycholesterol 1:62 Æ 0:05 1:48 Æ 0:29 1:86 Æ 0:29 -Epoxycholesterol 128 Æ 26a 123 Æ 42a 252 Æ 38b -Epoxycholesterol 1:15 Æ 0:09 1:96 Æ 0:35 1:97 Æ 0:26 -Cholestanetriol 29:6 Æ 6:626:4 Æ 4:225:1 Æ 7:5 -Cholestanetriol 0:18 Æ 0:04a 0:14 Æ 0:02a 0:47 Æ 0:05b -Cholestanetriol 29:3 Æ 0:128:7 Æ 8:934:4 Æ 7:8 -Cholestanetriol n.d.