Food Chemistry 169 (2015) 277–282
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Plasma cholesterol-raising potency of dietary free cholesterol versus cholesteryl ester and effect of b-sitosterol Yuwei Liu a, Lin Lei a, Xiaobo Wang a, Ka Ying Ma a, Yuk Man Li a, Lijun Wang a, Sun Wa Man a, Yu Huang b, Zhen-Yu Chen a,⇑ a b
Food & Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 24 July 2014 Accepted 25 July 2014 Available online 7 August 2014 Keywords: Cholesterol Cholesteryl ester Lipoprotein profile NPC1L1 b-Sitosterol
a b s t r a c t The present study (i) compared plasma cholesterol-raising activity of free cholesterol (FC) with that of cholesteryl palmitate (CP) and (ii) examined plasma cholesterol-reducing activity of b-sitosterol in FCinduced and CP-induced hypercholesterolemia. Male hamsters were divided into five groups and fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S), respectively, for 8 weeks. Hamsters fed diet C had plasma TC of 317.5 mg/dl whereas hamsters fed diet CP has plasma TC of 281.3 mg/dl. b-Sitosterol reduced plasma TC by 17.4% and 11.6%, respectively, in FC-induced and CP-induced hypercholesterolemia (not significant). It was concluded that plasma cholesterol-raising activity of dietary cholesterol was a function of its chemical forms in diet, and b-sitosterol could similarly suppress the hypercholesterolemia induced by both dietary FC and CP. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction It is well documented that individuals with elevated concentrations of plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) as well as reduced concentration of high-density lipoprotein cholesterol (HDL-C) have a higher risk of developing coronary heart disease (Daniels, Killinger, Michal, Wright, & Jiang, 2009; Sharrett et al., 2001). It is recommended that daily cholesterol consumption shall not be more than 300 mg per person. On an average, dietary surveys have demonstrated that humans consume cholesterol ranging 200–500 mg per day (Centers for Disease Control, 2000; Elmadfa & Weichselbaum, 2005; Food & Nutrition Department, 1994; Schmidhuber, 2007; Woo, Leung, Ho, Lam, & Janus, 1998; Zhao et al., 2009). Cholesterol in human diet exists in two forms, namely free cholesterol (FC) and cholesteryl esters (CE), with the latter accounting for approximately up to 30% total cholesterol (Awad, Bennink, & Smith, 1997; Bitman & Wood, 1980). Although adverse effect of dietary cholesterol on cardiovascular health has been extensively investigated, the effect of dietary CE versus dietary FC on plasma TC has not been fully explored. Phytosterols have become popular as a health supplement in reducing plasma TC and LDL-C (Lingberg et al., 2008; Vanstone, ⇑ Corresponding author. Tel.: +852 3943 6382; fax: +852 2603 7246. E-mail address: [email protected] (Z.-Y. Chen). http://dx.doi.org/10.1016/j.foodchem.2014.07.123 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Raeini-Sarjaz, Parsons, & Jones, 2002). In the intestinal lumen, they displace cholesterol from mixed micelles and inhibit cholesterol absorption due to their poor absorption and structural similarity with that of cholesterol (Smet, Mensink, & Plat, 2012). In humans, about 40–70% dietary cholesterol can be absorbed whereas only 1– 2% dietary phytosterols is absorbed. Both cholesterol and phytosterols can enter enterocytes via transporter Niemann–Pick C1 like 1 (NPC1L1). However, phytosterols are prevented from being further absorbed because the ATP binding cassette transporters (ABCG5/8) return them to the lumen of the intestine (Ostlund & Lin, 2006). Intestinal acyl CoA: cholesterol acyltransferase (ACAT2) is responsible for conversion of FC to CE. Subsequently, CE is packed into chylomicrons for absorption by microsomal triglyceride protein (MTP). In this regard, ACAT2 prefers cholesterol to phytosterol for esterification, preventing phytosterols from the absorption (Temel, Gebre, Parks, & Rudel, 2003). Research has demonstrated plasma TC-lowering activity of phytosterol is mediated by not only its competition for incorporation into micelles but also its inhibition in gene expression of intestinal NPC1L1 (Jesch, Seo, Carr, & Lee, 2009). However, it remains unexplored how dietary phytosterols interact with dietary CE versus dietary FC in the intestinal absorption process. b-Sitosterol is the major phytosterol while cholesteryl palmitate (CP) is the major CE in human diets in addition to free cholesterol. The present study was designed to (i) compare plasma TC-raising potency of dietary CP with that of dietary FC; and (ii) compare
278
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
plasma TC-suppressing activity of b-sitosterol in dietary FCinduced hypercholesterolemia compared with that in CP-induced hypercholesterolemia. 2. Materials and methods
Stanbio Laboratories (Boerne, TX), respectively. For measurement of HDL-C, non-HDL cholesterol (non-HDL-C) was firstly precipitated with phosphotungstic acid and magnesium chloride using a commercial kit (Stanbio Laboratories) and HDL-C in the supernatant was determined similarly as for TC (Jiao et al., 2013).
2.1. Diets
2.4. Determination of cholesterol in the liver
Diets were prepared by modifying the formulation as we had previously described (Jiao et al., 2013). The basal diet containing no cholesterol (NC) was prepared by mixing the following ingredients (per kilogram diet): cornstarch, 508 g; casein, 242 g; sucrose, 119 g; lard, 50 g; mineral mix 40 g; vitamin mix 20 g; gelatin, 20 g; DL-methionine, 1 g. The four experimental diets were prepared by adding 2.6 mmol of free cholesterol (C), 2.6 mmol free cholesterol plus 2.6 mmol of b-sitosterol (C+S), 2.6 mmol of cholesteryl palmitate (CP), and 2.6 mmol of cholesteryl plamitate plus 2.6 mmol of b-sitosterol (CP+S), into the basal diet, respectively (Table 1).
Hepatic cholesterol was determined according to the method as we previously described (Liang et al., 2011). Total lipids were extracted into chloroform:methanol (2:1, v/v) followed by saponification. Cholesterol in the fraction of non-saponifiable substances was converted to its TMS-ether derivative by a commercial TMS reagent (Sigma-Sil-A; Sigma). The analysis of cholesterol TMS-ether derivative was performed in a SACTM-5 column (Bellefonte, USA) using a Shimadzu GC-14 B GLC equipped with a flame ionisation detector. Hepatic cholesterol was quantified according to the amount of internal standard 5a-cholestane added during the extraction.
2.2. Hamsters
2.5. Determination of faecal neutral and acidic sterols
Male Golden Syrian hamsters (n = 56) were divided into five groups with NC having 8 hamsters and the other groups having 12 hamsters each. All hamsters were housed in an animal room at 23 °C with 12/12-h light–dark cycles. The fresh diets were given to the hamsters daily, and uneaten food was discarded. Food intake was measured daily and body weight was recorded twice a week. The hamsters were allowed free access food and water. Each hamster was bled from the retro-orbital sinus into a heparinized capillary tube under light anaesthesia using a mixture of ketamine, xylazine and saline (v/v/v; 4:1:5) after overnight fasting, respectively, at week 0 and 8. The blood was be centrifuged at 2300g for 10 min and the plasma was collected. At the end of week 8, all the hamsters were killed using carbon dioxide anaesthesia; the abdomen was cut open with blood being sampled from the aorta into syringe. The liver was then removed, washed with saline, weighed and frozen in liquid nitrogen. The first 5 cm of duodenum was discarded, and the next 30 cm of the small intestine was kept. All samples were stored at a 80 °C freezer prior to cholesterol analysis. All the faeces from each hamster were also collected and pooled at week 1 and 8 followed by being freeze-dried, ground and saved for neutral and acidic sterol analyses. Experiments were conducted following the approval and in accordance with the guidelines set by the Animal Experimental Ethical Committee, The Chinese University of Hong Kong.
2.6. Measurement of atherosclerotic plaque The atherosclerotic plaque in aorta was determined as previously described (Chan et al., 1999). In brief, aorta artery was cut opened vertically and then stained with 0.05 g oil red in 1 ml isopropanol for 30 min. The endothelial layer of aorta was washed with isopropanol and distiled water for 3 times and scanned with a table scanner. The area of atherosclerotic plaque was measured with the aid of computer images analysing program ‘‘Sigma Scan Pro 5.0’’ (SPSS, Inc., Chicago, USA). 2.7. Measurement of mRNA of intestinal NPC1L1, ABCG5, ABCG8, ACAT2 and MTP
2.3. Analysis of plasma lipids Plasma TC and total triacylglycerols (TG) were measured using the commercial enzymatic kits from Infinity (Waltham, MA) and Table 1 Composition of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S). Ingredients (per kg diet)
NC
C
C+S
CP
CP+S
Corn starch (g) Casein (g) Sucrose (g) Lard (g) Mineral mixture AIN-76 (g) Vitamin mixture AIN-76A (g) Gelatin (g)
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
DL-Methionine (g) Cholesterol (mmol) Cholesteryl palmitate (mmol) b-Sitosterol (mmol)
Neutral and acidic sterols in the faeces were quantified as we previously described (Chan et al., 1999). Total faecal sample from each hamster was freeze-dried, ground and well mixed. Faecal sample (300 mg) was weighed and then saponified. The total neutral sterols were extracted into cyclohexane and then were converted to their corresponding TMS-ether derivatives for GC analysis. The remaining aqueous layer was saponified using NaOH, then neutralise with HCl and extracted with diethyl ether twice. The acidic sterols were similarly converted to their TMS-ether derivatives for GC analysis. Total and individual neutral and acidic sterols were quantified according to internal standards 5a-cholestane and hyodeoxycholic acid added into the faecal sample during the extraction.
0 0 0
2.6 0 0
2.6 0 2.6
0 2.6 0
0 2.6 2.6
Real-time PCR was employed to quantify mRNA of intestinal NPC1L1, ABCG5/8, ACAT2, and MTP (Ma et al., 2011). Total intestinal mRNA was extracted and converted to complementary DNA (cDNA) using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Reverse transcription was carried out in a thermocycler (Gene AmpÒ PCR system 9700, Applied Biosystems). Real-time PCR analysis was carried out on a Fast Real-time PCR System 7500 (Applied Biosystems). To measure intestinal NPC1L1, ABCG5, ABCG8, ACAT2, MTP and Cyclophilin, SYBR green was used as a fluorophore. Data were analysed using the Sequence Detection Software version 1.3.1.21 (Applied Biosystems). 2.8. Statistics Data were expressed as mean ± standard deviation (SD). Following the analysis of variance (ANOVA), post hoc LSD test was
279
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
carried out to detect the differences among the group means using the SigmaStat Advisory Statistical Software (SigmaStat version 18.0, SPSS Inc., Chicago, USA). Significance was defined as p-value less than 0.05. 3. Results 3.1. Food intake, body and organ weight No difference in food intake, initial and final body weight was seen among the five groups (Table 2). When the organ weights were expressed as the percentage of total body weight, no significant difference was seen among the five groups. 3.2. Plasma TC, HDL-C, TG and non-HDL-C Five groups of hamsters had similar levels of plasma TC, HDL-C and TG at the beginning of the experiment (week 0). Two major changes were observed after a period of 8 week feeding. First, plasma lipids were compared among the three groups of NC, C and CP at the end of week 8 (Table 3). Results showed that group C had plasma TC of 317.5 mg/dl and group CP had TC of 281.3 mg/dl, while TC in group NC was only 202.9 mg/dl (p < 0.05, group C versus group CP versus group NC). These observations showed that dietary CP had lesser potency in raising
plasma TC compared with dietary FC. Second, plasma TC was compared among four groups of C, C+S, CP and CP+S. It was found that plasma TC in group C+S was 262.2 mg/dl versus 317.5 mg/dl in group C, implying that addition of b-sitosterol into diet reduced plasma TC by 17.4%. In contrast, plasma TC in group CP+S was 248.3 mg/dl while that in group CP was 281.3 mg/dl, showing that addition of b-sitosterol into diet could reduce plasma TC by 11.6% (Table 3). However, this percentage decrease (17.4% versus 11.6%) was statistically insignificant. A similar pattern in changes of plasma TG was seen at the end of week 8 (Table 3). First, addition of free cholesterol into diet led plasma TG to increase by 233% (74.6 mg/dl in group NC versus 248.1 mg/dl in group C), whereas addition of CP in diet increased plasma TG only by 150% (74.6 mg/dl in group NC versus 186.3 mg/dl in group CP), finding dietary FC was more effective in raising plasma TG than dietary CP. Second, addition of b-sitosterol into diet containing 2.6 mmol free cholesterol decreased plasma TG by 22.5% (248.1 mg/dl in group C versus 192.1 mg/dl in group C+S), while addition of b-sitosterol in diet containing 2.6 mmol of CP decreased plasma TG by 6.3% (186.3 mg/dl in group CP versus 174.5 mg/dl in group CP+S). 3.3. Liver cholesterol Results demonstrated that both dietary free cholesterol and CP were effective in raising hepatic cholesterol (Table 3). It was found
Table 2 Changes in food consumption, body weight and relative organ weights in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC Daily food intake (g) Body weight (g) Initial Final Relative organ weight (% body weight) Liver Kidney Heart Testis Epididymal fat pad Perirenal fat pad
C
C+S
CP
CP+S
10.9 ± 0.8
11.4 ± 0.7
11.2 ± 0.8
10.9 ± 0.7
11.0 ± 0.5
104.6 ± 5.5 118.7 ± 6.4
108.3 ± 6.7 128.7 ± 8.6
105.8 ± 6.0 125.2 ± 9.1
107.4 ± 7.1 129.8 ± 8.8
105.1 ± 6.3 126.3 ± 10.2
4.4 ± 0.8 1.1 ± 0.2 0.5 ± 0.2 2.6 ± 0.9 1.6 ± 0.2 1.2 ± 0.3
4.9 ± 0.4 0.9 ± 0.2 0.3 ± 0.1 2.5 ± 0.9 1.6 ± 0.2 1.0 ± 0.1
5.1 ± 0.4 0.9 ± 0.2 0.4 ± 0.1 2.6 ± 0.8 1.6 ± 0.1 1.0 ± 0.2
4.9 ± 0.5 0.9 ± 0.2 0.5 ± 0.1 2.5 ± 0.9 1.7 ± 0.2 1.0 ± 0.2
5.0 ± 0.6 0.9 ± 0.2 0.3 ± 0.1 2.5 ± 0.9 1.7 ± 0.2 0.9 ± 0.1
Values are mean ± SD, n = 8–12.
Table 3 Changes in plasma total cholesterol (TC), total triacylglycerols (TG), high density lipoprotein cholesterol (HDL-C), non-HDL cholesterol (non HDL-C), HDL-C:TC, liver cholesterol and aortic plaque in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC
Week 0 TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) Non-HDL-C (mg/dl) Non-HDL-C/HDL-C Week 8 TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) Non-HDL-C (mg/dl) Non-HDL-C/HDL-C Liver cholesterol (mg/g) Aortic plaque (%)
Free cholesterol
Cholesteryl ester
C
C+S
% Change
CP
CP+S
% Change
158.4 ± 25.1 53.5 ± 7.7 98.5 ± 21.6 59.9 ± 13.5 0.64 ± 0.18
156.4 ± 11.2 61.1 ± 11.4 96.8 ± 7.6 59.6 ± 15.1 0.63 ± 0.20
156.1 ± 15.0 57.6 ± 6.9 97.0 ± 5.9 59.0 ± 13.5 0.61 ± 0.15
NA NA NA NA NA
155.7 ± 11.5 56.2 ± 6.3 98.3 ± 5.6 57.4 ± 10.8 0.59 ± 0.12
157.1 ± 18.8 59.0 ± 8.4 100.5 ± 8.1 56.5 ± 15.1 0.56 ± 0.14
NA NA NA NA NA
202.9 ± 28.2d 74.6 ± 22.9c 127.0 ± 26.7b 74.1 ± 11.3d 0.63 ± 0.26b 6.6 ± 2.1c 7.1 ± 1.0b
317.5 ± 29.7a 248.1 ± 78.1a 161.0 ± 11.8a 156.5 ± 31.6a 0.98 ± 0.24a 63.2 ± 5.9a 18.1 ± 5.3a
262.4 ± 37.6bc 192.1 ± 31.1b 148.4 ± 15.8a 114.0 ± 46.1bc 0.80 ± 0.41ab 48.3 ± 7.1b 7.4 ± 2.9b
281.3 ± 28.2b 186.3 ± 43.1b 145.7 ± 24.4a 135.5 ± 46.8ab 0.99 ± 0.40a 54.0 ± 9.0ab 15.9 ± 4.2a
248.3 ± 37.2c 174.5 ± 58.9b 147.2 ± 20.1a 101.0 ± 23.9cd 0.69 ± 0.16ab 49.3 ± 4.7b 10.6 ± 3.8ab
11.6 6.3 +1.0 25.6 30.4 8.6 33.4
Values are mean ± SD, n = 8–12. Means in a row for a given week with different letters differ significantly, p < 0.05.
a,b,c
17.4 22.5 7.9 27.1 18.4 23.6 59.4
280
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
that addition of b-sitosterol in diet containing 2.6 mmol free cholesterol could effectively decrease hepatic cholesterol concentration by 23.6% (63.2 mg/g in group C versus 48.3 mg/g in group C+S, p < 0.05). In contrast, addition of b-sitosterol into diet containing 2.6 mmol CP could only decrease hepatic cholesterol concentration by 8.6% (54.0 mg/g in group CP versus 49.3 mg/g in CP+S). However, the percentage suppression (23.6% versus 8.6%) was statistically insignificant. 3.4. Atherosclerotic plaque As shown in Table 3, incorporation of b-sitosterol into diet containing 2.6 mmol free cholesterol could attenuate the formation of atherosclerotic plaque on the endothelial layer by 59.4% (18.1% in group C versus 7.4% in group C+S). When b-sitosterol was added into diet containing 2.6 mmol CP, it was also able to reduce the formation of atherosclerotic plaque by 33.4% (15.9% in group CP versus 10.6% in group CP+S). No significant difference was seen in percentage suppression on atherosclerosis when C+S group was compared with CP+S hamsters.
namely coprostanol, coprostanone and dihydrocholesterol (Tables 4 and 5). When group CP was compared with group C, the former had greater excretion of faecal total neutral sterols (week 1 in Table 4: 2.44 mg/g versus 1.59 mg/g; week 8 in Table 5: 3.11 mg/ g versus 1.90 mg/g), demonstrating dietary CP was less absorbed than dietary free cholesterol. When group C and group C+S were compared with group CP and group CP+S, it was found that addition of b-sitosterol into diet was more effective in increasing the neutral sterol excretion in C-fed than CP-fed hamsters. To be specific, b-sitosterol increased the neutral sterol excretion in C-fed hamsters by 62.03% at week 1 and by 38.18% at week 8, while this percentage increase in CP-fed hamsters was only 21.43% at week 1 and 9.54% at week 8. Faecal bile acids are mainly consisting of cholic, lithocholic, chenodeoxycholic, deoxycholic and ursodeoxycholic acid. Both diets C and CP increased the excretion of bile acids when compared with diet NC. Addition of b-sitosterol into diets further increased the excretion of faecal total bile acids similarly in C diet-induced and CP diet-induced hypercholesterolemia. 3.6. Intestinal mRNA of NPC1L1, ABCG5, ABCG8, ACAT2 and MTP
3.5. Faecal total sterol excretion The major neutral sterols present in faeces consist of mainly cholesterol and three major microbial transformed derivatives
Intestinal mRNA NPC1L1 level was increased in group C compared with that in group NC followed by a decrease in group C+S, demonstrating b-sitosterol was capable of down-regulating
Table 4 Faecal individual neutral and acidic sterols and their derivatives at week 1 in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC
Free cholesterol
Cholesteryl ester
C
C+S
% Change
CP
CP+S
% Change
Faecal cholesterol and its derivatives (mg/g) Cholesterol 0.29 ± 0.11b Coprostanol 0.66 ± 0.09c Coprostanone 0.05 ± 0.02 Dihydrocholesterol 0.11 ± 0.02c Total 1.12 ± 0.25c b-Sitosterol 0.01 ± 0.01b
0.35 ± 0.13b 0.77 ± 0.03c 0.06 ± 0.04 0.40 ± 0.02b 1.59 ± 0.14c 0.01 ± 0.01b
0.97 ± 0.15a 0.94 ± 0.08b 0.06 ± 0.03 0.61 ± 0.08a 2.57 ± 0.24b 2.96 ± 0.23a
+177.14 +22.08 +0.00 +52.75 +62.03 NA
0.88 ± 0.11a 1.03 ± 0.23ab 0.08 ± 0.03 0.45 ± 0.04b 2.44 ± 0.28b 0.01 ± 0.01b
0.95 ± 0.10a 1.30 ± 0.34a 0.07 ± 0.02 0.64 ± 0.05a 2.96 ± 0.37a 2.84 ± 0.45a
+7.95 +26.21 12.09 +42.35 +21.43 NA
Faecal bile acids (mg/g) Lithocholic acid Deoxycholic acid Chenodeoxycholic+cholic acid Ursodeoxycholic acid Total
0.57 ± 0.23a 0.16 ± 0.07 0.34 ± 0.12b 0.06 ± 0.01b 1.13 ± 0.34b
0.61 ± 0.18a 0.15 ± 0.06 0.97 ± 0.24a 0.26 ± 0.05a 1.99 ± 0.38a
+7.38 6.88 +184.50 +329.51 +76.11
0.59 ± 0.23a 0.17 ± 0.08 0.38 ± 0.17b 0.06 ± 0.02b 1.20 ± 0.43b
0.62 ± 0.24a 0.17 ± 0.05 0.88 ± 0.12a 0.29 ± 0.14a 1.91 ± 0.45a
+5.10 +0.00 +130.97 +356.25 +59.21
0.34 ± 0.19b 0.19 ± 0.15 0.49 ± 0.17b 0.04 ± 0.01b 1.16 ± 0.44b
Values are mean ± SD, n = 8–12. Means in a row for a given week with different letters differ significantly, p < 0.05. NA, not applicable.
a,b,c
Table 5 Faecal individual neutral and acidic sterols and their derivatives at week 8 in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC
Free cholesterol
Cholesteryl ester
C
C+S
% Change
CP
CP+S
% Change
Faecal cholesterol and its derivatives (mg/g) Cholesterol 0.41 ± 0.23c Coprostanol 0.70 ± 0.19b Coprostanone 0.05 ± 0.04 Dihydrocholesterol 0.29 ± 0.08c Total 1.46 ± 0.43c b-Sitosterol 0.01 ± 0.01b
0.56 ± 0.09c 0.81 ± 0.14b 0.05 ± 0.04 0.48 ± 0.05b 1.90 ± 0.26c 0.01 ± 0.01b
0.99 ± 0.23b 1.04 ± 0.27a 0.06 ± 0.03 0.54 ± 0.07b 2.63 ± 0.37b 3.16 ± 0.23a
+76.33 +28.01 +24.48 +12.89 +38.18 NA
1.47 ± 0.21b 1.15 ± 0.30a 0.08 ± 0.04 0.44 ± 0.12b 3.11 ± 0.79a 0.01 ± 0.01b
1.48 ± 0.17a 1.20 ± 0.32a 0.07 ± 0.02 0.65 ± 0.04a 3.41 ± 0.62a 3.24 ± 0.45a
+0.74 +4.97 9.88 +48.29 +9.54 NA
Faecal bile acids (mg/g) Lithocholic acid Deoxycholic acid Chenodeoxycholic+cholic acid Ursodeoxycholic acid Total
0.72 ± 0.34 0.14 ± 0.09 0.36 ± 0.10b 0.06 ± 0.01b 1.28 ± 0.45b
0.76 ± 0.38 0.16 ± 0.07 1.08 ± 0.34a 0.31 ± 0.05a 2.31 ± 0.42a
+5.56 +15.60 +201.17 +409.84 +80.77
0.69 ± 0.33 0.18 ± 0.08 0.40 ± 0.16b 0.06 ± 0.02b 1.33 ± 0.48b
0.70 ± 0.38 0.17 ± 0.05 1.13 ± 0.32a 0.27 ± 0.07a 2.27 ± 0.53a
+1.89 5.03 +182.46 +359.32 +70.58
0.62 ± 0.29 0.18 ± 0.15 0.54 ± 0.24b 0.08 ± 0.04b 1.42 ± 0.35b
Values are mean ± SD, n = 8–12. Means in a row for a given week with different letters differ significantly, p < 0.05. NA, not applicable.
a,b,c
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
281
Fig. 1. Effect of b-sitosterol on mRNA levels of intestinal Niemann–Pick C1 like 1 (NPC1L1), acyl coenzyme A: cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), ATP binding cassette transporter (ABCG8) in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. Data are normalised with cyclophilin. Values are expressed as means ± S.D (n = 8–12). a,b,c Means with different superscript letters differ significantly, p < 0.05. Other abbreviations: C, free cholesterol; CE, cholesteryl esters; CMs, chylomicrons; FC, free cholesterol; PCE, pancreatic cholesterol esterase.
NPC1L1 in the intestine (Fig. 1). However, no such observation was seen when comparison was made among groups C, CP and CP+S. Intestinal mRNA ACAT-2 level was up-regulated when group C was compared with group NC. No effect on mRNA ACAT-2 was seen when b-sitosterol was added into diets containing free cholesterol or CP (Fig. 1). Intestinal mRNA MTP was up-regulated when group C was compared with group NC. Addition of b-sitosterol had a trend of down-regulating intestinal mRNA MTP in both free cholesterol-fed and CP-fed hamsters, however, the effect was not statistically significant. Intestinal mRNA ABCG8 showed an upregulating trend when groups C and C+S were compared with group NC. Addition of b-sitosterol in diet containing CP was able to up-regulate intestinal mRNA ABCG8.
4. Discussion Humans consume diets containing not only free cholesterol but also cholesteryl esters. The present study clearly demonstrated both dietary free cholesterol and CP were capable of raising plasma TC significantly. However, CP was less potent than dietary free cholesterol, suggesting that for a given amount, total cholesterol in different foods may exhibit a varying plasma TC-raising potency dependent on the ratio of free to esterified cholesterol in diets. Less plasma TC-raising potency associated with CP was perhaps due to its difference in absorption compared with that for FC. Absorption of FC and CE is a complex process (Fig. 1). In this regard,
transporter NPC1L1 can directly carry FC from the lumen into enterocytes, while CE needs to be hydrolysed by pancreatic cholesterol esterase before absorption (Grober, Lucas, & Sorhede-Winsell, 2003; Howles, Carter, & Hui, 1996). It was believed that the incomplete hydrolysis or the slow rate of hydrolysis of CP in the intestine might contribute to its lesser potency in raising plasma TC. This hypothesis was supported by the following observations. Firstly, the present result is in agreement with that in a previous study showing cholesterol palmitate and cholesteryl stearate had a slower rate of hydrolysis by pancreatic cholesterol esterase (Jiao et al., 2013). Secondly, group CP had greater excretion of faecal cholesterol and total neutral sterols compared with group C (Tables 4 and 5), proving that CP had the incomplete hydrolysis and was less absorbed. Thirdly, the hypothesis was further supported by the observation that plasma TC-raising activity of dietary free cholesterol was associated with up-regulation of intestinal NPC1L1, ACAT-2 and MTP, but no such up-regulations were seen with dietary CP. This indicated that CP was incompletely hydrolysed to release free cholesterol in the intestinal lumen to reach a threshold concentration which was high enough to up-regulate the intestinal NPC1L1, ACAT-2 and MTP. b-Sitosterol as one of the major phytosterols in human diets has been widely used as a health supplement in treatment of hypercholesterolemia. The present study was the first time to investigate the effect of b-sitosterol on plasma TC in hypercholesterolemia hamsters induced by dietary cholesteryl ester compared with that induced by dietary free cholesterol. It demonstrated that
282
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
b-sitosterol was slightly better in suppressing the hypercholesterolemia induced by dietary free cholesterol than that induced by CP (17.4% versus 11.6%, Table 3). However, the difference was statistically insignificant. Regarding effect of b-sitosterol on the faecal sterol excretion, bsitosterol added into diets containing free cholesterol (diets C and C+S) increased the faecal excretion of neutral sterols greater (week 1, 62.03% versus 21.43%; week 8, 38.18% versus 9.54%) than it was added into diets containing cholesteryl ester (diets CP and CP+S). Intestinal NPC1L1 can transport both cholesterol and b-sitosterol and facilitate the uptake of sterols into enterocyte (Davis et al., 2004; Yamanashi, Takada, & Suzuki, 2007). Because b-sitosterol and cholesterol both have one free hydroxyl group and the same ring structure, they compete for the active side of NPC1L1 and interfere with the absorption of each other. In contrast, CP has a hydroxyl group esterified with palmitic acid and thus b-sitosterol has less competition with CP for NPC1L1. It was therefore expected that addition of b-sitosterol in diets containing only free cholesterol decreased the uptake of cholesterol into enterocytes, leading to a greater excretion of faecal cholesterol and its microbial derivatives, while addition of b-sitosterol in diets containing CP is expected to have a lesser percentage increase in excretion of faecal total neutral sterols. 5. Conclusion In summary, humans daily consume about 200–500 mg cholesterol with an approximate of 30% being present in a form of cholesteryl esters. Although the data could not be directly extrapolated to what happen in humans, the present study demonstrated in hamster model that diets containing cholesteryl esters, particularly CP, was less potent in raising plasma TC compared with diets containing free cholesterol. b-Sitosterol could suppress the hypercholesterolemia induced by both a free cholesterol diet and a cholesteryl ester diet, however, the difference was insignificant. b-Sitosterol relatively increased the faecal excretion of neutral sterols in hamsters given a free cholesterol diet more than it did in hamsters given a cholesteryl ester diet. Conflict of interest We have no conflict of interest in this research. Acknowledgement This project is supported by Hong Kong GRF grant (Project Number CUHK 461112). References Awad, A. C., Bennink, M. R., & Smith, D. M. (1997). Composition and functional properties of cholesterol reduced egg yolk. Poultry Science, 76, 649–653. Bitman, J., & Wood, D. L. (1980). Cholesterol and cholesteryl esters of eggs from various avian species. Poultry Science, 59, 2014–2023. Centers for Disease Control and Prevention. (2000). National Health and Nutrition Examination Survey: Intake of calories and selected nutrients for the United States population. US Department of Health and Human Services.
Chan, P. T., Fong, W. P., Cheung, Y. L., Huang, Y., Ho, W. K., & Chen, Z. Y. (1999). Jasmine green tea epicatechins are hypolipidemic in hamster (Mesocricetus auratus) fed a high fat diet. Journal of Nutrition, 129, 1094–1101. Daniels, T. F., Killinger, K. M., Michal, J. J., Wright, R. W., & Jiang, Z. (2009). Lipoproteins, cholesterol homeostasis and cardiac health. International Journal of Biological Sciences, 5, 474–488. Davis, H. R., Zhu, L. J., Hoos, L. M., Tetzloff, G., Maguire, M., Liu, J., et al. (2004). Niemann–Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. The Journal of Biological Chemistry, 279, 33586–33592. Elmadfa, I., & Weichselbaum, E. (2005). On the nutrition and health situation in the European Union. Journal of Public Health, 13, 62–68. Food and Nutrition Department (1994). Food consumption study 1993. Singapore: Ministry of Health. Grober, J., Lucas, S., & Sorhede-Winsell, M. (2003). Hormone-sensitive lipase is a cholesterol esterase of the intestinal mucosa. The Journal of Biological Chemistry, 278, 6510–6515. Howles, P. N., Carter, C. P., & Hui, D. Y. (1996). Dietary free and esterified cholesterol absorption in cholesterol esterase (bile salt-stimulated lipase) gene-targeted mice. The Journal of Biological Chemistry, 271, 7196–7202. Jesch, E. D., Seo, J. M., Carr, T. P., & Lee, J. Y. (2009). Sitosterol reduces messenger RNA and protein expression levels of Niemann–Pick C1-like 1 in FHs 74 Int cells. Nutrition Research, 29, 859–866. Jiao, R., Chen, J., Peng, C., Liang, Y., Ma, K. Y., Wang, X., et al. (2013). Cholesterol ester species differently elevate plasma cholesterol in hamsters. Journal of Agricultural and Food Chemistry, 61, 11041–11047. Liang, Y. T., Wong, W. T., Guan, L., Tian, X. Y., Ma, K. Y., Huang, Y., et al. (2011). Effect of phytosterols and their oxidation products on lipoprotein profiles and vascular function in hamster fed a high cholesterol diet. Atherosclerosis, 219, 124–133. Lingberg, S., Ellegård, L., Johansson, I., Hallmans, G., Weinehall, L., & Andersson, H. (2008). Inverse relation between dietary intake of naturally occurring plant sterols and serum cholesterol in northern Sweden. The American Journal of Clinical Nutrition, 87, 993–1001. Ma, K. Y., Yang, N., Jiao, R., Peng, C., Guan, L., Huang, Y., et al. (2011). Dietary calcium decreases plasma cholesterol by down-regulation of intestinal Niemann–Pick C1 like 1 and microsomal triacylglycerol transport protein and up-regulation of CYP7A1 and ABCG 5/8 in hamsters. Molecular Nutrition & Food Research, 55, 247–258. Ostlund, R. E., & Lin, X. (2006). Regulation of cholesterol absorption by phytosterols. Current Atherosclerosis Reports, 8, 487–491. Schmidhuber, J. (2007). The EU Diet – Evolution, evaluation and impacts of the CAP global perspectives studies unit. FAO. Sharrett, A. R., Ballantyne, C. M., Coady, S. A., Heiss, G., Sorlie, P. D., & Catellier, D. (2001). Coronary heart disease prediction from lipoprotein cholesterol levels, triglycerides, lipoprotein(a), apolipoproteins A-I and B, and HDL density subfractions: The Atherosclerosis Risk in Communities (ARIC) study. Circulation, 104, 1108–1113. Smet, E., Mensink, R. P., & Plat, J. (2012). Effects of plant sterols and stanols on intestinal cholesterol metabolism: Suggested mechanisms from past to present. Molecular Nutrition & Food Research, 56, 1058–1072. Temel, R. E., Gebre, A. K., Parks, J. S., & Rudel, L. L. (2003). Compared with acylCoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol. The Journal of Biological Chemistry, 278, 47594–47601. Vanstone, C. A., Raeini-Sarjaz, M., Parsons, W. E., & Jones, P. J. (2002). Unesterified plant sterols and stanols lower LDL-cholesterol concentrations equivalently in hypercholesterolemic persons. The American Journal of Clinical Nutrition, 76, 1272–1278. Woo, J., Leung, S. S., Ho, S. C., Lam, T. H., & Janus, E. D. (1998). Dietary intake and practices in the Hong Kong Chinese population. Journal of Epidemiology and Community Health, 52, 631–637. Yamanashi, Y., Takada, T., & Suzuki, H. (2007). Niemann–Pick C1-like 1 overexpression facilitates ezetimibe-sensitive cholesterol and b-sitosterol uptake in CaCo-2 cells. Journal of Pharmacology and Experimental Therapeutics, 320, 559–564. Zhao, L. C., Hu, J. H., Zheng, R. P., Tian, X. Z., Ren, F. X., & Wu, Y. F. (2009). The trends of dietary cholesterol intake and its food sources among workers and farmers in Beijing. Acta Nutrimenta Sinica, 31, 556–559.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Plasma cholesterol-raising potency of dietary free cholesterol versus cholesteryl ester and effect of b-sitosterol Yuwei Liu a, Lin Lei a, Xiaobo Wang a, Ka Ying Ma a, Yuk Man Li a, Lijun Wang a, Sun Wa Man a, Yu Huang b, Zhen-Yu Chen a,⇑ a b
Food & Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 24 July 2014 Accepted 25 July 2014 Available online 7 August 2014 Keywords: Cholesterol Cholesteryl ester Lipoprotein profile NPC1L1 b-Sitosterol
a b s t r a c t The present study (i) compared plasma cholesterol-raising activity of free cholesterol (FC) with that of cholesteryl palmitate (CP) and (ii) examined plasma cholesterol-reducing activity of b-sitosterol in FCinduced and CP-induced hypercholesterolemia. Male hamsters were divided into five groups and fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S), respectively, for 8 weeks. Hamsters fed diet C had plasma TC of 317.5 mg/dl whereas hamsters fed diet CP has plasma TC of 281.3 mg/dl. b-Sitosterol reduced plasma TC by 17.4% and 11.6%, respectively, in FC-induced and CP-induced hypercholesterolemia (not significant). It was concluded that plasma cholesterol-raising activity of dietary cholesterol was a function of its chemical forms in diet, and b-sitosterol could similarly suppress the hypercholesterolemia induced by both dietary FC and CP. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction It is well documented that individuals with elevated concentrations of plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) as well as reduced concentration of high-density lipoprotein cholesterol (HDL-C) have a higher risk of developing coronary heart disease (Daniels, Killinger, Michal, Wright, & Jiang, 2009; Sharrett et al., 2001). It is recommended that daily cholesterol consumption shall not be more than 300 mg per person. On an average, dietary surveys have demonstrated that humans consume cholesterol ranging 200–500 mg per day (Centers for Disease Control, 2000; Elmadfa & Weichselbaum, 2005; Food & Nutrition Department, 1994; Schmidhuber, 2007; Woo, Leung, Ho, Lam, & Janus, 1998; Zhao et al., 2009). Cholesterol in human diet exists in two forms, namely free cholesterol (FC) and cholesteryl esters (CE), with the latter accounting for approximately up to 30% total cholesterol (Awad, Bennink, & Smith, 1997; Bitman & Wood, 1980). Although adverse effect of dietary cholesterol on cardiovascular health has been extensively investigated, the effect of dietary CE versus dietary FC on plasma TC has not been fully explored. Phytosterols have become popular as a health supplement in reducing plasma TC and LDL-C (Lingberg et al., 2008; Vanstone, ⇑ Corresponding author. Tel.: +852 3943 6382; fax: +852 2603 7246. E-mail address: [email protected] (Z.-Y. Chen). http://dx.doi.org/10.1016/j.foodchem.2014.07.123 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Raeini-Sarjaz, Parsons, & Jones, 2002). In the intestinal lumen, they displace cholesterol from mixed micelles and inhibit cholesterol absorption due to their poor absorption and structural similarity with that of cholesterol (Smet, Mensink, & Plat, 2012). In humans, about 40–70% dietary cholesterol can be absorbed whereas only 1– 2% dietary phytosterols is absorbed. Both cholesterol and phytosterols can enter enterocytes via transporter Niemann–Pick C1 like 1 (NPC1L1). However, phytosterols are prevented from being further absorbed because the ATP binding cassette transporters (ABCG5/8) return them to the lumen of the intestine (Ostlund & Lin, 2006). Intestinal acyl CoA: cholesterol acyltransferase (ACAT2) is responsible for conversion of FC to CE. Subsequently, CE is packed into chylomicrons for absorption by microsomal triglyceride protein (MTP). In this regard, ACAT2 prefers cholesterol to phytosterol for esterification, preventing phytosterols from the absorption (Temel, Gebre, Parks, & Rudel, 2003). Research has demonstrated plasma TC-lowering activity of phytosterol is mediated by not only its competition for incorporation into micelles but also its inhibition in gene expression of intestinal NPC1L1 (Jesch, Seo, Carr, & Lee, 2009). However, it remains unexplored how dietary phytosterols interact with dietary CE versus dietary FC in the intestinal absorption process. b-Sitosterol is the major phytosterol while cholesteryl palmitate (CP) is the major CE in human diets in addition to free cholesterol. The present study was designed to (i) compare plasma TC-raising potency of dietary CP with that of dietary FC; and (ii) compare
278
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
plasma TC-suppressing activity of b-sitosterol in dietary FCinduced hypercholesterolemia compared with that in CP-induced hypercholesterolemia. 2. Materials and methods
Stanbio Laboratories (Boerne, TX), respectively. For measurement of HDL-C, non-HDL cholesterol (non-HDL-C) was firstly precipitated with phosphotungstic acid and magnesium chloride using a commercial kit (Stanbio Laboratories) and HDL-C in the supernatant was determined similarly as for TC (Jiao et al., 2013).
2.1. Diets
2.4. Determination of cholesterol in the liver
Diets were prepared by modifying the formulation as we had previously described (Jiao et al., 2013). The basal diet containing no cholesterol (NC) was prepared by mixing the following ingredients (per kilogram diet): cornstarch, 508 g; casein, 242 g; sucrose, 119 g; lard, 50 g; mineral mix 40 g; vitamin mix 20 g; gelatin, 20 g; DL-methionine, 1 g. The four experimental diets were prepared by adding 2.6 mmol of free cholesterol (C), 2.6 mmol free cholesterol plus 2.6 mmol of b-sitosterol (C+S), 2.6 mmol of cholesteryl palmitate (CP), and 2.6 mmol of cholesteryl plamitate plus 2.6 mmol of b-sitosterol (CP+S), into the basal diet, respectively (Table 1).
Hepatic cholesterol was determined according to the method as we previously described (Liang et al., 2011). Total lipids were extracted into chloroform:methanol (2:1, v/v) followed by saponification. Cholesterol in the fraction of non-saponifiable substances was converted to its TMS-ether derivative by a commercial TMS reagent (Sigma-Sil-A; Sigma). The analysis of cholesterol TMS-ether derivative was performed in a SACTM-5 column (Bellefonte, USA) using a Shimadzu GC-14 B GLC equipped with a flame ionisation detector. Hepatic cholesterol was quantified according to the amount of internal standard 5a-cholestane added during the extraction.
2.2. Hamsters
2.5. Determination of faecal neutral and acidic sterols
Male Golden Syrian hamsters (n = 56) were divided into five groups with NC having 8 hamsters and the other groups having 12 hamsters each. All hamsters were housed in an animal room at 23 °C with 12/12-h light–dark cycles. The fresh diets were given to the hamsters daily, and uneaten food was discarded. Food intake was measured daily and body weight was recorded twice a week. The hamsters were allowed free access food and water. Each hamster was bled from the retro-orbital sinus into a heparinized capillary tube under light anaesthesia using a mixture of ketamine, xylazine and saline (v/v/v; 4:1:5) after overnight fasting, respectively, at week 0 and 8. The blood was be centrifuged at 2300g for 10 min and the plasma was collected. At the end of week 8, all the hamsters were killed using carbon dioxide anaesthesia; the abdomen was cut open with blood being sampled from the aorta into syringe. The liver was then removed, washed with saline, weighed and frozen in liquid nitrogen. The first 5 cm of duodenum was discarded, and the next 30 cm of the small intestine was kept. All samples were stored at a 80 °C freezer prior to cholesterol analysis. All the faeces from each hamster were also collected and pooled at week 1 and 8 followed by being freeze-dried, ground and saved for neutral and acidic sterol analyses. Experiments were conducted following the approval and in accordance with the guidelines set by the Animal Experimental Ethical Committee, The Chinese University of Hong Kong.
2.6. Measurement of atherosclerotic plaque The atherosclerotic plaque in aorta was determined as previously described (Chan et al., 1999). In brief, aorta artery was cut opened vertically and then stained with 0.05 g oil red in 1 ml isopropanol for 30 min. The endothelial layer of aorta was washed with isopropanol and distiled water for 3 times and scanned with a table scanner. The area of atherosclerotic plaque was measured with the aid of computer images analysing program ‘‘Sigma Scan Pro 5.0’’ (SPSS, Inc., Chicago, USA). 2.7. Measurement of mRNA of intestinal NPC1L1, ABCG5, ABCG8, ACAT2 and MTP
2.3. Analysis of plasma lipids Plasma TC and total triacylglycerols (TG) were measured using the commercial enzymatic kits from Infinity (Waltham, MA) and Table 1 Composition of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S). Ingredients (per kg diet)
NC
C
C+S
CP
CP+S
Corn starch (g) Casein (g) Sucrose (g) Lard (g) Mineral mixture AIN-76 (g) Vitamin mixture AIN-76A (g) Gelatin (g)
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
508 242 119 50 40 20 20 1
DL-Methionine (g) Cholesterol (mmol) Cholesteryl palmitate (mmol) b-Sitosterol (mmol)
Neutral and acidic sterols in the faeces were quantified as we previously described (Chan et al., 1999). Total faecal sample from each hamster was freeze-dried, ground and well mixed. Faecal sample (300 mg) was weighed and then saponified. The total neutral sterols were extracted into cyclohexane and then were converted to their corresponding TMS-ether derivatives for GC analysis. The remaining aqueous layer was saponified using NaOH, then neutralise with HCl and extracted with diethyl ether twice. The acidic sterols were similarly converted to their TMS-ether derivatives for GC analysis. Total and individual neutral and acidic sterols were quantified according to internal standards 5a-cholestane and hyodeoxycholic acid added into the faecal sample during the extraction.
0 0 0
2.6 0 0
2.6 0 2.6
0 2.6 0
0 2.6 2.6
Real-time PCR was employed to quantify mRNA of intestinal NPC1L1, ABCG5/8, ACAT2, and MTP (Ma et al., 2011). Total intestinal mRNA was extracted and converted to complementary DNA (cDNA) using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Reverse transcription was carried out in a thermocycler (Gene AmpÒ PCR system 9700, Applied Biosystems). Real-time PCR analysis was carried out on a Fast Real-time PCR System 7500 (Applied Biosystems). To measure intestinal NPC1L1, ABCG5, ABCG8, ACAT2, MTP and Cyclophilin, SYBR green was used as a fluorophore. Data were analysed using the Sequence Detection Software version 1.3.1.21 (Applied Biosystems). 2.8. Statistics Data were expressed as mean ± standard deviation (SD). Following the analysis of variance (ANOVA), post hoc LSD test was
279
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
carried out to detect the differences among the group means using the SigmaStat Advisory Statistical Software (SigmaStat version 18.0, SPSS Inc., Chicago, USA). Significance was defined as p-value less than 0.05. 3. Results 3.1. Food intake, body and organ weight No difference in food intake, initial and final body weight was seen among the five groups (Table 2). When the organ weights were expressed as the percentage of total body weight, no significant difference was seen among the five groups. 3.2. Plasma TC, HDL-C, TG and non-HDL-C Five groups of hamsters had similar levels of plasma TC, HDL-C and TG at the beginning of the experiment (week 0). Two major changes were observed after a period of 8 week feeding. First, plasma lipids were compared among the three groups of NC, C and CP at the end of week 8 (Table 3). Results showed that group C had plasma TC of 317.5 mg/dl and group CP had TC of 281.3 mg/dl, while TC in group NC was only 202.9 mg/dl (p < 0.05, group C versus group CP versus group NC). These observations showed that dietary CP had lesser potency in raising
plasma TC compared with dietary FC. Second, plasma TC was compared among four groups of C, C+S, CP and CP+S. It was found that plasma TC in group C+S was 262.2 mg/dl versus 317.5 mg/dl in group C, implying that addition of b-sitosterol into diet reduced plasma TC by 17.4%. In contrast, plasma TC in group CP+S was 248.3 mg/dl while that in group CP was 281.3 mg/dl, showing that addition of b-sitosterol into diet could reduce plasma TC by 11.6% (Table 3). However, this percentage decrease (17.4% versus 11.6%) was statistically insignificant. A similar pattern in changes of plasma TG was seen at the end of week 8 (Table 3). First, addition of free cholesterol into diet led plasma TG to increase by 233% (74.6 mg/dl in group NC versus 248.1 mg/dl in group C), whereas addition of CP in diet increased plasma TG only by 150% (74.6 mg/dl in group NC versus 186.3 mg/dl in group CP), finding dietary FC was more effective in raising plasma TG than dietary CP. Second, addition of b-sitosterol into diet containing 2.6 mmol free cholesterol decreased plasma TG by 22.5% (248.1 mg/dl in group C versus 192.1 mg/dl in group C+S), while addition of b-sitosterol in diet containing 2.6 mmol of CP decreased plasma TG by 6.3% (186.3 mg/dl in group CP versus 174.5 mg/dl in group CP+S). 3.3. Liver cholesterol Results demonstrated that both dietary free cholesterol and CP were effective in raising hepatic cholesterol (Table 3). It was found
Table 2 Changes in food consumption, body weight and relative organ weights in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC Daily food intake (g) Body weight (g) Initial Final Relative organ weight (% body weight) Liver Kidney Heart Testis Epididymal fat pad Perirenal fat pad
C
C+S
CP
CP+S
10.9 ± 0.8
11.4 ± 0.7
11.2 ± 0.8
10.9 ± 0.7
11.0 ± 0.5
104.6 ± 5.5 118.7 ± 6.4
108.3 ± 6.7 128.7 ± 8.6
105.8 ± 6.0 125.2 ± 9.1
107.4 ± 7.1 129.8 ± 8.8
105.1 ± 6.3 126.3 ± 10.2
4.4 ± 0.8 1.1 ± 0.2 0.5 ± 0.2 2.6 ± 0.9 1.6 ± 0.2 1.2 ± 0.3
4.9 ± 0.4 0.9 ± 0.2 0.3 ± 0.1 2.5 ± 0.9 1.6 ± 0.2 1.0 ± 0.1
5.1 ± 0.4 0.9 ± 0.2 0.4 ± 0.1 2.6 ± 0.8 1.6 ± 0.1 1.0 ± 0.2
4.9 ± 0.5 0.9 ± 0.2 0.5 ± 0.1 2.5 ± 0.9 1.7 ± 0.2 1.0 ± 0.2
5.0 ± 0.6 0.9 ± 0.2 0.3 ± 0.1 2.5 ± 0.9 1.7 ± 0.2 0.9 ± 0.1
Values are mean ± SD, n = 8–12.
Table 3 Changes in plasma total cholesterol (TC), total triacylglycerols (TG), high density lipoprotein cholesterol (HDL-C), non-HDL cholesterol (non HDL-C), HDL-C:TC, liver cholesterol and aortic plaque in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC
Week 0 TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) Non-HDL-C (mg/dl) Non-HDL-C/HDL-C Week 8 TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) Non-HDL-C (mg/dl) Non-HDL-C/HDL-C Liver cholesterol (mg/g) Aortic plaque (%)
Free cholesterol
Cholesteryl ester
C
C+S
% Change
CP
CP+S
% Change
158.4 ± 25.1 53.5 ± 7.7 98.5 ± 21.6 59.9 ± 13.5 0.64 ± 0.18
156.4 ± 11.2 61.1 ± 11.4 96.8 ± 7.6 59.6 ± 15.1 0.63 ± 0.20
156.1 ± 15.0 57.6 ± 6.9 97.0 ± 5.9 59.0 ± 13.5 0.61 ± 0.15
NA NA NA NA NA
155.7 ± 11.5 56.2 ± 6.3 98.3 ± 5.6 57.4 ± 10.8 0.59 ± 0.12
157.1 ± 18.8 59.0 ± 8.4 100.5 ± 8.1 56.5 ± 15.1 0.56 ± 0.14
NA NA NA NA NA
202.9 ± 28.2d 74.6 ± 22.9c 127.0 ± 26.7b 74.1 ± 11.3d 0.63 ± 0.26b 6.6 ± 2.1c 7.1 ± 1.0b
317.5 ± 29.7a 248.1 ± 78.1a 161.0 ± 11.8a 156.5 ± 31.6a 0.98 ± 0.24a 63.2 ± 5.9a 18.1 ± 5.3a
262.4 ± 37.6bc 192.1 ± 31.1b 148.4 ± 15.8a 114.0 ± 46.1bc 0.80 ± 0.41ab 48.3 ± 7.1b 7.4 ± 2.9b
281.3 ± 28.2b 186.3 ± 43.1b 145.7 ± 24.4a 135.5 ± 46.8ab 0.99 ± 0.40a 54.0 ± 9.0ab 15.9 ± 4.2a
248.3 ± 37.2c 174.5 ± 58.9b 147.2 ± 20.1a 101.0 ± 23.9cd 0.69 ± 0.16ab 49.3 ± 4.7b 10.6 ± 3.8ab
11.6 6.3 +1.0 25.6 30.4 8.6 33.4
Values are mean ± SD, n = 8–12. Means in a row for a given week with different letters differ significantly, p < 0.05.
a,b,c
17.4 22.5 7.9 27.1 18.4 23.6 59.4
280
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
that addition of b-sitosterol in diet containing 2.6 mmol free cholesterol could effectively decrease hepatic cholesterol concentration by 23.6% (63.2 mg/g in group C versus 48.3 mg/g in group C+S, p < 0.05). In contrast, addition of b-sitosterol into diet containing 2.6 mmol CP could only decrease hepatic cholesterol concentration by 8.6% (54.0 mg/g in group CP versus 49.3 mg/g in CP+S). However, the percentage suppression (23.6% versus 8.6%) was statistically insignificant. 3.4. Atherosclerotic plaque As shown in Table 3, incorporation of b-sitosterol into diet containing 2.6 mmol free cholesterol could attenuate the formation of atherosclerotic plaque on the endothelial layer by 59.4% (18.1% in group C versus 7.4% in group C+S). When b-sitosterol was added into diet containing 2.6 mmol CP, it was also able to reduce the formation of atherosclerotic plaque by 33.4% (15.9% in group CP versus 10.6% in group CP+S). No significant difference was seen in percentage suppression on atherosclerosis when C+S group was compared with CP+S hamsters.
namely coprostanol, coprostanone and dihydrocholesterol (Tables 4 and 5). When group CP was compared with group C, the former had greater excretion of faecal total neutral sterols (week 1 in Table 4: 2.44 mg/g versus 1.59 mg/g; week 8 in Table 5: 3.11 mg/ g versus 1.90 mg/g), demonstrating dietary CP was less absorbed than dietary free cholesterol. When group C and group C+S were compared with group CP and group CP+S, it was found that addition of b-sitosterol into diet was more effective in increasing the neutral sterol excretion in C-fed than CP-fed hamsters. To be specific, b-sitosterol increased the neutral sterol excretion in C-fed hamsters by 62.03% at week 1 and by 38.18% at week 8, while this percentage increase in CP-fed hamsters was only 21.43% at week 1 and 9.54% at week 8. Faecal bile acids are mainly consisting of cholic, lithocholic, chenodeoxycholic, deoxycholic and ursodeoxycholic acid. Both diets C and CP increased the excretion of bile acids when compared with diet NC. Addition of b-sitosterol into diets further increased the excretion of faecal total bile acids similarly in C diet-induced and CP diet-induced hypercholesterolemia. 3.6. Intestinal mRNA of NPC1L1, ABCG5, ABCG8, ACAT2 and MTP
3.5. Faecal total sterol excretion The major neutral sterols present in faeces consist of mainly cholesterol and three major microbial transformed derivatives
Intestinal mRNA NPC1L1 level was increased in group C compared with that in group NC followed by a decrease in group C+S, demonstrating b-sitosterol was capable of down-regulating
Table 4 Faecal individual neutral and acidic sterols and their derivatives at week 1 in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC
Free cholesterol
Cholesteryl ester
C
C+S
% Change
CP
CP+S
% Change
Faecal cholesterol and its derivatives (mg/g) Cholesterol 0.29 ± 0.11b Coprostanol 0.66 ± 0.09c Coprostanone 0.05 ± 0.02 Dihydrocholesterol 0.11 ± 0.02c Total 1.12 ± 0.25c b-Sitosterol 0.01 ± 0.01b
0.35 ± 0.13b 0.77 ± 0.03c 0.06 ± 0.04 0.40 ± 0.02b 1.59 ± 0.14c 0.01 ± 0.01b
0.97 ± 0.15a 0.94 ± 0.08b 0.06 ± 0.03 0.61 ± 0.08a 2.57 ± 0.24b 2.96 ± 0.23a
+177.14 +22.08 +0.00 +52.75 +62.03 NA
0.88 ± 0.11a 1.03 ± 0.23ab 0.08 ± 0.03 0.45 ± 0.04b 2.44 ± 0.28b 0.01 ± 0.01b
0.95 ± 0.10a 1.30 ± 0.34a 0.07 ± 0.02 0.64 ± 0.05a 2.96 ± 0.37a 2.84 ± 0.45a
+7.95 +26.21 12.09 +42.35 +21.43 NA
Faecal bile acids (mg/g) Lithocholic acid Deoxycholic acid Chenodeoxycholic+cholic acid Ursodeoxycholic acid Total
0.57 ± 0.23a 0.16 ± 0.07 0.34 ± 0.12b 0.06 ± 0.01b 1.13 ± 0.34b
0.61 ± 0.18a 0.15 ± 0.06 0.97 ± 0.24a 0.26 ± 0.05a 1.99 ± 0.38a
+7.38 6.88 +184.50 +329.51 +76.11
0.59 ± 0.23a 0.17 ± 0.08 0.38 ± 0.17b 0.06 ± 0.02b 1.20 ± 0.43b
0.62 ± 0.24a 0.17 ± 0.05 0.88 ± 0.12a 0.29 ± 0.14a 1.91 ± 0.45a
+5.10 +0.00 +130.97 +356.25 +59.21
0.34 ± 0.19b 0.19 ± 0.15 0.49 ± 0.17b 0.04 ± 0.01b 1.16 ± 0.44b
Values are mean ± SD, n = 8–12. Means in a row for a given week with different letters differ significantly, p < 0.05. NA, not applicable.
a,b,c
Table 5 Faecal individual neutral and acidic sterols and their derivatives at week 8 in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. NC
Free cholesterol
Cholesteryl ester
C
C+S
% Change
CP
CP+S
% Change
Faecal cholesterol and its derivatives (mg/g) Cholesterol 0.41 ± 0.23c Coprostanol 0.70 ± 0.19b Coprostanone 0.05 ± 0.04 Dihydrocholesterol 0.29 ± 0.08c Total 1.46 ± 0.43c b-Sitosterol 0.01 ± 0.01b
0.56 ± 0.09c 0.81 ± 0.14b 0.05 ± 0.04 0.48 ± 0.05b 1.90 ± 0.26c 0.01 ± 0.01b
0.99 ± 0.23b 1.04 ± 0.27a 0.06 ± 0.03 0.54 ± 0.07b 2.63 ± 0.37b 3.16 ± 0.23a
+76.33 +28.01 +24.48 +12.89 +38.18 NA
1.47 ± 0.21b 1.15 ± 0.30a 0.08 ± 0.04 0.44 ± 0.12b 3.11 ± 0.79a 0.01 ± 0.01b
1.48 ± 0.17a 1.20 ± 0.32a 0.07 ± 0.02 0.65 ± 0.04a 3.41 ± 0.62a 3.24 ± 0.45a
+0.74 +4.97 9.88 +48.29 +9.54 NA
Faecal bile acids (mg/g) Lithocholic acid Deoxycholic acid Chenodeoxycholic+cholic acid Ursodeoxycholic acid Total
0.72 ± 0.34 0.14 ± 0.09 0.36 ± 0.10b 0.06 ± 0.01b 1.28 ± 0.45b
0.76 ± 0.38 0.16 ± 0.07 1.08 ± 0.34a 0.31 ± 0.05a 2.31 ± 0.42a
+5.56 +15.60 +201.17 +409.84 +80.77
0.69 ± 0.33 0.18 ± 0.08 0.40 ± 0.16b 0.06 ± 0.02b 1.33 ± 0.48b
0.70 ± 0.38 0.17 ± 0.05 1.13 ± 0.32a 0.27 ± 0.07a 2.27 ± 0.53a
+1.89 5.03 +182.46 +359.32 +70.58
0.62 ± 0.29 0.18 ± 0.15 0.54 ± 0.24b 0.08 ± 0.04b 1.42 ± 0.35b
Values are mean ± SD, n = 8–12. Means in a row for a given week with different letters differ significantly, p < 0.05. NA, not applicable.
a,b,c
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
281
Fig. 1. Effect of b-sitosterol on mRNA levels of intestinal Niemann–Pick C1 like 1 (NPC1L1), acyl coenzyme A: cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), ATP binding cassette transporter (ABCG8) in hamsters fed one of the five diets containing no cholesterol (NC), 2.6 mmol cholesterol (C), 2.6 mmol cholesterol plus 2.6 mmol b-sitosterol (C+S), 2.6 mmol cholesteryl palmitate (CP), and 2.6 mmol CP plus 2.6 mmol b-sitosterol (CP+S) per kilogram diet, respectively. Data are normalised with cyclophilin. Values are expressed as means ± S.D (n = 8–12). a,b,c Means with different superscript letters differ significantly, p < 0.05. Other abbreviations: C, free cholesterol; CE, cholesteryl esters; CMs, chylomicrons; FC, free cholesterol; PCE, pancreatic cholesterol esterase.
NPC1L1 in the intestine (Fig. 1). However, no such observation was seen when comparison was made among groups C, CP and CP+S. Intestinal mRNA ACAT-2 level was up-regulated when group C was compared with group NC. No effect on mRNA ACAT-2 was seen when b-sitosterol was added into diets containing free cholesterol or CP (Fig. 1). Intestinal mRNA MTP was up-regulated when group C was compared with group NC. Addition of b-sitosterol had a trend of down-regulating intestinal mRNA MTP in both free cholesterol-fed and CP-fed hamsters, however, the effect was not statistically significant. Intestinal mRNA ABCG8 showed an upregulating trend when groups C and C+S were compared with group NC. Addition of b-sitosterol in diet containing CP was able to up-regulate intestinal mRNA ABCG8.
4. Discussion Humans consume diets containing not only free cholesterol but also cholesteryl esters. The present study clearly demonstrated both dietary free cholesterol and CP were capable of raising plasma TC significantly. However, CP was less potent than dietary free cholesterol, suggesting that for a given amount, total cholesterol in different foods may exhibit a varying plasma TC-raising potency dependent on the ratio of free to esterified cholesterol in diets. Less plasma TC-raising potency associated with CP was perhaps due to its difference in absorption compared with that for FC. Absorption of FC and CE is a complex process (Fig. 1). In this regard,
transporter NPC1L1 can directly carry FC from the lumen into enterocytes, while CE needs to be hydrolysed by pancreatic cholesterol esterase before absorption (Grober, Lucas, & Sorhede-Winsell, 2003; Howles, Carter, & Hui, 1996). It was believed that the incomplete hydrolysis or the slow rate of hydrolysis of CP in the intestine might contribute to its lesser potency in raising plasma TC. This hypothesis was supported by the following observations. Firstly, the present result is in agreement with that in a previous study showing cholesterol palmitate and cholesteryl stearate had a slower rate of hydrolysis by pancreatic cholesterol esterase (Jiao et al., 2013). Secondly, group CP had greater excretion of faecal cholesterol and total neutral sterols compared with group C (Tables 4 and 5), proving that CP had the incomplete hydrolysis and was less absorbed. Thirdly, the hypothesis was further supported by the observation that plasma TC-raising activity of dietary free cholesterol was associated with up-regulation of intestinal NPC1L1, ACAT-2 and MTP, but no such up-regulations were seen with dietary CP. This indicated that CP was incompletely hydrolysed to release free cholesterol in the intestinal lumen to reach a threshold concentration which was high enough to up-regulate the intestinal NPC1L1, ACAT-2 and MTP. b-Sitosterol as one of the major phytosterols in human diets has been widely used as a health supplement in treatment of hypercholesterolemia. The present study was the first time to investigate the effect of b-sitosterol on plasma TC in hypercholesterolemia hamsters induced by dietary cholesteryl ester compared with that induced by dietary free cholesterol. It demonstrated that
282
Y. Liu et al. / Food Chemistry 169 (2015) 277–282
b-sitosterol was slightly better in suppressing the hypercholesterolemia induced by dietary free cholesterol than that induced by CP (17.4% versus 11.6%, Table 3). However, the difference was statistically insignificant. Regarding effect of b-sitosterol on the faecal sterol excretion, bsitosterol added into diets containing free cholesterol (diets C and C+S) increased the faecal excretion of neutral sterols greater (week 1, 62.03% versus 21.43%; week 8, 38.18% versus 9.54%) than it was added into diets containing cholesteryl ester (diets CP and CP+S). Intestinal NPC1L1 can transport both cholesterol and b-sitosterol and facilitate the uptake of sterols into enterocyte (Davis et al., 2004; Yamanashi, Takada, & Suzuki, 2007). Because b-sitosterol and cholesterol both have one free hydroxyl group and the same ring structure, they compete for the active side of NPC1L1 and interfere with the absorption of each other. In contrast, CP has a hydroxyl group esterified with palmitic acid and thus b-sitosterol has less competition with CP for NPC1L1. It was therefore expected that addition of b-sitosterol in diets containing only free cholesterol decreased the uptake of cholesterol into enterocytes, leading to a greater excretion of faecal cholesterol and its microbial derivatives, while addition of b-sitosterol in diets containing CP is expected to have a lesser percentage increase in excretion of faecal total neutral sterols. 5. Conclusion In summary, humans daily consume about 200–500 mg cholesterol with an approximate of 30% being present in a form of cholesteryl esters. Although the data could not be directly extrapolated to what happen in humans, the present study demonstrated in hamster model that diets containing cholesteryl esters, particularly CP, was less potent in raising plasma TC compared with diets containing free cholesterol. b-Sitosterol could suppress the hypercholesterolemia induced by both a free cholesterol diet and a cholesteryl ester diet, however, the difference was insignificant. b-Sitosterol relatively increased the faecal excretion of neutral sterols in hamsters given a free cholesterol diet more than it did in hamsters given a cholesteryl ester diet. Conflict of interest We have no conflict of interest in this research. Acknowledgement This project is supported by Hong Kong GRF grant (Project Number CUHK 461112). References Awad, A. C., Bennink, M. R., & Smith, D. M. (1997). Composition and functional properties of cholesterol reduced egg yolk. Poultry Science, 76, 649–653. Bitman, J., & Wood, D. L. (1980). Cholesterol and cholesteryl esters of eggs from various avian species. Poultry Science, 59, 2014–2023. Centers for Disease Control and Prevention. (2000). National Health and Nutrition Examination Survey: Intake of calories and selected nutrients for the United States population. US Department of Health and Human Services.
Chan, P. T., Fong, W. P., Cheung, Y. L., Huang, Y., Ho, W. K., & Chen, Z. Y. (1999). Jasmine green tea epicatechins are hypolipidemic in hamster (Mesocricetus auratus) fed a high fat diet. Journal of Nutrition, 129, 1094–1101. Daniels, T. F., Killinger, K. M., Michal, J. J., Wright, R. W., & Jiang, Z. (2009). Lipoproteins, cholesterol homeostasis and cardiac health. International Journal of Biological Sciences, 5, 474–488. Davis, H. R., Zhu, L. J., Hoos, L. M., Tetzloff, G., Maguire, M., Liu, J., et al. (2004). Niemann–Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. The Journal of Biological Chemistry, 279, 33586–33592. Elmadfa, I., & Weichselbaum, E. (2005). On the nutrition and health situation in the European Union. Journal of Public Health, 13, 62–68. Food and Nutrition Department (1994). Food consumption study 1993. Singapore: Ministry of Health. Grober, J., Lucas, S., & Sorhede-Winsell, M. (2003). Hormone-sensitive lipase is a cholesterol esterase of the intestinal mucosa. The Journal of Biological Chemistry, 278, 6510–6515. Howles, P. N., Carter, C. P., & Hui, D. Y. (1996). Dietary free and esterified cholesterol absorption in cholesterol esterase (bile salt-stimulated lipase) gene-targeted mice. The Journal of Biological Chemistry, 271, 7196–7202. Jesch, E. D., Seo, J. M., Carr, T. P., & Lee, J. Y. (2009). Sitosterol reduces messenger RNA and protein expression levels of Niemann–Pick C1-like 1 in FHs 74 Int cells. Nutrition Research, 29, 859–866. Jiao, R., Chen, J., Peng, C., Liang, Y., Ma, K. Y., Wang, X., et al. (2013). Cholesterol ester species differently elevate plasma cholesterol in hamsters. Journal of Agricultural and Food Chemistry, 61, 11041–11047. Liang, Y. T., Wong, W. T., Guan, L., Tian, X. Y., Ma, K. Y., Huang, Y., et al. (2011). Effect of phytosterols and their oxidation products on lipoprotein profiles and vascular function in hamster fed a high cholesterol diet. Atherosclerosis, 219, 124–133. Lingberg, S., Ellegård, L., Johansson, I., Hallmans, G., Weinehall, L., & Andersson, H. (2008). Inverse relation between dietary intake of naturally occurring plant sterols and serum cholesterol in northern Sweden. The American Journal of Clinical Nutrition, 87, 993–1001. Ma, K. Y., Yang, N., Jiao, R., Peng, C., Guan, L., Huang, Y., et al. (2011). Dietary calcium decreases plasma cholesterol by down-regulation of intestinal Niemann–Pick C1 like 1 and microsomal triacylglycerol transport protein and up-regulation of CYP7A1 and ABCG 5/8 in hamsters. Molecular Nutrition & Food Research, 55, 247–258. Ostlund, R. E., & Lin, X. (2006). Regulation of cholesterol absorption by phytosterols. Current Atherosclerosis Reports, 8, 487–491. Schmidhuber, J. (2007). The EU Diet – Evolution, evaluation and impacts of the CAP global perspectives studies unit. FAO. Sharrett, A. R., Ballantyne, C. M., Coady, S. A., Heiss, G., Sorlie, P. D., & Catellier, D. (2001). Coronary heart disease prediction from lipoprotein cholesterol levels, triglycerides, lipoprotein(a), apolipoproteins A-I and B, and HDL density subfractions: The Atherosclerosis Risk in Communities (ARIC) study. Circulation, 104, 1108–1113. Smet, E., Mensink, R. P., & Plat, J. (2012). Effects of plant sterols and stanols on intestinal cholesterol metabolism: Suggested mechanisms from past to present. Molecular Nutrition & Food Research, 56, 1058–1072. Temel, R. E., Gebre, A. K., Parks, J. S., & Rudel, L. L. (2003). Compared with acylCoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol. The Journal of Biological Chemistry, 278, 47594–47601. Vanstone, C. A., Raeini-Sarjaz, M., Parsons, W. E., & Jones, P. J. (2002). Unesterified plant sterols and stanols lower LDL-cholesterol concentrations equivalently in hypercholesterolemic persons. The American Journal of Clinical Nutrition, 76, 1272–1278. Woo, J., Leung, S. S., Ho, S. C., Lam, T. H., & Janus, E. D. (1998). Dietary intake and practices in the Hong Kong Chinese population. Journal of Epidemiology and Community Health, 52, 631–637. Yamanashi, Y., Takada, T., & Suzuki, H. (2007). Niemann–Pick C1-like 1 overexpression facilitates ezetimibe-sensitive cholesterol and b-sitosterol uptake in CaCo-2 cells. Journal of Pharmacology and Experimental Therapeutics, 320, 559–564. Zhao, L. C., Hu, J. H., Zheng, R. P., Tian, X. Z., Ren, F. X., & Wu, Y. F. (2009). The trends of dietary cholesterol intake and its food sources among workers and farmers in Beijing. Acta Nutrimenta Sinica, 31, 556–559.
