Plant Sterol Consumption Frequency Affects Plasma Lipid Levels and Cholesterol Kinetics in Humans

ORIGINAL ARTICLE Plant sterol consumption frequency affects plasma lipid levels and cholesterol kinetics in humans

SS AbuMweis1, CA Vanstone1, AH Lichtenstein2 and PJH Jones1,3

1School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, Montre´al, Quebec, Canada; 2Cardiovascular Nutrition Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA, USA and 3Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Manitoba, Canada

Background/Objectives: To compare the efficacy of single versus multiple doses of plant sterols on circulating lipid level and cholesterol trafficking. Subjects/Methods: A randomized, placebo-controlled, three-phase (6 days/phase) crossover, supervised feeding trial was conducted in 19 subjects. Subjects were provided (i) control margarine with each meal; (ii) 1.8 g/day plant sterols in margarine with breakfast (single-BF) and control margarine with lunch and supper or (iii) 1.8 g/day plant sterols in margarine divided equally at each of the three daily meals (three times per day). Results: Relative to control, end point plasma low-density lipoprotein (LDL) cholesterol concentrations were lower (Po0.05) after consuming plant sterols three times per day but were not different when consumed once per day (3.43±0.62, 3.22±0.58 and 3.30±0.65 mmol/l, control, three times per day and single-BF, respectively). Relative to the control, end point LDL level was 0.21±0.27 mmol/l (6%) lower (Po0.05) at the end of the three times per day phase. Cholesterol fractional synthesis rate was highest (Po0.05) after the three times per day phase (0.0827±0.0278, 0.0834±0.0245 and 0.0913±0.0221 pool/day, control, single-BF and three times per day, respectively). Cholesterol-absorption efficiency decreased (Po0.05) by 36 and 39% after the three times per day and single-BF phase, respectively, relative to control. Conclusions: Present data indicate that to obtain optimal cholesterol-lowering impact, plant sterols should be consumed as smaller doses given more often, rather than one large dose. European Journal of Clinical Nutrition (2009) 63, 747–755; doi:10.1038/ejcn.2008.36; published online 4 June 2008

Keywords: plant sterols; single dose; LDL cholesterol; diet; lipoproteins

Introduction sterols and stanols suppress cholesterol absorption consequently increasing cholesterol synthesis, with a net effect of The National Cholesterol Education Panel and the American reducing LDL-cholesterol concentrations by about 10 per- Heart Association have suggested the use of plant sterols and cent (Gylling and Miettinen, 1994; Jones et al., 2000; stanols, the hydrogenated form of sterols, as an approach to Vanstone et al., 2002; Katan et al., 2003). control circulating levels of low-density lipoprotein (LDL) The majority of studies have shown efficacy of plant sterol cholesterol (Cleeman et al., 2001; Fletcher et al., 2005). Plant supplementation when distributed in two (Weststrate and Meijer, 1998; Hallikainen et al., 2000a; Maki et al., 2001; Correspondence: Dr PJH Jones, Richardson Centre for Functional Foods and Ntanios, 2001) or three (Jones et al., 2000; Plat and Mensink, Nutraceuticals, University of Manitoba, 196 Innovation Drive, Smartpark, 2000; Mensink et al., 2002; Vanstone et al., 2002) doses per Winnipeg, Manitoba R3T 6C5, Canada. day. Discordant data exist, however, on the effect of a single E-mail: [email protected] dose of plant sterols on plasma cholesterol concentrations. Contributors: PJHJ planned the study and edited the article. SSA was responsible for study planning and execution, cholesterol kinetics analysis A single dose of plant sterol has been shown to lower and statistical analysis, data interpretation, and writing the paper. CAV cholesterol levels to a similar extent as multiple doses of contributed to the study design, helped in screening of the subjects and the same amount (Plat et al., 2000). Plat et al. (2000) editing of the article. AHL was responsible for lipid profile analysis and edited hypothesized that plant stanols remain in the intestinal the paper. Received 3 October 2007; revised 10 March 2008; accepted 29 March 2008; lumen for extended periods where they decrease cholesterol published online 4 June 2008 absorption, thus it is not necessary to consume plant stanol Plant sterols and cholesterol trafficking SS AbuMweis et al 748 ester products throughout the day. It has also been reported times per day). Each phase was separated by a washout that a single dose of plant sterol failed to lower LDL- period of 2 weeks during which time the subjects consumed cholesterol concentrations when given as a single morning their habitual diets. Subjects were randomly assigned to one administration (AbuMweis et al., 2006). Results from the of six predetermined treatment sequences using a Latin study by AbuMweis et al. (2006) do not support the square design. hypothesis by Plat et al. (2000) and suggest that the dosing Diets were prepared in the metabolic kitchen of the Mary frequency of plant sterols or the time of intake of single dose Emily Clinical Nutrition Research Unit where foods were of plant sterols could affect their cholesterol-lowering action. weighed to the nearest 0.5 g. Three isocaloric meals were Nonetheless, current marketing of plant sterol-containing prepared each day for the five study days for every subject products is largely directed toward consumption once a day during each phase. Total caloric intake was matched to each with breakfast meals. To date, no studies have examined the subject’s energy requirements. Diets were designed to effects of plant sterol consumption frequency on circulating contain 55% of energy (E) from carbohydrates, 15% E from lipid profiles and cholesterol kinetics as part of a controlled protein and 30% E from fat, 80 mg/1000 kcal of cholesterol low-fat, low-cholesterol diet. The objective of this study was, and 12 g/1000 kcal of fibers. During the study period, therefore, to compare under controlled and supervised subjects weighed themselves every morning. Subjects con- conditions the effect of a standardized dose of plant sterols sumed breakfasts and the suppers at the Mary Emily Clinical given once in the morning or three times throughout the Nutrition Research Unit under supervision. The lunch meals day on plasma lipids and cholesterol kinetics. were packed to be taken out. Plant sterol-enriched margarine (Take Control-Unilever Inc., Vlaardingen, The Netherlands) was used to provide a dose of 1.8 g/day of plant sterols, and Subjects and methods the same margarine without plant sterols served as the control margarine (Table 1). For the single-BF phase, Subjects the plant sterol-enriched margarine was incorporated in the Male and postmenopausal female subjects 40–80 years with a breakfast meal and the control margarine was incorporated body mass index between 22 and 32 kg/m2 were recruited in the lunch and supper meals. During the divided phase, from a database of subjects who had participated in previous plant sterol-enriched margarine was divided equally among studies at the Mary Emily Clinical Nutrition Research Unit the breakfast, lunch and supper meals. of McGill University. Respondents underwent a thorough Subjects consumed a controlled diet including the historical and physical examination to exclude those with assigned study treatment from day 1 to day 5. On thyroid disease, diabetes mellitus, kidney disease, liver the second day (24 h) of each phase, subjects ingested disease, smokers or consumption of alcohol at 414 drinks 75 mg [3, 4-13C] cholesterol (CDN Isotopes; Montreal) to per week. Volunteers taking medication known to affect lipid metabolism, high-dose dietary supplements, fish oil capsules or plant sterol for 3 months or more prior to the start of the Table 1 Nutrient and plant sterol composition of control and sterol- study were excluded from participation. At the first visit, enriched margarinea volunteers were screened for LDL cholesterol 43.0 mmol/l. Eligible volunteers were asked to return for a second Component (per 100 g) Control Sterol-enriched margarine margarine screening to measure complete blood counts, serum bio- chemistry and lipid profiles that included total cholesterol, Energy (kcal) 327 327 triacylglycerol, LDL-cholesterol and high-density lipoprotein Total fat (g) 35 35 cholesterol levels. Saturated fatty acids (g) 8.2 8.2 Polyunsaturated fatty acids (g) 18.0 18.0 Monounsaturated fatty acids (g) 8.3 8.3 Trans fatty acids (g) 0.5 0.5 Study design and protocol Protein (g) 0.1 0.1 A randomized, single-blind crossover placebo-controlled Carbohydrate (g) 3.2 3.2 clinical trial was carried out at the Mary Emily Clinical Fiber (g) 0.3 0.3 Total plant sterols (g) 0.01 8.0 Nutrition Research Unit. The study consisted of three phases of 6 days each. During day 1 through to day 5, subjects Plant sterol composition (% w/w) consumed a precisely controlled weight-maintaining diet. Brassicasterol — 2.9 At the end of each phase, that is day 6, blood samples were Campesterol — 22.2 Campestanol — 0.6 collected but no diets were provided. The three phases of Stigmasterol — 9.6 treatments included control phase: no plant sterols were b-Sitosterol — 58.1 consumed (control); single morning dose phase: plant sterol b-Sitostanol — 3.6 dose was given only at breakfast (single-BF); and three times D5-Avenasterol — 0.9 Other — 1.9 dose phase: the same plant sterol dose was divided equally among three daily meals, breakfast, lunch and supper (three aAs provided by the margarine supplier.

European Journal of Clinical Nutrition Plant sterols and cholesterol trafficking SS AbuMweis et al 749 measure cholesterol absorption over the following 72 h. The Endogenous cholesterol-synthesis determination. Cholesterol 13C-cholesterol was dissolved in 5 g of warmed margarine synthesis rates were assessed after 24 h of deuterium water and consumed on a slice of toast. On day 5 of each feeding administration, using the deuterium incorporation approach period, approximately 25 ml of deuterium oxide was given (Jones et al., 1993). This method measures cholesterol orally to each subject before breakfast to measure endogen- synthesis as the rate of deuterium incorporation from body ous cholesterol synthesis. Fasting blood samples were taken water into RBC membrane free cholesterol over a 24-h at baseline prior to isotope administration (24 h) as well as period. The deuterium incorporation method has been on days 3, 4, 5 and 6 to monitor enrichment levels of both validated against the sterol balance method (Jones et al., isotopes. 1998a) and mass isotopomer distribution analysis (Di Buono et al., 2000). Deuterium enrichment was measured in both RBC free cholesterol and plasma water. Enrichments were expressed relative to standard mean ocean water and a series Analyses of standards of known enrichment. Plasma lipid profile. Fasting blood samples collected on days The measurement of free-cholesterol deuterium enrich- 5 and 6 were used to measure circulating lipid concentra- ment was performed using online gas chromatography/ tions. Blood was centrifuged for 20 min at 520 Â g and 4 1Cto pyrolysis/isotope ratio mass spectrometry as described above. separate plasma from red blood cells (RBCs). Plasma total Lipids were extracted from RBCs as described above for and high-density lipoprotein cholesterol, and triacylglycerol cholesterol-absorption determination. Isotope abundance, concentrations were measured using standardized proce- expressed in d%, was calculated using H2 as reference gas. dures as previously described (Lichtenstein et al., 2006). The The d values were further expressed relative to standard Friedewald equation was used to calculate LDL-cholesterol mean ocean water using a calibration curve of working concentrations (Friedewald et al., 1972). standards. Fractional synthesis rate (FSR) is taken to represent RBC free-cholesterol deuterium enrichment values relative to the Cholesterol-absorption determination. Cholesterol absorption corresponding mean plasma water sample enrichment after was assessed using the stable isotope single-tracer method as correcting for the free-cholesterol pool. FSR represents that previously described (Ostlund et al., 1999, 2002; Spilburg fraction of the cholesterol pool that is synthesized in 24 h et al., 2003; Shin et al., 2005; Varady and Jones, 2006). It was and is calculated as per the equation (Jones et al., 1993): reported that the area under the 13C-enrichment curve, as = del = del : measured by the single-tracer method, correlates with FSR ðpools dayÞ¼ cholesterol ð plasmaÃ0 478Þð1Þ cholesterol absorption rate as measured by the dual stable where del for deuterium is the enrichment difference isotope ratio method (Wang et al., 2004). Compared to the between free cholesterol and plasma water between 72 h dual stable isotope ratio method, the single stable isotope (day 5) and 96 h (day 6) in parts per thousand relative to a single-tracer method is less invasive. standard mean ocean water standard. The factor 0.478 Free cholesterol extracted (Folch et al., 1957) from RBCs accounts for the ratio of labelled H atoms replaced by was used to determine 13C-cholesterol enrichments using deuterium (22/46) during in vivo biosynthesis. Cholesterol on-line gas chromatography/combustion/isotope ratio mass absolute synthesis rates (ASR) were calculated as follows: spectrometry. Lipids extracted were dissolved in hexane and injected into a gas chromatograph (Agilent 6890N) inter- ASRðg=dayÞ¼FSR Ã M1 Ã 0:33 ð2Þ faced with a Finnigan Delta V Plus isotope ratio mass spectrometer (Bremen, Germany) through a Finnigan combustion interface (Combustion Interface III; Bremen). Iso- tope abundance, expressed in delta (d) per mil (%), was Statistics calculated using CO2 as a reference gas. The d values were All data are expressed as means±s.d. Statistical analysis further expressed relative to the international reference was carried out using SAS (version 8.0; SAS Institute Inc., standard, Pee Dee Belemnite limestone, using a calibration Cary, NC, USA). The principal statistical model used was the curve of working standards that were analyzed previously analysis of variance taking into account subject as a random using a conventional isotope ratio mass spectrometry (SIRA effect, study treatment effect, period effect, sequence effect 12; Isomass, Cheshire, United Kingdom). From 24 to 96 h and carryover effect. Residuals of every outcome were (72 h post-13C-cholesterol ingestion), two kinetic parameters checked for normality using Shapiro–Wilk test. If the were calculated using NCSS (version 2000; NCSS Statistical normality assumption was violated for a variable, then log Software, Kaysville, UT, USA) software: area under the transformation was performed. In the event of a statistically 13C-enrichment curve versus time curve (AUC (24–96 h)) significant treatment effect (P-valueo0.05), pair-wise com- and maximum 13C-cholesterol enrichment (E-max). The parisons were performed using Tukey’s test. Tests for AUC (24–96 h) corrected for baseline values was calculated associations between variables were performed using Pearson with the use of the trapezoidal rule. Correlation Coefficient analyses.

European Journal of Clinical Nutrition Plant sterols and cholesterol trafficking SS AbuMweis et al 750 Results Table 3. End point plasma LDL-cholesterol concentrations were 3.43±0.62, 3.22±0.58 and 3.30±0.65 mmol/l after Characteristics of the study subjects are provided in Table 2. control, three times per day and single-BF, respectively. There were no significant weight changes across any of the Relative to the control, end point plasma LDL-cholesterol study phases. Concentrations of plasma lipids and lipopro- level was 0.21±0.27-mmol/l (6%) lower (Po0.05) at the end teins at the end of each treatment phase are shown in of the three times per day phase and not significantly different at the end of the single-BF phase compared to Table 2 Characteristics of the subjects at the time of screeninga control. Total cholesterol tended to be lowest (P ¼ 0.0639) during the three times per day phase whereas plasma high- Variable density lipoprotein and triacylglycerol concentrations were Age (years) 55±8 Weight (kg) 80±15 not different among the study treatments. The total BMI (kg/m2)26±4 cholesterol/high-density lipoprotein ratio was lower (Po0.05) at the end of the three times per day phase by Lipids (mmol/l) 4.4%, relative to control phase. Total cholesterol 5.10±0.52 LDL cholesterol 3.44±0.41 Cholesterol absorption AUC (24–96 h) of RBC cholesterol HDL cholesterol 1.11±0.23 during the single-BF phase and three times per day phase was Triacylglycerol 1.16±0.45 lower by 39 and 36%, respectively, compared to the control phase (Table 4). Similarly, E-max values were lower Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein. (Po0.0001) during the single-BF phase and three times per aAll values are mean±s.d.; n ¼ 19 (16 male and 3 female subjects). day phase than the control phase. When subjects were

Table 3 Lipid and lipoprotein concentrations at the end of each experimental diet phasea

Parameter Control Single-BF Three times per day P-value

Total cholesterol (mmol/l) 5.29±0.72 5.14±0.68 5.05±0.71 0.0637 HDL cholesterol (mmol/l) 1.25±0.21 1.24±0.21 1.25±0.22 0.9145 LDL cholesterol (mmol/l)b 3.43±0.62a 3.30±0.65a,b 3.22±0.58b 0.0452 Total cholesterol/HDLc 4.32±0.80a 4.22±0.77a,b 4.13±0.79b 0.0380 Triacylglycerol (mmol/l) 1.32±0.66 1.30±0.64 1.26±0.61 0.5024

Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein. aAll values are mean±s.d.; n ¼ 19; weight change was entered as a covariant. Single-BF, plant sterols were consumed only with breakfast; three times per day, plant sterol-enriched margarine was divided equally among the three meals—breakfast, lunch and supper. Within a row, values with different letters are significantly different. bP-values for pair-wise comparisons adjusted by Tukey’s test are as follows: P ¼ 0.2616 control vs single-BF; P ¼ 0.0371 control vs three times per day; P ¼ 0.5860 single-BF vs three times per day. cP-values for pairwise comparisons adjusted by Tukey’s test are as follows: P ¼ 0.3729 control vs single-BF; P ¼ 0.0292 control vs three times per day; P ¼ 0.3874 single-BF vs three times per day.

Table 4 Cholesterol kinetics as measured from free cholesterol from RBCs in study subjects during each of dietary phasea

Parameter Control Single-BF Three times per day P-value

Cholesterol absorption AUC24–96 h (per mil Â h) 398±79a 242±61b 253±58b o0.0001 E-max (per mil) 6.3±1.3a 3.9±1.0b 4.1±1.0b o0.0001

Cholesterol synthesis Fractional synthesis rate (pool/day)b,c 0.0827±0.0278a 0.0834±0.0245a,b 0.0913±0.0221b 0.0219 Absolute synthesis rate (g/day)b,d 0.7492±0.3185a 0.7326±0.2186a,b 0.8101±0.2262b 0.0266

Abbreviation: RBCs, red blood cells. aAll values are mean±s.d.; n ¼ 19. Single-BF, plant sterols were consumed only with breakfast; three times per day, plant sterol-enriched margarine was divided 13 equally among the three meals—breakfast, lunch and supper; AUC24À96 h, area under the curve of C enrichment of RBCs cholesterol during a 24- to 96-h period following the oral dose of 13C-cholesterol; E-max; maximum 13C-cholesterol enrichment in RBCs. Within a row, values with different letters are significantly different. bAll values were log-transformed prior to statistical analysis; arithmetic means are reported. Weight change was entered as a covariant. cP-values for pair-wise comparisons adjusted by Tukey’s test are as follows: P ¼ 0.9097 control vs single-BF; P ¼ 0.0281 control vs three times per day; P ¼ 0.0764 single-BF vs three times per day. dP-values for pair-wise comparisons adjusted by Tukey’s test are as follows: P ¼ 0.9109 control vs single-BF; P ¼ 0.0266 control vs three times per day; P ¼ 0.0718 single-BF vs three times per day.

European Journal of Clinical Nutrition Plant sterols and cholesterol trafficking SS AbuMweis et al 751 analyzed by treatment grouping, the change in LDL relative single-BF phase (P ¼ 0.4858). Maximum values for 13C to the control phase tended to be associated with the change enrichment were attained at around 48 h (Figure 1). Choles- in AUC (24–96 h) relative to the control phase for the three terol FSR and ASR values were highest (Po0.05) during the times per day phase (r ¼ 0.43, P ¼ 0.0623), but not for the plant sterol three times per day phase (Table 4).

Control 7.0

6.0

5.0 a 4.0

3.0

2.0 C Enrichment (per mil) 13 1.0

0.0 0 24487296 Time (h)

Single-BF phase 7.0

6.0

5.0

4.0

3.0

2.0 b C Enrichment (per mil) 13 1.0

0.0 0 24487296 Time (h)

Three times per day phase 7.0

6.0

5.0

4.0

3.0 b

C Enrichment (per mil) 2.0 13 1.0

0.0 0 24487296 Time (h) Figure 1 Enrichment of 13C in RBC cholesterol at the end of control phase and plant sterol single-BF and three times per day phases. Values are mean±s.e. Areas under the curve with different superscripts are significantly different (Po0.05). RBC, red blood cell.

European Journal of Clinical Nutrition Plant sterols and cholesterol trafficking SS AbuMweis et al 752 Discussion the three times per day phase. Previous studies utilizing different experimental approaches have shown that con- The novel finding of this study is that consumption of a sumption of plant sterols/stanols reduced cholesterol absorp- single morning dose of plant sterols was not as efficacious in tion and consequently increased cholesterol synthesis, with lowering LDL concentrations as was the three times per day a net reduction in LDL-cholesterol concentrations in hyper- dosing pattern. Most noticeably, a reduction in plasma LDL cholesterolemic individuals (Gylling and Miettinen, 1994; concentration was achieved after a relatively short interven- Jones et al., 2000; Vanstone et al., 2002). The regulatory tion period. Previous studies have reported a reduction in process behind this phenomenon is not clearly understood cholesterol concentrations after 1 day (Miettinen et al., yet. 2000), 8 days (Hallikainen et al., 2002) and 10 days (Jones Plant sterols consumed during the single-BF phase reduced et al., 1998b) of plant sterol/stanol consumption. However, cholesterol-absorption efficiency, as measured by the stable the single-day study by Miettinen et al. (2000) was carried isotope single-tracer method, but did not reduce LDL- out in colectomized individuals whose cholesterol metabo- cholesterol concentrations compared to control. It is possible lism differed from that of healthy individuals. The 8-day that gut cholesterol pools did not have enough time to study of Hallikainen et al. (2002) lacked a control group, and adjust for changing in cholesterol absorption caused by the the reduction in serum cholesterol concentrations by stanol consumption of plant sterols at different dosage frequency. ester consumption was relative to baseline values; thus a The discrepancy may be due to the method used to assess time effect could have contributed to the reduction in cholesterol absorption efficiency. The single-tracer method cholesterol concentrations. Although the duration of the measures only the efficiency of cholesterol absorption rather present study was relatively short, plant sterol ester lowered than the absolute amount of absorbed cholesterol that LDL-cholesterol concentrations by 0.21 mmol/l (6%). The depends on endogenous biliary cholesterol secretion magnitude of reduction in LDL-cholesterol concentrations (Ostlund et al., 2002). Some studies have shown that the observed in the current study is consistent with that consumption of plant sterols/stanols increases biliary secre- typically observed after several 3- to 6-week intervention tion of cholesterol (Becker et al., 1992; Gylling and periods (Hendriks et al., 1999; Sierksma et al., 1999; Colgan Miettinen, 1994; Miettinen and Vanhanen, 1994). Whether et al., 2004; Jakulj et al., 2005; Kratz et al., 2007) or even for the frequency of intake of plant sterols affects the absolute 52 weeks (Hendriks et al., 2003). Nonetheless, there is some amount of cholesterol absorbed and/or bile acid excretion evidence that even this modest reduction in circulating was not assessed. It is our assumption that if the dietary levels of cholesterol may decrease the risk of developing intake of cholesterol and fat was maintained constant across cardiovascular disease (Muldoon et al., 1990). Also, the periods, then it would be anticipated that the rate of rise of addition of plant sterol/stanol margarine to statin therapy tracer in blood should reflect not only fractional absorption has an additive effect on reduction in LDL-cholesterol levels, but also total mass of sterol taken up from the gut. It is, serving as an alternative approach to doubling the statin however, understood that shifts, for instance, in biliary sterol dose, without increasing costs, and in side effects that excretion could impact the net amount of cholesterol usually accompany an increase in statin dose (Vorlat et al., absorbed. Additional experiments should be carried out 2003). Doubling the dose of statin normally produces a using the intestinal infusion technique to assess whether further reduction of about 6% in LDL-cholesterol levels (Blair the frequency of intake of plant sterols affects the absolute et al., 2000). amount of cholesterol absorbed and/or bile acid excretion. Limited data are available on the effect of dosing In addition, the observed reduction in efficiency of dietary frequency of plant sterols on lipid profiles and cholesterol cholesterol absorption may not always reflect the magnitude kinetics. An earlier study demonstrated that 2.5 g/day of of reduction in circulating cholesterol. This explanation is plant stanol consumed for 4 weeks once per day at lunch or supported by a finding from previous work. Jones et al. divided among three meals, 0.42 g at breakfast, 0.84 g at (2000) reported no difference in cholesterol absorption lunch and 1.25 g at supper, lowered LDL-cholesterol con- between plant sterol- and plant stanol-containing diets, centrations to a similar extent, about 10% (Plat et al., 2000). although the decline in LDL-cholesterol levels was greater at A more recent study reported that consuming a single dose the end of plant sterol, compared to the plant stanol phase. of plant sterols provided in a yoghurt drink with lunch Gylling et al. (1997) have shown that the sitostanol-induced resulted in a larger decrease in LDL-cholesterol concentra- changes in LDL cholesterol were significantly associated with tions than consuming the same dose with breakfast the change in cholesterol-absorption efficiency (r ¼ 0.443). (Doornbos et al., 2006). Likewise, Jones et al. (2000) reported an r ¼ 0.530 between In addition to monitoring changes in plasma cholesterol plasma total cholesterol concentrations and cholesterol- concentration, the mechanism(s) accounting for these absorption coefficient with plant sterol treatment. In the changes were also assessed. In accordance with previous present study, the changes in LDL-cholesterol concentration work, administration of plant sterols at a dose of 1.8 g/day tended to be associated with changes in AUC of 13C- was shown to significantly decrease intestinal cholesterol- cholesterol when plant sterols were consumed three times absorption efficiency. Cholesterol FSR increased only during a day. The proportion of the total variation in circulating

European Journal of Clinical Nutrition Plant sterols and cholesterol trafficking SS AbuMweis et al 753 total and LDL-cholesterol concentrations that is explained LDL levels to different frequencies of plant sterol interven- by cholesterol-absorption efficiency in the present study and tion. Few studies have looked at genetic variation in genes prior studies (Gylling et al., 1997; Jones et al., 2000) ranges involved in sterol metabolism and heterogeneity in plasma from 18 to 28%. These findings suggest that either the plant sterols and LDL responsiveness to dietary intervention methods used to measure absorption are insensitive to with plant sterol-enriched products. Apolipoprotein E changes in cholesterol-absorption efficiency or plant sterols/ phenotype, which has been shown to be correlated with stanols reduce circulating levels by other means. Notably, cholesterol absorption (Kesaniemi et al., 1987), has been cholesterol FSR was highest during the three times per day reported to affect plant sterol cholesterol-lowering efficacy in phase, likely in response to the decrease in cholesterol- one study (Vanhanen et al., 1993), but not in others (Plat and absorption efficiency. On the contrary, no reciprocal increase Mensink, 2000; Hallikainen et al., 2000b). In the other work, in cholesterol synthesis in response to the decrease in the genotype of apolipoprotein A-IV, scavenger receptor BI, absorption during the single-BF phase was observed. It is 3-hydroxy-3-methyl-coenzyme A reductase and cholesterol likely that many changes were taking place in the process of ester transfer did not affect cholesterol-lowering effects of cholesterol absorption and these changes were not detected plant stanol (Plat and Mensink, 2002). In response to dietary by the single-tracer method. It remains to be assessed if the plant sterols, the T400K variant in ABCG8 accounted for data obtained in the present investigation will be different if differences in serum sterol concentration and predicted the the oral isotope is to be given in three divided daily doses subject responsiveness to changes in serum sterols, but not and not as a single bolus. The single stable isotope single- serum lipoprotein profile (Plat et al., 2005). Unfortunately, tracer method has not been validated under conditions due to the limited sample size, we were not able to further that might affect cholesterol absorption, including effect of explore the effect of single-nucleotide polymorphisms. multiple oral dosages, time of intake of single oral dose, the In summary, results from this study indicate that dosing effect of low and high cholesterol diets and the effect of pre- frequency of plant sterols may affect their action as and post-test dietary intake of cholesterol. cholesterol-lowering agents. Consumption of 1.8 g/day of Nevertheless, results of this study have implications for the plant sterols distributed over the day was more effective in mechanism of action of plant sterols as cholesterol-lowering lowering LDL cholesterol than the same dose once a day with agents. In contrast to the hypothesis that plant sterols/ breakfast, in spite of a reduction in cholesterol-absorption stanols are retained in the enterocyte, it does not appear that efficiency. Moreover, cholesterol FSR increased when plant a single dose consumed early in the morning is as efficacious sterols were consumed in divided doses, which may be as the same dose divided across multiple meals. Recent attributed to feedback upregulation of cholesterol FSR in work suggests that the AUC (24–96 h) of RBC 13C-cholesterol response to reduced sterol absorption. Present data indicate and E-max values when single dose of plant sterols was that to obtain optimal cholesterol-lowering impact, plant consumed with supper was similar to control values sterols should be consumed as smaller doses given more (unpublished data, S AbuMweis et al.). Cholesterol absorp- often, rather than one large dose. Therefore, health claims tion is a gradual process as labeled cholesterol usually peaks that are to be issued for the consumption of plant sterols/ after 48 h of administration (Bosner et al., 1993), and the stanols should be based on clinical trials with dosing ingested dietary cholesterol is secreted by the small intestine regimens that mirror real-life usage of such materials. in chylomicrons into the circulation during X3 subsequent postprandial periods in human (Beaumier-Gallon et al., 2001). Thus, it appears that plant sterols are not retained within the intestinal lumen or enterocytes and do not Acknowledgements prevent the absorption of previously ingested dietary cholesterol. We wish to thank Unilever Research for providing the sterol- Finally, the study population is also an important limita- enriched and control margarines in-kind. We also thank the tion in the context of its clinical implications. Subjects study subjects for their enthusiastic participation. involved in this study were middle-aged males and postmenopausal females. Study subjects were overweight with borderline high levels of LDL cholesterol who were not References taking any drug known to affect lipid metabolism. The study population in this study may have responded differently to AbuMweis SS, Vanstone CA, Ebine N, Kassis A, Ausman LM, Jones et al. some of the parameters measured, compared with the PJH (2006). Intake of a single morning dose of standard and novel plant sterol preparations for 4 weeks does not dramatically general population. Therefore, results obtained in this study affect plasma lipid concentrations in humans. JNutr136, may be representative of middle-aged adults with moderate 1012–1016. hypercholesterolemia, and hence care must be taken in Beaumier-Gallon G, Dubois C, Senft M, Vergnes MF, Pauli AM, Portugal H et al. (2001). Dietary cholesterol is secreted in extrapolation of these results to other individuals such as intestinally derived chylomicrons during several subsequent obese and/or diabetic subjects. Another concern is the effect postprandial phases in healthy humans. Am J Clin Nutr 73, of the genetic make up of subjects on the responsiveness of 870–877.

European Journal of Clinical Nutrition Plant sterols and cholesterol trafficking SS AbuMweis et al 754 Becker M, Staab D, Vonbergmann K (1992). Long-term treatment of combination of dietary plant sterols and ezetimibe: effects on severe familial hypercholesterolemia in children: effect of sitosterol plasma lipid levels. J Lipid Res 46, 2692–2698. and bezafibrate. Pediatrics 89, 138–142. Jones PJ, Raeini-Sarjaz M, Ntanios FY, Vanstone CA, Feng JY, Parsons Blair SN, Capuzzi DM, Gottlieb SO, Nguyen T, Morgan JM, Cater NB WE (2000). Modulation of plasma lipid levels and cholesterol (2000). Incremental reduction of serum total cholesterol and low- kinetics by phytosterol versus phytostanol esters. J Lipid Res 41, density lipoprotein cholesterol with the addition of plant stanol 697–705. ester-containing spread to statin therapy. Am J Cardiol 86, 46–52. Jones PJH, Ausman LM, Croll DH, Feng JY, Schaefer EA, Lichtenstein Bosner MS, Ostlund RE, Osofisan O, Grosklos J, Fritschle C, Lange LG AH (1998a). Validation of deuterium incorporation against sterol (1993). Assessment of percent cholesterol absorption in humans balance for measurement of human cholesterol biosynthesis. with stable isotopes. J Lipid Res 34, 1047–1053. J Lipid Res 39, 1111–1117. Cleeman JI, Grundy SM, Becker D, Clark LT, Cooper RS, Denke MA Jones PJH, Howell T, MacDougall DE, Feng JY, Parsons W (1998b). et al. (2001). Executive summary of the Third Report of the Short-term administration of tall oil phytosterols improves plasma National Cholesterol Education Program (NCEP) expert panel on lipid profiles in subjects with different cholesterol levels. Metab- detection, evaluation, and treatment of high blood cholesterol in Clin Exp 47, 751–756. adults (Adult Treatment Panel III). JAMA 285, 2486–2497. Jones PJH, Leitch CA, Li ZC, Connor WE (1993). Human cholesterol- Colgan HA, Floyd S, Noone EJ, Gibney MJ, Roche HM (2004). synthesis measurement using deuterated water—theoretical and Increased intake of fruit and vegetables and a low-fat diet, with procedural considerations. Arterioscler Thromb 13, 247–253. and without low-fat plant sterol-enriched spread consumption: Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti R effects on plasma lipoprotein and carotenoid metabolism. JHum (2003). Efficacy and safety of plant stanols and sterols in Nutr Diet 17, 561–569. the management of blood cholesterol levels. Mayo Clin Proc 78, Di Buono M, Jones PJH, Beaumier L, Wykes LJ (2000). Comparison of 965–978. deuterium incorporation and mass isotopomer distribution ana- Kesaniemi YA, Ehnholm C, Miettinen TA (1987). Intestinal choles- lysis for measurement of human cholesterol biosynthesis. J Lipid terol absorption efficiency in man is related to apoprotein E Res 41, 1516–1523. phenotype. J Clin Invest 80, 578–581. Doornbos AME, Meynen EM, Duchateau G, van der Knaap HCM, Kratz M, Kannenberg F, Gramenz E, Berning B, Trautwein E, Assmann Trautwein EA (2006). Intake occasion affects the serum cholesterol G et al. (2007). Similar serum plant sterol responses of human lowering of a plant sterol-enriched single-dose yoghurt drink subjects heterozygous for a mutation causing sitosterolemia and in mildly hypercholesterolaemic subjects. Eur J Clin Nutr 60, controls to diets enriched in plant sterols or stanols. Eur J Clin Nutr 325–333. 61, 896–905. Fletcher B, Berra K, Ades P, Braun LT, Burke LE, Durstine JL et al. Lichtenstein AH, Matthan NR, Jalbert SM, Resteghini NA, Schaefer (2005). Managing abnormal blood lipids—A collaborative EJ, Ausman LM (2006). Novel soybean oils with different fatty acid approach. Circulation 112, 3184–3209. profiles alter cardiovascular disease risk factors in moderately Folch J, Lees M, Stanley GHS (1957). A simple method for the hyperlipidemic subjects. Am J Clin Nutr 84, 497–504. isolation and purification of total lipids from animal tissues. J Biol Maki KC, Davidson MH, Umporowicz DM, Schaefer EJ, Dicklin MR, Chem 226, 497–509. Ingram KA et al. (2001). Lipid responses to plant-sterol-enriched Friedewald WT, Fredrick DS, Levy RI (1972). Estimation of concen- reduced-fat spreads incorporated into a National Cholesterol tration of low-density lipoprotein cholesterol in plasma, without Education Program Step I diet. Am J Clin Nutr 74, 33–43. use of preparative ultracentrifuge. Clin Chem 18, 499–502. Mensink RP, Ebbing S, Lindhout M, Plat J, van Heugten MMA (2002). Gylling H, Miettinen TA (1994). Serum-cholesterol and cholesterol Effects of plant stanol esters supplied in low-fat yoghurt on serum and cipoprotein metabolism in hypercholesterolemic NIDDM lipids and lipoproteins, non-cholesterol sterols and fat soluble patients before and during sitostanol ester-margarine treatment. antioxidant concentrations. Atherosclerosis 160, 205–213. Diabetologia 37, 773–780. Miettinen TA, Vanhanen H (1994). Dietary sitostanol related to Gylling H, Radhakrishnan R, Miettinen TA (1997). Reduction of absorption, synthesis and serum Level of cholesterol in different serum cholesterol in postmenopausal women with previous apolipoprotein-E phenotypes. Atherosclerosis 105, 217–226. myocardial infarction and cholesterol malabsorption induced by Miettinen TA, Vuoristo M, Nissinen M, Jarvinen HJ, Gylling H dietary sitostanol ester margarine—women and dietary sitostanol. (2000). Serum, biliary, and fecal cholesterol and plant sterols in Circulation 96, 4226–4231. colectomized patients before and during consumption of stanol Hallikainen M, Sarkkinen E, Wester I, Uusitupa M (2002). Short-term ester margarine. Am J Clin Nutr 71, 1095–1102. LDL cholesterol-lowering efficacy of plant stanol esters. BMC Muldoon MF, Manuck SB, Matthews KA (1990). Lowering cholesterol Cardiovascular Disordorders 2, 14. concentrations and mortality—a quantitative review of primary Hallikainen MA, Sarkkinen ES, Gylling H, Erkkila AT, Uusitupa MIJ prevention trials. BMJ 301, 309–314. (2000a). Comparison of the effects of plant sterol ester and plant Ntanios F (2001). Plant sterol-ester-enriched spreads as an example of stanol ester-enriched margarines in lowering serum cholesterol a new functional food. Eur J Lipid Sci Technol 103, 102–106. concentrations in hypercholesterolaemic subjects on a low-fat Ostlund RE, Racette SB, Okeke A, Stenson WF (2002). Phytosterols diet. Eur J Clin Nutr 54, 715–725. that are naturally present in commercial corn oil significantly Hallikainen MA, Sarkkinen ES, Uusitupa MIJ (2000b). Plant stanol reduce cholesterol absorption in humans. Am J Clin Nutr 75, esters affect serum cholesterol concentrations of hypercholestero- 1000–1004. lemic men and women in a dose-dependent manner. J Nutr 130, Ostlund RE, Spilburg CA, Stenson WF (1999). Sitostanol adminis- 767–776. tered in lecithin micelles potently reduces cholesterol absorption Hendriks HFJ, Brink EJ, Meijer GW, Princen HMG, Ntanios FY (2003). in humans. Am J Clin Nutr 70, 826–831. Safety of long-term consumption of plant sterol esters-enriched Plat J, Bragt MCE, Mensink RP (2005). Common sequence variations spread. Eur J Clin Nutr 57, 681–692. in ABCG8 are related to plant sterol metabolism in healthy Hendriks HFJ, Weststrate JA, van Vliet T, Meijer GW (1999). Spreads volunteers. J Lipid Res 46, 68–75. enriched with three different levels of vegetable oil sterols and Plat J, Mensink RP (2000). Vegetable oil based versus wood based the degree of cholesterol lowering in normocholesterolaemic stanol ester mixtures: effects on serum lipids and hemostatic and mildly hypercholesterolaemic subjects. Eur J Clin Nutr 53, factors in non-hypercholesterolemic subjects. Atherosclerosis 148, 319–327. 101–112. Jakulj L, Trip MD, Sudhop T, von Bergmann K, Kastelein JJP, Plat J, Mensink RP (2002). Relationship of genetic variation in genes Vissers MN (2005). Inhibition of cholesterol absorption by the encoding apolipoprotein A-IV, scavenger receptor BI, HMG-CoA

European Journal of Clinical Nutrition Plant sterols and cholesterol trafficking SS AbuMweis et al 755 reductase, CETP and apolipoprotein E with cholesterol metabo- apoe phenotypes during dietary Sitostanol ester treatment. J Lipid lism and the response to plant stanol ester consumption. Eur J Clin Res 34, 1535–1544. Invest 32, 242–250. Vanstone CA, Raeini-Sarjaz M, Parsons WE, Jones PJ (2002). Plat J, van Onselen ENM, van Heugten MMA, Mensink RP (2000). Unesterified plant sterols and stanols lower LDL-cholesterol Effects on serum lipids, lipoproteins and fat soluble antioxidant concentrations equivalently in hypercholesterolemic persons. concentrations of consumption frequency of margarines and Am J Clin Nutr 76, 1272–1278. shortenings enriched with plant stanol esters. Eur J Clin Nutr 54, Varady KA, Jones PJH (2006). Decrease in intestinal cholesterol 671–677. absorption by plant sterols and exercise improves lipid profiles in Shin MJ, Lee JH, Jang Y, Lee-Kim YC, Park E, Kim KM et al. (2005). hypercholesterolemic adults. FASEB J 20, A1025. Micellar phytosterols effectively reduce cholesterol absorption at Vorlat A, Conraads VM, Vrints CJ (2003). Regular use of margarine- low doses. Ann Nutr Metab 49, 346–351. containing stanol/sterol esters reduces total and low-density Sierksma A, Weststrate JA, Meijer GW (1999). Spreads enriched with lipoprotein (LDL) cholesterol and allows reduction of statin plant sterols, either esterified 4,4-dimethylsterols or free 4- therapy after cardiac transplantation: preliminary observations. desmethylsterols, and plasma total- and LDL-cholesterol concen- J Heart Lung Transplant 22, 1059–1062. trations. Br J Nutr 82, 273–282. Wang YW, Vanstone CA, Parsons WD, Jones PJH (2004). Validation of Spilburg CA, Goldberg AC, McGill JB, Stenson WF, Racette SB, a single-isotope-labeled cholesterol tracer approach for measuring Bateman J et al. (2003). Fat-free foods supplemented with soy human cholesterol absorption. Lipids 39, 87–91. stanol-lecithin powder reduce cholesterol absorption and LDL Weststrate JA, Meijer GW (1998). Plant sterol-enriched cholesterol. J Am Diet Assoc 103, 577–581. margarines and reduction of plasma total- and LDL- Vanhanen HT, Blomqvist S, Ehnholm C, Hyvonen M, Jauhiainen M, cholesterol concentrations in normocholesterolaemic and Torstila I et al. (1993). Serum-cholesterol, cholesterol precursors, mildly hypercholesterolaemic subjects. Eur J Clin Nutr 52, and plant sterols in hypercholesterolemic subjects with different 334–343.

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