European Journal of Clinical (2010) 64, 1481–1487 & 2010 Macmillan Publishers Limited All rights reserved 0954-3007/10 www.nature.com/ejcn

ORIGINAL ARTICLE The effects of present in natural matrices on and LDL-cholesterol: a controlled feeding trial

X Lin1, SB Racette2,1, M Lefevre3,5, CA Spearie4, M Most3,6,LMa1 and RE Ostlund Jr1

1Division of Endocrinology, Metabolism and Research, Department of Medicine, Washington University School of Medicine, St Louis, MO, USA; 2Program in Physical Therapy, Washington University School of Medicine, St Louis, MO, USA; 3Pennington Biomedical Research Center, Baton Rouge, LA, USA and 4Center for Applied Research Sciences, Washington University School of Medicine, St Louis, MO, USA

Background/Objectives: Extrinsic phytosterols supplemented to the reduce intestinal cholesterol absorption and plasma low-density lipoprotein (LDL)-cholesterol. However, little is known about their effects on cholesterol metabolism when given in native, unpurified form and in amounts achievable in the diet. The objective of this investigation was to test the hypothesis that intrinsic phytosterols present in unmodified alter whole-body cholesterol metabolism. Subjects/Methods: In all, 20 out of 24 subjects completed a randomized, crossover feeding trial wherein all were provided by a metabolic kitchen. Each subject consumed two diets for 4 weeks each. The diets differed in content (phytosterol-poor diet, 126 mg phytosterols/2000 kcal; phytosterol-abundant diet, 449 mg phytosterols/2000 kcal), but were otherwise matched for content. Cholesterol absorption and excretion were determined by gas chromatography/mass spectrometry after oral administration of stable isotopic tracers. Results: The phytosterol-abundant diet resulted in lower cholesterol absorption (54.2±2.2% (95% confidence interval 50.5%, 57.9%) vs 73.2±1.3% (69.5%, 76.9%), Po0.0001) and 79% higher fecal cholesterol excretion (1322±112 (1083.2, 1483.3) vs 739±97 mg/day (530.1, 930.2), Po0.0001) relative to the phytosterol-poor diet. Plasma /cholesterol ratio rose by 82% (from 0.71±0.11 (0.41, 0.96) to 1.29±0.14 mg/mg (0.98, 1.53), Po0.0001). LDL-cholesterol was similar between diets. Conclusions: Intrinsic phytosterols at levels present in a are biologically active and have large effects on whole-body cholesterol metabolism not reflected in circulating LDL. More work is needed to assess the effects of phytosterol-mediated fecal cholesterol excretion on coronary heart disease risk in humans. European Journal of Clinical Nutrition (2010) 64, 1481–1487; doi:10.1038/ejcn.2010.180; published online 1 September 2010

Keywords: diets; absorption; mass spectrometry; deuterium

Introduction (Peterson, 1951; Pollak, 1953). Specifically, phytosterols reduce intestinal cholesterol absorption (Norme´n et al., Phytosterols are that are structurally similar to 2000) and plasma low-density lipoprotein (LDL)-cholesterol cholesterol. It has been several decades since phytosterols (Piironen et al., 2000; Ostlund, 2002). These actions form the were first reported to have lipid-lowering health effects basis for the recommendation by the National Cholesterol Education Program’s Adult Treatment Panel III for adults to Correspondence: Dr RE Ostlund Jr, Division of Endocrinology, Metabolism and consume 2 g phytosterols/day to reduce LDL-cholesterol and Lipid Research, Department of Medicine, Washington University School of risk (Expert Panel on Detection, 2001). Medicine, Campus Box 8127, 660 South Euclid Avenue, St Louis, MO 63110, Likewise, the American Heart Association’s 2006 Diet and USA. E-mail: [email protected] Lifestyle Recommendations (Lichtenstein et al., 2006) pro- 5Current address: Center for Advanced Nutrition, Utah State University, 4715 mote the consumption of phytosterols daily as a therapeutic Old Main Hill, Logan, UT 84322-4715, USA. option among individuals with elevated LDL-cholesterol. 6 Current address: Department of & Food, School of Human The potential cardiovascular health benefits of phytosterol Ecology, Louisiana State University, Baton Rouge, LA 70803, USA. Received 22 January 2010; revised 15 June 2010; accepted 17 June 2010; supplementation have driven interest in enriching foods published online 1 September 2010 with phytosterols to reach the recommended 2-g/day dose, Natural phytosterols and cholesterol metabolism XLinet al 1482 which cannot be achieved with natural foods without dairy products, poultry, meat, fish, eggs and refined ) extrinsic supplementation. Consequently, little is known and ingredients (that is, white-rice flour, and about the effects of the intake of intrinsic phytosterols tapioca flour) were incorporated. Third, oil (Bakers present in non-enriched foods (Piironen and Lampi, 2004). & Chefs, Sam’s Club, Maplewood, MO, USA) used in the Phytosterols present in fed to normolipidemic menus was purified to remove most phytosterols (Ostlund subjects partially accounted for the differences in plasma et al., 2003). To test the effects of phytosterols present in cholesterol levels when compared with oil (Howell natural forms in the diet without using supplements, a et al., 1998). A vegan diet containing 732-mg phytosterols/ phytosterol-abundant diet was also developed. Foods or day reduced total and LDL-cholesterol in patients with ingredients naturally high in phytosterols were incorporated rheumatoid arthritis when compared with a normal diet into the menu. As current food databases contain limited with unspecified phytosterol amounts (Agren et al., 2001). information on phytosterol content, it was necessary to Corn oil purified to remove sterols increased cholesterol analyze several individual foods by gas chromatography/ absorption when compared with the original corn oil, mass spectrometry (Racette et al., 2009) when designing the suggesting that intrinsic phytosterols present in corn oil menu. reduce cholesterol absorption in humans (Ostlund et al., Both phytosterol-poor and naturally phytosterol-abun- 2002). However, the potential physiological benefits of dant diets were developed as a 5-day menu cycle at six intrinsic phytosterols, provided entirely from natural energy levels (1000, 1500, 2000, 2500, 3000 and 3500 kcal), foods in amounts achievable in a healthy diet, are not which conformed to the macronutrient recommendations of currently known. the Dietary Guidelines for Americans (US Department of Extensive research has consistently shown that phyto- Health and Human Services and US Department of Agricul- supplements reduce cholesterol absorption. However, ture, 2005). Unit foods of 100 kcal each, which matched the most previous studies have not eliminated or considered the nutrient composition of the diets, were used to achieve the amount of phytosterols present in the background diet, appropriate energy prescription for each subject. The 5-day making it difficult to assess the effects of phytosterols at menu cycle was repeated throughout the 4-week feeding levels typically achievable in the US diet. We hypothesized periods. Table 1 shows the nutrient composition of the diets. that phytosterols present in natural food matrices alter The phytosterol-poor diet included 5 g of soluble cholesterol metabolism when compared with a novel (Benefiber, Novartis Consumer Health, Inc., Parsippany, NJ, phytosterol-poor diet. USA), 8 g of insoluble fiber (Solka-Floc, International Fiber Corporation, North Tonawanda, NY, USA), 600 mg potas- sium and 81 mg per 2000 kcal given as supple- Materials and methods ments. The soluble fiber was mixed into beverages and incorporated into three recipes. The insoluble fiber was Subjects incorporated into eight recipes, including baked goods, Volunteers were recruited from the greater Baton Rouge area salads and casseroles, to match the fiber content of the and the study was conducted at the Pennington Biomedical Research Center (PBRC). Inclusion criteria were healthy Table 1 Nutrient content of diets individuals aged 18–81 years with plasma LDL-cholesterol Phytosterol-poor Phytosterol-abundant between 100 and 189 mg per 100 ml, less (per 2000 kcal) (per 2000 kcal) than 250 mg per 100 ml, resting blood pressure lower than 160/95 mm Hg and body mass index between 20 and 35 kg/m2. Phytosterols, mg 126 449 Cholesterol, mg 161 155 Participants could not have active medical or surgical illnesses , g (% of energy) 276 (55%) 280 (56%) and should not have been taking prescription medicines or Protein, g (% of energy) 75.2 (15.0%) 76.8 (15.0%) dietary supplements known to affect lipid metabolism, Fat, g (% of energy) 66.9 (30.0%) 68.7% (31.0%) although oral contraceptives were allowed. Women who were Saturated 19.5 (8.8%) 17.2 (7.7%) pregnant, breast feeding or perimenopausal were excluded. The Monounsaturated 21.1 (9.5%) 20.8 (9.3%) study was approved by the PBRC Institutional Review Board. All Polyunsaturated 21.0 (9.4%) 21.6 (9.7%) participants provided written informed consent. Soluble fiber, g 6.7a 6.6 Insoluble fiber, g 20.4b 20.8 , mg 2365 2864 c Phytosterol-poor and phytosterol-abundant diets , mg 2166 2308 Magnesium, mg 202d 236 To provide a control condition for investigating the effects of , mg 657 616 phytosterols, a phytosterol-poor diet was developed (Racette a et al., 2009). First, high-phytosterol-containing foods, such This value includes 5 g of soluble fiber provided as a supplement mixed as oils, whole and other whole grains, into beverages and unit foods. bThis value includes 8 g of insoluble fiber incorporated into baked goods. and , were avoided or minimized in cThis value includes 600 mg of supplemental potassium. the menus. Second, very-low-phytosterol foods (that is, dThis value includes 81 mg of supplemental magnesium.

European Journal of Clinical Nutrition Natural phytosterols and cholesterol metabolism XLinet al 1483 phytosterol-abundant diet. Composites of each day’s lathosterol were measured as markers for cholesterol absorption plan were analyzed for total phytosterol content by gas and biosynthesis, respectively, and normalized after dividing by chromatography/mass spectrometry after both acid and the total plasma cholesterol concentration (Miettinen et al., alkaline hydrolysis. 1989). Samples were drawn in the morning after a 10-h fast on days 24 and 28 of each feeding period for the determination of total, HDL-, and LDL-cholesterol and triglycerides. Total Clinical protocol cholesterol and -blanked triglycerides were measured All participants received both the phytosterol-poor and by automated enzymatic commercialkits.HDL-cholesterolwas the phytosterol-abundant diet in a randomized, crossover measured in serum after precipitation of apolipoprotein B- design. Each diet was administered for 4 weeks, with up to 1 containing lipoproteins by sulfate and magnesium week between feeding periods. All foods and beverages (Warnick et al., 1982). LDL-cholesterol was calculated using the were prepared in the PBRC Metabolic Kitchen. Participants Friedewald equation (Friedewald et al., 1972). Results for days 24 were allowed a limited choice of additional seasonings and and 28 were averaged. beverages. They were required to consume breakfast and dinner at the feeding center during weekdays, with the rest of the meals taken outside. Participants began on an energy Statistical analyses level that most closely matched their energy requirement Statistical analyses were conducted using SAS (version 9.2, based on their estimated resting metabolic rate and physical SAS Institute Inc., Cary, NC, USA). Proc Mixed was employed activity level (Harris and Benedict, 1919; Food and Agri- to analyze the fixed effects of phytosterol intake, period and cultural Organization/World Health Organization/United interaction between phytosterol intake and period to assess Nations University, 1985). Energy levels were adjusted if carryover effects. No significant carryover effects were body weight changed. observed for any of the outcome measures. The statistical Participants were instructed to consume all food and power to detect a 5% difference in LDL-cholesterol between beverages provided. Compliance was determined by weigh- the two diets at a significance level of 0.05 assuming a 5% ing any food and beverages that were not consumed, by coefficient of variation is 85%. obtaining daily logs of self-reported intake and by measuring plasma phytosterol concentrations during each diet period. Results

Outcome measurements Phytosterol-poor and phytosterol-abundant diets Cholesterol absorption and excretion. Fecal cholesterol excre- The phytosterol-poor and phytosterol-abundant diets con- tion and intestinal cholesterol absorption were determined tained 126±8 and 449±19 mg of phytosterols per 2000 kcal, by administering capsules containing 2 mg 25,26, respectively (Table 1). , and sitos- 2 26,26,27,27,27-( H7)cholesterol (CDN Isotopes, Quebec, terol accounted for 16.0±0.4, 14.6±0.8 and 69.4±0.5% of 2 Canada) and 1 mg 5,6,22,23-( H4)sitostanol (Medical Iso- total phytosterols, respectively, for the phytosterol-poor diet; topes, Pelham, NH, USA) dissolved in twice daily corresponding values for the phytosterol-abundant diet were for the last 5 days of each 28-day feeding period. Stool 17.0±0.6, 9.8±0.2 and 73.2±0.5%. By design, average samples were collected on days 27 and 28, saponified, phytosterol intake during the phytosterol-abundant diet extracted and analyzed for cholesterol, coprostanol, copros- (512±23 mg/day) was 3.6-fold that of the phytosterol-poor tanone and sitostanol content (Lutjohann et al., 1993) using diet (140±7 mg/day). negative and positive ion chemical ionization gas chromato- graphy/mass spectrometryin the Washington University Mass Spectrometry Resource. The results for days 27 and 28 Compliance were averaged for use in all subsequent analyses. Excretion of In all, 24 subjects (18F, 6M) were randomized. Three subjects fecal cholesterol and its metabolite coprostanol was cor- dropped out during the first week of the first dietary period rected for recovery of the non-absorbable and non-degrad- and another during the first week of the second dietary 2 able marker ( H4)sitostanol. Fecal coprostanone ranged from period. Twenty subjects (15F, 5M) completed both diet undetectable to less than 1.0% of total fecal sterols and was periods and are the subject of this report. Baseline character- not used in the calculations. Results for cholesterol excretion istics of the participants are listed in Table 2. Body weight, include coprostanol excretion. Percentage cholesterol absor- blood pressure, pulse and fasting plasma were stable 2 ption was calculated as 100  (1À(( H7cholesterolfeces þ throughout the study. 2 2 2 2 H7coprostanolfeces)/ H4sitostanolfeces)/( H7cholesterolcapsule/ H4- Only 8 out of 1600 meals (0.5%) that were supposed to be sitostanolcapsule)). Dietary cholesterol excretion was calculated as eaten at the feeding center were missed and 6 of these 8 were cholesteroldietÀ(cholesteroldiet  percent cholesterol absorbed) owing to excused absences. Energy intake during the two and endogenous cholesterol excretion as total cholesterol feeding periods averaged 2317±17 kcal/day; the prescribed excretedÀdietary cholesterol excreted. Plasma cholestanol and energy intake averaged 2322±17 kcal/day. As expected, total

European Journal of Clinical Nutrition Natural phytosterols and cholesterol metabolism XLinet al 1484 Table 2 Participant characteristics at baseline Table 3 Cholesterol metabolism and lipid levels

Women/men 15/5 Variables Intrinsic dietary phytosterols White/Black 14/6 (mg/2000 kcal) Age, years 57.5±3.0 Weight, kg 74.4±2.9 126 449 BMI, kg/m2 26.7±0.8 Cholesterol metabolism Percent cholesterol absorption, % 73.2±1.3 54.2±2.2z Total cholesterol, mg per 100 ml 220±6 Total cholesterol absorbed, mg/day 1977±228 1627±183 LDL-cholesterol, mg per 100 ml 140±4 Total cholesterol excreted, mg/day 739±97 1322±112z HDL-cholesterol, mg per 100 ml 62±3 , mg per 100 ml 92±8 Plasma sterol ratios Total cholesterol: HDL-cholesterol 3.7±0.2 Phytosterols/cholesterol, mg/mg 2.90±0.28 4.88±0.40z Glucose 94±2 Campesterol/cholesterol, mg/mg 1.80±0.16 2.84±0.22z Stigmasterol/cholesterol, mg/mg 0.050±0.005 0.070±0.006w Blood pressure Sitosterol/cholesterol, mg/mg 1.05±0.12 1.97±0.18z Systolic, mm Hg 116±3 ± ± Diastolic, mm Hg 73±2 Cholestanol/cholesterol, mg/mg 1.37 0.12 0.75 0.06z Lathosterol/cholesterol, mg/mg 0.71±0.10 1.29±0.14z Values are means±s.e. Serum lipid concentrations Total cholesterol, mg per 100 ml 198±6 200±6 plasma phytosterols normalized to cholesterol increased by LDL-cholesterol, mg per 100 ml 128±5 127±5 68% during the phytosterol-abundant diet as compared with HDL-cholesterol, mg per 100 ml 53±355±3 ± ± the phytosterol-poor diet (Po0.0001, Table 3). Plasma Triglycerides, mg per 100 ml 88 6948 campesterol (Po0.0001), stigmasterol (Po0.001) and sito- Values are means±s.e. The levels of statistical significance relative to the w sterol (Po0.0001) were significantly higher on the phyto- phytosterol-poor diet are indicated by Po0.001 or zPo0.0001. Total sterol-abundant diet (Table 3). cholesterol excreted includes coprostanol.

terol and triglycerides were also stable for both diet periods (Table 3). Whole-body cholesterol metabolism Percentage cholesterol absorption determined by gas chro- matography/mass spectrometry was 26.0% lower with the Discussion phytosterol-abundant diet relative to the phytosterol-poor diet (Po0.0001, Table 3). As shown in Figure 1a, this effect We tested the hypothesis that intrinsic phytosterols present was consistent across subjects. In agreement with these in natural food matrices reduce fractional cholesterol findings, the ratio of plasma cholestanol to plasma choles- absorption and increase fecal cholesterol excretion when terol (an indicator of fractional cholesterol absorption) consumed in modest amounts achievable in a natural diet. was 45% lower (Po0.0001) on the phytosterol-abundant Using specially designed diets that differed only in phytos- diet. Absolute cholesterol absorption, expressed as mg total terol content, we demonstrated that 512 mg/day intrinsic cholesterol absorbed/day, was not significantly different phytosterols alter whole-body cholesterol metabolism when during the two diet periods despite large differences in compared with a phytosterol-poor diet. Although most absorption efficiency. previous studies have used phytosterol supplements of 2 g The greatest effects of the phytosterol-abundant diet were daily added to an ad libitum diet of uncontrolled phytosterol on cholesterol excretion. Fecal cholesterol excretion was content, the present work used only naturally occurring 79% higher on the abundant diet (Po0.0001, Table 3 and phytosterols in a modest dose that was substantially lower Figure 2). Although both dietary and endogenous cholesterol than those of supplement studies, and phytosterols in both excretion were higher, endogenous cholesterol excretion diets were carefully quantified and controlled. accounted for most of the differences between diet condi- Our results confirm and extend previous single-meal tions. The ratio of plasma lathosterol to cholesterol, an studies that show that selected food phytosterols delivered indicator of cholesterol biosynthesis, was 82% larger on the in natural matrices are bioactive (Ostlund, 2002; Ostlund phytosterol-abundant diet (Table 3). et al., 2003; Lin et al., 2009). The general question of whether food phytosterols are bioactive is important because some forms of the phytosterols (for example, purified phytosterols Lipoprotein concentrations in crystalline form) appear to have little or no biological Despite large differences in cholesterol absorption and activity (Denke, 1995; Ostlund et al., 1999). cholesterol excretion between the two diet periods, LDL- Fractional cholesterol absorption was substantially lower cholesterol concentrations were similar (Table 3 and on the phytosterol-abundant diet, an effect that was Figure 1b). Concentrations of total cholesterol, HDL-choles- expected based on phytosterols’ mechanism of action. The

European Journal of Clinical Nutrition Natural phytosterols and cholesterol metabolism XLinet al 1485

Figure 1 Individual percentage cholesterol absorption (a) and LDL-cholesterol (b) in response to two levels of phytosterol intake from natural foods. Cholesterol absorption and LDL-cholesterol were determined as described in Materials and methods. Individual values are presented for 20 subjects who received two levels of phytosterol intake (126 or 449 mg phytosterols per 2000 kcal) for 4 weeks each in a crossover design.

lathosterol has been reported previously (Nestel et al., 2001) and our work extends that finding to comparatively lower amounts of phytosterols in foods. Circulating LDL-cholesterol concentration was not lower on the phytosterol-abundant diet. This is not surprising in light of the following factors. First, the phytosterol intake of 512 mg/day was based on the amount achievable in a natural diet, but was not optimized for lowering LDL-cholesterol. Phytosterol supplement studies show that doses of 2 g/day are needed for optimum reduction of LDL-cholesterol (Demonty et al., 2009), whereas the US Food and Drug Administration requires products to provide at least 800 mg/day in order to include a on their labels (US Food and Drug Administration, 2008). A recent controlled feeding study comparing 459 and 59 mg/day phytosterols given as phytoster- ol ester supplements showed a non-significant 5% reduction in Figure 2 Fecal cholesterol excretion (dietary and endogenous) LDL-cholesterol. Second, a feeding period longer than 4 weeks in response to two levels of phytosterol intake from natural may be necessary to observe an effect on LDL-cholesterol foods. Excretion of dietary (hatched bars) and endogenous concentration. In support of this, a previous trial using 2 g/day (white bars) cholesterol was measured as described in Materials and methods in 20 subjects in a two-treatment (phytosterol- of phytosterols showed that LDL-cholesterol concentrations poor diet: 126 mg/2000 kcal and phytosterol-abundant diet: were unchanged after 4 weeks, but were reduced significantly 449 mg/2000 kcal)/two-period crossover study. Each phytosterol after 8 or 12 weeks (Devaraj et al., 2006). Third, the intake period lasted for 4 weeks. The level of statistical significance compensatory increase in whole-body cholesterol synthesis, as is indicated by **Po0.0001. demonstrated by higher plasma lathosterol concentrations, may have prevented a potential reduction in LDL-cholesterol. relatively high percentage of cholesterol absorption Thus, a combination of inhibiting cholesterol synthesis and (73.2±1.3%) that we observed with the phytosterol-poor inhibiting cholesterol absorption may be more effective in diet is higher than the 60% mean reported in the literature reducing LDL-cholesterol (Turley and Dietschy, 2003) than (Lutjohann et al., 1993), which is likely explained by its either approach alone. Taken together, these data suggest that unusually low phytosterol content. phytosterols are unlikely to have significant effects on LDL- Fecal cholesterol elimination (predominantly endogenous cholesterol after only 4 weeks when consumed at levels much cholesterol) was significantly greater with the phytosterol- less than 2 g/day. abundant diet (Figure 2). Increased cholesterol excretion was Our results suggest that phytosterols are bioactive in their paralleled by increases in cholesterol biosynthesis, indicated natural food matrices and in amounts (around 500 mg/day) by plasma lathosterol, showing that the increased cholester- that can be readily achieved in the diet without supplemen- ol excretion was sufficient to generate a strong compensa- tation (Nair et al., 1984; Ostlund 2002; Racette et al., 2009). tory response due to relative tissue cholesterol deficiency. We could not attribute the cholesterol metabolic differences The ability of phytosterol supplements to increase plasma to differences in macronutrients, cholesterol, fat quality,

European Journal of Clinical Nutrition Natural phytosterols and cholesterol metabolism XLinet al 1486 or . In order to match the fiber contents of represent a potentially new approach to improve dietary the two diets, the phytosterol-poor diet was supplemented treatment and prevention of coronary heart disease and that with soluble and insoluble fiber by incorporating it into this approach requires additional research on two fronts. many foods. It is possible that supplemented First, accurate analyses of food phytosterols are critical for may differ from that contained in natural food matrices, the design of dietary trials. Second, the relationship of representing a potential limitation of the study. Although it reverse cholesterol transport and phytosterols with coronary is possible that the differences observed in fecal cholesterol heart disease risk needs further study. Most investi- excretion were due to another unmeasured dietary compo- gations focus on the pharmaceutical approach of lowering nent, phytosterols are the principal dietary factor known to LDL-cholesterol, but it may be advantageous to use an old regulate cholesterol absorption. A similar controlled feeding and well-known nutrient, phytosterols, to test the hypoth- study using 459 mg purified phytosterols given in phytoster- esis that whole-body cholesterol metabolism is related ol ester supplements produced reductions in fractional to heart disease risk independently of circulating LDL- cholesterol absorption (10%) and increases in fecal choles- cholesterol in humans. terol excretion (36%) (Racette et al., 2010). The somewhat larger reductions in fractional cholesterol absorption (26%) and increases in fecal cholesterol excretion (79%) observed Conflict of interest in the present study may be attributable to greater bioavail- ability of intrinsic food phytosterols as compared with Washington University and Dr Ostlund have a financial phytosterol supplements, or with other experimental differ- interest in Lifeline Technologies, Inc., a startup company ences between the studies. commercializing emulsified phytosterols. (Emulsified phy- The effects of phytosterols on cholesterol absorption are tosterols and Lifeline products were not used in this work.) well established, yet comparatively little is known regarding The remaining authors declare no conflict of interest. phytosterol’s effects on or clinical out- comes. In apoEÀ/À mice, disrupting Npc1l1 or treating with ezetimibe decreased cholesterol absorption as well as Acknowledgements circulating cholesterol and prevented atherosclerosis (Davis et al., 2007), suggesting that reducing cholesterol absorption This work was supported by NIH grants R01 HL050420, MO1 has the potential to favorably affect atherosclerosis. Accu- RR-00036, RR-00954, P60-DK020579-30 and P30 DK056341. mulating evidence, however, supports the notion that We thank Robin Fitzgerald and Michele Burton for excellent interventions that hinder atherosclerosis progression and/ technical assistance. or enhance atherosclerosis regression can act independently of the effects on LDL-cholesterol. Constrictive remodeling of the arterial wall with therapy was not associated with References changes in LDL-cholesterol, but was linked to anti-inflam- matory effects (Schoenhagen et al., 2006). Similarly, the anti- Agren JJ, Tvrzicka E, Nenonen MT, Helve T, Hanninen O (2001). atherosclerotic action of the synthetic LXR agonists (which Divergent changes in serum sterols during a strict uncooked vegan inhibits the development of atherosclerosis) in apoEÀ/À and diet in patients with rheumatoid arthritis. Br J Nutr 85, 137–139. LDL-receptor-deficient mice was independent of changes in Davis Jr HR, Hoos LM, Tetzloff G, Maguire M, Zhu LJ, Graziano MP et al. (2007). Deficiency of Niemann-Pick C1 Like 1 prevents plasma lipid profiles (Joseph et al., 2002; Terasaka et al., atherosclerosis in ApoEÀ/À mice. Arterioscler Thromb Vasc Biol 27, 2003). Higher cholesterol absorption but lower cholesterol 841–849. synthesis (estimated by plasma cholestanol and phytosterols, Demonty I, Ras RT, van der Knaap HCM, Duchateau GSMJE, Meijer L, et al. and lathosterol levels, respectively) was observed in cardio- Zock PL (2009). Continuous dose-response relationship of the LDL-cholesterol-lowering effect of phytosterol intake. J Nutr vascular disease cases compared with controls in the 139, 271–284. Framingham Offspring Study. LDL-cholesterol concentra- Denke MA (1995). Lack of efficacy of low-dose sitostanol therapy as tions were similar and these biomarkers were better pre- an adjunct to a cholesterol-lowering diet in men with moderate . Am J Clin Nutr 61, 392–396. dictors of cardiovascular disease than traditional lipid risk Devaraj S, Autret BC, Jialal I (2006). Reduced-calorie juice factors (Matthan et al., 2009), suggesting that changes in beverage with plant sterols lowers C-reactive protein concentra- cholesterol homeostasis without changes in LDL-cholesterol tions and improves the lipid profile in human volunteers. Am J might be related to cardiovascular disease risk. Clin Nutr 84, 756–761. Currently, the phytosterol content of foods is not taken Expert Panel on Detection (2001). Executive summary of the third report of the national cholesterol education program (NCEP) into consideration in prescribed therapeutic diets and is not Expert Panel on Detection, Evaluation, and Treatment of High consistently included in nutrient databases. In fact, design- Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA ing the phytosterol-poor and phytosterol-abundant diets was 285, 2486–2497. Food and Agricultural Organization/World Health Organization/ a challenge for the investigators due to the paucity of United Nations University (1985). Energy and protein require- phytosterol data, requiring substantial chemical analysis and ments. Report of a Joint FAO/WHO/UNU Expert Consultation. WHO: recipe experimentation. We suggest that food phytosterols Geneva, Switzerland, pp 1–206.

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