Effects of Low-Fat Hard Cheese Enriched with Plant Stanol Esters on Serum Lipids and Apolipoprotein B in Mildly Hypercholesterolaemic Subjects

Home , Stanol ester

ORIGINAL ARTICLE Effects of low-fat hard cheese enriched with plant stanol esters on serum lipids and apolipoprotein B in mildly hypercholesterolaemic subjects

T Jauhiainen1,2, P Salo3, L Niittynen4, T Poussa5 and R Korpela1,2,6

1Valio Ltd, Helsinki, Finland; 2Institute of Biomedicine, University of Helsinki, Helsinki, Finland; 3Raisio Research and Development, Raisio plc, Finland; 4Nutritionist Leena Niittynen, Vihti, Finland; 5Stat-Consulting, Tampere, Finland and 6Foundation for Nutrition Research, Helsinki, Finland

Objective: To investigate the cholesterol-lowering effects of a low-fat cheese enriched with plant stanol esters in mildly hypercholesterolaemic subjects, as part of their normal diet. Design: A randomized double-blind parallel-group study. Setting: Valio Ltd, Helsinki. Subjects: Sixty-seven mildly hypercholesterolaemic volunteers (24 men, 43 women) participated in the study, which all of them completed. Interventions: The subjects were randomly assigned to the plant stanol ester group or the control group. During the 5-week intervention, the subjects in the stanol group consumed a cheese enriched with 2 g of plant stanols per day, and the subjects in the control group, a control cheese with no plant stanols. Results: In the stanol ester group, as compared to the control group, both serum total and low-density lipoprotein (LDL) cholesterol decreased significantly, that is, by 5.8% (À0.32 mmol/l, 95% CI À0.50 to À0.15 mmol/l, Po0.001) and 10.3% (À0.36 mmol/l, 95% CI À0.53 to À0.18 mmol/l, Po0.001), respectively. There were no significant changes in high-density lipoprotein cholesterol (HDL), triglycerides or apolipoprotein B concentrations between the groups. Conclusion: Cheese enriched with 2 g of plant stanol in the form of fatty acid esters decreases serum total and LDL cholesterol significantly. European Journal of Clinical Nutrition (2006) 60, 1253–1257. doi:10.1038/sj.ejcn.1602445; published online 24 May 2006

Keywords: plant stanols; hypercholesterolaemia; serum cholesterol; apolipoprotein B; cheese

Introduction lowering elevated serum cholesterol levels. Various drugs are used for patients with severe hypercholesterolaemia, whereas Hypercholesterolaemia is one of the major risk factors for for those with mild or moderate hypercholesterolaemia, coronary heart disease (Stamler et al., 1999, 2000), and dietary therapy is used to lower serum cholesterol levels. considerable efforts have focused on different methods of Non-pharmacological treatment is also considered to be an important alternative for patients with mild or moderate hypercholesterolaemia. One interesting alternative among non-pharmacological Correspondence: Dr R Korpela, Foundation for Nutrition Research, PO Box 30, treatments is the use of plant sterols. Plant sterols are a group FIN-00039 Helsinki, Finland. of compounds very similar in structure to cholesterol (Ikeda E-mail: [email protected] et al., 1989). The most common plant sterols are b-sitosterol, Guarantor: R Korpela. Contributors: TJ conducted the study and was responsible for the study design, campesterol and stigmasterol (Pollak, 1985). The saturation to which PS also contributed. TJ, PS, LN and TP all contributed to the of the double bonds of plant sterols results in the formation interpretation of the data. TJ and LN were responsible for the actual writing of of plant stanols. Plant stanols reduce the absorption of both the manuscript, and TP was responsible for the statistical analyses. RK is the dietary and biliary cholesterol by displacing cholesterol from leader of the group and acted as adviser throughout the work. Received 7 July 2005; revised 16 January 2006; accepted 2 March 2006; the micellar phase (Ikeda et al., 1989). When the absorption published online 24 May 2006 of cholesterol decreases, the synthesis of cholesterol in the Effects of cheese enriched with plant stanols on serum lipids T Jauhiainen et al 1254 liver increases, thus enhancing cholesterol turnover (Gylling Study design and diet et al., 1999). Several studies have shown that 2–3 g of plant This study was a randomized, double-blind, parallel-group stanols per day reduces total and low-density lipoprotein study. The sample size calculation was based on previous cholesterol (LDL-C) concentrations without changing high- studies on the effect of stanol esters on cholesterol (the risk density lipoprotein cholesterol (HDL-C) and triglyceride level is 5% and the power is 80% when the expected (TG) concentrations (Miettinen et al., 1995; Hallikainen difference between the groups is 6.5% in total cholesterol et al., 2000a; Plat et al., 2000; Nestel et al., 2001; Mensink (TC) and 11% in LDL-C). The subjects visited the health et al., 2002). centre five times: at the beginning of the run-in period In most studies, the cholesterol-lowering effect of plant (week -1) and the treatment period (week 0), and during the stanol ester has been studied with plant stanols incorporated treatment at weeks 3, 4 and 5. in an ordinary or low-fat margarine (Gylling and Miettinen, After the run-in period, the subjects were randomly 1994; Gylling et al., 1995, 1997; Miettinen et al., 1995; assigned to one of the treatment groups: the subjects in Hallikainen and Uusitupa, 1999; Hallikainen et al., 2000a, b, the stanol group ingested 50 g of low-fat cheese enriched 2002; Plat et al., 2000; Plat and Mensink, 2000; Tammi et al., with plant stanol ester per day, and the subjects in the 2002) or mayonnaise (Vanhanen et al., 1993; Miettinen and control group ingested a control cheese without plant Vanhanen, 1994). Mensink et al. (2002) also reported that a stanols. During the 5-week treatment period, the subjects yoghurt enriched with 3 g/day of plant stanols reduced LDL consumed the daily dose of the cheese product either once a cholesterol significantly. The effects of dairy products other day with lunch or in two separate portions with their two than yoghurt, enriched with plant stanol esters, have not so main meals. The control and experimental cheeses were far been published. In the West, milk products have an similar in colour and taste. The nutrient content of the important role in the diet. control cheese (1 dose ¼ 50 g) was as follows: energy 600 kJ, The purpose of this study was to investigate the effect of a carbohydrates 0 g, protein 17 g, fat 8.5 g, saturated fat 4.5 g. low-fat hard cheese enriched with plant stanol ester on The stanol cheese was similar in every way to the control serum cholesterol levels in mildly hypercholesterolaemic cheese, except that it also contained 3.4 g of plant stanol subjects, as part of their normal diet. esters per dose. Two grams of plant stanol mixture containing sitostanol and campestanol was transesterified with 1.4 g of rapeseed oil fatty acids (Raisio group, Raisio, Finland) and the plant stanol esters were then mixed with the study cheese. Subjects and methods The subjects were instructed to maintain their normal dietary habits and lifestyle. Weight was measured at each Subjects visit using a digital scale. Dietary intake was assessed by Sixty-seven mildly hypercholesterolaemic subjects applied means of a food frequency questionnaire during the first and for the study. The inclusion criteria were serum total last study visits. Another structured questionnaire collected cholesterol concentration of between 5.0 and 6.5 mmol/l information on smoking habits and physical activities and total TG concentration below 3.0 mmol/l at screening during the study. Information on the occurrence and (week À1), age 25–65 years, no lipid-lowering medication severity of gastrointestinal symptoms and other adverse and no unstable coronary artery diseases. Subjects with effects was collected by means of a structured questionnaire diabetes mellitus, malignant diseases, alcohol abuse (over during the two last study visits. In addition, at each visit, the four portions per day) or milk allergy, or who were pregnant subjects were asked about current medication and any were also excluded. Subjects were not allowed to consume changes in medication. any products enriched with plant stanol ester or plant sterol during the study and for 3 weeks before the study. The inclusion criteria were checked during the screening visit Laboratory measurements before randomization. Sixty-seven subjects (24 men, 43 At each study visit, blood samples were taken after a 12-h women) fulfilled these criteria and were randomly assigned overnight fast. The measurements were taken at United to the plant stanol ester group (n ¼ 33) or to the control Laboratories Ltd, Helsinki, Finland. Serum TC, HDL-C and group (n ¼ 34). At the beginning of the study, their mean age TG concentrations were measured by an enzymatic method. was 43.379.5 years (mean7s.d.). Two subjects were taking LDL-C was calculated according to Friedewald’s equation antiasthma medication, one subject, postmenopausal estro- (Friedewald et al., 1972). This method is generally accepted, gen therapy, four had thyroxin therapy for hypothyroidism and is accurate when total TG concentration is below and six, blood pressure-lowering medicine. Nine subjects 4.0 mmol/l, as was the case with our subjects. Apolipoprotein were smokers. The subjects gave their written consent to the B (apo B) was determined at the second and the last study study, and the study protocol was approved by the Ethics visits by an immunoturbiometric method. Routine labora- Committee at the Department of Medicine, Helsinki tory measurements and safety parameters (gamma-glutamyl- University Central Hospital. transferase, creatinine, glucose, uric acid) were measured at

European Journal of Clinical Nutrition Effects of cheese enriched with plant stanols on serum lipids T Jauhiainen et al 1255 the beginning and at the end of the study to make sure that between the groups remained stable from then on, a result the subjects maintained their normal health status. confirmed by analysis of variance for repeated measures (weeks 3, 4 and 5; time effect P ¼ 0.986 for TC and P ¼ 0.317 for LDL-C). The cholesterol-lowering effect of stanol treat- Statistical analyses ment was similar in both men and women (data not shown). Baseline cholesterol levels for TC, HDL, LDL and TG were There were no significant differences in HDL-C and TG defined as the mean of the values measured at weeks 1 and 0. between the groups (Table 1). The mean values of the serum lipid measurements after 4 There were no statistically significant differences between weeks and 5 weeks of treatment were used to estimate the the effects on apo B of the stanol ester and control cheeses cholesterol level at the end of the intervention. As the (Table 1). However, apo B decreased by 11.4% in the stanol primary analysis, the changes from baseline to the end of the group and by 6.7% in the control group. Compared to the treatment period were calculated for both groups and were control group, apo B decreased by 4.7% (95% CI À10.0 to compared between the groups by an independent samples 0.6; P ¼ 0.080) in the stanol group. t-test. The results are given as means and s.e.m.s. The treatment differences (stanol vs control) are given as means with 95% confidence intervals. As the secondary analysis, Discussion the changes from baseline to weeks 3, 4 and 5 were analysed separately using the independent samples t-test. All statis- This randomized double-blind study showed that a low-fat tical analyses were performed with the SPSS statistics hard cheese enriched with plant stanol ester lowered serum program (Version 12.0; SPSS Inc., Chicago, IL, USA). TC concentrations by nearly 6% and LDL-C concentrations by 10%. This result is similar to those of earlier studies, in which plant stanol esters (2–3 g/day of plant stanols) have Results reduced LDL-C by 9–15% (Vanhanen et al., 1993; Gylling and Miettinen, 1994; Gylling et al., 1995, 1997, 1999; General Miettinen et al., 1995; Hallikainen and Uusitupa, 1999; All 24 men and 43 women completed the study. The mean Hallikainen et al., 2000a; Plat and Mensink, 2000; Mensink body weight at the beginning of the study was 74.0 kg in the et al., 2002). Our result also confirms the fact that plant stanol group and 76.3 kg in the control group. Weight stanol ester does not affect serum HDL-C or TG. decreased by 0.5 kg during the study in both groups. Two In the present study, the treatment period was 5 weeks, subjects took up smoking during the study. There were no which was estimated to be long enough for a significant significant changes in the dietary intake or physical activity reduction of serum cholesterol levels. In earlier clinical in either of the groups (data not shown). studies, the ingestion of plant stanols for 4–7 weeks has Compliance with the cheese was excellent: 99% of the reduced serum cholesterol levels significantly (Gylling and cheese was eaten. During the study, 14 subjects in the stanol Miettinen, 1994; Gylling et al., 1995, 1997; Hallikainen et al., group and 21 subjects in the control group reported that 2000a, b; Plat et al., 2000; Mensink et al., 2002). It has also they suffered adverse effects such as headaches, tiredness, been reported that the full cholesterol-lowering effect can be flatulence, diarrhoea and constipation. Most of these reached within 1–2 weeks with the use of plant stanol symptoms were mild and occurred only occasionally, but (Hallikainen et al., 2002; Mensink et al., 2002). In our study, one subject in the control group reported that constipation the maximal reduction in cholesterol levels was also reached and abdominal discomfort were very disturbing during the by the first measurement, that is, after 3 weeks of treatment, first 3 weeks of the study. There were no significant and the difference between the groups remained stable differences in the changes in routine safety parameters thereafter. As was seen in earlier studies (Hallikainen et al., between the groups (data not shown). 2000a; Tammi et al., 2002), the cholesterol-lowering effect of plant stanol esters did not differ between the genders. Because the plant stanols were transesterified with rape- Serum lipids and apolipoprotein B seed oil fatty acids, the amount of fat was slightly higher in Table 1 shows TC, LDL-C, HDL-C, TG and apo B at baseline the stanol cheese than in the control cheese. However, this and at the end of the study in the stanol (n ¼ 33) and the small quantity of rapeseed oil (1.4 g/day) cannot have been control (n ¼ 34) groups. Compared to the control group, sufficient to cause the cholesterol-lowering effect observed serum TC decreased by 5.8% (95% CI À8.8 to À2.8%; after ingestion of the stanol cheese. Thus one can with Po0.001) and LDL-C by 10.3% (95% CI À14.9 to À5.7%; confidence claim that the reduction in serum lipids in the Po0.001) in the stanol group. The differences between the stanol group was almost certainly owing to the plant stanols. stanol group and the control group in TC and LDL-C were The daily intake of plant stanol was 2 g, which was, on the statistically significant. The maximal difference in TC and basis of the earlier studies (Vanhanen et al., 1993; Gylling LDL-C (Figure 1) between the groups had already been and Miettinen, 1994; Gylling et al., 1995, 1997, 1999; reached after only 3 weeks of treatment. The difference Miettinen et al., 1995; Hallikainen and Uusitupa, 1999; Plat

European Journal of Clinical Nutrition Effects of cheese enriched with plant stanols on serum lipids T Jauhiainen et al 1256 Table 1 Serum lipids (mmol/l) and apolipoprotein B (g/l) during the study in the stanol (n ¼ 33) and control (n ¼ 34) groups

Stanol group Control group Treatment difference

Mean7s.e.m. Mean7s.e.m. Mean 95% CI P-value

TC (mmol/l) Baseline 5.6570.09 5.7970.09 End 5.1470.10 5.6070.11 Change À0.5170.06 À0.1970.06 À0.32 À0.50 to –0.15 0.0004*

LDL (mmol/l) Baseline 3.5870.09 3.6070.10 End 3.1070.10 3.4870.10 Change À0.4870.06 À0.1270.06 À0.36 À0.53 to –0.18 0.0001*

HDL (mmol/l) Baseline 1.6170.06 1.6570.07 End 1.6070.06 1.6370.07 Change À0.0170.02 À0.0370.02 0.01 À0.05 to 0.07 0.713

TG (mmol/l) Baseline 1.0270.06 1.1870.10 End 1.0170.06 1.1770.09 Change À0.0170.06 À0.0170.05 0.001 À0.15 to 0.15 0.994

Apo B (g/l) Baseline 1.1070.03 1.1770.05 End 0.9770.03 1.0770.04 Change À0.1370.02 À0.1070.03 À0.04 À0.11 to 0.04 0.328

Abbreviations: Apo B, apolipoprotein B; LDL, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides. Values are mean7s.e.m. Baseline cholesterol and apo B levels were defined as the mean of the values measured at weeks –1 and 0. The levels at the end of the study were calculated from the values measured at weeks 4 and 5. The changes were compared by the independent samples t-test. *Significant difference between the stanol and control treatments.

4.0 has been reported that plant stanols, either consumed once a Stanol day or divided between the meals, resulted in a similar 3.8 Control decrease in serum lipids (Plat et al., 2000). It seems unlikely, therefore, that the cholesterol-lowering effect of this stanol 3.6 ester cheese would have been stronger if the cheese had been divided between the meals. In contrast to most other studies for testing the cholesterol- 3.4 lowering effect of a margarine enriched with plant stanols (Hallikainen et al., 2000a, b, 2002; Plat et al., 2000; Tammi 3.2 * et al., 2002), we investigated the effect of a dairy product ** ** LDL cholesterol (mmol/l) enriched with these stanols. In another study, which tested 3.0 the cholesterol-lowering effect of a dairy product, a low-fat yoghurt enriched with plant stanols (3 g/day) reduced serum 2.8 TC and LDL-C by 8.7 and 13.7%, respectively (Mensink et al., -1012345 2002). In our study, plant stanol esters were ingested in a Weeks cheese with a higher fat content (17%). It seems that the Figure 1 LDL-C at the beginning of the run-in (week À1) and cholesterol-lowering effect of plant stanols is very similar treatment (week 0) periods and during the treatment period (weeks regardless of the food matrix in which it is ingested. 3, 4 and 5) in the stanol (n ¼ 33, dark circles) and control (n ¼ 34, light circles) groups. Values are mean7s.e.m. (indicated by vertical Although an increased concentration of LDL-C is the main lines). **Po0.001, *Po0.01, significant differences between the risk factor for coronary heart disease, the constituents of stanol and control groups. LDL-C, apolipoproteins, may also play an important role in atherosclerosis (Homma, 2004). In earlier studies, serum apo and Mensink, 2000; Hallikainen et al., 2000a; Mensink et al., B concentration has decreased significantly after the use of 2002), estimated to be adequate.The daily dose of the cheese plant stanols (Gylling and Miettinen, 1994; Plat and product was taken in one or two portions with main meals. It Mensink, 2000; Hallikainen et al., 2000b; Homma et al.,

European Journal of Clinical Nutrition Effects of cheese enriched with plant stanols on serum lipids T Jauhiainen et al 1257 2003), and these decreases have been parallel to those of concentrations in hypercholesteroleamic subjects on a low-fat serum LDL-C concentrations (Hallikainen et al., 2000a; diet. Eur J Clin Nutr 54, 715–725. Homma et al., 2003), possibly implying that plant stanols Hallikainen MA, Sarkkinen ES, Uusitupa MIJ (2000b). Plant stanol esters affect serum cholesterol concentrations of hypercholestero- reduce large, less dense LDL particles and small, dense LDL lemic men and women in a dose-dependent manner. J Nutr 130, particles equally (Homma et al., 2003). Small, dense LDL 767–776. particles are rich in apo B and thus are considered to be Hallikainen MA, Uusitupa MIJ (1999). Effects of 2 low-fat stanol ester-containing margarines on serum cholesterol concentrations extremely atherogenic (Homma, 2004). Compared to the as part of a low-fat diet in hypercholesterolemic subjects. Am J Clin control treatment, we found no statistically significant Nutr 69, 403–410. changes in serum apo B concentration after ingestion of Homma Y (2004). Predictors of atherosclerosis. J Atheroscler Thromb plant stanol esters. However, serum apo B decreased in both 11, 265–270. Homma Y, Ikeda I, Ishikawa T, Tateno M, Sugano M, Nakamura H groups, and the reduction in apo B concentrations was (2003). Decrease in plasma low-density lipoprotein cholesterol, parallel to the reduction of serum LDL-C concentrations. It apolipoprotein B, cholesteryl ester transfer protein, and oxidized seems, therefore, that the cheese enriched with stanol ester low-density lipoprotein by plant stanol ester-containing spread: a had beneficial effects on both serum lipid and apo B randomized, placebo-controlled trial. Nutrition 19, 369–374. Ikeda I, Tanabe Y, Sugano M (1989). Effects of sitosterol and concentrations. sitostanol on micellar solubility of cholesterol. J Nutr Sci Vitaminol In the present study, mild side effects such as gastro- 35, 361–369. intestinal symptoms were fairly common. These symptoms Mensink RP, Ebbing S, Lindhout M, Plat J, van Heugten MMA (2002). were similar to those found in earlier studies (Hallikainen Effects of plant stanol esters supplied in low-fat yoghurt on serum lipids and lipoproteins, non-cholesterol sterols and fat soluble et al., 2000b; Neil et al., 2001) and occurred occasionally in antioxidant concentrations. Atherosclerosis 160, 205–213. both groups. As in earlier studies (Gylling et al., 1995, 1999; Miettinen TA, Puska PP, Gylling H, Vanhanen H, Vartiainen E (1995). Miettinen et al., 1995; Mensink et al., 2002), no serious Reduction of serum cholesterol with sitostanol-ester margarine in adverse effects were reported. In addition, we found no a mildly hypercholesterolemic population. N Engl J Med 333, 1308–1312. changes in the safety parameters. Miettinen TA, Vanhanen H (1994). Dietary sitostanol related to In conclusion, a cheese enriched with plant stanol ester absorption, synthesis and serum level of cholesterol in different reduces serum total cholesterol and LDL cholesterol levels to apolipoprotein E phenotypes. Atherosclerosis 105, 217–226. the same extent as oil-based products, and also has a Neil HAW, Meijer GW, Roe LS (2001). Randomised, controlled trial of use by hypercholesterolaemic patients of a vegetable oil sterol- beneficial effect on serum apo B concentration. Thus, a enriched fat spread. Atherosclerosis 156, 329–337. cheese enriched with plant stanol ester can be recommended Nestel P, Cehun M, Pomeroy S, Abbey M, Weldon G (2001). for hypercholesterolaemic subjects despite its high content Cholesterol-lowering effects of plant sterol esters and non- of saturated fat and cholesterol. esterified stanols in margarine, butter and low-fat foods. Eur J Clin Nutr 55, 1084–1090. Plat J, Mensink RP (2000). Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects. Atherosclerosis 148, References 101–112. Plat J, van Onselen ENM, van Heugten MMA, Mensink RP (2000). Friedewald WT, Levy RI, Fredrickson DS (1972). Estimation of the Effects on serum lipids, lipoproteins and fat soluble antioxidant concentration of low-density lipoprotein cholesterol in plasma, concentrations of consumption frequency of margarines and without use of the preparative ultracentrifuge. Clin Chem 18, 449– shortenings enriched with plant stanol esters. Eur J Clin Nutr 54, 502. 671–677. Gylling H, Miettinen TA (1994). Serum cholesterol and cholesterol Pollak OJ (1985). Effect of plant sterol on serum lipids and and lipoprotein metabolism in hypercholesterolaemic NIDDM atherosclerosis. Pharmacol Ther 31, 177–208. patients before and during sitostanol ester-margarine treatment. Stamler J, Daviglus ML, Garside D, Dyer AR, Neaton JD (2000). Diabetologia 37, 773–780. Relationship of baseline serum cholesterol level in 3 large cohorts Gylling H, Puska P, Vartiainen E, Miettinen TA (1999). Serum sterols of younger men to long-term coronary, cardiovascular, and all- during stanol ester feeding in a mildly hypercholesterolemic cause mortality and to longvity. JAMA 284, 311–318. population. J Lipid Res 40, 593–600. Stamler J, Stamler R, Neaton JD, Wentworth D, Daviglus ML, Gylling H, Radhakrishnan R, Miettinen TA (1997). Reduction of Garside D et al. (1999). Low risk-factor profile and long-term serum cholesterol in postmenopausal women with previous cardiovascular mortality and life expectancy: findings for 5 large myocardial infarction and cholesterol malabsorption induced by cohorts of young adult and middle-aged men and women. JAMA dietary sitostanol ester margarine. Circulation 96, 4226–4231. 282, 2012–2018. Gylling H, Siimes MA, Miettinen TA (1995). Sitostanol ester Tammi A, Ro¨nnemaa T, Miettinen TA, Gylling H, Rask-Nissila¨ L, margarine in dietary treatment of children with familial hyper- Viikari J et al. (2002). Effects of gender, apolipoprotein E cholesterolemia. J Lipid Res 36, 1807–1812. phenotype and cholesterol-lowering by plant stanol esters in Hallikainen M, Sarkkinen E, Wester I, Uusitupa M (2002). Short-term children: the STRIP study. Acta Paediatr 91, 1155–1162. LDL cholesterol-lowering efficacy of plant stanol esters. BMC Vanhanen HT, Blomqvist S, Ehnholm C, Hyvo¨nen M, Jauhiainen M, Cardiovasc Disord 2,14. Torstila I et al. (1993). Serum cholesterol, cholesterol precursors, Hallikainen MA, Sarkkinen ES, Gylling H, Erkkila¨ AT, Uusitupa MIJ and plant sterols in hypercholesterolemic subjects with different (2000a). Comparison of the effects of plant sterol ester and plant apoE phenotypes during dietary sitostanol ester treatment. J Lipid stanol ester-enriched margarines in lowering serum cholesterol Res 34, 1535–1544.

European Journal of Clinical Nutrition