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The effect of a combination of plant sterol-enriched foods in mildly hypercholesterolemic subjects
ARTICLE IN PRESS Clinical Nutrition (2007) 26, 792–798
Available at www.sciencedirect.com
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
The effect of a combination of plant sterol-enriched foods in mildly hypercholesterolemic subjects Martin B. Madsen, Anne-Mette Jensen, Erik B. Schmidt ˚rhus University Hospitals, Aalborg Hospital, Sdr. Skovvej 15, Department of Cardiology, Center for Cardiovascular Research, A 9000 Aalborg, Denmark Received 5 December 2006; accepted 24 May 2007
KEYWORDS LDL cholesterol; Apolipoprotein B; Apolipoprotein A-I; Phytosterols; Coronary heart disease
Summary Background & aims: The purpose of this study was to evaluate the effect of low-fat products enriched with plant sterols in addition to a National Cholesterol Education Program step 1 diet on serum lipids and lipoproteins. Methods: This study was a double-blind, randomised, placebo-controlled cross-over design with a run-in period and 2 intervention periods, each lasting 4 weeks. A total of 46 mildly hypercholesterolemic subjects (age 50.679.8) completed the trial. The study products consisted of 20 g low-fat margarine (35% fat) and 250 ml low-fat milk (0.7% fat), in total delivering 2.3 g plant sterols/d. Results: Serum total and low-density lipoprotein cholesterol were significantly reduced by 5.5% (po0.001, 95% CI: 2.5; 8.3) and 7.7% (p ¼ 0.001, 95% CI: 3.4; 11.9), respectively, by plant sterol-enriched products compared to placebo. Serum apolipoprotein B was significantly reduced by 4.6% (po0.05, 95% CI: 1.7; 7.5), and apolipoprotein B/ apolipoprotein A-I by 3.4% (po0.05, 95% CI: 0.1; 6.6) after plant sterol intake compared to the placebo supplement. Conclusions: A combination of low-fat margarine and milk enriched with plant sterols significantly reduced low-density lipoprotein cholesterol, apolipoprotein B and the ratio of apolipoprotein B to apolipoprotein A-I in mildly hypercholesterolemic subjects, but had no effect on C-reactive protein and lipoprotein (a) concentrations. Sponsorship: Unilever Denmark A/S. & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Abbreviations: Apo, apolipoprotein; HDL, high-density lipoprotein; HsCRP, high sensitivity C-reactive protein; LDL, low-density lipoprotein; Lp(a), lipoprotein (a); NCEP, National Cholesterol Education Program Corresponding author. Tel.: +45 99326899; fax: +45 99326804. E-mail address: [email protected] (E.B. Schmidt). 0261-5614/$ - see front matter & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2007.05.008
ARTICLE IN PRESS Consumption of 2.3 g plant sterols/d reduces CHD risk factors
Introduction Plasma cholesterol is a major risk factor for coronary heart disease (CHD) and lowering of low-density lipoprotein (LDL) cholesterol concentrations significantly reduces CHD mortality.1 The first step to reduce LDL cholesterol levels is by lifestyle measures including dietary changes, in particular a reduction in the intake of total and saturated fat.2 The effect of such changes is variable, with a mean reduction in LDL cholesterol in the order of 5–10%.3 Consumption of 2 g plant sterols/day is also among the recommendations from the National Cholesterol Education Program (NCEP) to lower elevated LDL cholesterol concentrations,2 as plant sterols and stanols inhibit dietary and biliary cholesterol absorption in the gut.4 On average, a dose of 2 g plant sterols/day have resulted in a 10% reduction in LDL cholesterol which is additive to the cholesterol-lowering effect of a low-fat diet.5 Plants sterols naturally occur in foods in low concentrations, with an estimated daily intake below 400 mg.6 In addition to fatty foods such as margarines, plant sterols and stanols have also been recently incorporated in low-fat food formats, such as low-fat yoghurts,7–9 milk9–11 and yoghurt minidrinks.12,13 While these new food formats offer alternatives to lowering of cholesterol concentrations, only a few studies have evaluated the impact of combinations of plant sterol-enriched foods on LDL cholesterol levels.10,14,15 There is a growing body of evidence suggesting that apolipoprotein B/apolipoprotein A-I (apo B/apo A-I) ratio is a better marker of CHD risk than lipid concentrations per se and lipid ratios such as LDL:HDL cholesterol and total:HDL cholesterol.16,17 In the few studies that investigated the effect of plant sterol undertaken so far, evaluation of CHD risk has been limited to traditional risk factors such as lipid concentrations and ratios.10,14,15 Another emerging risk factor related to the inflammatory hypothesis of CHD is C-reactive protein (CRP).18–20 Data on the potential impact of plant sterol supplementation on this marker are, however, very scarce.21 The objective of the present study was therefore to evaluate the impact of combining two different plant sterol-enriched foods as part of a low-fat diet on CHD risk, with a particular attention to emerging risk factors for CHD such as apo B/apo A-I ratio and CRP.
Subjects and methods Subjects Fifty volunteers were recruited by advertisement among patients from the outpatient Lipid Clinic and employees of Aalborg Hospital, and among employees of a local insurance company. Moderately hypercholesterolemic (LDL cholesterol 3.0–5.5 mmol/l), otherwise healthy men and women aged 20–70 y, with a body mass index (BMI) of 20–35 kg/m2 were eligible to participate in the study. For subjects on medication, including statins, the type and dosage had to be stable for at least 6 weeks prior to screening. Individuals who reported having taken medications known to interfere with cholesterol absorption (e.g. ezetimibe, bile acid sequestrants) or had used plant sterol- or stanol-enriched
793 products during the previous 6 weeks were excluded. Subjects suffering from serious illnesses, diabetes mellitus, familial hypercholesterolemia, gastrointestinal disorder or lactose intolerance were excluded, as well as those consuming more than 21 units of alcohol per week (one unit corresponding to 12 g alcohol). Pregnant women and those planning to be pregnant or breastfeeding, and subjects who had participated in another trial within 3 months of study entry or were engaged in intense physical activities (410 h per week) were not eligible. All subjects provided written informed consent before entering the study. The study protocol was approved by the regional Ethics Committee.
Design and diets The study was a randomised, double-blind, placebo-controlled, cross-over trial consisting of two 4-week intervention phases. During both the placebo and the plant sterol phases participants followed the NCEP step 1 diet.2 In order to allow for stabilisation of dietary patterns prior to the start of the treatments, a 4-week run-in period preceded the trial. All subjects were provided dietary advice by an experienced clinical dietician before the start of the run-in period in order to meet the guidelines of the NCEP step 1 diet (total fat contributing to a maximum of 30% of total energy intake, saturated fat contributing to less than 10% of energy, not more than 300 mg cholesterol/d, 15% and 55% of energy provided by proteins and carbohydrates, respectively). An adequate fruit and vegetable intake was recommended, including more than one serving per day of carotenoid-rich foods. After inclusion the subjects were asked to use one portion of 20 g low-fat spread and one 250 ml serving of low-fat milk, to be consumed on three different occasions each day, with main meals. They were encouraged to maintain their normal activity pattern and body weight. At the end of the run-in period, half of the participants were assigned to consuming the placebo products for 4 additional weeks, whilst the other half was assigned to the plant sterol-enriched margarine and milk. At the end of the first phase, subjects on the plant sterolenriched diet switched to the placebo products, and those on the placebo switched to the plant sterol-enriched foods for 4 weeks without wash-out between treatments. The placebo and the plant sterol-enriched spread and milks were provided by Unilever Denmark and were all packed in black containers and coded such that both the study investigators and the participants were blinded. Randomisation was done by Unilever Research, The Netherlands, and treatment codes were broken after the statistical analyses were performed. The composition of the placebo and the plant sterol-enriched spread and milk is presented in Table 1. The placebo and plant sterol-enriched products had a similar composition, except for the content of plant sterols. The plant sterol-enriched margarine (20 g/d) provided an equivalent of 1.6 g free plant sterols, and the plant sterol-enriched milk (250 ml/d) contributed 0.7 g plant sterols, for a total intake of 2.3 g plant sterols/day. The spread was a regular low-fat plant sterol-enriched spread similar to the one available in the Danish market. The plant sterols in the spread and milk were esterified to sunflower
ARTICLE IN PRESS 794
Table 1
M.B. Madsen et al.
Nutritional composition of the investigational products used in the trial.
Per 100 g
Energy (kJ) Proteins (g) Carbohydrates (g) Fat (g) PUFA (g) MUFA (g) SAFA (g) Plant sterols (g)
Margarine
Milk
Placebo
Plant sterol-enriched
Placebo
Plant sterol-enriched
1385a 0.1a 5.5a 37.4 17.4 9.9 10.8 0.1
1365a 0.1a 4.0a 36.8 17.0 9.9 8.4 8.0
146 3.5 5.1 o0.1 o0.1 o0.1 o0.1 o0.01
168 3.6 5.0 0.6 0.4 0.2 0.2 0.3
PUFA: polyunsaturated fatty acids, MUFA: monounsaturated fatty acids, SAFA: saturated fatty acids. Note: The values for polyunsaturated fat, monounsaturated fat, and saturated fat have been calculated from the % of PUFA, MUFA and SAFA of all fatty acids (46.5%, 26.6%, 23.2% of PUFA, MUFA and SAFA, respectively, in the control spread, and 46.1%, 26.8%, and 22.8% of PUFA, MUFA and SAFA in the plant sterol-enriched spread). a Calculated values from the recipe; otherwise, measured values.
based on data obtained by Dual Energy X-ray Absorptiometry (DXA).
oil fatty acids and consisted mainly of 69% b-sitosterol, 15% campesterol and 8% stigmasterol, with other plant sterols contributing to the rest. Dietary intake was assessed by food records completed on three consecutive days (including one week-end day) before screening (start of the run-in period), at randomisation (baseline) and at the end of the first and second study phases. Nutrient intakes were analysed using the Master Diaetist System version 1.223, 2006 Master Data I/S, Copenhagen, Denmark. A questionnaire was given to the subjects at the beginning of the run-in phase and again at the beginning of each treatment phase to record every day consumption of the study products. All product packages were returned to the investigators and counted. Compliance was calculated from both the records and the returned packages as the number of servings consumed relative to scheduled servings. A subject was considered compliant if he/she had consumed 80% or more of the study products. Weight and body fat percentage were measured at run-in, baseline and after each intervention period, whereas height and blood pressure were recorded only at baseline.
Fifty subjects were included in the trial, based on an 80% power to detect a 10% decrease in LDL cholesterol with the plant sterol-enriched vs the placebo treatment, and allowing for a 10% drop-out rate. A per-protocol analysis was performed using the subjects that completed the study according to the protocol. The change in concentration of serum parameters measured at the end of the placebo and the plant sterol phases were compared both on an absolute scale (using paired t-tests on a log-scale). We assumed that there was no carry-over effect.23 The analyses were carried out both on an absolute scale (original scale) and a relative scale (log-scale). Model diagnostics have included histograms, quantile–quantile plots and Bland–Altman plots and revealed for all variables that both scales could in principle be used. A p-value o0.05 (two-tailed) was considered statistically significant.
Analytical procedures
Results
Blood was collected in the morning after at least 10 h of fasting and the serum samples were stored at 80 1C until final analyses. Serum total cholesterol, HDL cholesterol (precipitation method) and triglycerides were measured enzymatically while LDL cholesterol concentrations were calculated according to the Friedewald equation.22 Apo B and apo A-I were measured using the respective antibodies from DAKO on an Advia 1650 (Bayer, Diagnostics, NY, USA). Lipoprotein (a) (Lp(a)) concentrations were determined using an ELISA kit from Mercodia. High sensitivity C-reactive protein (hsCRP) concentrations were measured by an immunotorbidimetric assay from Randox on an Advia 1650. Body fat percentage was determined using a Tanita BC418MA Body Composition Analyzer which calculates the body fat percentage by using Bioelectrical Impedance Analysis
A total of 46 of the 50 participants completed the study. During the run-in period, one subject dropped out due to disliking of the margarine and another was excluded due to lack of compliance. These two subjects were not included in the final data analysis. During the first trial arm, one subject receiving the plant sterol-enriched products was excluded due to an intercurrent disease considered unrelated to the study products, and one subject receiving the placebo products left the study because of disliking the margarine. Characteristics of the 48 participants at baseline are presented in Table 2. No significant change in weight, BMI or body fat percentage was observed during the trial between the subjects receiving intervention products and the subjects receiving placebo products (data not shown).
Statistical analysis
ARTICLE IN PRESS Consumption of 2.3 g plant sterols/d reduces CHD risk factors There was no significant change in fat and total energy intake during the trial between the participants in the intervention and placebo phase (Table 3). During the plant Table 2 pants.
Baseline characteristics of the study partici-
Group
AB
BA
n
24 50.879.6 9/15 5
24 50.3710.3 3/21 3
76.3710.4 25.972.8 30.878.5
68.2713.6 24.173.3 3276.1
5.170.7 3.370.6 1.270.3 3.870.7
5.870.9 3.770.8 1.570.4 4.270.9
1.270.6 1.070.2 1.370.2 0.870.2 121.5 (6–1177) 1.41 (0–28)
1.170.5 1.170.2 1.570.2 0.870.2 178 (11–1999) 0.675 (0–3)
137719 8379
131721 81710
Age (years) Gender (men/women) Smokers (n) Anthropometric data Body weight (kg) BMI (kg/m2) Body fat (%) Biochemistry Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Non-HDL cholesterol (mmol/l) Triglycerides (mmol/l) Apolipoprotein B (g/l) Apolipoprotein A-I (g/l) Apo B/apo A-I ratio Lipoprotein (a) (IU/L) hsCRP (mg/l) Blood pressure Systolic BP (mmHg) Diastolic BP (mmHg)
Values are means7SD, median (range) or numbers (n) of subjects. AB: subjects receiving plant sterol-enriched products in the first intervention period, BA: subjects receiving plant sterol-enriched products in the second intervention period, BMI: body mass index, LDL: low-density lipoprotein, HDL: high-density lipoprotein, hsCRP: high sensitivity Creactive protein, BP: blood pressure.
Table 3
795 sterol phase, the subjects significantly decreased their intake of dietary fibre by 7.6% (po0.05, 95% CI: 1.2; 13.6) compared to the placebo phase, while other dietary variables did not significantly differ between the interventions. All 46 participants completing the trial were compliant and no adverse effects were reported, but some of the participants reported that 20 g margarine was difficult to consume. The changes in serum lipid concentrations are given in Table 4. Serum total cholesterol and LDL cholesterol decreased significantly by 5.5% and 7.7%, respectively, after consumption of plant sterol-enriched products compared to placebo. The individual results are given in Fig. 1 showing a lowering in LDL cholesterol in 31 of the 46 patients completing the trial, no effect in 6 and an increase in LDL cholesterol in 9 out of the 46 subjects. One patient had an unexplained increase in LDL cholesterol from 2.1 mmol/l at the end of the placebo phase to 3.5 mmol/l (data not shown) at the end of the plant sterol phase but was kept in the analysis. No difference in LDL cholesterol reduction was seen whether the subjects started with either plant sterolenriched or placebo. The reduction in LDL cholesterol or apo B did not depend on the initial LDL cholesterol levels, fat consumption or total energy intake. Non-HDL cholesterol significantly decreased 7.3%. There was no significant change in HDL cholesterol and triglycerides between subjects in the intervention phase and the placebo phase. Serum apo B decreased significantly by 4.6% and apo B/apo A-I by 3.4% after plant sterol-enriched consumption compared to placebo, while neither apo A-I, Lp(a) nor hsCRP was affected by any of the supplements.
Discussion Our results show that including 2.3 g plant sterols per day in the form of plant sterol-enriched margarine and milk in subjects already advised to eat according to a NCEP step 1 diet lowered LDL cholesterol a further 7.7% in mildly hypercholesterolemic subjects. As a new target in the AHA/ACC guideline,24 non-HDL cholesterol was determined
The daily intake of dietary variables at baseline, end of placebo phase and after plant sterol-enriched phase.
n Energy (kJ) Protein (E%) Fat (E%) Carbohydrate E%) Alcohol (E%) PUFA (g) MUFA (g) SAFA (g) Dietary fibres (g) Cholesterol (mg)
Baseline
End of placebo phase
End of plant sterol-enriched phase
48 8 5107267 (2 046764 kcal) 16.770.3 30.570.8 48.370.9 4.470.6 12.270.2 24.571.1 23.271.5 21.470.9 290719
46 8 6887315 (2 079775 kcal) 16.370.3 31.770.9 47.270.9 4.870.6 12.570.5 25.371.3 26.171.7 20.770.8 296719
46 8 9307348 (2 136783 kcal) 16.070.4 33.170.9 45.871.1 5.270.6 13.170.4 27.271.3 26.971.7 19.470.9 304719
Values are means7SEM. PUFA: polyunsaturated fatty acids, MUFA: monounsaturated fatty acids, SAFA: saturated fatty acids.
ARTICLE IN PRESS 796
Table 4
M.B. Madsen et al.
The effect of plant sterols on serum lipids, lipoproteins and hsCRP.
Variable
Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l) LDL cholesterol/HDL cholesterol Total cholesterol/HDL cholesterol Non-HDL cholesterol (mmol/l) Apolipoprotein B (g/l) Apolipoprotein A-I (g/l) Lipoprotein (a) (IU/l) Apolipoprotein B/ Apolipoprotein A-I hsCRP (mg/l)
End of placebo phase
End of plant sterolenriched phase
Difference (95% CI)
Relative change (%) (95% CI)
5.4570.13 3.4570.12 1.4670.05 1.1870.08 2.5470.15
5.1470.11 3.1670.09 1.4770.06 1.0970.07 2.3370.12
0.31c (0.47; 0.14) 0.29c (0.45; 0.14) 0.01 (0.04; 0.06) 0.09 (0.20; 0.01) 0.21b (0.35; 0.08)
5.5c (8.3; 2.5) 7.7b (11.9; 3.4) 0.2 (2.8; 3.0) 6.2 (14.2; 2.6) 7.6b (12.4; 2.4)
3.9570.17
3.7270.15
0.23b (0.38; 0.08)
5.3b (8.7; 1.8)
3.9870.14
3.6770.11
0.32c (0.47; 0.16)
7.3c (11.1; 3.4)
1.0570.03 1.4170.03 301.4758.4 0.7670.03
1.0070.03 1.4070.03 289.3756.3 0.7370.02
0.05b (0.08; 0.02) 0.02 (0.05; 0.02) 12.07 (29.77; 56.37) 0.03a (0.05; -0.003)
4.6b (7.5; 1.7) 1.3 (3.2; 0.8) 5.0 (12.1; 2.6) 3.4a (6.6; 0.1)
1.3970.25
1.5270.24
0.12 (0.18; 0.43)
3.4 (11.8; 21.1)
LDL: low-density lipoprotein, HDL: high-density lipoprotein, hsCRP: high-sensitivity C-reactive protein. a po0.05, b po0.01, c po0.001.
Figure 1 The individual response to the plant sterol-enriched products (difference in LDL cholesterol on sterols vs. placebo). The individual response to the plant sterol-enriched low-fat margarine and milk compared to placebo. Each column represents one individual (in total all 46 subjects concluding the trial). The y-axis is a relative scale i.e. the individual effect is given as % reduction/increase. LDL: low-density lipoprotein.
and was reduced significantly by 7.3%. Apo B was also significantly reduced by 4.6% and the ratio of apo B to apo AI was significantly decreased by 3.4%. There was a decrease in dietary fibre intake in the subjects receiving the intervention products, but it was probably too small to have had an impact on the results. The observed lowering of LDL cholesterol is in accordance with a recent study by Noakes et al.,10 where a combination of milk and margarine-enriched with plant sterols reduced LDL cholesterol by 11.4%. Their study was a single-blind, placebo-controlled cross-over study with 39 participants completing the trial showing no difference in cholesterol
reduction whether a plant sterol-enriched spread (2 g), a plant sterol-enriched milk (2 g) or a combination (4 g) was used. Likewise, our study is in accordance with other studies using plant sterol and stanol intakes of approximately 2 g/d leading to a reduction of 10% in LDL cholesterol.5 Studies with low-fat dairy products have shown similar effects.11,13,25 Thus it seems that the effect of plant sterols and stanols are similar for spreads and dairy products. However, one trial testing plant sterols in low-fat and no-fat beverages did not show a significant reduction in LDL cholesterol,26 in a trial where free plant sterols and not plant sterol esters were used. The fact that free plant sterols were used may not be responsible for the lower efficacy, some studies have shown that both free plant sterols and plant sterol esters decrease intestinal cholesterol absorption.27 Recent data suggest that apo B, apo A-I and hsCRP are important risk factors for CHD.20,28 To our knowledge, only one study with plant sterols has evaluated all these parameters.21 Their findings suggested the same as ours, i.e. that a daily intake of 2 g plant sterols has no effect on apo A-I, Lp(a) and hsCRP, but significantly decreases apo B. Apo B is a strong predictor of coronary events,29,30 and the fact that apo B concentrations were reduced by 4.6% indicates that plant sterol esters do not solely reduce the cholesterol content of LDL cholesterol particles, but also reduce the total number of circulating atherogenic particles. Furthermore, the apo B to apo A-I ratio decreased by 3.4% and, interestingly, this ratio has recently been proposed the strongest marker of risk for vascular disease.28–30 One of the strengths of our study was that the participants were given dietary guidance by an experienced dietician. All the participants filled questionnaires on their diet, ensuring as high an accuracy as possible regarding their
ARTICLE IN PRESS Consumption of 2.3 g plant sterols/d reduces CHD risk factors nutritional intake. Also, this is only the second trial studying the combined effect of plant sterol-enriched margarine and milk, an important feature because patient acceptance of available products are essential for their ultimate use. Finally, the trial is one of only a few studies with evaluation of apolipoproteins and hsCRP. Plant sterols are part of the NCEP2 and the AHA/ACC1 guidelines for secondary prevention of CHD. For each percentage of LDL cholesterol reduction, the risk for CHD disease is reduced by 2–3%. Our study showed that a combination of dietary intervention and low-fat margarine and milk enriched with plant sterols significantly reduced LDL cholesterol, apo B and the apo B to apo A-I ratio. Lowfat products enriched with plant sterols may therefore play a role in managing higher than desirable cholesterol levels.
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Acknowledgements 11.
Unilever Denmark A/S delivered all trial products and partly sponsored the trial. MSc, PhD, Biostatistician Claus Dethlefsen, Center for Cardiovascular Research, Aalborg Hospital, Århus University Hospitals, Denmark, for statistical analysis. Janne Danielsen, Department of Cardiology, Aalborg Hospital, Århus University Hospitals, Denmark, for dietary calculations. Michelle Gordon, Unilever Food and Health Research Institute, for scientific advise. Isabelle Demonty, Unilever Food and Health Research Institute, for having critically read and commented this manuscript. A-M Jensen and EB Schmidt were responsible for the design and conduct of the study. MB Madsen and EB Schmidt were responsible for the analysis and interpretation of the data. MB Madsen drafted the manuscript. All authors took part in critical revision of the manuscript.
References 1. De Backer G, Ambrosioni E, Borch-Johnsen K, Brotons C, Cifkova R, Dallongeville J, et al. European guidelines on cardiovascular disease prevention in clinical practice Third Joint Task Force of European and other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of eight societies and by invited experts). Atherosclerosis 2004;173:379–89. 2. Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection. Evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). J Am Med Assoc 2001; 285: 2486–97. 3. Hunninghake DB, Stein EA, Dujovne CA, Harris WS, Feldman EB, Miller VT, et al. The efficacy of intensive dietary therapy alone or combined with lovastatin in outpatients with hypercholesterolemia. N Engl J Med 1993;328:1213–9. 4. Normen L, Dutta P, Lia A, Andersson H. Soy sterol esters and beta-sitostanol ester as inhibitors of cholesterol absorption in human small bowel. Am J Clin Nutr 2000;71:908–13. 5. Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti R. Efficacy and safety of plant stanols and sterols in the management of blood cholesterol levels. Mayo Clin Proc 2003;78:965–78. 6. de Vries JHM, Jansen A, Kromhout D, van de Bovenkamp P, van Staveren WA, Mensink RP, et al. The fatty acid and sterol content of food composites of middle-aged men in seven
12. 13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
countries. Journal of Food Composition and Analysis 1997; 10:115–41. Mensink RP, Ebbing S, Lindhout M, Plat J, van Heugten MM. Effects of plant stanol esters supplied in low-fat yoghurt on serum lipids and lipoproteins, non-cholesterol sterols and fat soluble antioxidant concentrations. Atherosclerosis 2002;160: 205–13. Hyun YJ, Kim OY, Kang JB, Lee JH, Jang Y, Liponkoski L, et al. Plant stanol esters in low-fat yogurt reduces total and lowdensity lipoprotein cholesterol and low-density lipoprotein oxidation in normocholesterolemic and mildly hypercholesterolemic subjects. Nutr Res 2005;25:743–53. Clifton PM, Noakes M, Sullivan D, Erichsen N, Ross D, Annison G, et al. Cholesterol-lowering effects of plant sterol esters differ in milk, yoghurt, bread and cereal. Eur J Clin Nutr 2004; 58:503–9. Noakes M, Clifton PM, Doornbos AM, Trautwein EA. Plant sterol ester-enriched milk and yoghurt effectively reduce serum cholesterol in modestly hypercholesterolemic subjects. Eur J Nutr 2005;44:214–22. Thomsen AB, Hansen HB, Christiansen C, Green H, Berger A. Effect of free plant sterols in low-fat milk on serum lipid profile in hypercholesterolemic subjects. Eur J Clin Nutr 2004;58: 860–70. Salo P, Wester I. Low-fat formulations of plant stanols and sterols. Am J Cardiol 2005;96:51D–4D. Volpe R, Niittynen L, Korpela R, Sirtori C, Bucci A, Fraone N, et al. Effects of yoghurt enriched with plant sterols on serum lipids in patients with moderate hypercholesterolaemia. Br J Nutr 2001;86:233–9. Nestel P, Cehun M, Pomeroy S, Abbey M, Weldon G. Cholesterollowering effects of plant sterol esters and non-esterified stanols in margarine, butter and low-fat foods. Eur J Clin Nutr 2001; 55:1084–90. Tikkanen MJ, Hogstrom P, Tuomilehto J, Keinanen-Kiukaanniemi S, Sundvall J, Karppanen H. Effect of a diet based on low-fat foods enriched with nonesterified plant sterols and mineral nutrients on serum cholesterol. Am J Cardiol 2001;88: 1157–62. Sniderman AD, Jungner I, Holme I, Aastveit A, Walldius G. Errors that result from using the TC/HDL C ratio rather than the apoB/ apoA-I ratio to identify the lipoprotein-related risk of vascular disease. J Intern Med 2006;259:455–61. Walldius G, Aastveit AH, Jungner I. Stroke mortality and the apoB/apoA-I ratio: results of the AMORIS prospective study. J Intern Med 2006;259:259–66. Kim JS, Kang TS, Kim JB, Seo HS, Park S, Kim C, et al. Significant association of C-reactive protein with arterial stiffness in treated non-diabetic hypertensive patients. Atherosclerosis 2006; available online 19 June 2006. Lloyd-Jones DM, Liu K, Tian L, Greenland P. Narrative review: assessment of C-reactive protein in risk prediction for cardiovascular disease. Ann Intern Med 2006;145:35–42. Ridker PM, Rifai N, Cook NR, Bradwin G, Buring JE. Non-HDL cholesterol, apolipoproteins A-I and B100, standard lipid measures, lipid ratios, and CRP as risk factors for cardiovascular disease in women. J Am Med Assoc 2005;294:326–33. AbuMweis SS, Vanstone CA, Ebine N, Kassis A, Ausman LM, Jones PJ, et al. Intake of a single morning dose of standard and novel plant sterol preparations for 4 weeks does not dramatically affect plasma lipid concentrations in humans. J Nutr 2006; 136:1012–6. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18:499–502. Senn S. Cross-over trials in clinical research. 2nd ed. ISBN0-47149653-7; 2002.
ARTICLE IN PRESS 798 24. Smith Jr. SC, Allen J, Blair SN, Bonow RO, Brass LM, Fonarow GC, et al. AHA/ACC guidelines for secondary prevention for patients with coronary and other atherosclerotic vascular disease: 2006 update: endorsed by the National Heart, Lung, and Blood Institute. Circulation 2006;113: 2363–72. 25. Korpela R, Tuomilehto J, Hogstrom P, Seppo L, Piironen V, SaloVaananen P, et al. Safety aspects and cholesterol-lowering efficacy of low fat dairy products containing plant sterols. Eur J Clin Nutr 2006;60:633–42. 26. Jones PJ, Vanstone CA, Raeini-Sarjaz M, St-Onge MP. Phytosterols in low- and nonfat beverages as part of a controlled diet fail to lower plasma lipid levels. J Lipid Res 2003;44:1713–9. 27. Richelle M, Enslen M, Hager C, Groux M, Tavazzi I, Godin JP, et al. Both free and esterified plant sterols reduce cholesterol
M.B. Madsen et al. absorption and the bioavailability of beta-carotene and alphatocopherol in normocholesterolemic humans. Am J Clin Nutr 2004;80:171–7. 28. Yusuf S, Hawken S, Ounpuu S, Dans T, Avezum A, Lanas F, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet 2004;364:937–52. 29. Sniderman AD, Furberg CD, Keech A, Roeters van Lennep JE, Frohlich J, Jungner I, et al. Apolipoproteins versus lipids as indices of coronary risk and as targets for statin treatment. Lancet 2003;361:777–80. 30. Walldius G, Jungner I, Holme I, Aastveit AH, Kolar W, Steiner E. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet 2001;358:2026–33.
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ORIGINAL ARTICLE
The effect of a combination of plant sterol-enriched foods in mildly hypercholesterolemic subjects Martin B. Madsen, Anne-Mette Jensen, Erik B. Schmidt ˚rhus University Hospitals, Aalborg Hospital, Sdr. Skovvej 15, Department of Cardiology, Center for Cardiovascular Research, A 9000 Aalborg, Denmark Received 5 December 2006; accepted 24 May 2007
KEYWORDS LDL cholesterol; Apolipoprotein B; Apolipoprotein A-I; Phytosterols; Coronary heart disease
Summary Background & aims: The purpose of this study was to evaluate the effect of low-fat products enriched with plant sterols in addition to a National Cholesterol Education Program step 1 diet on serum lipids and lipoproteins. Methods: This study was a double-blind, randomised, placebo-controlled cross-over design with a run-in period and 2 intervention periods, each lasting 4 weeks. A total of 46 mildly hypercholesterolemic subjects (age 50.679.8) completed the trial. The study products consisted of 20 g low-fat margarine (35% fat) and 250 ml low-fat milk (0.7% fat), in total delivering 2.3 g plant sterols/d. Results: Serum total and low-density lipoprotein cholesterol were significantly reduced by 5.5% (po0.001, 95% CI: 2.5; 8.3) and 7.7% (p ¼ 0.001, 95% CI: 3.4; 11.9), respectively, by plant sterol-enriched products compared to placebo. Serum apolipoprotein B was significantly reduced by 4.6% (po0.05, 95% CI: 1.7; 7.5), and apolipoprotein B/ apolipoprotein A-I by 3.4% (po0.05, 95% CI: 0.1; 6.6) after plant sterol intake compared to the placebo supplement. Conclusions: A combination of low-fat margarine and milk enriched with plant sterols significantly reduced low-density lipoprotein cholesterol, apolipoprotein B and the ratio of apolipoprotein B to apolipoprotein A-I in mildly hypercholesterolemic subjects, but had no effect on C-reactive protein and lipoprotein (a) concentrations. Sponsorship: Unilever Denmark A/S. & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Abbreviations: Apo, apolipoprotein; HDL, high-density lipoprotein; HsCRP, high sensitivity C-reactive protein; LDL, low-density lipoprotein; Lp(a), lipoprotein (a); NCEP, National Cholesterol Education Program Corresponding author. Tel.: +45 99326899; fax: +45 99326804. E-mail address: [email protected] (E.B. Schmidt). 0261-5614/$ - see front matter & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2007.05.008
ARTICLE IN PRESS Consumption of 2.3 g plant sterols/d reduces CHD risk factors
Introduction Plasma cholesterol is a major risk factor for coronary heart disease (CHD) and lowering of low-density lipoprotein (LDL) cholesterol concentrations significantly reduces CHD mortality.1 The first step to reduce LDL cholesterol levels is by lifestyle measures including dietary changes, in particular a reduction in the intake of total and saturated fat.2 The effect of such changes is variable, with a mean reduction in LDL cholesterol in the order of 5–10%.3 Consumption of 2 g plant sterols/day is also among the recommendations from the National Cholesterol Education Program (NCEP) to lower elevated LDL cholesterol concentrations,2 as plant sterols and stanols inhibit dietary and biliary cholesterol absorption in the gut.4 On average, a dose of 2 g plant sterols/day have resulted in a 10% reduction in LDL cholesterol which is additive to the cholesterol-lowering effect of a low-fat diet.5 Plants sterols naturally occur in foods in low concentrations, with an estimated daily intake below 400 mg.6 In addition to fatty foods such as margarines, plant sterols and stanols have also been recently incorporated in low-fat food formats, such as low-fat yoghurts,7–9 milk9–11 and yoghurt minidrinks.12,13 While these new food formats offer alternatives to lowering of cholesterol concentrations, only a few studies have evaluated the impact of combinations of plant sterol-enriched foods on LDL cholesterol levels.10,14,15 There is a growing body of evidence suggesting that apolipoprotein B/apolipoprotein A-I (apo B/apo A-I) ratio is a better marker of CHD risk than lipid concentrations per se and lipid ratios such as LDL:HDL cholesterol and total:HDL cholesterol.16,17 In the few studies that investigated the effect of plant sterol undertaken so far, evaluation of CHD risk has been limited to traditional risk factors such as lipid concentrations and ratios.10,14,15 Another emerging risk factor related to the inflammatory hypothesis of CHD is C-reactive protein (CRP).18–20 Data on the potential impact of plant sterol supplementation on this marker are, however, very scarce.21 The objective of the present study was therefore to evaluate the impact of combining two different plant sterol-enriched foods as part of a low-fat diet on CHD risk, with a particular attention to emerging risk factors for CHD such as apo B/apo A-I ratio and CRP.
Subjects and methods Subjects Fifty volunteers were recruited by advertisement among patients from the outpatient Lipid Clinic and employees of Aalborg Hospital, and among employees of a local insurance company. Moderately hypercholesterolemic (LDL cholesterol 3.0–5.5 mmol/l), otherwise healthy men and women aged 20–70 y, with a body mass index (BMI) of 20–35 kg/m2 were eligible to participate in the study. For subjects on medication, including statins, the type and dosage had to be stable for at least 6 weeks prior to screening. Individuals who reported having taken medications known to interfere with cholesterol absorption (e.g. ezetimibe, bile acid sequestrants) or had used plant sterol- or stanol-enriched
793 products during the previous 6 weeks were excluded. Subjects suffering from serious illnesses, diabetes mellitus, familial hypercholesterolemia, gastrointestinal disorder or lactose intolerance were excluded, as well as those consuming more than 21 units of alcohol per week (one unit corresponding to 12 g alcohol). Pregnant women and those planning to be pregnant or breastfeeding, and subjects who had participated in another trial within 3 months of study entry or were engaged in intense physical activities (410 h per week) were not eligible. All subjects provided written informed consent before entering the study. The study protocol was approved by the regional Ethics Committee.
Design and diets The study was a randomised, double-blind, placebo-controlled, cross-over trial consisting of two 4-week intervention phases. During both the placebo and the plant sterol phases participants followed the NCEP step 1 diet.2 In order to allow for stabilisation of dietary patterns prior to the start of the treatments, a 4-week run-in period preceded the trial. All subjects were provided dietary advice by an experienced clinical dietician before the start of the run-in period in order to meet the guidelines of the NCEP step 1 diet (total fat contributing to a maximum of 30% of total energy intake, saturated fat contributing to less than 10% of energy, not more than 300 mg cholesterol/d, 15% and 55% of energy provided by proteins and carbohydrates, respectively). An adequate fruit and vegetable intake was recommended, including more than one serving per day of carotenoid-rich foods. After inclusion the subjects were asked to use one portion of 20 g low-fat spread and one 250 ml serving of low-fat milk, to be consumed on three different occasions each day, with main meals. They were encouraged to maintain their normal activity pattern and body weight. At the end of the run-in period, half of the participants were assigned to consuming the placebo products for 4 additional weeks, whilst the other half was assigned to the plant sterol-enriched margarine and milk. At the end of the first phase, subjects on the plant sterolenriched diet switched to the placebo products, and those on the placebo switched to the plant sterol-enriched foods for 4 weeks without wash-out between treatments. The placebo and the plant sterol-enriched spread and milks were provided by Unilever Denmark and were all packed in black containers and coded such that both the study investigators and the participants were blinded. Randomisation was done by Unilever Research, The Netherlands, and treatment codes were broken after the statistical analyses were performed. The composition of the placebo and the plant sterol-enriched spread and milk is presented in Table 1. The placebo and plant sterol-enriched products had a similar composition, except for the content of plant sterols. The plant sterol-enriched margarine (20 g/d) provided an equivalent of 1.6 g free plant sterols, and the plant sterol-enriched milk (250 ml/d) contributed 0.7 g plant sterols, for a total intake of 2.3 g plant sterols/day. The spread was a regular low-fat plant sterol-enriched spread similar to the one available in the Danish market. The plant sterols in the spread and milk were esterified to sunflower
ARTICLE IN PRESS 794
Table 1
M.B. Madsen et al.
Nutritional composition of the investigational products used in the trial.
Per 100 g
Energy (kJ) Proteins (g) Carbohydrates (g) Fat (g) PUFA (g) MUFA (g) SAFA (g) Plant sterols (g)
Margarine
Milk
Placebo
Plant sterol-enriched
Placebo
Plant sterol-enriched
1385a 0.1a 5.5a 37.4 17.4 9.9 10.8 0.1
1365a 0.1a 4.0a 36.8 17.0 9.9 8.4 8.0
146 3.5 5.1 o0.1 o0.1 o0.1 o0.1 o0.01
168 3.6 5.0 0.6 0.4 0.2 0.2 0.3
PUFA: polyunsaturated fatty acids, MUFA: monounsaturated fatty acids, SAFA: saturated fatty acids. Note: The values for polyunsaturated fat, monounsaturated fat, and saturated fat have been calculated from the % of PUFA, MUFA and SAFA of all fatty acids (46.5%, 26.6%, 23.2% of PUFA, MUFA and SAFA, respectively, in the control spread, and 46.1%, 26.8%, and 22.8% of PUFA, MUFA and SAFA in the plant sterol-enriched spread). a Calculated values from the recipe; otherwise, measured values.
based on data obtained by Dual Energy X-ray Absorptiometry (DXA).
oil fatty acids and consisted mainly of 69% b-sitosterol, 15% campesterol and 8% stigmasterol, with other plant sterols contributing to the rest. Dietary intake was assessed by food records completed on three consecutive days (including one week-end day) before screening (start of the run-in period), at randomisation (baseline) and at the end of the first and second study phases. Nutrient intakes were analysed using the Master Diaetist System version 1.223, 2006 Master Data I/S, Copenhagen, Denmark. A questionnaire was given to the subjects at the beginning of the run-in phase and again at the beginning of each treatment phase to record every day consumption of the study products. All product packages were returned to the investigators and counted. Compliance was calculated from both the records and the returned packages as the number of servings consumed relative to scheduled servings. A subject was considered compliant if he/she had consumed 80% or more of the study products. Weight and body fat percentage were measured at run-in, baseline and after each intervention period, whereas height and blood pressure were recorded only at baseline.
Fifty subjects were included in the trial, based on an 80% power to detect a 10% decrease in LDL cholesterol with the plant sterol-enriched vs the placebo treatment, and allowing for a 10% drop-out rate. A per-protocol analysis was performed using the subjects that completed the study according to the protocol. The change in concentration of serum parameters measured at the end of the placebo and the plant sterol phases were compared both on an absolute scale (using paired t-tests on a log-scale). We assumed that there was no carry-over effect.23 The analyses were carried out both on an absolute scale (original scale) and a relative scale (log-scale). Model diagnostics have included histograms, quantile–quantile plots and Bland–Altman plots and revealed for all variables that both scales could in principle be used. A p-value o0.05 (two-tailed) was considered statistically significant.
Analytical procedures
Results
Blood was collected in the morning after at least 10 h of fasting and the serum samples were stored at 80 1C until final analyses. Serum total cholesterol, HDL cholesterol (precipitation method) and triglycerides were measured enzymatically while LDL cholesterol concentrations were calculated according to the Friedewald equation.22 Apo B and apo A-I were measured using the respective antibodies from DAKO on an Advia 1650 (Bayer, Diagnostics, NY, USA). Lipoprotein (a) (Lp(a)) concentrations were determined using an ELISA kit from Mercodia. High sensitivity C-reactive protein (hsCRP) concentrations were measured by an immunotorbidimetric assay from Randox on an Advia 1650. Body fat percentage was determined using a Tanita BC418MA Body Composition Analyzer which calculates the body fat percentage by using Bioelectrical Impedance Analysis
A total of 46 of the 50 participants completed the study. During the run-in period, one subject dropped out due to disliking of the margarine and another was excluded due to lack of compliance. These two subjects were not included in the final data analysis. During the first trial arm, one subject receiving the plant sterol-enriched products was excluded due to an intercurrent disease considered unrelated to the study products, and one subject receiving the placebo products left the study because of disliking the margarine. Characteristics of the 48 participants at baseline are presented in Table 2. No significant change in weight, BMI or body fat percentage was observed during the trial between the subjects receiving intervention products and the subjects receiving placebo products (data not shown).
Statistical analysis
ARTICLE IN PRESS Consumption of 2.3 g plant sterols/d reduces CHD risk factors There was no significant change in fat and total energy intake during the trial between the participants in the intervention and placebo phase (Table 3). During the plant Table 2 pants.
Baseline characteristics of the study partici-
Group
AB
BA
n
24 50.879.6 9/15 5
24 50.3710.3 3/21 3
76.3710.4 25.972.8 30.878.5
68.2713.6 24.173.3 3276.1
5.170.7 3.370.6 1.270.3 3.870.7
5.870.9 3.770.8 1.570.4 4.270.9
1.270.6 1.070.2 1.370.2 0.870.2 121.5 (6–1177) 1.41 (0–28)
1.170.5 1.170.2 1.570.2 0.870.2 178 (11–1999) 0.675 (0–3)
137719 8379
131721 81710
Age (years) Gender (men/women) Smokers (n) Anthropometric data Body weight (kg) BMI (kg/m2) Body fat (%) Biochemistry Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Non-HDL cholesterol (mmol/l) Triglycerides (mmol/l) Apolipoprotein B (g/l) Apolipoprotein A-I (g/l) Apo B/apo A-I ratio Lipoprotein (a) (IU/L) hsCRP (mg/l) Blood pressure Systolic BP (mmHg) Diastolic BP (mmHg)
Values are means7SD, median (range) or numbers (n) of subjects. AB: subjects receiving plant sterol-enriched products in the first intervention period, BA: subjects receiving plant sterol-enriched products in the second intervention period, BMI: body mass index, LDL: low-density lipoprotein, HDL: high-density lipoprotein, hsCRP: high sensitivity Creactive protein, BP: blood pressure.
Table 3
795 sterol phase, the subjects significantly decreased their intake of dietary fibre by 7.6% (po0.05, 95% CI: 1.2; 13.6) compared to the placebo phase, while other dietary variables did not significantly differ between the interventions. All 46 participants completing the trial were compliant and no adverse effects were reported, but some of the participants reported that 20 g margarine was difficult to consume. The changes in serum lipid concentrations are given in Table 4. Serum total cholesterol and LDL cholesterol decreased significantly by 5.5% and 7.7%, respectively, after consumption of plant sterol-enriched products compared to placebo. The individual results are given in Fig. 1 showing a lowering in LDL cholesterol in 31 of the 46 patients completing the trial, no effect in 6 and an increase in LDL cholesterol in 9 out of the 46 subjects. One patient had an unexplained increase in LDL cholesterol from 2.1 mmol/l at the end of the placebo phase to 3.5 mmol/l (data not shown) at the end of the plant sterol phase but was kept in the analysis. No difference in LDL cholesterol reduction was seen whether the subjects started with either plant sterolenriched or placebo. The reduction in LDL cholesterol or apo B did not depend on the initial LDL cholesterol levels, fat consumption or total energy intake. Non-HDL cholesterol significantly decreased 7.3%. There was no significant change in HDL cholesterol and triglycerides between subjects in the intervention phase and the placebo phase. Serum apo B decreased significantly by 4.6% and apo B/apo A-I by 3.4% after plant sterol-enriched consumption compared to placebo, while neither apo A-I, Lp(a) nor hsCRP was affected by any of the supplements.
Discussion Our results show that including 2.3 g plant sterols per day in the form of plant sterol-enriched margarine and milk in subjects already advised to eat according to a NCEP step 1 diet lowered LDL cholesterol a further 7.7% in mildly hypercholesterolemic subjects. As a new target in the AHA/ACC guideline,24 non-HDL cholesterol was determined
The daily intake of dietary variables at baseline, end of placebo phase and after plant sterol-enriched phase.
n Energy (kJ) Protein (E%) Fat (E%) Carbohydrate E%) Alcohol (E%) PUFA (g) MUFA (g) SAFA (g) Dietary fibres (g) Cholesterol (mg)
Baseline
End of placebo phase
End of plant sterol-enriched phase
48 8 5107267 (2 046764 kcal) 16.770.3 30.570.8 48.370.9 4.470.6 12.270.2 24.571.1 23.271.5 21.470.9 290719
46 8 6887315 (2 079775 kcal) 16.370.3 31.770.9 47.270.9 4.870.6 12.570.5 25.371.3 26.171.7 20.770.8 296719
46 8 9307348 (2 136783 kcal) 16.070.4 33.170.9 45.871.1 5.270.6 13.170.4 27.271.3 26.971.7 19.470.9 304719
Values are means7SEM. PUFA: polyunsaturated fatty acids, MUFA: monounsaturated fatty acids, SAFA: saturated fatty acids.
ARTICLE IN PRESS 796
Table 4
M.B. Madsen et al.
The effect of plant sterols on serum lipids, lipoproteins and hsCRP.
Variable
Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l) LDL cholesterol/HDL cholesterol Total cholesterol/HDL cholesterol Non-HDL cholesterol (mmol/l) Apolipoprotein B (g/l) Apolipoprotein A-I (g/l) Lipoprotein (a) (IU/l) Apolipoprotein B/ Apolipoprotein A-I hsCRP (mg/l)
End of placebo phase
End of plant sterolenriched phase
Difference (95% CI)
Relative change (%) (95% CI)
5.4570.13 3.4570.12 1.4670.05 1.1870.08 2.5470.15
5.1470.11 3.1670.09 1.4770.06 1.0970.07 2.3370.12
0.31c (0.47; 0.14) 0.29c (0.45; 0.14) 0.01 (0.04; 0.06) 0.09 (0.20; 0.01) 0.21b (0.35; 0.08)
5.5c (8.3; 2.5) 7.7b (11.9; 3.4) 0.2 (2.8; 3.0) 6.2 (14.2; 2.6) 7.6b (12.4; 2.4)
3.9570.17
3.7270.15
0.23b (0.38; 0.08)
5.3b (8.7; 1.8)
3.9870.14
3.6770.11
0.32c (0.47; 0.16)
7.3c (11.1; 3.4)
1.0570.03 1.4170.03 301.4758.4 0.7670.03
1.0070.03 1.4070.03 289.3756.3 0.7370.02
0.05b (0.08; 0.02) 0.02 (0.05; 0.02) 12.07 (29.77; 56.37) 0.03a (0.05; -0.003)
4.6b (7.5; 1.7) 1.3 (3.2; 0.8) 5.0 (12.1; 2.6) 3.4a (6.6; 0.1)
1.3970.25
1.5270.24
0.12 (0.18; 0.43)
3.4 (11.8; 21.1)
LDL: low-density lipoprotein, HDL: high-density lipoprotein, hsCRP: high-sensitivity C-reactive protein. a po0.05, b po0.01, c po0.001.
Figure 1 The individual response to the plant sterol-enriched products (difference in LDL cholesterol on sterols vs. placebo). The individual response to the plant sterol-enriched low-fat margarine and milk compared to placebo. Each column represents one individual (in total all 46 subjects concluding the trial). The y-axis is a relative scale i.e. the individual effect is given as % reduction/increase. LDL: low-density lipoprotein.
and was reduced significantly by 7.3%. Apo B was also significantly reduced by 4.6% and the ratio of apo B to apo AI was significantly decreased by 3.4%. There was a decrease in dietary fibre intake in the subjects receiving the intervention products, but it was probably too small to have had an impact on the results. The observed lowering of LDL cholesterol is in accordance with a recent study by Noakes et al.,10 where a combination of milk and margarine-enriched with plant sterols reduced LDL cholesterol by 11.4%. Their study was a single-blind, placebo-controlled cross-over study with 39 participants completing the trial showing no difference in cholesterol
reduction whether a plant sterol-enriched spread (2 g), a plant sterol-enriched milk (2 g) or a combination (4 g) was used. Likewise, our study is in accordance with other studies using plant sterol and stanol intakes of approximately 2 g/d leading to a reduction of 10% in LDL cholesterol.5 Studies with low-fat dairy products have shown similar effects.11,13,25 Thus it seems that the effect of plant sterols and stanols are similar for spreads and dairy products. However, one trial testing plant sterols in low-fat and no-fat beverages did not show a significant reduction in LDL cholesterol,26 in a trial where free plant sterols and not plant sterol esters were used. The fact that free plant sterols were used may not be responsible for the lower efficacy, some studies have shown that both free plant sterols and plant sterol esters decrease intestinal cholesterol absorption.27 Recent data suggest that apo B, apo A-I and hsCRP are important risk factors for CHD.20,28 To our knowledge, only one study with plant sterols has evaluated all these parameters.21 Their findings suggested the same as ours, i.e. that a daily intake of 2 g plant sterols has no effect on apo A-I, Lp(a) and hsCRP, but significantly decreases apo B. Apo B is a strong predictor of coronary events,29,30 and the fact that apo B concentrations were reduced by 4.6% indicates that plant sterol esters do not solely reduce the cholesterol content of LDL cholesterol particles, but also reduce the total number of circulating atherogenic particles. Furthermore, the apo B to apo A-I ratio decreased by 3.4% and, interestingly, this ratio has recently been proposed the strongest marker of risk for vascular disease.28–30 One of the strengths of our study was that the participants were given dietary guidance by an experienced dietician. All the participants filled questionnaires on their diet, ensuring as high an accuracy as possible regarding their
ARTICLE IN PRESS Consumption of 2.3 g plant sterols/d reduces CHD risk factors nutritional intake. Also, this is only the second trial studying the combined effect of plant sterol-enriched margarine and milk, an important feature because patient acceptance of available products are essential for their ultimate use. Finally, the trial is one of only a few studies with evaluation of apolipoproteins and hsCRP. Plant sterols are part of the NCEP2 and the AHA/ACC1 guidelines for secondary prevention of CHD. For each percentage of LDL cholesterol reduction, the risk for CHD disease is reduced by 2–3%. Our study showed that a combination of dietary intervention and low-fat margarine and milk enriched with plant sterols significantly reduced LDL cholesterol, apo B and the apo B to apo A-I ratio. Lowfat products enriched with plant sterols may therefore play a role in managing higher than desirable cholesterol levels.
797
7.
8.
9.
10.
Acknowledgements 11.
Unilever Denmark A/S delivered all trial products and partly sponsored the trial. MSc, PhD, Biostatistician Claus Dethlefsen, Center for Cardiovascular Research, Aalborg Hospital, Århus University Hospitals, Denmark, for statistical analysis. Janne Danielsen, Department of Cardiology, Aalborg Hospital, Århus University Hospitals, Denmark, for dietary calculations. Michelle Gordon, Unilever Food and Health Research Institute, for scientific advise. Isabelle Demonty, Unilever Food and Health Research Institute, for having critically read and commented this manuscript. A-M Jensen and EB Schmidt were responsible for the design and conduct of the study. MB Madsen and EB Schmidt were responsible for the analysis and interpretation of the data. MB Madsen drafted the manuscript. All authors took part in critical revision of the manuscript.
References 1. De Backer G, Ambrosioni E, Borch-Johnsen K, Brotons C, Cifkova R, Dallongeville J, et al. European guidelines on cardiovascular disease prevention in clinical practice Third Joint Task Force of European and other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of eight societies and by invited experts). Atherosclerosis 2004;173:379–89. 2. Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection. Evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). J Am Med Assoc 2001; 285: 2486–97. 3. Hunninghake DB, Stein EA, Dujovne CA, Harris WS, Feldman EB, Miller VT, et al. The efficacy of intensive dietary therapy alone or combined with lovastatin in outpatients with hypercholesterolemia. N Engl J Med 1993;328:1213–9. 4. Normen L, Dutta P, Lia A, Andersson H. Soy sterol esters and beta-sitostanol ester as inhibitors of cholesterol absorption in human small bowel. Am J Clin Nutr 2000;71:908–13. 5. Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti R. Efficacy and safety of plant stanols and sterols in the management of blood cholesterol levels. Mayo Clin Proc 2003;78:965–78. 6. de Vries JHM, Jansen A, Kromhout D, van de Bovenkamp P, van Staveren WA, Mensink RP, et al. The fatty acid and sterol content of food composites of middle-aged men in seven
12. 13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
countries. Journal of Food Composition and Analysis 1997; 10:115–41. Mensink RP, Ebbing S, Lindhout M, Plat J, van Heugten MM. Effects of plant stanol esters supplied in low-fat yoghurt on serum lipids and lipoproteins, non-cholesterol sterols and fat soluble antioxidant concentrations. Atherosclerosis 2002;160: 205–13. Hyun YJ, Kim OY, Kang JB, Lee JH, Jang Y, Liponkoski L, et al. Plant stanol esters in low-fat yogurt reduces total and lowdensity lipoprotein cholesterol and low-density lipoprotein oxidation in normocholesterolemic and mildly hypercholesterolemic subjects. Nutr Res 2005;25:743–53. Clifton PM, Noakes M, Sullivan D, Erichsen N, Ross D, Annison G, et al. Cholesterol-lowering effects of plant sterol esters differ in milk, yoghurt, bread and cereal. Eur J Clin Nutr 2004; 58:503–9. Noakes M, Clifton PM, Doornbos AM, Trautwein EA. Plant sterol ester-enriched milk and yoghurt effectively reduce serum cholesterol in modestly hypercholesterolemic subjects. Eur J Nutr 2005;44:214–22. Thomsen AB, Hansen HB, Christiansen C, Green H, Berger A. Effect of free plant sterols in low-fat milk on serum lipid profile in hypercholesterolemic subjects. Eur J Clin Nutr 2004;58: 860–70. Salo P, Wester I. Low-fat formulations of plant stanols and sterols. Am J Cardiol 2005;96:51D–4D. Volpe R, Niittynen L, Korpela R, Sirtori C, Bucci A, Fraone N, et al. Effects of yoghurt enriched with plant sterols on serum lipids in patients with moderate hypercholesterolaemia. Br J Nutr 2001;86:233–9. Nestel P, Cehun M, Pomeroy S, Abbey M, Weldon G. Cholesterollowering effects of plant sterol esters and non-esterified stanols in margarine, butter and low-fat foods. Eur J Clin Nutr 2001; 55:1084–90. Tikkanen MJ, Hogstrom P, Tuomilehto J, Keinanen-Kiukaanniemi S, Sundvall J, Karppanen H. Effect of a diet based on low-fat foods enriched with nonesterified plant sterols and mineral nutrients on serum cholesterol. Am J Cardiol 2001;88: 1157–62. Sniderman AD, Jungner I, Holme I, Aastveit A, Walldius G. Errors that result from using the TC/HDL C ratio rather than the apoB/ apoA-I ratio to identify the lipoprotein-related risk of vascular disease. J Intern Med 2006;259:455–61. Walldius G, Aastveit AH, Jungner I. Stroke mortality and the apoB/apoA-I ratio: results of the AMORIS prospective study. J Intern Med 2006;259:259–66. Kim JS, Kang TS, Kim JB, Seo HS, Park S, Kim C, et al. Significant association of C-reactive protein with arterial stiffness in treated non-diabetic hypertensive patients. Atherosclerosis 2006; available online 19 June 2006. Lloyd-Jones DM, Liu K, Tian L, Greenland P. Narrative review: assessment of C-reactive protein in risk prediction for cardiovascular disease. Ann Intern Med 2006;145:35–42. Ridker PM, Rifai N, Cook NR, Bradwin G, Buring JE. Non-HDL cholesterol, apolipoproteins A-I and B100, standard lipid measures, lipid ratios, and CRP as risk factors for cardiovascular disease in women. J Am Med Assoc 2005;294:326–33. AbuMweis SS, Vanstone CA, Ebine N, Kassis A, Ausman LM, Jones PJ, et al. Intake of a single morning dose of standard and novel plant sterol preparations for 4 weeks does not dramatically affect plasma lipid concentrations in humans. J Nutr 2006; 136:1012–6. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18:499–502. Senn S. Cross-over trials in clinical research. 2nd ed. ISBN0-47149653-7; 2002.
ARTICLE IN PRESS 798 24. Smith Jr. SC, Allen J, Blair SN, Bonow RO, Brass LM, Fonarow GC, et al. AHA/ACC guidelines for secondary prevention for patients with coronary and other atherosclerotic vascular disease: 2006 update: endorsed by the National Heart, Lung, and Blood Institute. Circulation 2006;113: 2363–72. 25. Korpela R, Tuomilehto J, Hogstrom P, Seppo L, Piironen V, SaloVaananen P, et al. Safety aspects and cholesterol-lowering efficacy of low fat dairy products containing plant sterols. Eur J Clin Nutr 2006;60:633–42. 26. Jones PJ, Vanstone CA, Raeini-Sarjaz M, St-Onge MP. Phytosterols in low- and nonfat beverages as part of a controlled diet fail to lower plasma lipid levels. J Lipid Res 2003;44:1713–9. 27. Richelle M, Enslen M, Hager C, Groux M, Tavazzi I, Godin JP, et al. Both free and esterified plant sterols reduce cholesterol
M.B. Madsen et al. absorption and the bioavailability of beta-carotene and alphatocopherol in normocholesterolemic humans. Am J Clin Nutr 2004;80:171–7. 28. Yusuf S, Hawken S, Ounpuu S, Dans T, Avezum A, Lanas F, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet 2004;364:937–52. 29. Sniderman AD, Furberg CD, Keech A, Roeters van Lennep JE, Frohlich J, Jungner I, et al. Apolipoproteins versus lipids as indices of coronary risk and as targets for statin treatment. Lancet 2003;361:777–80. 30. Walldius G, Jungner I, Holme I, Aastveit AH, Kolar W, Steiner E. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet 2001;358:2026–33.