European Journal of Clinical Nutrition (2008) 62, 263–273 & 2008 Nature Publishing Group All rights reserved 0954-3007/08 $30.00 www.nature.com/ejcn

ORIGINAL ARTICLE Effects of plant sterol and stanol ester consumption on lipid , status and markers of oxidative stress, endothelial function and low-grade inflammation in patients on current treatment

A De Jong1, J Plat1, A Bast2, RWL Godschalk3, S Basu4 and RP Mensink1

1Department of Human Biology, Maastricht University, Maastricht, The Netherlands; 2Department of Pharmacology, Maastricht University, Maastricht, The Netherlands; 3Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands and 4Sections of Geriatrics and Clinical Nutrition Research, Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Sweden

Objective: The present study was designed to examine for the first time, side-by-side, the effects of plant sterol and stanol consumption on lipid metabolism and markers of antioxidant status, oxidative stress, endothelial dysfunction and low-grade inflammation in subjects on stable statin–treatment. Design: Double-blind, randomized, placebo-controlled, intervention trial. Setting: University. Subjects: Forty-five patients on current statin treatment were recruited via newspaper advertisements. Data of 41 patients were used in statistical analysis. Intervention: Subjects consumed with no added plant sterols or stanols for 4 weeks and were then divided into three groups of 15 subjects. For the next 16 weeks, one group continued with the control margarine and the other two groups with either a plant sterol- or stanol (2.5 g/day)-enriched margarine. was sampled at the end of the run-in and intervention periods. Results: Plant sterol and stanol consumption significantly (P ¼ 0.026) reduced low-density (LDL) by 0.34 mmol/l (95% confidence interval (CI), À0.67 to À0.04 mmol/l). No effects were shown on enzymatic and non-enzymatic and markers of oxidative modification of lipids and DNA. In addition, no effect was found on soluble adhesion molecules, C-reactive protein and monocyte chemotactic protein-1 concentrations. Conclusions: We conclude that 16 weeks of plant sterol or stanol consumption did not affect markers of antioxidant status, oxidative stress, endothelial dysfunction and low-grade inflammation in patients on stable statin treatment, despite a significant reduction of LDL cholesterol. European Journal of Clinical Nutrition (2008) 62, 263–273; doi:10.1038/sj.ejcn.1602733; published online 9 May 2007

Keywords: plant sterols; plant stanols; ; cardiovascular disease; endothelial dysfunction; low-grade inflammation

Correspondence: Professor RP Mensink, Department of Human Biology, Maastricht University, PO Box 616, Maastricht, Limburg, 6200 MD, Introduction The Netherlands. E-mail: [email protected] Atherosclerosis, the underlying cause of cardiovascular Guarantor: J Plat. Contributors: AJ conducted the study and statistically analysed the data. RPM diseases (CVDs), is related to low-grade systemic inflamma- and JP designed and supervised the study. AB was responsible for the analyses tion initiated by endothelial dysfunction, caused by factors of the enzymatic antioxidants and MDA. RWLG conducted the 8-oxo-dG such as elevated concentrations of oxidized cholesterol, analysis. SB was responsible for the analysis of 15-PDGH. All authors smoking, obesity and diabetes. These factors may increase contributed to the writing of the paper. Received 27 April 2006; revised 10 January 2007; accepted 19 February 2007; the permeability of the vessel wall for mononuclear cells as published online 9 May 2007 well as the production of cellular adhesion molecules, Effects of plant sterol and stanol ester consumption A De Jong et al 264 cytokines and growth factors, and thus initiate the process subjects were interested and received an information of low-grade inflammation in the arteries (Ross, 1999). In brochure about the purpose and the protocol of the study. support, increased plasma concentrations of circulating Subjects came to the university for two screening visits with adhesion molecules and markers of low-grade systemic an interval of at least 3 days. On both visits, fasting blood inflammation such as soluble vascular cell adhesion mole- was sampled for analyses of total cholesterol and cule 1 (sVCAM-1) (Peter et al., 1997), intercellular adhesion triacylglycerol concentrations, blood pressure was measured molecule 1 (sICAM-1) and C-reactive protein (CRP) (Ridker three times (of which the average was calculated) and height et al., 2000) are associated with future events of CVD. As well and body weight were determined. Furthermore, subjects treatment with drugs, such as the cholesterol-lowering had to complete a medical and general questionnaire. After statins (Liao and Laufs, 2005) and nutritional interventions, screening, 45 patients on stable statin treatment met all of such as a diet high in a-linolenic acid, have been shown to our criteria and started the study. Two subjects dropped out lower circulating concentrations of sICAM, sVCAM and CRP during the run-in period, because they could not consume (Zhao et al., 2004), indicating the possibility to reverse the requested amount of margarine. Thus, 43 subjects (22 endothelial dysfunction in response to interventions. male and 21 female) completed the study. All women, except Plant sterols and stanols, which are structurally related one, were postmenopausal. The Ethics Committee of the to cholesterol, lower serum low-density lipoprotein (LDL) Maastricht University had approved the protocol and all cholesterol levels and are nowadays added to a wide variety subjects signed an informed consent. of foods known as functional foods. Moreover, they obtained a prominent position in the National Cholesterol Education Program guidelines for lowering coronary heart disease risk Diets and design (Expert Panel on Detection, 2001). However, not much is Subjects were asked to replace their own margarine or butter known about the effects of plant sterols and stanols on with the ‘light’ (40% fat) of which 30 g per day markers of endothelial function or low-grade inflammation. should be consumed, divided over at least two meals. For the Furthermore, plant sterols and stanols not only lower serum first 4 weeks, subjects used control margarine without added LDL cholesterol but may also decrease lipid-standardized plant sterols or stanols (run-in period). At the end of the run- concentrations of diet-derived hydrocarbon in period, subjects were randomly allocated to one of the (Katan et al., 2003). Whether the decrease in these anti- three experimental groups, stratified for sex and age. The oxidants has any further consequences is not known. In first group continued with the control margarine, the second theory, it is possible that increased activity of enzymatic group with a plant sterol-enriched margarine and the last antioxidants compensate for the reduction in diet-derived group with a plant stanol-enriched margarine for 16 weeks. carotenoids. However, it is also possible that markers of lipid For the second and third groups, the daily intake of 30 g of peroxidation (oxidized LDL (oxLDL), malondialdehyde margarine equaled an intake of 2.5 g plant sterols or stanols a (MDA) and 15-keto-dihydro-prostaglandin F2a (15-PGDH)) day. Plant sterols and stanols were provided as and DNA damage 7-hydro-8-oxo-2-deoxyguanosine (8-oxo-dG) esters by transesterification of free plant sterols and stanols increase. with rapeseed oil fatty acids. The plant mixtures Therefore, in the present study, we have examined the mainly contained sitosterol ester (49%), campesterol ester effects of plant sterol or stanol consumption, not only on (31%) and stigmasterol ester (16%) and plant stanol mixtures lipid and lipoprotein metabolism and plasma hydrocarbon were obtained by saturation of these sterols, giving sitostanol concentrations, but also on enzymatic anti- ester (69%) and campestanol ester (31%) (Raisio Group, oxidant systems, plasma markers of oxidized lipids, DNA Raisio, Finland). The margarine was packed in tubs of 220 g damage and markers of endothelial function and low-grade each, equivalent to margarine for 7 days. All products were inflammation in subjects on current statin treatment. coded with a color label to blind the subjects and the investigators. The volunteers came to the university at least every 3 weeks to receive the products and for measuring Subjects and methods body weight (without shoes and heavy clothing). At the end of the week, the used tubs had to be set aside and returned to Subjects the department to be weighed back for the calculation of Subjects were recruited via posters in the university and total margarine intake per week. At the end of the study, this hospital buildings and via local newspaper advertisements. information was used to calculate the average daily intake. Inclusion criteria were: current treatment with a 3-hydroxy- At the end of the run-in and experimental periods, subjects 3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitor completed a food frequency questionnaire (Plat and Mensink, (statins), age 18–65 years, body mass index p32 kg/m2,no 2000), in which they recorded their food intake from the proteinuria or glucosuria, diastolic blood pressure p95 mm previous 4 weeks. The dietician checked these questionnaires Hg and systolic blood pressure p200 mm Hg. Subjects with and calculated the composition of the diet according to the clinical manifestations of liver disorders, CVD (o6 months) Dutch food-composition table to estimate the energy and and type II diabetes mellitus were excluded. Seventy-five nutrient intake of the subjects. In a diary, the subjects wrote

European Journal of Clinical Nutrition Effects of plant sterol and stanol ester consumption A De Jong et al 265 down any signs of illness, change of medication and the variation coefficients within runs were 3.2 for total choles- amount of margarine that was used per day. In addition, terol, 2.8 for triacylglycerol, 5.8 for HDL cholesterol, 2.6 for subjects were asked not to change their habitual diet, level of apoA-I and 1.6% for ApoB. physical exercise, smoking habits or use of alcohol during the study. Plant sterols and cholesterol precursors Serum plant sterol (sitosterol, campesterol), plant stanol Blood sampling (sitostanol, campestanol) and cholesterol precursor (latho- Fasting blood samples were taken by venipuncture in weeks sterol) concentrations were analyzed by gas chromatography 0, 3, 4, 7, 19 and 20. Subjects were not allowed to eat after as described before (Plat and Mensink, 2001). Serum samples 20.00 h the day preceding blood sampling, smoke on the of weeks 3 and 4 and weeks 19 and 20 were pooled before morning of the blood sampling and use alcohol 24 h before analysis. Samples of one subject were analyzed in the same blood sampling. Venipuncture was performed in the ante- analytical run. Plant sterol and stanol concentrations were cubital vein with a vacutainer system and 0.8 Â 38 mm sterile expressed per mmol total cholesterol. needles. Blood was sampled in 10 ml serum separator tubes for analysis of lipids and (apo), serum plant sterol and stanols and clinical safety measurements (aspar- LDL-receptor mRNA agines–aminotransferase (ASAT), alanine-aminotransferase PBMCs were isolated from 20 ml EDTA blood by Lympho- (ALAT), g-glutamyl transpeptidase (g-GT), total bilirubin prep density gradient centrifugation (Nycomed Pharma AS, and creatinine). Serum was obtained by centrifugation at Oslo, Norway) according to instructions of the manufacturer. 2000g for 30 min at 41C, minimally 1 h after blood sampling. The isolated cell pellet was taken up in 1500 ml Trizol Next, three 10 ml ethylenediaminetetraacetic acid (EDTA) (GIBCO, Invitrogen, Breda, The Netherlands) and immedi- tubes were drawn for RNA isolation from peripheral blood ately stored at –801C until further isolation according mononuclear cells (PBMCs), for hematological parameters standard protocols. Following DNAse treatment (Promega and for preparing EDTA plasma. Plasma, obtained by Benelux B.V., Leiden, The Netherlands), cDNA was synthe- centrifugation of the EDTA tube at 2000g for 30 min at 41C, sized as described before (Plat and Mensink, 2002b). LDL- was analyzed for fat-soluble antioxidants, soluble adhesion receptor mRNA expression was determined by real-time molecules, CRP and monocyte chemotactic protein (MCP)-1. PCR. Oligonucleotide primers and probes for both the Finally, 4 ml heparin blood was sampled and analyzed for LDL receptor (forward 50-GAGAAGAAGCCCAGTAGCGTG, enzymatic antioxidants concentrations of the red blood cells reverse 50-GCTGTTGATGTTCTTAAGCCGC, probe 50-TGTCC (RBCs). All tubes and needles were from Becton, Dickinson TCCCCATCGTGCTCCTC) and b-actin as housekeeping gene and Company (NJ, USA). All plasma and serum samples were (forward 50-AGCCTCGCCTTTGCCGA, reverse 50-CTGGT snap-frozen and stored in small aliquots directly after GCCTGGGGCG, probe 50-CCGCCGCCCGTCCACACCCG sampling at À801C. CC) were purchased from Sigma Genosys (UK). LDL receptor and b-actin mRNA concentrations from one subject before and after the intervention were always analyzed in duplo in Lipids and apolipoproteins the same analytical run. The temperature program consisted Total cholesterol, high-density lipoprotein (HDL) cholesterol of 2 min at 501C, 10 min at 951C, and 45 cycles of 15 s at 951C and triacylglycerol concentrations were analyzed in all and 1 min at 601C. To quantify the LDL-receptor expression, serum samples using a semiautomatic COBAS Mira analyzer the comparative CT method (User Bulletin #2; ABI Prism (Roche, Basel, Switzerland) by using commercial available 7700 Sequence Detection System, Applied Biosystems), in kits (cholesterol: CHOD-PAP method; Roche Diagnostic which the expression of the LDL receptor gene was normal- Systems, Hofmann-La Roche Ltd, Basel, Switzerland; triacyl- ized against the housekeeping gene b-actin, was used. The glycerol: GPO-trinder method; Sigma Diagnostics, St Louis, given difference resulted in the reported DDCT value. USA). HDL was determined by the precipitation method by adding a mixture of phosphotungstic acid and magnesium ions to the sample and the CHOD-PAP method (Monotest Antioxidants and markers of endothelial function and low-grade cholesterol; Boehringer Mannheim GmbH, Mannheim, inflammation Germany). LDL cholesterol was calculated by Friedewald At the end of the run-in period (weeks 3 and 4) and the formula (Friedewald et al., 1972). experimental period (weeks 19 and 20) a broad spectrum of Apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB) antioxidants and markers reflecting low-grade inflammation were measured using an immunoturbidimetric reaction and endothelial (dys)function were measured in EDTA (UNI-KIT apoA-I and UNI-KIT apoB; Roche, Basel, Swiss). plasma. Soluble E-selectin and soluble ICAM were measured Samples of weeks 3 and 4 and samples of weeks 19 and 20 by enzyme-linked immunosorbent assay (ELISA) as described were pooled before analyses of apoA-I and apoB. All samples by Bouma et al. (1997) and Leeuwenberg et al. (1992). Soluble from one subject were analyzed within the same run. The VCAM-1 and MCP-1 were measured with commercially

European Journal of Clinical Nutrition Effects of plant sterol and stanol ester consumption A De Jong et al 266 available ELISA kits (R&D Systems Europe Ltd, Abingdon, Counter (Coulter MD series, Beckmann Coulter Inc., Miami, UK), according to the manufacturer’s instructions. Hs-CRP FL, USA). None of these parameters were affected by the was measured on Cobas Mira with a commercially available treatments (data not shown). kit (Kamiya Biomedical Company, Seattle, WA, USA). For all performed assays, samples were analyzed in duplo. Plasma-oxidized LDL concentrations were measured by a Statistics commercially available sandwich ELISA (Mercodia, Uppsala, Forty-three subjects completed the study. The results of two Sweden) with the specific murine monoclonal antibody subjects were however excluded from the statistical analyses. mAb-4E6 as described by Holvoet et al. (1998). For one subject from the control group, the statin dose was and carotenoids were analyzed by reversed-phase high- doubled (from 20 to 40 mg simvastatin) during the study, performance liquid chromatography (HPLC) as described whereas a second subject from the control group was before (Plat and Mensink, 2001). As a marker for enzymatic excluded because he did not consume the requested amount lipid peroxidation, 15-PGDH was measured by radioimmuno- of margarine and did not come to the appointments of the assay as described before (Basu, 1998). MDA was measured in last 4 weeks. Thus, the statistical analyses were performed on plasma using a fluorescent thiobarbituric acid assay as the results of 41 subjects, 20 males and 21 females. described before (Lepage et al., 1991) (Guillen-Sans and Guz- The responses to treatment for each subject were calcu- man-Chozas, 1998). To assess oxidative DNA damage, DNA was lated as the difference between values obtained at the end of isolated from PBMCs by the QIAmp DNA Blood Midi Kit the experimental period (means of values of weeks 19 and (QIAgen Benelux B.V., Venlo, The Netherlands) and subse- 20) and the run-in period (means of values of weeks 3 and 4). quently 8-oxo-dG was determined by HPLC with electroche- Differences in changes between the treatment groups were mical detection (Briede et al., 2004). Finally, erythrocyte analyzed by one-way analysis of variance. When a significant samples were analyzed for erythrocyte glutathione concentra- diet effect was found, treatments were compared pair wise tions as described by Paglia and Valentine (1967) and Flohe and and corrected for three group comparisons (a ¼ 0.017) using Gunzler (1984). The analyses of catalase concentrations (Aebi, the Bonferroni multicomparison test. Contrast analysis was 1984) and superoxide dismutase (SOD) activities (Kirkova et al., performed to analyze the effects of plant sterol and stanol 1999) were performed as described before. consumption as a group against the control group (a ¼ 0.05). All statistical analyses were performed with SPSS 11.0 for Mac Os X (SPSS Inc., Chigaco, CA, USA). Clinical safety parameters and hematological measurements Concentrations of liver and kidney enzymes (total bilirubin, ASAT, ALAT, alcalic phosphatase, g-GT and creatinine) were Results determined at the Department of Clinical Chemistry, University Hospital Maastricht, Maastricht, The Netherlands Dietary intakes, margarine consumption and body weight (Beckman Synchron CX7 Clinical Systems, Beckman). Table 1 shows that baseline characteristics were not sig- Various hematological parameters (white blood cell count, nificantly different between subjects of the control and percentages and numbers of lymphocytes, mononuclear cells intervention groups. Energy intake and the proportions of and granulocytes, RBC count, hemoglobin concentration, energy from carbohydrates, fatty acids and proteins as well as hematocrit, mean corpuscular volume, platelet count and cholesterol intake did not change significantly during the platelet volume) were determined by using a Coulter study (Table 2). Mean estimated daily intake of the

Table 1 Subject baseline characteristics

Control group Plant sterol group Plant stanol group

Male/female 4/7 8/7 8/7 Age (years) 57.875.8a 58.479.9 58.777.8 BMI (kg/m2) 27.372.4 26.472.8 26.872.9 Smoking 0 3 0 Total cholesterol (mmol/l) 5.4270.67 5.7571.10 5.1971.05 HDL cholesterol (mmol/l) 1.5270.40 1.2870.25 1.5870.45 LDL cholesterol (mmol/l) 3.2070.55 3.5771.04 3.4470.80 Triacylglycerol (mmol/l) 1.5570.53 1.9470.70 1.3070.79 Type of statin Six atorvastatin Six atorvastatin Seven atorvastatin Four simvastatin Five simvastatin Four simvastatin One pravastatin Four pravastatin Four pravastatin

Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein. ax7s.d. (all such values).

European Journal of Clinical Nutrition Effects of plant sterol and stanol ester consumption A De Jong et al 267 Table 2 Daily intake of energy and nutrients during the study Serum lipids and apolipoproteins Plant sterols and stanols lowered serum total cholesterol Control group Plant sterol group Plant stanol group concentrations by 0.39 mmol/l or 6.9% as compared with the Energy (MJ) control group (P ¼ 0.052 for the difference in absolute Run in 8.9271.59a 8.9071.47 9.3572.08 changes; 95% CI, À0.78 to 0 mmol/l) and LDL cholesterol Experimental 8.4071.44 8.7771.99 9.2172.63 by 0.35 mmol/l or 10.3% (P ¼ 0.028; 95% CI, À0.67 to 7 7 7 Change À0.53 1.39 À0.13 1.67 À0.14 1.48 À0.04 mmol/l). Triacylglycerol (P ¼ 0.365) and HDL choles- Fat (energy %) terol (P ¼ 0.571) concentrations were not significantly Run in 34.5675.49 34.9877.14 36.0573.77 changed (Table 3). No significant difference in cholesterol- Experimental 34.4674.30 34.8576.87 35.0575.61 lowering effect was found between plant sterols and stanols. 7 7 7 Change À0.09 4.38 À0.13 5.24 À1.00 3.26 ChangesinconcentrationsofapoAIandapoB100werenot SAFA (energy %) significantly different between the control and plant sterol and Run in 12.2972.22 12.4073.30 12.3472.26 stanol groups. However, baseline concentrations (r ¼ 0.757, Experimental 12.3072.86 12.3973.05 12.2572.58 Po0.001) as well as changes in concentrations (r ¼ 0.795, Change 0.0172.12 0.0072.46 À0.0971.40 Po0.001) of LDL cholesterol and apoB correlated positively. MUFA (energ y %) Run in 12.3072.42 12.2972.64 13.0071.73 Experimental 12.1071.71 12.2272.68 12.4772.68 Plant sterols and cholesterol precursors 7 7 7 Change À0.20 1.59 À0.07 2.32 À0.53 1.54 As expected, plant sterol consumption increased cholesterol- PUFA (energy %) standardized campesterol concentrations significantly by Run in 7.6271.75 7.9571.65 8.2771.72 59% (P ¼ 0.004) as compared with changes in the control Experimental 7.7370.87 7.9371.47 7.9671.87 group. Also cholesterol-standardized sitosterol concentra- 7 7 7 Change 0.11 1.29 À0.02 1.04 À0.32 1.84 tions increased by 42% as compared with the control group Protein (energy %) (P ¼ 0.022). In contrast, plant stanol consumption decreased Run in 16.9871.95 16.6172.01 17.5772.62 cholesterol-standardized concentrations of campesterol Experimental 17.0471.78 17.0673.54 17.0572.59 (84%) and sitosterol (64%) significantly (po0.001) as 7 7 7 Change 0.06 1.97 0.45 2.62 À0.52 1.75 compared with the plant sterol group. Cholesterol-standar- Carbohydrates (energy %) dized sitostanol and campestanol concentrations did not Run in 46.7076.47 46.1878.48 44.1776.81 change significantly in any of the groups. Changes in Experimental 47.1973.96 46.4977.84 46.3978.32 cholesterol-standardized lathosterol concentrations were not 7 7 7 Change 0.49 5.54 0.32 5.34 2.22 3.17 significantly different between the three groups (P ¼ 0.860). Cholesterol (mg/MJ) Run in 24.0474.93 22.1675.38 24.4875.11 Experimental 23.1074.90 22.7778.54 23.4977.40 LDL-receptor mRNA 7 7 7 Change À0.95 3.11 0.61 7.92 À0.99 3.60 Expression of LDL-receptor mRNA did not change in the Abbreviations: MUFA, mono unsaturated fatty acids; PUFA, poly unsaturated plant sterol and stanol groups as compared with the control fatty acids; SAFA, saturated fatty acids. group (P ¼ 0.929). The DDCT value for the control group was a x7s.d. (all such values). 1.1170.84 and for the experimental groups 1.1470.62. margarines approximated the targeted amount of 30 g and Antioxidants and markers of endothelial function and low-grade was 29.171.1 g for the control group, 29.671.3 g for the inflammation plant sterol group and 30.171.0 g for the plant stanol group, Plant sterol and stanol consumption did not affect LDL– which was not significantly different between the groups cholesterol-standardized concentrations of the hydro- (P ¼ 0.110). This means that total daily plant sterol and carbon carotenoids (Table 4), that is, a-carotene (P ¼ 0.273), stanol intake was 2.5 g per day. During the study, body b-carotene (P ¼ 0.815) and lycopene (P ¼ 0.320). Also, the weight changed in the control group by þ 0.471.3 kg, in the sum of these hydrocarbon carotenoids (P ¼ 0.677) as well as plant sterol group by À0.271.4 kg and in the plant stanol concentrations of the other lipid soluble antioxidants group by –0.771.6 kg (P ¼ 0.227 for treatment effects). (oxygenated carotenoids and tocopherols) did not change. Inspections of the diaries did not reveal any deviations from With respect to the enzymatic antioxidants (Table 5), plant the protocol that may have affected the results. At the end of sterol and stanol consumption did not affect RBC catalase the experimental period, subjects were asked what kind of (P ¼ 0.693) and SOD (P ¼ 0.742) concentrations or glu- margarine they had consumed. This question was answered tathione peroxidase (GpX) activity (P ¼ 0.606). correctly by 17% of the control group, 7% of the plant sterol Plant sterol and stanol consumption had no significant and 0% of the plant stanol group, illustrating that blinding effects on markers of oxidative stress, that is, oxLDL, MDA, had been successful. 15-PGDH and 8-oxo-dG concentrations. When changes in

European Journal of Clinical Nutrition Effects of plant sterol and stanol ester consumption A De Jong et al 268 Table 3 Effects of plant sterol and stanol esters on serum lipid and apolipoprotein concentrations during the study

Control group Plant sterol group Plant stanol group P-value control vs plant sterol/stanol P-value plant sterol vs stanol

Total cholesterol (mmol/l) Run in 5.4270.671 5.7571.10 5.5970.89 Experimental 5.4670.77 5.4571.11 5.1971.05 Change 0.0470.42 À0.3070.56 À0.4070.61 0.052 0.628

HDL cholesterol (mmol/l) Run in 1.5270.40 1.2870.25 1.5870.45 Experimental 1.5170.35 1.3270.35 1.5970.59 Change À0.0170.17 0.0470.16 0.170.12 0.571 0.628

Triacylglycerol (mmol/l) Run in 1.5570.51 1.7570.70 1.3070.79 Experimental 1.6271.10 1.6371.04 1.2770.75 Change 0.0870.29 À0.1270.64 À0.0370.31 0.365 0.630

LDL cholesterol (mmol/l) Run in 3.2070.57 3.5771.04 3.4470.80 Experimental 3.2270.43 3.3071.01 3.0270.88 Change 0.0170.37 À0.2770.39 À0.4270.53 0.027 0.339

ApoA1 (mg/dl) Run in 161727 146721 155725 Experimental 159727 147723 161728 Change À2715 1710 6711 0.280 0.248

ApoB (mg/dl) Run in 103716 105719 103721 Experimental 102713 101722 95721 Change À177 À4712 À7713 0.250 0.390

TC/HDL ratio (units) Run in 3.770.7 4.671.1 3.871.2 Experimental 3.770.7 4.371.1 3.571.1 Change 0.070.4 À0.370.5 À0.370.6 0.061 0.983

Abbreviations: ApoA-I, apolipoprotein A-I; ApoB, apolipoprotein B; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol. ax7s.d. (all such values).

concentrations of oxidized LDL were standardized for sterol and stanol consumption for 16 weeks did not change changes in apoB100, still no significant effects was found markers of low-grade inflammation and endothelial function (Table 6). Baseline oxLDL concentrations correlated with in subjects on stable statin therapy. Also, concentrations of catalase concentrations (r ¼ 0.389, P ¼ 0.017) and GpX lipid soluble and enzymatic antioxidants as well as markers activities (r ¼ 0.606, Po0.001). Baseline CRP concentrations reflecting oxidative modification of lipids and DNA, did not also correlated with catalase concentrations (r ¼ 0.523; change. P ¼ 0.001). No other significant correlations were found. Numerous intervention trials in healthy subjects as well The results for markers of low-grade inflammation, as in various patient groups have consistently shown that endothelial function and oxidative damage can be found plant sterol and stanol esters lower LDL cholesterol, when in Table 6. Plant sterol and stanol consumption did not incorporated into a wide variety of food products. Several change plasma concentrations of the soluble adhesion side-by-side comparisons further suggest that plant sterol molecules ICAM, VCAM-1 and E-selectin. Also, plasma and stanol esters are equally effective, at least in normo- and concentrations of MCP-1 and CRP were not significantly hypercholesterolemic subjects not using cholesterol-lower- changed after the intervention period. ing medication (Hallikainen et al., 2000); (Weststrate and Meijer, 1998; Normen et al., 2000). In seven ileostomy patients’, for example, plant sterols and stanols (1.5 g/day) Discussion lowered cholesterol absorption to the same extent (Normen et al., 2000). In hypercholesterolemic subjects, plant sterol In the present study, we found that despite a significant or stanol consumption (2 g/day) resulted in a LDL cholesterol reduction in serum LDL cholesterol concentrations, plant reduction of 10.4 and 12.7%, respectively (Hallikainen et al.,

European Journal of Clinical Nutrition Effects of plant sterol and stanol ester consumption A De Jong et al 269 Table 4 Effects of plant sterol and stanol esters on lipid-soluble antioxidant concentrations during the study

Control group Plant sterol group Plant stanol group P-value ANOVA P-value contrast

Hydrocarbon carotenoids Run in 0.4370.12a 0.3370.07 0.4370.15 Experimental 0.4070.10 0.3670.12 0.4270.22 Change À0.0370.07 0.0370.11 À0.0170.13 0.625 0.690

Lycopene Run in 0.2370.12 0.1670.05 0.2070.07 Experimental 0.2170.13 0.1770.06 0.2070.10 Change À0.0270.07 À0.0270.07 0.0070.09 0.516 0.320 a-Carotene Run in 0.0670.02 0.0670.03 0.0670.03 Experimental 0.0770.02 0.0670.04 0.0770.04 Change 0.0070.01 0.0170.02 0.0170.01 0.517 0.273 b-Carotene Run in 0.1570.03 0.1170.04 0.1770.09 Experimental 0.1570.04 0.1270.05 0.1570.11 Change 0.0070.03 0.0170.04 À0.0170.04 0.309 0.815

Oxygenated carotenoids Run in 0.2670.05 0.2770.13 0.2570.06 Experimental 0.2670.07 0.2770.13 0.2870.08 Change 0.0070.04 0.0070.08 0.0370.07 0.584 0.530

Phytofluene Run in 3.8172.58 2.4470.83 3.3471.20 Experimental 3.6672.50 2.8671.42 2.9770.96 Change À0.1670.97 0.4271.14 À0.3771.19 0.155 0.648

Lutein Run in 0.1470.03 0.1470.07 0.1470.04 Experimental 0.1470.04 0.1570.07 0.1570.04 Change 0.0070.03 0.0070.05 0.0270.04 0.691 0.675

Cryptoxantin Run in 0.1170.04 0.1170.08 0.1070.05 Experimental 0.1070.04 0.1170.07 0.1170.06 Change À0.0170.03 0.0070.05 0.0170.04 0.399 0.443

Total Run in 10.5572.29 10.2572.87 8.8571.64 Experimental 10.1372.41 10.9774.94 9.9772.93 Change À0.4371.36 0.8172.92 0.9471.82 0.168 0.240

Abbreviation: ANOVA, analysis of variance. ax7s.d. (all such values).

2000). Finally, in normo- and mildly hypercholesterolemic (Ketomaki et al., 2005). In our study – also a side-by-side subjects, plant sterols (3.2 g/day) and stanols (2.7 g/day) comparison in subjects on stable statin treatment – LDL lowered LDL cholesterol by 13.1 and 11.9%, respectively cholesterol was lowered by 12.6% in the plant stanol (2.5 (Weststrate and Meijer, 1998). However, only two small g/day) and 8.1% in the plant sterol group (2.5 g/day) as studies have compared side-by-side the effects of plant sterol compared with the control group. This response is smaller and stanol esters in subjects on statin therapy (Ketomaki than that in the studies of Ketomaki et al. (2004, 2005), et al., 2004, 2005). In five subjects with familial hypercho- whereas the intake of plant sterols and stanols was higher. lesterolemia (FH), plant stanol ester consumption (2 g/day) However, in those studies there was no run-in period, which lowered LDL cholesterol by 14.6%. For plant sterol esters means that, unlike in our study, the response may include (2 g/day), a reduction of 16.4% was observed compared with the effect of a change in background diet during the baseline. In their second study in 18 FH subjects on statin intervention. However, the most important aspect of these therapy, plant stanol and sterol consumption (2 g/day) studies is that the combination of plant sterols or stanols lowered LDL cholesterol by 15.3 and 14.2%, respectively with statins was more effective in terms of LDL-cholesterol

European Journal of Clinical Nutrition Effects of plant sterol and stanol ester consumption A De Jong et al 270 Table 5 Effects of plant sterol and stanol esters on enzymatic antioxidant concentrations or activity during the study

Control group Plant sterol group Plant stanol group P-value ANOVA P-value contrast-analysis

Catalase (k/g Hb) Run in 174.5718.3a 184.3739.6 165.7721.1 Experimental 169.8736.3 175.1746.0 173.5723.2 Change À4.8737.7 À9.3738.2 7.8725.9 0.178 0.693

GpX activity (mmol NADPH/g Hb min) Run in 0.0270.01 0.0270.01 0.0270.00 Experimental 0.0270.00 0.0370.01 0.0270.00 Change 0.0070.00 0.0070.00 0.0070.00 0.606 0.961

SOD (U/mg Hb) Run in 2.970.3 2.970.4 2.870.3 Experimental 2.970.4 2.870.3 2.870.4 Change À0.8719.9 À7.0721.5 À1.3711.9 0.742 0.990

Abbreviations: ANOVA, analysis of variance; GpX, glutathione peroxidase; Hb, hemoglobin; NADPH, nicotinamide adenosine dinucleotide phosphate; SOD, super oxide dismutase. ax7s.d. (all such values).

lowering than could be expected from doubling the dose of The change in LDL-receptor expression and cholesterol statins. In general, this results only in an extra 5–7% LDL synthesis can thus be seen as a consequence of the effects cholesterol reduction (Jones et al., 1998; Blair et al., 2000). of plant sterols and stanols in the intestine and not as the The mechanism by which plant sterols and stanols lower cause of the serum LDL cholesterol reduction. Of course, LDL cholesterol becomes more and more clear. It is well the extent of the compensatory reactions, that is, elevated known that these components lower intestinal cholesterol synthesis and LDL-receptor expression, may influence the absorption. To compensate for the reduced cholesterol flux overall reduction in serum LDL cholesterol concentrations. into cells, both endogenous cholesterol synthesis and LDL In addition to the primary effect in the intestine, a decrease receptor expression increase, at least in healthy subjects (Plat in very low density lipoprotein (VLDL) synthesis can add to and Mensink, 2002a). However, plant sterol and stanol the cholesterol-lowering effect. As plant sterols or stanols consumption also lowers LDL cholesterol in FH children and decrease intestinal cholesterol absorption, less cholesterol adults (Ketomaki et al., 2005) who do not have functional will appear into the circulation and enter the liver. A smaller LDL receptors. This indicates that these dietary components cholesterol pool is then available for incorporation into can lower LDL cholesterol independent of LDL-receptor VLDL particles. However, this reduction in pool size will upregulation. Moreover, in the study with FH adults partly be compensated for by elevated endogenous choles- (Ketomaki et al., 2005), plant sterol and stanol consumption terol production. Whether VLDL production was changed at increased endogenous cholesterol synthesis despite statin all and whether this effect does depend on statin treatment treatment. This agrees with findings in non-statin-treated should be unraveled in future studies. subjects and can be explained by the fact that statins do not Plant sterol or stanol consumption can decrease plasma completely block HMG-CoA reductase (Ketomaki et al., concentrations of the hydrocarbon carotenes: a-carotene, 2005). In our study, LDL-receptor expression also did not b-carotene and lycopene. As these fat-soluble antioxidants change, whereas cholesterol synthesis was elevated – though are carried by lipoproteins, correction for changes in total or nonsignificantly – by 42% in the plant sterol group and by LDL cholesterol concentrations is required. It has been 15% in the plant stanol group. The finding that serum LDL estimated that at intakes of plant sterols or stanol 41.5 g/ cholesterol can be decreased and endogenous cholesterol day, total serum cholesterol standardized b-carotene con- synthesis can increase, without a change in LDL receptor centrations decrease by 12.1% on average (Katan et al., activity, seems controversial. The question then arises what 2003). We hypothesized that the decrease in b-carotene mechanism can explain the observed LDL cholesterol would affect enzymatic antioxidant systems. However, reduction in our study. The most likely explanation may be concentrations of the fat-soluble diet-derived antioxidants found in the primary location of the cholesterol-lowering did not change and also activity or concentrations of the effect, that is, the intestine. We have earlier hypothesized enzymatic antioxidants GpX, SOD and catalase did not that the effect of plant sterols and stanols on cholesterol is change. It is possible that the statin background of our because of the effects inside the enterocytes; most likely they subjects interfered with the effects of plant sterols or stanols increase cholesterol efflux back into the intestinal lumen on fat-soluble antioxidant concentrations. In a study of 12 (Plat and Mensink, 2002b). This can also occur in FH months of statin therapy, after the first 12 weeks serum lipid- patients, as it does not involve LDL-receptor functioning. soluble antioxidants were reduced. After 52 weeks, however,

European Journal of Clinical Nutrition Effects of plant sterol and stanol ester consumption A De Jong et al 271 Table 6 Effects of plant sterol and stanol esters on markers of low-grade inflammation and endothelial function

Control group Plant sterol group Plant stanol group P-value ANOVA P-value contrast analysis

OxLDL (pg/ml) Run in 42.9710.8a 43.5711.6 39.5713.5 Experimental 44.8710.0 42.5712.0 37.9712.6 Change 1.974.6 À1.176.6 À1.677.0 0.157 0.807

OxLDL/apoB Run in 0.470.1 0.470.1 0.470.1 Experimental 0.470.1 0.470.1 0.470.1 Change 0.070.1 0.070.1 0.070.1 0.442 0.842

15-PDGH (pg/ml) Run in 49.5713.1 48.979.6 46.6711.3 Experimental 51.2714.0 49.6713.6 43.9712.0 Change 1.778.2 0.8712.9 À2.7713.5 0.607 0.532

TBARS (mmol/l) Run in 0.770.2 0.670.1 0.870.3 Experimental 0.870.2 0.770.1 0.770.2 Change 0.170.2 0.170.1 À0.170.2 0.395 0.179

ICAM (ng/ml) Run in 184.4757.9 178.4752.5 186.1752.4 Experimental 184.4756.2 184.8756.8 185.8741.5 Change À0.0720.5 6.4721.3 À0.4728.2 0.718 0.441

VCAM (ng/ml) Run in 268.8778.8 287.67112.6 280.4797.3 Experimental 261.3786.1 278.67106.6 268.8774.3 Change À7.5749.1 À9.0729.6 À11.6745.2 0.847 0.864

E-selectin (ng/ml) Run in 78.6726.5 80.4747.8 90.1749.7 Experimental 80.7729.1 78.3749.9 87.3742.2 Change 2.174.3 À2.178.8 À2.8712.5 0.183 0.853

MCP-1 (pg/ml) Run in 62.3730.4 74.3735.7 71.0737.3 Experimental 67.7730.1 78.6733.3 70.5738.0 Change 5.3710.0 4.3716.7 À0.5717.1 0.533 0.400

CRP (mg/l) Run in 1.671.8 2.272.4 1.171.3 Experimental 1.871.7 2.372.2 1.171.2 Change 0.271.0 À0.371.2 À0.170.5 0.462 0.495

8-oxo-dG (8-oxo-dG/106dG) Run in 30.5711.5 37.6717.6 33.8716.9 Experimental 30.3712.0 28.5713.1 33.8716.9 Change À0.278.1 À9.1716.5 À5.5721.8 0.412 0.358

Abbreviations: ANOVA, analysis of variance; CRP, C-reactive protein; ICAM, intercellular adhesion molecule; MCP-1, monocyte chemotactic protein-1; 15-keto- dihydro-prostaglandin F2a; 8-oxo-dG, 7-hydro-8-oxo-2-deoxyguanosine; OxLDL; oxidized LDL; TBAR, thiobarbituric acid reactants. ax7s.d. (all such values).

the b-carotene, a-tocopherol and g-tocopherol ratios to lipoproteins, antioxidant concentrations and markers of in LDL-–holesterol were normalized or even increased (Vasankari vivo oxidative stress, we also evaluated markers reflecting et al., 2004). This may indicate that an interaction exists low-grade systemic inflammation and/or endothelial (dys)- between statin treatment and antioxidant concentrations, function. No effects on plasma concentrations of sICAM, which overrules effects of plant sterol or stanol consumption. sVCAM-1, sE-selectin, MCP-1 and CRP were observed. As Also, diet-sensitive markers of in vivo oxidative stress such as 8- hypercholesterolemia is linked to endothelial dysfunction oxo dG (DNA damage) and oxLDL, MDA and 15-PGDH (lipid (Landmesser et al., 2000), it can be hypothesized that lowering peroxidation) did not change. Besides effects on serum LDL cholesterol reduces the low-grade systemic inflammatory

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Am J Cardiol 81, 582–587. Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti R This work was sponsored by the Netherlands Organization (2003). Efficacy and safety of plant stanols and sterols in the for Health Research and Development (Program Nutrition: management of blood cholesterol levels. Mayo Clin Proc 78, Health, Safety and Sustainability, Grant 014-12-010). 965–978.

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