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1 From the School of Dietetics and Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, Montreal (KAV, NE, CAV, and PJHJ), and the Veterans' Hospital, Sainte Anne de Bellevue, Quebec (WEP)
2 Supported by the Heart and Stroke Foundation of Canada. 3 Address reprint requests to PJH Jones, School of Dietetics and Human Nutrition, McGill University, 21,111 Lakeshore Road, Sainte Anne de Bellevue, Quebec, Canada H9X 3V9. E-mail: jonesp{at}macdonal.mcgill.ca.
ABSTRACT
Background: Plant sterol supplementation was shown to reduce total and LDL-cholesterol concentrations, whereas endurance training was shown to increase HDL-cholesterol concentrations and decrease triacylglycerol concentrations.
Objective: The objective was to examine the effect of plant sterols, endurance training, and the combination of plant sterols and endurance training on plasma lipid and lipoprotein cholesterol concentrations, sterol concentrations, and cholesterol precursor concentrations in previously sedentary hypercholesterolemic adults.
Design: In an 8-wk, placebo-controlled, parallel-arm clinical trial, 84 subjects were randomly assigned to receive 1 of 4 interventions: 1) combination of sterols and exercise, 2) exercise, 3) sterols, or 4) control treatment.
Results: Sterol supplementation significantly (P < 0.01) decreased total cholesterol concentrations by 8.2% from baseline. In addition, sterols significantly (P < 0.01) lowered absolute LDL-cholesterol concentrations after treatment but had no effect on the percentage change from the beginning to the end of the trial. Exercise significantly (P < 0.01) increased HDL-cholesterol concentrations by 7.5% and decreased triacylglycerol concentrations by 13.3% from baseline. Moreover, sterol supplementation significantly (P < 0.05) increased lathosterol, campesterol, and ß-sitosterol concentrations after treatment. Exercise significantly (P < 0.01) decreased percentage of body fat by 3.9% from the beginning to the end of the trial.
Conclusions: In comparison with plant sterols or exercise alone, the combination of plant sterols and exercise yields the most beneficial alterations in lipid profiles. Implementation of such a combination therapy could improve lipid profiles in those at risk of coronary artery disease.
Key Words: Plant sterols • exercise • LDL cholesterol • HDL cholesterol • hypercholesterolemia • sedentary humans
INTRODUCTION
Coronary artery disease (CAD) is a leading cause of death in the developed world today. It is well established that increased total, LDL-cholesterol, and triacylglycerol concentrations, as well as decreased HDL-cholesterol concentrations, are strong independent predictors of CAD (1). It can, therefore, be assumed that an intervention, which combines the lowering of total, LDL-cholesterol, and triacylglycerol concentrations with the raising of HDL-cholesterol concentrations, would be highly preventive against CAD. Such dietary and behavioral changes to promote heart health were put forth by the National Cholesterol Education Program Adult Treatment Panel III and are included in the new therapeutic lifestyle change guidelines (1). These guidelines include recommendations to increase physical activity and to implement plant sterols as therapeutic dietary options to favorably alter lipid profiles.
Plant sterols were shown to decrease total and LDL-cholesterol concentrations in several population groups (2-5). Including 2 g plant sterols/d in a typical diet can lead to reductions in total and LDL-cholesterol concentrations up to 13% and 16%, respectively (6). However, although most trials testing the efficacy of plant sterols in lowering plasma lipids observed reductions in total and LDL-cholesterol concentrations, HDL-cholesterol and triacylglycerol concentrations seem to be unaffected. In contrast, recent results suggest that endurance training improves lipoprotein profiles by increasing HDL cholesterol while decreasing triacylglycerol concentrations, but it has no significant effect on total or LDL-cholesterol concentrations (7-10). More specifically, exercise was shown to increase HDL-cholesterol concentrations by up to 14% and decrease triacylglycerol concentrations by up to 21% (11). One study showed that such lipid-altering effects occur within a 12–50 wk period of moderate aerobic training (10). However, to our knowledge, no study to date has tested the effect of a short-term moderate intensity exercise program, ie, training for an 8-wk period, on lipid and lipoprotein response.
Therefore, although the effects of these individual interventions are well established, the complementary effects of these 2 therapies on lipid profiles when placed in combination has yet to be tested. Thus, the aim of the present research trial was to examine the effect of plant sterols, endurance training, and the combination of plant sterols and endurance training on plasma lipid and lipoprotein cholesterol, as well as sterol and cholesterol precursor concentrations, in previously sedentary hypercholesterolemic adults at risk of developing CAD.
SUBJECTS AND METHODS
Subjects
Subjects were recruited from the greater Montreal area by means of advertisements placed in local newspapers. A total of 142 persons expressed interest in the study, but only 84 were deemed eligible after the preliminary questionnaire, blood screening, and physical examination. Key inclusion criteria were as follows: age, 40–70 y; previously sedentary, defined as <1 h/wk of light intensity exercise at 2.5–4.0 metabolic equivalents for the 3 mo before the study (12); total cholesterol concentrations > 4.5 mmol/L; nonsmoking; free of cardiovascular disease; nondiabetic; body mass index (in kg/m2) between 18 and 40; not taking lipid- or glucose-lowering medications; normotensive or hypertensive controlled by medications not affecting lipid or glucose metabolism; free of other medical conditions that would preclude subjects from participating in a moderate-intensity endurance exercise program. In addition, women of menopausal age were either premenopausal or postmenopausal (absence of menses for >2 y) and were required to maintain their current hormone replacement therapy regimen for the duration of the study. The experimental protocol was approved by the Human Ethical Review Committee of the Faculty of Agricultural and Environmental Sciences for the School of Dietetics and Human Nutrition at McGill University. All volunteers gave their written informed consent to participate in the trial before the commencement of the study.
Experimental design
An 8-wk, randomized, single-blind, placebo-controlled, parallel-arm clinical intervention trial was implemented as a means of testing the study objectives. Subjects were randomly assigned by way of a stratified random sample and were divided into strata according to total cholesterol concentrations and age. Subjects from each stratum were then randomly assigned into the following 4 intervention groups: 1) combination group (administered sterol-enriched margarine with exercise intervention), 2) exercise group (administered placebo margarine with exercise intervention), 3) sterol group (administered sterol-enriched margarine with no exercise intervention), and 4) control group (administered placebo margarine with no exercise intervention).
Exercise protocol
Subjects assigned to the exercise intervention groups trained at a moderate intensity (13) 3 times/wk under supervised conditions in the research laboratory. Control subjects were asked to maintain their regular level of activity throughout the course of the 8-wk trial. Endurance training was performed with the use of stair-stepping machines and stationary bicycles. Training intensity was estimated for each subject with the use of an age-predicted heart rate maximum (HRmax) equation [209 - (0.7 x age)] (14). Initial exercise sessions consisted of 25 min of exercise corresponding to 60% of each subject's HRmax. Training duration and intensity increased incrementally at week 2, week 4, and week 6, by 5 min and 5% HRmax. Thus, at week 6, the participants trained for a 40-min duration at an intensity of 75% HRmax. Subjects wore Polar Heart Rate Monitors (Polar USA Inc, Woodbury, NY) while training to estimate their training intensity. Heart rates were assessed every 5 min throughout the training session to ensure that the subjects were exercising within safe limits. Compliance was assessed by recording the subject's attendance at each session. If a training session was missed, the subject was required to make up for the missed session during that same week.
Plant sterol protocol
Throughout the study, subjects were asked to replace their habitual margarine intake with the experimental margarine provided. On day 0 of the trial, subjects were given a 1500-g container of unlabeled margarine along with a standardized utensil that measured 5.5 g margarine per scoop. The subjects were instructed to consume 4 level scoops of the margarine/d on a bread product of their choice. Subjects randomly assigned to the sterol supplement groups consumed daily 22 g Proactive margarine (Unilever BestFoods, Purfleet, United Kingdom), corresponding to an intake of 1.8 g plant sterols/d. Subjects randomly assigned to receive the control margarine consumed daily 22 g Flora Light (VandenBergh Foods, Crawley, United Kingdom), a spread not fortified with sterols. The nutrient distribution of the control and sterol-enriched margarines were similar with respect to total energy, fat, carbohydrate, protein, and fiber (Table 1). The study was single-blinded such that the subjects did not know whether they were receiving the control or sterol-enriched margarine. Compliance with the margarine protocol was assessed by weighing the containers on days 0 and 56, and the calculated difference was taken to represent the amount of margarine consumed. In addition, subjects were required to complete a "Daily Margarine Diary," indicating the number of scoops consumed per day. Subjects were asked to maintain their regular diet regimens throughout the course of the trial.
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TABLE 1. Nutrient composition of control and sterol-enriched spreads per 100-g serving
Blood collection protocol
Twelve-hour fasting blood samples were collected on the mornings of days 0, 53, 54, and 55 of the trial. Blood was centrifuged for 15 min at 520 x g and 4 °C to separate plasma from red blood cells and was stored at –20 °C until analyzed.
Assessment of body weight and percentage of body fat
Body weight was assessed on days 0 and 55. Percentage of body fat (%BF) was assessed in triplicate on days 0 and 55 with the use of a hand-held bioelectrical impedance analyzer (Omron BF302; Omron Healthcare, Kyoto, Japan) (15). The instrument recorded impedance from hand to hand and consequently calculated %BF from the impedance value and the pre-entered personal particulars (weight, height, age, and sex). The within-run CV for %BF was 2.9%.
Analyses
Plasma lipid profile determination
Plasma total cholesterol, HDL-cholesterol, and triacylglycerol concentrations were measured in duplicate with the use of enzymatic kits, standardized reagents, and standards with the use of a VP Autoanalyzer (Abbott Laboratories, North Chicago, IL). LDL-cholesterol concentrations were calculated with the use of the Friedwald equation (16). The within-run CVs were 2.1% for total cholesterol concentrations, 1.9% for HDL-cholesterol concentrations, and 3.2% for triacylglycerol concentrations.
Plasma cholesterol precursor and plant sterol determination
Plasma plant sterol concentrations were determined in duplicate by gas-liquid chromatography from the nonsaponifiable material of plasma lipid as reported previously (17). Briefly, 1-mL plasma samples were saponified with 0.5 mol methanolic KOH/L for 1 h at 100 °C, and the nonsaponifiable materials were extracted with petroleum ether. 5-Cholestane was used as an internal standard. After extraction, samples were derivatized with 1.5 mL TMSi reagent [pyridine-hexamethyldisilazan-trimethylchlorosilane (9:3:1, vol:vol)] (18). Samples were injected into a gas-liquid chromatograph equipped with a flame ionization detector (HP 5890 Series II; Hewlett-Packard, Palo Alto, CA) and with a 30-m capillary column (SAC-5; Supelco, Bellefont, PA). Lathosterol, campesterol, and ß-sitosterol peaks were identified by comparison with authenticated standards (Sigma-Aldrich Canada Ltd, Oakville, Canada).
Statistics
Results are presented as means ± SEMs. Differences between groups at baseline were analyzed with the use of a one-way analysis of variance (ANOVA) model. When a significant difference was found between groups, a Tukey post hoc test was performed to determine the differences between group means. When baseline differences were noted for a specific variable, analysis of covariance was performed with the baseline value as a covariate. Differences between group posttreatment values and percentage of change from the beginning to the end of the trial were analyzed with the use of a two-factor ANOVA model, which identified sterol and exercise effects and their interactions. A level of statistical significance at P < 0.05 was used in all analyses. Tests for normality were included in the model. Sample size was calculated with the assumption of a 10% change in LDL-cholesterol concentrations, with a power of 80% and an risk of 5%. Data were analyzed by using SAS software (version 8.0; SAS Institute Inc, Cary, NC).
RESULTS
Subject dropout and compliance
Eighty-four subjects commenced the study, with 74 completing the entire 8-wk trial. Eight subjects dropped out because of time constraints, and 2 others dropped out because of injuries not resulting from participation in the study. After loss because of dropouts, the remaining subjects in each intervention group were as follows: combination group (n = 18), exercise group (n = 18), sterol group (n = 18), and control group (n = 20). The mean attendance at the 24 exercise sessions was 23.4 and 23.2 sessions attended for the combination and exercise groups, respectively. The mean daily margarine consumption for the combination, exercise, sterol, and control groups was 21.7, 21.7, 21.6, and 21.9 g/d, respectively. With respect to blinding, subjects were not able to identify which margarine they were consuming. Furthermore, during the study, no changes were reported with regard to diet or lifestyle habits.
Subject baseline characteristics
Baseline characteristics of the subjects who completed the 8-wk trial are presented in Table 2. Lipid concentrations denoted in the table are based on the values obtained from the initial blood screen. On average, the subjects within each intervention group were hypercholesterolemic (total cholesterol concentrations >5.2 mmol/L). No significant difference was noted at the beginning of the study between the groups with regard to age, body mass index, plasma lipid concentrations, and exercise level. Furthermore, no differences were noted between those participants who completed the trial and those participants who did not.
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TABLE 2. Baseline characteristics of the subjects in the 4 intervention groups who completed the 8-wk trial1
Plasma lipid profiles
Mean plasma lipid concentrations over the 8-wk trial are presented in Table 3. No significant difference was observed between groups in mean total cholesterol concentrations at baseline. When these data were analyzed with the use of two-factor ANOVA, sterol-by-exercise interactions were not significant. In addition, no significant main effect was observed for either sterols or exercise on posttreatment absolute total cholesterol concentrations. However, when total cholesterol concentrations were expressed as the difference between pretreatment and posttreatment concentrations, a significant (P < 0.01) main effect of sterols was noted. After correction for the changes in the control group, total cholesterol concentrations for the combination, exercise, and sterol groups were –5.4%, 2.1%, and –7.1%, respectively.
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TABLE 3. Plasma lipid concentrations at baseline and after treatment1
No significant difference was noted between groups for mean LDL-cholesterol concentrations at baseline. In addition, no significant sterol-by-exercise interaction was noted for this lipid parameter. However, a significant (P < 0.01) main effect of sterols was noted for absolute LDL-cholesterol concentrations after treatment. With regard to percent of change from the beginning to the end of the trial, no significant main effects of sterols or exercise were noted. After correcting for the changes in the control group, LDL-cholesterol concentrations for the combination, exercise, and sterol groups were –5.9%, 6.9%, and –11.3%, respectively.
Mean HDL-cholesterol concentrations at baseline did not differ significantly between groups. In addition, sterol-by-exercise interactions were not significant. Moreover, no significant main effects of sterols or exercise were noted for absolute HDL-cholesterol concentrations after treatment. When HDL-cholesterol concentrations were expressed as the difference between pretreatment and posttreatment values, a significant (P < 0.01) main effect of exercise was observed. After correction for the changes in the control group, HDL-cholesterol concentrations for the combination, exercise, and sterol groups were 9.2%, 11.2%, and 5.8%, respectively.
Triacylglycerol concentrations were shown to be significantly (P < 0.05) different between groups at baseline. After further analysis, it was shown that the mean baseline concentrations of the sterol group were significantly higher than those of the combination, exercise, and control groups. Results of the two-factor ANOVA showed no significant sterol-by-exercise interactions. However, a significant (P < 0.05) main effect of sterols was noted with respect to absolute triacylglycerol concentrations after treatment. In addition, with regard to the percentage of change from the beginning to the end of the trial, a significant (P < 0.01) main effect of exercise was observed. After correction for the changes in the control group, triacylglycerol concentrations for the combination, exercise, and sterol groups were –9.7%, –14.5%, and –1.3%, respectively.
Plasma cholesterol precursor and plant sterols
Plasma cholesterol precursor and plant sterol concentrations over the 8-wk trial are shown in Table 4. No significant difference was seen between groups in mean lathosterol concentrations at baseline. When these data were analyzed with the use of two-factor ANOVA, sterol-by-exercise interactions were not significant. However, significant main effects of both sterols (P < 0.01) and exercise (P < 0.05) were noted for posttreatment absolute lathosterol values. When lathosterol concentrations were expressed as the difference between pretreatment and posttreatment values, a significant (P < 0.01) main effect of sterols was observed. After correction for the changes in the control group, lathosterol concentrations for the combination, exercise, and sterol groups were 20.2%, 3.2%, and 14.8%, respectively.
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TABLE 4. Plasma concentrations of cholesterol precursor and plant sterols at baseline and after treatment1
Campesterol concentrations were not significantly different between groups at baseline. In addition, sterol-by-exercise interactions were not significant. No significant main effects of sterols or exercise were noted for absolute campesterol concentrations after treatment. With regard to the percentage of change from the beginning to the end of the trial, a significant (P < 0.01) main effect of sterols was noted. After correction for the changes in the control group, campesterol concentrations for the combination, exercise, and sterol groups were 44.2%, –0.3%, and 49.1%, respectively.
No significant difference was noted between groups for mean ß-sitosterol concentrations at baseline. Moreover, no significant sterol-by-exercise interactions were observed. In addition, no significant main effects were observed for either sterols or exercise on posttreatment absolute ß-sitosterol values. When ß-sitosterol concentrations were expressed as the difference between pretreatment and posttreatment values, a significant (P < 0.05) main effect of sterols was observed. After correction for the changes in the control group, ß-sitosterol concentrations for the combination, exercise, and sterol groups were 20.1%, 3.9%, and 27.0%, respectively.
Body weight and percentage of body fat
Changes in body weight and %BF over the 8-wk trial are presented in Table 5. No significant difference in body weight at baseline was observed between the groups. When these data were analyzed with the use of two-factor ANOVA, sterol-by-exercise interactions were not significant. In addition, no significant main effects of sterols or exercise were noted on posttreatment body weight values. With respect to change in body weight from the beginning to the end of the trial, a significant (P < 0.05) main effect of exercise was observed. After correction for the changes in the control group, the changes in body weight in the combination and exercise groups were –1.4% and –1.2%, respectively.
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TABLE 5. Body weight and percentage of body fat at baseline and after treatment1
%BF was shown to be significantly different (P < 0.01) between groups at the beginning of the trial. After further analysis, it was shown that the %BF of the combination and exercise groups was significantly higher than that of the sterol and control groups. Sterol-by-exercise interactions were not significant. For posttreatment %BF values, a significant (P < 0.01) main effect of exercise was observed. In addition, a significant (P < 0.01) main effect of exercise was noted when %BF was expressed as the difference between pretreatment and posttreatment values. After correction for the changes in the control group, the changes in %BF in the combination and exercise groups were 4.2% and 3.1%, respectively.
DISCUSSION
The present study is the first to show that the combination of plant sterols and endurance training results in greater lipid-altering effects than that of each intervention alone. In addition, to our knowledge, the present research is the first to demonstrate that favorable alterations in HDL-cholesterol and triacylglycerol concentrations can result from short-term (ie, 8 wk) supervised endurance exercise.
Despite the lack of a controlled diet regimen, total cholesterol concentrations were shown to be substantially lower in the 2 groups consuming the sterol-enriched margarine than in the control group. Because the 2 groups consuming the placebo margarine only experienced marginal changes in total cholesterol, the extent to which this lipid marker was lowered can be attributed primarily to the plant sterol intervention. The consumption of sterol-enriched margarine without the implementation of a controlled diet has resulted in similar lipid-lowering effects in other recent studies (3, 19). After a shorter duration of sterol supplementation, total cholesterol concentrations decreased by 7.4%, without the implementation of a controlled diet (19). However, the lack of a control diet in the present study could potentially account for the slight decreases seen in both total and LDL-cholesterol concentrations in the control group. Because the diet of these volunteers was not rigorously monitored, it is possible that these subjects altered their diet patterns during the course of the trial in a way that produced beneficial effects on their lipid profiles. With respect to LDL cholesterol, sterols were shown to significantly lower absolute concentrations after treatment but had no effect on percent of change from the beginning to the end of the trial. Although not statistically significant, relative to the control group, LDL-cholesterol concentrations in the combination and sterol groups experienced decreases of 5.9% and 11.3%, respectively, whereas the concentration in the exercise group increased by 6.9% relative to the control group. These results suggest that exercise could potentially decrease the LDL cholesterol-lowering effect of plant sterols.
Results from the present study indicate that HDL-cholesterol concentrations increased, whereas triacylglycerol concentrations decreased in response to training. Similar findings were observed in both groups partaking in the training component of the study. In contrast, the 2 groups not involved in the exercise intervention showed no change in either of these lipid markers. Although participation in the training intervention could not be blinded, the lack of effect on HDL-cholesterol and triacylglycerol concentrations within the control and sterol groups suggests that these subjects maintained their regular activity habits throughout the 8-wk trial period. Similar effects on HDL-cholesterol and triacylglycerol concentrations in response to endurance training were observed in previous studies (20, 21). After endurance training for a slightly longer period of time, HDL-cholesterol concentrations increased by 2.6%, whereas triacylglycerol concentrations decreased by 18.8% in mildly hypercholesterolemic men and women (20). Contrary to previous work, the present study was able to show comparable lipid alterations within a much shorter trial duration. The more favorable alterations seen in the current study could potentially be attributed to the tightly controlled exercise intervention. In this way, it is probable that other studies did not see the same degree of an effect on these lipid markers because compliance with the training protocol was not rigorously monitored.
In accordance with previous work, concentrations of the cholesterol precursor, lathosterol, were shown to increase as a result of sterol supplementation. Similar increases in lathosterol concentrations were observed in other studies that supplemented plant sterols at a comparable daily dose (22, 23). Increases in lathosterol concentrations are associated with the partial desuppression of cholesterol synthesis by the liver (24, 25). This slight increase in endogenous synthesis is thought to arise from decreased intestinal absorption of cholesterol. However, this increase in synthesis does not fully compensate for the decrease in cholesterol absorbed, thus allowing the net effect of plant sterol intake to result in an overall decrease in circulating cholesterol concentrations.
In the present study, plasma campesterol and ß-sitosterol concentrations were significantly higher at the end of the trial in the 2 groups consuming the sterol-enriched margarine than in the control group. The degree to which campesterol and ß-sitosterol concentrations increased as a result of sterol supplementation is consistent with previous studies (22, 26). These findings not only indicate that the compliance of the volunteers was good but also could have implications on the change in cholesterol metabolism as a result of plant sterol supplementation. It was proposed that plant sterols reduce circulating cholesterol concentrations by inhibiting cholesterol absorption (27). Both campesterol and ß-sitosterol are more hydrophobic than cholesterol and, therefore, have a higher affinity for micelles (27). Thus, plant sterols can displace cholesterol from micelles, which, in turn, results in less cholesterol being absorbed. When this theory is applied to the present study, it is possible to assume that the increased concentrations of campesterol could reflect the decreased absorption of cholesterol. This mechanism could potentially account for the decrease in circulating lipid concentrations seen in the sterol-supplemented groups.
Previous research suggests that a decrease in %BF as a result of training can have favorable effects on lipid profiles (28, 29). Thus, the decrease in %BF seen in both the combination group and the exercise group could potentially confound the degree to which blood lipids were altered as a result of training. However, because the precise dose-response relation between decrease in %BF and its effect on lipid concentrations has yet to be elucidated, the degree to which the change in %BF confounds the lipid results of the present study is not easily delineated. On this note, future research should aim to define the dose–response relation between decrease in %BF as a result of training and its effect on lipid profiles.
In summary, the results of the present study indicate that the combination of plant sterols and endurance training results in greater lipid-altering effects than does either intervention alone. This combined therapy favorably altered lipid profiles by decreasing total, LDL-cholesterol, and triacylglycerol concentrations and increasing HDL-cholesterol concentrations. Moreover, our results show that short-term supervised training for an 8-wk period can significantly increase HDL-cholesterol concentrations and decrease triacylglycerol concentrations. The present findings suggest that the implementation of such a combination therapy could improve lipid profiles in previously sedentary hypercholesterolemic adults and, therefore, could reduce the risk of CAD in this population.
ACKNOWLEDGMENTS
KAV conducted the clinical trial, performed the laboratory analyses, and prepared the manuscript. NE provided technical assistance during the analysis phase of the experiment and was a valuable resource while preparing the manuscript. PJHJ and CAV assisted in the design of the experiment and provided support throughout the course of the trial and analysis. WEP is a family physician who assisted with the physical examinations during the screening phase of the trial. None of the authors had a financial interest in the margarine products used or in the companies that supplied the products.
REFERENCES