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1 From the Laboratory for Atherosclerosis and Metabolic Research and General Clinical Research Center, University of California Davis Medical Center, Sacramento, CA
2 Supported by the Beverage Institute for Health & Wellness, The Coca-Cola Company, and the National Institutes of Health K-24 AT00596 (to IJ). 3 Address reprint requests to S Devaraj, Laboratory for Atherosclerosis and Metabolic Research, Department of Pathology, UC Davis Medical Center, 4635 Second Avenue, Res 1 Building, Room 3000, Sacramento, CA 95817. E-mail: sridevi.devaraj{at}ucdmc.ucdavis.edu.
ABSTRACT
Background: Dietary plant sterols effectively reduce LDL cholesterol when incorporated into fat matrices. We showed previously that supplementation with orange juice containing plant sterols (2 g/d) significantly reduced LDL cholesterol. Inflammation is pivotal in atherosclerosis. High-sensitivity C-reactive protein (hs-CRP), the prototypic marker of inflammation, is a cardiovascular disease risk marker; however, there is a paucity of data on the effect of plant sterols on CRP concentrations.
Objective: The aim of this study was to examine whether plant sterols affect CRP concentrations and the lipoprotein profile when incorporated into a reduced-calorie (50 calories/240 mL) orange juice beverage.
Design: Seventy-two healthy subjects were randomly assigned to receive a reduced-calorie orange juice beverage either without (Placebo Bev) or with (1 g/240 mL; Sterol Bev) plant sterols twice a day with meals for 8 wk. Fasting blood was obtained at baseline and after 8 wk of Placebo Bev or Sterol Bev supplementation.
Results: Sterol Bev supplementation significantly reduced total cholesterol (5%; P < 0.01) and LDL cholesterol (9.4%; P < 0.001) compared with both baseline and Placebo Bev (P < 0.05). HDL cholesterol increased significantly with Sterol Bev (P < 0.02). No significant changes in triacylglycerol, glucose, or liver function tests were observed with Sterol Bev. Sterol Bev supplementation resulted in no significant change in vitamin E and carotenoid concentrations. Sterol Bev supplementation resulted in a significant reduction of CRP concentrations compared with baseline and Placebo Bev (median reduction: 12%; P < 0.005).
Conclusion: Supplementation with a reduced-calorie orange juice beverage containing plant sterols is effective in reducing CRP and LDL cholesterol and could be incorporated into the dietary portion of therapeutic lifestyle changes.
Key Words: Inflammation • C-reactive protein • phytosterol • plant sterol • cholesterol • lipid profile • nonfat beverage • diet
INTRODUCTION
Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the United States. High concentrations of LDL cholesterol are associated with an increased incidence of CVD. Dietary recommendations from the National Cholesterol Education Panel (NCEP) and the US Food and Drug Administration have emphasized the utility of supplementation with plant sterols or stanols in reducing LDL-cholesterol concentrations and possibly reducing the risk of CVD (1, 2). Stanols and sterols, found in fat-soluble fractions of plants, are structurally related to cholesterol and originate mainly from the diet because they cannot be synthesized by humans. Plant sterols exert their cholesterol-lowering action presumably by suppressing intestinal absorption and increasing ATP-binding cassette transporter expression, which promotes cholesterol efflux (3-7). Consumption of plant sterols, in free and esterified form, reduces plasma total and LDL-cholesterol concentrtions in human subjects, especially in fat matrices. Meta-analyses suggest that ingestion of 2 g plant sterols/d incorporated into dietary fat vehicles such as margarine yields a 10% reduction in LDL-cholesterol concentrations in patients with hypercholesterolemia (3, 4). Because of their cholesterol-lowering effects, plant sterols are now incorporated into many functional foods. We recently reported that in a nonfat matrix, ie, orange juice (OJ) with plant sterols (2 g/d) significantly reduced LDL-cholesterol concentrations in mildly hypercholesterolemic subjects when consumed twice a day with meals (8). However, OJ contains 110 calories and 27 g total carbohydrate per 240-mL serving. To expand the usefulness of such nonfat moieties in reducing LDL cholesterol, it is desirable to provide an option for those persons interested in reducing caloric intake. Inflammation is pivotal in all stages of atherosclerosis, and high concentrations of C-reactive protein (CRP) have been shown to confer an increased risk of CVD in several prospective studies (9). A paucity of data exists with regard to the effect of plant sterols on CRP concentrations. The hypotheses we tested were that a reduced-calorie beverage with plant sterols would reduce not only LDL cholesterol but also CRP concentrations. Therefore, the main objective of the present study was to examine the efficacy of supplementation with a reduced-calorie OJ beverage containing added plant sterols (Sterol Bev, 1g sterols/240 mL beverage, 50 calories) on the lipoprotein profile and CRP concentrations in healthy human volunteers in a parallel, placebo-controlled, double-blind, randomized trial.
SUBJECTS AND METHODS
Seventy-seven subjects aged 19–74 y participated in the present placebo-controlled, double-blind, randomized trial. All subjects gave informed consent, and the study was approved by the Institutional Review Board of the University of California at Davis Medical Center.
Adults with a normal complete blood count, LDL cholesterol >100 mg/dL, normal liver and renal function (normal transaminases, alkaline phosphatase, and creatinine), no bleeding diathesis, and normal thyroid function were included in the study. Secondary causes of hypercholesterolemia, such as nephrotic syndrome, cholestasis, and hypothyroidism, were ruled out.
The list of exclusion criteria were as follows: participation in an active weight-loss program; pregnancy or lactation; smoking; current use of vitamin supplements or alcohol intake >30 mL/d; history of CVD or chronic inflammatory diseases (eg, Crohn disease, rheumatoid arthritis, and systemic lupus erythematoses); recent bacterial infection (<2 wk); use of antiinflammatory steroidal or nonsteroidal medication, hypolipidemic or thyroid drugs, oral contraceptives, or anticoagulants; history of sitosterolemia; gastrointestinal problems; and concurrent or recent (within 30 d) participation in an intervention study.
Study design
Blood was drawn from the subjects after an overnight fast. The subjects were then randomly assigned in a blinded fashion to receive either Sterol Bev or Placebo Bev for the next 8 wk. Both the Placebo Bev and Sterol Bev were provided by The Coca-Cola Company (Houston, TX). Sterol Bev consisted of plant sterol with the targeted particle size distribution suspended in a reduced-calorie OJ beverage (Coca-Cola; patent pending). The beverage plant sterol was derived from vegetable oils, with the 3 major components distributed approximately as 40% ß-sitosterol, 25% campesterol, and 20% stigmasterol by weight. Calories were reduced by reducing the juice content of the beverage and adding back as much of the nutrients to the original OJ levels, with the exception of folate due to regulations regarding folate fortification in foods. The product was prepared and shipped by the supplier 1 wk before disbursement of juice to the subjects. The subjects were given enough juice to last 18 d, were asked to keep the juice refrigerated, and were instructed to shake the contents of the container before measuring their 240 mL serving. The study investigators were also blinded to protocol assignment until the end of the study. Each subject was asked to consume 240 mL juice twice a day with breakfast and dinner. This corresponded to 2 g sterol/d in the Sterol Bev (50 calories/240 mL); this dose was used because it has been shown to effectively reduce cholesterol concentrations and is the dose recommended by the NCEP Adult Treatment Panel III (ATPIII). The subjects were asked to refrain from consuming any source of margarines and spreads containing plant sterols, such as Benecol (MCNeil, Fort Washington, PA) or Take Control (Unilever, Englewood Cliffs, NJ), 4 wk before study entry and during the period of the study and were asked to adhere to their usual diet and exercise regimen for the duration of the study. Fasting blood was obtained from the subjects at baseline (average of 2 samples, 5–7 d apart) and after 4 and 8 wk of the study (average of 2 samples, 5–7 d apart). The subjects were asked to keep a 3-d diet record at the beginning and at the end of the study. The composition of the Placebo Bev and Sterol Bev are given in Table 1.
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TABLE 1. Composition of the beverages1
Analyses
Plasma was separated by centrifugation for 15 min at 4–12 °C and 600 x g. All analyses were carried out in the Clinical Pathology Laboratory at UC Davis Medical Center, Sacramento, CA. Total cholesterol and total triacylglycerol were analyzed on the Beckman Access autoanalyzer (Beckman Instruments, Brea, CA). LDL-cholesterol concentrations were calculated by using the Friedewald equation. HDL-cholesterol concentrations were analyzed by using the direct HDL-cholesterol assay. Apolipoprotein (apo) A and B concentrations were measured in the Clinical laboratory with the use of the Beckman Array (Beckman Instruments). The inter- and intraassay CVs for cholesterol and triacylglycerol assays were <4%. CRP concentrations were measured by using a high-sensitive assay (Beckman LxPro; Beckman Instruments), which has an inter- and intraassay CV of <5%. Vitamin E and carotenoid concentrations were assayed in plasma by HPLC. Diet analyses were performed with the use of the ESHA FOOD PROCESSOR program (version 7.4; ESHA Research, Salem, OR).
Statistical analysis
Data are expressed as means (±SDs) for parametric data and medians for nonparametric data. Statistical analyses were conducted with the use of GRAPHPAD PRISM software (version 4; GraphPad Software, San Diego, CA). Between-group and within-group differences were analyzed by 2-factor repeated-measures analysis of variance followed by Student's t tests for parametric data and Friedman test followed by Wilcoxon signed-rank tests for nonparametric data. For multiple comparisons, Bonferroni correction was performed on the Wilcoxon test. A P < 0.05 was considered significant. Spearman or Pearson correlations were performed to analyze for correlations in changes in the variables tested.
RESULTS
Although 77 subjects entered the study, 5 dropped out because of personal reasons (2 in the Sterol Bev group and 3 in the Placebo Bev group); therefore, 72 subjects (n = 36 per group) completed the study. Compliance was high and body weights were unchanged during the trial. The subjects in both groups (Placebo Bev and Sterol Bev) were matched for age, sex, ethnicity, and body mass index. Baseline subject characteristics and baseline lipid profiles are reported in Table 2. No significant differences in the baseline lipid profile, ie, total cholesterol, total triacylglycerols, HDL cholesterol, and LDL cholesterol, were observed between the 2 groups. Diet analyses uncovered no significant differences in the composition of the diet between the 2 groups before and after Sterol Bev and Placebo Bev supplementation (Table 3).
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TABLE 2. Baseline characteristics of the subjects1
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TABLE 3. Dietary composition before and after Sterol Bev and Placebo supplementation1
Sterol Bev supplementation resulted in no significant changes in body mass index, complete blood count, liver function tests, blood glucose concentrations, and renal function. Mean baseline and 4 and 8-wk concentrations of total cholesterol, LDL cholesterol, non-HDL cholesterol, HDL cholesterol, and total triacylglycerol are provided in Table 4. No significant changes in the lipid profile were observed with the Placebo Bev. A significant time-by-treatment interaction for total cholesterol and LDL-cholesterol concentrations was observed between the groups and between baseline and 8 wk in the Sterol Bev group (5.0% decrease in total cholesterol and 9.4% decrease in LDL cholesterol, P < 0.01, Table 4). As expected, non-HDL-cholesterol concentrations were reduced significantly (8.8%; time x treatment interaction, P <0.02) with Sterol Bev compared with baseline and Placebo Bev. No significant changes in triacylglycerol concentrations were observed. HDL-cholesterol concentrations were significantly increased in the Sterol Bev group at 8 wk compared with baseline (6% increase), but not compared with Placebo Bev (time x treatment interaction not significant; Table 4). Furthermore, although there was a significant reduction in apo B concentrations after supplementation with Sterol Bev compared with baseline and Placebo Bev, no significant changes in apo A1 concentrations were observed (P = 0.09 for Week 8 compared with baseline in the Sterol Bev group).
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TABLE 4. Effect of the reduced-calorie Sterol Bev on the lipoprotein profile1
Sterol Bev supplementation resulted in a significant reduction (12%) in CRP concentrations (time x treatment interaction, P < 0.02; Figure 1 A). No significant correlation between reductions in LDL-cholesterol and CRP concentrations were observed (r = 0.16, P > 0.05). To confirm the results of the present study, we also examined the effect of sterols on CRP concentrations in blood samples collected in an earlier study that was conducted with sterol-fortified OJ (110 calories/240 mL serving). The design and results of the previous study were reported previously (8). We also report for the first time that there was a significant reduction in CRP concentrations in the samples obtained from the earlier study (23% reduction; time x treatment interaction, P <0.01) (Figure 1B).
FIGURE 1.. Effect of a reduced-calorie orange juice beverage with plant sterols (Sterol Bev) on high-sensitivity C-reactive protein (hs-CRP) concentrations in the present study (A) and in an earlier study (B) with sterol-containing orange juice (sterol OJ) (8). Fasting blood samples were obtained at baseline and after 8 wk of supplementation with a placebo beverage (Placebo Bev) or Sterol Bev or as described in the previous study. All analyses were carried out as described in Methods. Data are presented as medians (25th and 75th percentiles). Two-factor nonparametric analyses (Friedman test) resulted in a significant time x treatment interaction. Significantly different from placebo: *P = 0.02, **P < 0.03.
We also examined the effects of Sterol Bev supplementation on plasma vitamin E and carotenoid concentrations. No significant differences in the concentrations of both vitamin E and carotenoids were observed after supplementation compared with Placebo Bev (Table 5).
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TABLE 5. Effect of the reduced-calorie sterol beverage on plasma vitamin E and carotenoid concentrations1
DISCUSSION
Dietary therapy is the cornerstone of strategies aimed at reducing LDL cholesterol and thereby reducing the risk of CVD (1). Incorporating foods fortified with plant sterols in the daily diet, in addition to other lifestyle modifications such as exercise, will greatly enhance the cholesterol-lowering effect of diet therapy. In the present placebo-controlled double-blind trial, we reported a significant improvement of the lipid profile in subjects who consumed a reduced-calorie beverage (Sterol Bev group), as evidenced by a significant reduction of total cholesterol and LDL cholesterol compared with placebo and a significant increase in HDL cholesterol compared with baseline. Furthermore, the addition of plant sterols to OJ or reduced-calorie (Sterol Bev) beverages resulted in a significant reduction in CRP concentrations.
Although several trials in different populations have shown that plant sterol consumption in fat matrices (margarine, butter, or dressing) results in reduced total and LDL-cholesterol concentrations (3.4–11.6% and 5.4–15.5%, respectively) (3, 4), the incorporation of plant sterols in reduced-fat matrices have yielded variable results. This could be due to a small sample size, lack of a placebo control, lack of ingestion of the supplement with meals, or other variables. Maki et al (10) reported that a 50% fat spread that provided 1.1 and 2.2 g plant sterols/d resulted in a respective 7.6% and 8.1% reduction in LDL-cholesterol. However, no difference in cholesterol concentrations was observed in another trial that compared the effects between consumption of plant sterols at 3 g/d in a reduced-fat spread, 6 g/d in a 28% fat dressing, and 9 g/d in reduced-fat spread and dressing (11). Daily consumption of low-fat (1%) yogurt containing 1g plant sterols significantly lowered total and LDL-cholesterol concentrations; however, the weakness of that study was that the placebo reduced total and LDL-cholesterol concentrations, albeit nonsignificantly, and comparisons with a placebo were not made (12). Mensink et al (13) also showed a 13.7% reduction in LDL cholesterol using esterified stanols (3 g/d) in low-fat yogurt. Jones et al (14) observed no significant differences in total and LDL cholesterol between the placebo and the low- or nonfat beverages containing free sterols, which were incorporated into a controlled diet regimen. The diet regimen itself resulted in a 5% reduction in the LDL-cholesterol concentration. Clifton et al (15) showed that the efficacy of plant sterols (1.6 g/d for 3 wk) consumed in low-fat milk was 3 times that of their consumption in bread and cereal. We previously showed that in a nonfat matrix (ie, OJ) containing 1 g sterols/240 mL consumed twice a day with meals lowered total and LDL-cholesterol concentrations (8). In a recent study conducted on modestly hypercholesterolemic subjects, Noakes et al (16) reported a significant reduction in LDL cholesterol (8–9%) with plant sterol esters (1.8-2 g/d) when incorporated in low-fat milk or yogurt. Because of the increase in the incidence of diabetes, metabolic syndrome, and obesity in the United States, it is desirable that a nonfat beverage with reduced calories and carbohydrate content that is also effective in improving the lipoprotein profile be available as an option for a heart-healthy diet. However, the efficacy of plant sterols incorporated into a different matrix in lowering total and LDL cholesterol needed to be assessed, as proposed by Katan et al (4). Also, whereas the concentrations appeared to be trending in the right direction at 4 wk, we saw no significant change in LDL cholesterol until after 8 wk supplementation with Sterol Bev compared with placebo. With an increased sample size, benefits may have been observed at 4 wk. Although it is hard to speculate on the exact mechanism by which the Sterol Bev reduces LDL cholesterol, its effects on cholesterol absorption and expression of ATP-binding cassette transporter G5 and 8 will be examined in future studies. Note that HDL-cholesterol concentrations increased in the Sterol Bev group but not compared with the Placebo Bev group. However, because apo A1 concentrations were not significantly different between the Placebo Bev and Sterol Bev groups, this needs to be confirmed with larger studies in patients with low HDL cholesterol.
Several lines of evidence provide support for the pivotal role of inflammation in atherosclerosis. Numerous prospective studies have shown that high concentrations of CRP predict increased cardiovascular events. Statins have been shown to have pleiotropic effects in addition to reducing LDL-cholesterol and CRP concentrations (17). Also, the cholesterol absorption inhibitor, ezetimibe, was shown to lower CRP concentrations when administered with a statin (18). Furthermore, in a small study, Cater et al (19) showed that combined administration of plant stanols with a statin significantly reduced CRP concentrations in patients with coronary artery disease; however, they found no significant change in CRP concentrations with plant stanol esters alone. Although statins produce greater reductions in CRP and LDL cholesterol, they are not tolerated by all persons. We showed for the first time that plant sterols added to a reduced-calorie OJ beverage as well as in regular OJ (from the previous study) effectively lower CRP concentrations in healthy human volunteers and could thus be added to the list of agents that can modulate CRP concentrations and possibly be considered antiinflammatory. This is particularly important because it has been previously shown that glucose intake increases oxidative stress and glucose infusion induces inflammatory responses (20, 21); however, the reduced-calorie Sterol Bev resulted in a significant reduction in both LDL-cholesterol and CRP concentrations without affecting blood glucose concentrations. Although more studies are needed to confirm the CRP-lowering action of plant sterols in different populations and examine the underlying mechanisms, this could have major implications with respect to the prevention of CVD because the concomitant reduction in LDL cholesterol and CRP with statin therapy was associated with the greatest benefit in terms of cardiovascular events (22, 23). A plausible mechanism for the antiinflammatory effect of plant sterols is the attenuation of the proinflammatory burden in the liver, which emanates from the gastrointestinal tract.
The concern with plant sterol supplementation is that it may not only reduce LDL-cholesterol concentrations by inhibiting cholesterol absorption but may also reduce other lipophilic compounds such as carotenoids and vitamin E at the same time (24). Lipid standardized concentrations of plasma -tocopherol, ß-carotene, and lycopene have been shown to be reduced after consumption of plant stanols or sterols in some studies but not in others. In our study, we observed no significant differences in concentrations of the different fat-soluble vitamins with Sterol Bev supplementation. This is probably due to the incorporation of these fat-soluble vitamins into the formulation in the free form. Previously, Richelle et al (24) showed that free sterols were less effective than sterol esters in reducing the bioavailability of vitamin E and ß-carotene.
In conclusion, the present study showed that a reduced-calorie nonfat OJ beverage significantly improved the lipid profile without compromising carotenoid and vitamin E status. In addition, it concomitantly reduced CRP concentrations, thus offering an attractive strategy to incorporate in the therapeutic lifestyle dietary regimen recommended by the NCEP/ATP III guidelines. Previously, Jenkins et al (25), using a portfolio diet high in plant sterols, soy protein, viscous fiber, and almonds, reported a significant reduction in LDL cholesterol and CRP. Although they could not ascribe the benefit to a particular dietary component, the study showed that diversifying cholesterol-lowering components in the same dietary portfolio increased the effectiveness of the diet in treating hypercholesterolemia and in attenuating inflammation. Such dietary therapies will go a long way in reducing cardiovascular burden, especially in subjects who are at an increased risk for the metabolic syndrome, diabetes, and CVD.
ACKNOWLEDGMENTS
We thank Carolyn Moore for discussions with regard to the beverage and for review of the manuscript.
SD conducted the study. BCA provided technical assistance. IJ provided overall supervision and the follow-up of the volunteers in the study. All authors approved the final version of the manuscript. None of the authors had any personal or financial conflict of interest.
REFERENCES