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ORIGINAL RESEARCH COMMUNICATION |
1 From the Service d'Endocrinologie-Métabolisme, AP-HP, Hôpital de la Pitié, Paris, France (BH and EB); Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 551 "Dyslipidemia and Atherosclerosis" and University Pierre and Marie Curie, Hôpital de la Pitié, Paris, France (BH, MJC, and EB); Danone Vitapole, Centre de Recherche Daniel Carasso, Nutrivaleur, Palaiseau, France (CN, FL, FT, and TL); OPTIMED, Gieres, France (YD); Service de Cardiologie, AP-HP, Centre Hospitalier Universitaire (CHU) Rangueil, Toulouse, France (JF); Service d'Endocrinologie et Nutrition, Hôpital hôtel Dieu, Nantes, France (MK); Service de Médecine Interne, CHU de Hautepierre, Strasbourg, France (J-LS); and Service Endocrinologie, Diabétologie et Maladies Métaboliques, Hôpital du Bocage, Dijon, France (BV)
2 Supported by a grant from Danone Research (Palaiseau, France). 3 Address reprint requests to B Hansel, Service d'Endocrinologie-Métabolisme, Pavillon Benjamin Delessert, Hôpital de la Pitié, 83 boulevard de l'Hôpital, 75651 Paris Cedex 13, France. E-mail: boris.hansel{at}psl.aphp.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: We examined the effect of daily consumption of PS-supplemented low-fat fermented milk (FM) on the plasma lipid profile and on systemic oxidative stress in hypercholesterolemic subjects.
Design: Hypercholesterolemic subjects (LDL-cholesterol concentrations 
130 and 
190 mg/dL; n = 194) consumed 2 low-fat portions of FM in the same meal daily for 6 wk. Subjects were randomly assigned to 2 groups: low-fat FM enriched with 0.8 g PS ester per portion or control FM. Plasma concentrations of lipids, oxidized LDL, ß-carotene, ß-sitosterol, campesterol, and high-sensitivity C-reactive protein were measured during the trial.
Results: Plasma LDL-cholesterol concentrations were reduced by 9.5% and 7.8% after 3 and 6 wk, respectively, in the 1.6-g/d PS group compared with the control group, whereas plasma triacylglycerol and HDL-cholesterol concentrations were not significantly affected. In addition, there were no significant changes in serum ß-carotene on normalization to LDL cholesterol during the study period in both groups, whereas plasma concentrations of oxidized LDL were reduced significantly in the PS group compared with the control group (–1.73 compared with 1.40 U/L, respectively; P < 0.05). Plasma sitosterol concentrations were increased by 35% (P < 0.001 compared with control); however, campesterol concentrations did not change during the study period.
Conclusion: Daily consumption of 1.6 g PS in low-fat FM efficiently lowers LDL cholesterol in subjects with moderate hypercholesterolemia without deleterious effects on biomarkers of oxidative stress.
Key Words: Plant sterol • hypercholesterolemia • oxidative stress • oxidized LDL • ß-carotene
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10% (1). Thus, a daily intake of PSs in the range of 1-2 g/d is now recommended for hypercholesterolemic patients (2). Indeed, the hypocholesterolemic effect of PSs is additive to that of other dietary measures, such as a reduction in saturated fat intake (3). PSs, which closely resemble cholesterol in their molecular structure, exert their hypocholesterolemic effect primarily through competitive inhibition of cholesterol micellar solubilization and, hence, of the intestinal absorption of both dietary and biliary cholesterol (4). In most previous studies, PSs were incorporated into high-fat foods, such as dressings, margarines, or spreads, to facilitate their solubility. In a recent meta-analysis, the consumption of >2 g/d of PS-enriched fat food products reduced LDL-cholesterol concentrations by 0.33-0.54 mmol/L (5). Furthermore, it has been suggested that the dispersion of PSs in different food forms may substantially affect the degree of LDL cholesterol reduction achieved (1). However, few studies have examined the hypocholesterolemic effect of PS supplementation in low-fat dairy products such as milk (6, 7), yogurt (6, 8-11), and other beverages (12, 13).
Atherosclerosis underlies most forms of cardiovascular disease, and both chronic low-level inflammation and oxidative stress are prominent features of its pathophysiology. Oxidative stress results from an imbalance between tissue prooxidants (free radicals or oxygen reactive species) and antioxidants (enzymes and vitamins; 14). LDL is preferentially deposited in the vascular wall at sites of endothelial dysfunction early in the course of the development of atherosclerotic lesions, where it is oxidized as a consequence of oxidative stress. Numerous in vitro or animal models of human atherosclerosis suggest that oxidized lipids derived from LDL (oxLDL) possess proinflammatory activity and play a major role not only at early stages of atherogenesis, but equally at late stages involving plaque rupture with ensuing clinical events (15). Recently, plasma concentrations of oxLDL were shown to be a strong predictor of acute coronary heart disease events (16). The potential effect of daily consumption of PS on the level of oxidative stress remains to be established. It could be speculated that dietary PS supplementation, which can mediate reduction in plasma concentrations of fat-soluble vitamins (5), may lead to global impairment of antioxidative defenses and thus to enhanced oxidative stress. In contrast, because dietary PS consumption leads to a reduction in the plasma concentrations of LDL cholesterol, thereby reflecting a decrease in the number of circulating LDL particles that are susceptible to oxidation, the formation of atherosclerotic lesions is potentially attenuated.
The objectives of the present study were 1) to examine the effect of daily consumption of 1.6 g PS in low-fat fermented milk (FM) on the plasma lipid profile in hypercholesterolemic subjects, 2) to quantify the influence of daily intake of PS-supplemented FM on oxidative stress (as assessed by plasma concentrations of ß-carotene and oxLDL) and on inflammation [as assessed by high-sensitivity C-reactive protein (hs-CRP)], and 3) to evaluate the influence of PS consumption on circulating PS concentrations.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subjects were eligible if they were between the ages of 18 and 75 y (inclusive), whether they were taking statins (but not other hypocholesterolemic drugs), or whether they were following a prescribed diet. To be included in the study, subjects had to have a serum LDL-cholesterol concentration of 130–190 mg/dL (3.35–4.9 mmol/L), a serum triacylglycerol concentration of <250 mg/dL (2.86 mmol/L), and not have diabetes; normal liver, kidney, and thyroid function and a body mass index (in kg/m2) between 19 and 30 were all inclusion criteria. Exclusion criteria were as follows: pregnancy or lactation, change of oral contraceptive formulation, the presence of severe disease able to influence the results (nephritic syndrome or cholestasis), a history of cardiovascular disease or chronic inflammatory disease, soy allergy, and hypersensitivity to milk proteins. On the basis of these criteria, 365 subjects were eligible; of these, 194 subjects were included in the study and were randomly assigned. One subject did not complete the study for personal reasons linked to adverse events not related to the study product. The baseline clinical and biological characteristics of all subjects (n = 194) are shown in Table 1
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Administration of FM
The PS-enriched FM and control FM were produced and prepared by Danone Research Center (Palaiseau, France). The compositions of the PS-enriched and control products are presented in Table 2. PSs were extracted from tall oil and were esterified with rapeseed oil. The PS-enriched FM contained mainly ß-sitosterol (75%) and campesterol (8.4%). One serving of FM supplied 0.8 g PS equivalent as free sterol. Subjects consumed 2 servings to provide a daily dose of 1.6 g PS equivalent as free sterol.
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Compliance
Compliance with the study product was evaluated by interviewing the subjects and by counting the unopened and unconsumed product packages returned to the clinic. Compliance was recorded as the percentage of the scheduled servings consumed. Noncompliance was defined as the consumption of <80% of the scheduled servings during the study period.
Statistical analyses
The number of subjects was calculated by taking into account a critical difference in LDL cholesterol of 0.32 ± 0.732 mmol/L between the active and the control groups with 
= 5% and a power of 80%. Given these constraints, 84 evaluable subjects per group to 168 in total were required. To take into account possible premature withdrawal and block size, a total of 200 subjects was planned to be included for random assignment.
Descriptive statistics are presented as means ± SDs or as medians and quartiles (with 95% CIs) for continuous data or as a percentage for qualitative variables. Comparisons between the active group and the control group were performed with statistical descriptive tests by using a significance level of 5% (2-sided) with appropriate methods according to the distribution (parametric or nonparametric or both). The hypocholesterolemic effect of PS was assessed after 3 and 6 wk of product consumption. Comparisons between 2 groups of continuous data were analyzed by using a mixed analysis of variance model (or appropriate nonparametric analysis). Analysis of variance was performed on raw data for cholesterol markers and on other markers if departure from normality was not established. In other cases, statistical analyses were performed on transformed data (logarithmic transformation as usually recommended for triacylglycerols or hs-CRP) or on rank data for other markers. Comparisons between 2 groups of qualitative data were analyzed by using a chi-square or Fisher's exact test, logistic regression analysis with a binary response, or a Cochran-Mantel-Haenzel test. For each subject and at each level of stratification, the change from baseline was calculated and expressed in absolute or relative change or both. Statistical analyses were performed on the individual data by using a general linear model with study product and stratification factors (center and statin level). Data analyses were performed by using SAS software (version 8.2; SAS Institute Inc, Cary, NC).
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RESULTS |
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There were no significant differences between the PS and the control groups at baseline in age, BMI, systolic and diastolic blood pressures (Table 1
), alcohol consumption (46.3% compared with 49.5%), and smoking habits (13.7% compared with 14.0%). Men and women were equally distributed in both groups (65% compared with 35% in the active group and 69% compared with 31% in the control group, respectively). Subjects were selected on the basis of screening values for mean fasting LDL-cholesterol concentrations between 130 and 190 mg/dL (3.35 and 4.90 mmol/L) and mean triacylglycerol concentrations <250 mg/dL (2.86 mmol/L). The mean plasma concentrations of LDL cholesterol were greater in the group consuming the PS-enriched product than in the control group (158.1 ± 13.4 compared with 151.8 ± 15.9 mg/dL, respectively; P < 0.05). Neither biological criteria nor dietary habits differed significantly at baseline. Only 14% and 15% of the control and PS groups, respectively, received statin treatment; none of those subjects discontinued statin treatment during the study. There were no significant changes in the dietary habits or levels of physical activity in either of the groups (data not shown).
Compliance and side effects during the study
Compliance for the product enriched in PS and for the control FM (defined as consumption of >80% of the scheduled servings during the study period) was highly satisfactory (95.5%) after 3 wk of consumption and attained 97.7% after 6 wk. All subjects followed the dietary recommendations. No adverse events related to the consumption of the product occurred during the experimental phase.
Serum lipid profile
The mean plasma lipid concentrations at baseline and after 3- and 6-wk consumption of the control FM and of the PS-supplemented FM are shown in Table 3. Most of the reduction in plasma LDL-cholesterol concentrations was achieved after 3 wk. The reduction in LDL cholesterol during the first 3 wk was 9.5% greater in the PS group than in the control group, and it corresponded with a 14.5-mg/dL decrease in LDL cholesterol (P < 0.001 between groups). After daily consumption of PS for 3 additional weeks, LDL-cholesterol concentrations were 8.4% and 0.7% lower than baseline in the PS and control groups, respectively. The mean reduction of LDL cholesterol after daily intake of 1.6 g PS was 12.4 mg/dL greater than that in the control group (P < 0.001 between groups). After 3-wk consumption of the PS-enriched FM, statin-treated and -untreated subjects had LDL-cholesterol concentrations that were reduced by 8.0 ± 2.1% and 8.4 ± 1.2%, respectively. Very little change was observed in the LDL-cholesterol concentrations of the statin-treated and -untreated subjects in the control group (2.3 ± 3.7% and 0.8 ± 1.2%, respectively). The same patterns were observed after 6 wk of FM consumption.
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): –6.2% reduction at the intermediate visit (P < 0.001 between groups) and –4.7% at the end of the intervention period (P < 0.001 between groups). HDL-cholesterol and plasma triacylglycerol concentrations were not significantly affected by the consumption of the PS-enriched FM (Table 3).
Plasma plant sterols
Variation in plasma PS concentrations during the study reflected the degree of absorption and transport of PSs in the systemic circulation (Table 4). The increase in plasma ß-sitosterol concentrations in the PS group was 35% greater than that in the control group at the end of the intervention period (0.81 compared with 0.06 mg/L). There was no significant change in plasma campesterol concentrations during the intervention. Plasma concentrations of ß-sitosterol plus campesterol increased significantly more in the PS group (14.5% compared with the control group, P < 0.001) after the 6-wk intervention period.
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): –0.08 compared with –0.02 mg/L between baseline and week 6 in the PS and the control groups, respectively; NS.
Oxidative stress and inflammation criteria
Absolute and LDL-cholesterol–adjusted ß-carotene concentrations are reported in Table 5. The reduction in plasma ß-carotene concentrations was 12% greater in the PS group than in the control group (P < 0.001). However, after standardization for LDL cholesterol, variation in ß-carotene concentrations during the study did not differ significantly between the 2 groups. Plasma concentrations of oxLDL were reduced significantly more in the group consuming PS (–1.73 compared with 1.40 U/L; P < 0.05; Table 5), but the decrease was not significant after standardization for LDL cholesterol. hs-CRP was unchanged after 6 wk of product consumption, and variation in this variable did not differ significantly between the 2 groups during the study (Table 5).
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DISCUSSION |
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In previous studies with products naturally containing fat, PSs were most frequently incorporated in fat-rich vehicles such as spreads and dressings. Two studies used a naturally fat-free orange juice as a vehicle for phytosterol ester and showed an LDL-cholesterol–lowering effect similar to that of PS-enriched fat products (17, 18). A meta-analysis (5) showed that daily consumption of >2 g PS in margarines, mayonnaise, butter, or olive oil can reduce LDL-cholesterol concentrations by 0.33-0.54 mmol/L in hypercholesterolemic subjects. Few data, however, are available on the plasma cholesterol–lowering properties of nonfat or low-fat PS-enriched foods. Daily consumption of a relatively low-fat (2%) yogurt containing 1 g PS ester significantly lowered total cholesterol and LDL-cholesterol concentrations (8); however, in this study, the control FM led to a decrease in total cholesterol and LDL-cholesterol concentrations, and comparisons with controls were not available. In a subsequent study conducted by Jones et al (13), daily ingestion of 1.8 g free PS in a low-fat beverage failed to decrease LDL-cholesterol concentrations in moderately hypercholesterolemic subjects. The authors hypothesized that the content of fat was not high enough to adequately solubilize phytosterols in the small intestine. On the contrary, other studies suggested that relatively low-fat dairy products may be an adequate vehicle for the delivery of esterified PS to effectively lower plasma cholesterol concentrations (6, 9-11, 19-21). The LDL-cholesterol reduction found in our study with PS-enriched FM agrees with the latter data. However, in these studies, the vector used to deliver PS was distinct (6, 9-11, 19-21). First, in the studies of Mensink et al (9) and of Doornbos et al (11), the experimental group received a daily intake of 3 g plant stanol esters, ie, about twice the dose of PS ingested in our study. In other studies, 2 g PS esters were consumed daily. Thus, the dose of PS administered in our protocol was the lowest among these studies to show a significant decrease in LDL cholesterol when incorporated in a low-fat dairy product. Another characteristic of the product tested in the present study is its very low fat content. In previous studies of "low-fat" dairy products (6, 10, 20), the fat content of the products tested was often relatively low but higher than that in the present study. The main dietary recommendation for patients with hypercholesterolemia is to limit their intake of saturated fats, including those of dairy products. In addition, patients may be reluctant to consume margarine either because they are not in the habit of using margarine or because of its taste. Thus, the use of a very-low-fat food to administer plant sterols at a sufficient dose is of particular interest in patients with hypercholesterolemia.
Compliance is obviously a key aspect of therapeutic strategies, which aim to treat chronic asymptomatic abnormalities. In the present study, compliance was excellent (reaching 97.7% at the end of the study), and FM consumption did not give rise to any clinical adverse events.
PS administered in food usually lowers plasma concentrations of LDL cholesterol within a short period of time. In the present study, the maximum change in LDL cholesterol (–9.5% compared with control) occurred after 3 wk of consumption of PS-enriched FM. No additional decrease in plasma LDL-cholesterol concentrations occurred after this period. Nguyen et al (22) reported a similar pattern of response to a spread enriched in plant stanol esters, with a maximum change after 2 wk.
It is well known that lipid and lipoprotein responses to a dietary change and particularly to sterol- or stanol-enriched products are heterogeneous (23-25). In our study, 67% of the patients in the sterol group showed a >5% decrease in LDL cholesterol. This finding suggests that most patients may exhibit a clinically relevant LDL cholesterol reduction derived from daily consumption of PS-enriched FM in addition to traditional diet recommendations.
The effect of PS-enriched margarine in patients taking statins has been shown to be additive rather than synergistic (26). It is generally assumed that dietary PS esters produce an additional reduction in LDL cholesterol of 6–8% (27); the design of our study did not allow us to accurately quantify this additive effect. However, the 7.8% LDL-cholesterol–lowering effect of PS-enriched FM was consistent in the subgroup of subjects treated with a statin. Concordantly, we did not observe any interaction between statin therapy and PS consumption. In line with previous results obtained with the use of margarines, the magnitude of the additional decrease in LDL cholesterol is slightly greater than that to be expected by doubling the dose of statin (28). Unlike PS, statins inhibit the synthesis of hepatic cholesterol but equally increase its intestinal absorption. These complementary effects suggest, therefore, that it may be of special interest to combine statin-mediated inhibition of cholesterol synthesis with the inhibition of cholesterol absorption by PS in the same subject.
One of the major concerns with respect to dietary PS supplementation involves the possibility that fat-soluble vitamin absorption is reduced (5). Because plasma lipoproteins (mainly VLDL and LDL) are the major transport vehicles for lipid-soluble antioxidants, a reduction in LDL concentrations may result in a decrease in fat-soluble vitamin concentrations. Fewer lipoprotein particles may, therefore, be available to carry carotenoids. Because it is common practice to express carotenoid concentrations relative to those of LDL cholesterol, we observed that the small decrease in plasma ß-carotene concentrations did not reach statistical significance after standardization for LDL-cholesterol concentrations. These observations are consistent with previous studies (7, 10, 29, 30).
Oxidative stress, defined as an imbalance between anti- and prooxidant factors, is implicated in the development of numerous chronic pathologies, including atherosclerosis. It has been suggested that even a minor decrease in ß-carotene concentrations may be proatherogenic. However, antioxidants include numerous substances or enzymes and liposoluble vitamins that may interact synergistically. Thus, analysis of one component independently of the others may not accurately reflect their combined action and may not, therefore, properly estimate systemic oxidative stress. Thus, we measured the effect of PS on the level of oxLDL, an integrative marker of systemic oxidative stress. In all likelihood, plasma oxLDL concentrations may depend on the capacity of in vivo antioxidants to protect LDL particles against oxidation in the arterial wall. Plasma concentrations of oxLDL were shown to be a strong predictor of acute coronary heart disease events (16). Interestingly, we report in the present study that consumption of PS was associated with a significant decrease in the plasma concentrations of oxLDL as compared with the consumption of a control dietary product (P = 0.03). Such a decrease paralleled the LDL-cholesterol–lowering effect. Thus, we interpreted our data to suggest that the slight decrease in systemic antioxidant concentrations reported with PS intake did not affect LDL oxidation. Our results confirm those of Homma et al (31), who reported a 20% decrease in oxLDL concentrations in patients consuming 2-3 g/d plant stanol in enriched spreads, as compared with the control group.
Another emergent question concerning the therapeutic use of PS has to do with the possibility that their accumulation in plasma may be atherogenic. In our study, plasma PS concentrations, notably, ß-sitosterol, significantly increased with PS-FM consumption (35% compared with control). However, the absolute changes in campesterol and ß-sitosterol concentrations were of a small magnitude, and the values in the experimental group were not significantly different from those in patients consuming a plant food–based diet. For example, in a population of healthy pure vegetarians, the plasma concentrations of ß-sitosterol plus campesterol were similar (6.0 mg/L) to those in patients consuming PS in our study (5.7 mg/L; 32). Moreover, these values are at least one-tenth to one-twentieth those typical of ß-sitosterolemia, which is characterized by elevated plasma concentrations of ß-sitosterol and accelerated atherosclerosis.
The main weakness of our study is its short duration. However, a large body of evidence now indicates that reductions in plasma cholesterol induced by PS consumption are sustained over the long term, especially when compliance is maintained. In addition, the low level of adverse effects suggests that PS consumption is safe over a relatively long period of time (33, 34). Finally, the decrease in hs-CRP observed in patients treated with PS-enriched FM was not significant, which can be explained by the large variability in hs-CRP at baseline and thus a lack of statistical power.
In summary, the present study showed that daily consumption of 1.6 g PS ester in a very-low-fat PS-enriched FM resulted in an LDL-cholesterol–lowering effect in mildly hypercholesterolemic subjects. Moreover, an absolute decrease in the plasma concentration of oxLDL was equally observed in both the group consuming PS and the control group. Subjects did not report any adverse effects of PS consumption. In addition, we did not observe alterations in adjusted plasma ß-carotene concentrations; equally, levels of oxidative stress as assessed by adjusted plasma concentrations of oxLDL were unchanged. Thus, the consumption of very-low-fat PS-enriched FM may represent a useful additive therapeutic measure to the classic hypocholesterolemic diet of the American Heart Association in hypercholesterolemic patients at high cardiovascular disease risk.
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ACKNOWLEDGMENTS |
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The authors' responsibilities were as follows—BH, YD, JF, MK, J-LS, and BV: collected data; and all authors: contributed to writing the manuscript. Statistical analyses were performed by OPTIMED. EB received honoraria for presentations and grants for research studies from Unilever, Danone, and Madaus. CN, FT, FL, and TL are employed by Danone. The other authors had no conflict of interest.
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