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Original Research |
Chicago Center for Clinical Research, Chicago, Illinois (M.H.D., K.C.M., D.M.U., K.A.I., M.R.D.)
Lipton, Englewood Cliffs, New Jersey (R.W.L., W.C.F.)
Boston VA Medical Center (S.J.R.), Boston, Massachusetts
Tufts University (E.S., J.R.M., J.D.R.-M., G.P.), Boston, Massachusetts
Address reprint requests to: Kevin C. Maki, PhD, Chicago Center for Clinical Research, 515 North State Street, Suite 2700, Chicago, Illinois 60610. E-mail: kmaki{at}protocare.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methods: Eighty-four free-living men and women consumed reduced-fat spread and salad dressing providing 0.0 g/day (n = 21), 3.0 g/day (n = 21), 6.0 g/day (n = 19) or 9.0 g/day (n = 23) of phytosterols as esters for an eight-week treatment period.
Results: Side effects did not differ among the groups during the study, and there were no study product-related serious adverse events. There were no changes in clinical laboratory values in response to phytosterol intake. Blood concentrations of all fat-soluble vitamins remained within normal reference ranges, and there were no differences in serum vitamin responses among the four groups. Alpha- and trans-ß-carotene levels were reduced in the 9.0 g/day group vs. control (p < 0.05), but all carotenoid values remained within normal ranges throughout the study. All groups receiving phytosterols had significant increases in serum campesterol vs. control (p < 0.001), but ß-sitosterol responses did not differ from control. Total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol responses did not differ significantly among the groups. The total:HDL cholesterol response in the 9.0 g/day group was significantly different from the control group response (-9.6% vs. 2.6%, p < 0.05). A median increase of 7.8% in serum triglycerides was observed in the control group, which differed significantly from the response in the 3.0 g/day arm (-13.3%, p < 0.05).
Discussion: The results of this study indicate that phytosterol esters are well tolerated and show no evidence of adverse effects at a daily intake of up to 9.0 g of phytosterols for eight weeks.
Key words: cholesterol, dietary management, sterol esters, hypercholesterolemia, lipoproteins
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INTRODUCTION |
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Results from the studies conducted to date indicate that plant sterol-ester containing foods are safe and effective for lowering cholesterol [4567]. The objective of the current study was to examine the effects of three levels of phytosterol esters, at and above those expected to be consumed during daily use by the average consumer of reduced-fat spreads and salad dressings, on subject-reported side effects, general markers of toxicity and blood concentrations of fat-soluble vitamins, carotenoids and sterols.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subjects
Participants were recruited from the metropolitan Chicago area utilizing the database of the Chicago Center for Clinical Research and radio and print advertisements. Men and women (18 to 65 years of age), pre-screened by telephone, were required to abstain from all dietary supplements for four weeks prior to reporting to the clinic for screening. At the screening visit, eligibility was further assessed by review of medical history and fasting serum lipid profile (total, calculated low-density lipoprotein [LDL] and high-density lipoprotein [HDL], cholesterol and triglycerides). To be eligible, subjects were required to have total cholesterol levels <7.8 mmol/L (300 mg/dL) and triglyceride levels <4.0 mmol/L (350 mg/dL).
Exclusion criteria included body mass index 
35.0 kg/m2, any medically unstable condition, pregnancy or lactation. Women of childbearing potential were required to utilize a medically approved method of contraception during the trial. Persons using hypolipidemic medications or therapies within the four weeks prior to the first clinic visit were excluded from participation. Individuals receiving drugs for regulating hemostasis, other than stable dose aspirin, were also excluded from participation, as were those using systemic corticosteroids, androgens, phenytoin, erythromycin or thyroid hormones (except stable-dose replacement therapy for 2 months prior to enrollment).
Study Products
Study products were provided by Lipton, Englewood Cliffs, NJ. Control and phytosterol ester-enriched products were similar in nutritional content (Table 1). The phytosterol esters were prepared with sterols from vegetable oil esterified with fatty acids from sunflower oil. These materials are generally recognized as safe (GRAS) by a committee of experts in food safety [8]. Throughout the trial subjects consumed 14 g reduced-fat spread (separated into two 7 g servings) and 46 g of reduced-fat ranch-flavored salad dressing per day, with food, as part of their usual diets. The active spread provided 3 g phytosterols/day and the active salad dressing 6 g/day. Subjects received individually packaged servings of the control or phytosterol ester-containing reduced-fat spreads and dressings at each clinic visit. Control and phytosterol ester-enriched products were similar in appearance and sensory quality, and both subjects and investigators were blinded as to group assignment.
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Subjects returned to the clinic at weeks 2, 4, 6 and 8 for assessment of vital signs and body weight. Serum chemistry, hematology, urinalysis and lipid profiles were assessed at weeks 2, 4 and 8. Additionally, at weeks 2, 4 and 8, blood levels of fat-soluble vitamins, carotenoids and sterols were measured. At the end of the eight-week intervention period, a physical examination was also conducted. Compliance with study product consumption was calculated as a percentage of scheduled servings of study product consumed, as evaluated by subject interview and counting of unopened study product packages returned to the clinic at weeks 2, 4, 6 and 8.
Diet Records
At the screening visit (week -1), all subjects were provided with instruction regarding completion of three-day diet records. Diet records were dispensed at weeks -1, 2, and 6 with instructions to record dietary intakes for three consecutive days (two weekdays [Monday through Friday] and one weekend day [Saturday or Sunday]) prior to the next clinic visit. Diet records were collected from subjects at weeks 0, 4, and 8 and were analyzed using the University of Minnesota Nutrition Data System for Research (NDS-R), Version 4.0 software (1998).
Analyses
Clinical Chemistry and Hematology.
Serum chemistry, hematology and urinalysis testing were completed during the screening period at weeks -6 and -1 and during treatment at weeks 2, 4 and 8. Analyses were conducted by Covance Central Laboratory Services, Indianapolis, IN, using a Hitachi 747-200 (Roche Diagnostics, formerly Boehringer Mannheim Diagnostics) analyzer for chemistry, Model 500 IRIS (International Remote Imaging Systems, Inc.) analyzer for urinalysis and Advia 120 (Bayer) analyzer for hematology.
Vitamin, Carotenoid, and Sterol Profiles.
Blood concentrations of fat-soluble vitamins (
- and 
-tocopherol, 25-hydroxyvitamin D, and phylloquinone), carotenoids (- and trans-ß-carotene, trans-lycopene, lutein, zeaxanthin, and cryptoxanthin) and sterols (total sterols [including cholesterol], total plant sterols, campesterol, and ß-sitosterol) at weeks -1, 0, 2, 4 and 8 were analyzed by Tufts University (Boston, MA). Samples for vitamin, carotenoid and sterol analyses were frozen at -80°C, and all measures for each subject were completed in the same run. Blood retinol and tocopherol concentrations were measured utilizing light-protected, reverse-phase, high performance liquid chromatography (HPLC) [9]. Following manual extraction, 25-hydroxyvitamin D concentrations were quantified utilizing a competitive protein binding reaction [10]. After purification, phylloquinone was quantified by HPLC employing post-column, on-line catalytic reduction and fluorescence detection [11]. All carotenoids were analyzed using HPLC techniques. For sterol analyses, following lipid extraction, saponification and re-extraction, free sterols were separated from fatty acids utilizing HPLC. Free sterols were then derivitized and quantified by HPLC.
Lipids.
Serum total cholesterol, HDL cholesterol and triglycerides were measured enzymatically in blood samples collected after fasting (>12 hours) at weeks -1, 0, 2, 4, and 8. Analyses were conducted by a laboratory (Covance Central Laboratory Services, Indianapolis, IN) that participates in the Centers for Disease Control and Prevention lipid measurement standardization program [12]. Low-density lipoprotein cholesterol in mg/dL was calculated using the Friedewald equation (LDL cholesterol = total cholesterol - HDL cholesterol - triglycerides/5) [13]. Since this equation is not valid when the triglyceride concentration is above 4.5 mmol/L (400 mg/dL), no LDL cholesterol value was calculated under these circumstances.
Statistical Analyses
Statistical analyses were conducted using the Statview 5.0 and SAS Version 6.12 statistical analyses packages (SAS Institute, Cary, NC). A two-tailed level of 0.05 was used to designate statistical significance for pairwise comparisons. Comparability of groups for baseline demographic, anthropometric and lipid values was assessed by analysis of variance (ANOVA), Kruskal-Wallis and chi-square tests as appropriate.
Statistical comparisons between groups in vitamin, carotenoid and sterol responses to intervention were conducted utilizing the Kruskal-Wallis test. In cases where the Kruskal-Wallis result was significant (p 
0.05), the data were ranked, and ANOVA was employed, followed by the Scheffé test. Differences in the incidence of adverse events and abnormal laboratory values were assessed with Fisher’s exact test. Evaluation of differences among groups in clinical laboratory value responses was completed using Kruskal-Wallis testing, followed by pairwise comparisons with the Mann-Whitney U test.
Analysis of variance was used to compare response to intervention (percent change from baseline to treatment) for total, LDL, HDL and total cholesterol/HDL cholesterol ratio. For lipid variables, "baseline" was the average of the values obtained at weeks -1 and 0, and "treatment" was the average of values obtained at weeks 4 and 8. If a subject terminated early, values from the last blood sample collected were carried forward. In cases where lipid testing was repeated, all values were averaged. Pairwise comparisons of lipid responses between groups were conducted with the Scheffé test. Triglyceride responses were not normally distributed; therefore, non-parametric tests, Kruskal-Wallis and Mann-Whitney U were performed.
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RESULTS |
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Demographic and anthropometric characteristics of the subjects are shown in Table 2. There were no significant differences among groups in baseline characteristics with the exception of race (p = 0.013). Subjects in the control, 3.0 g/day, and 9.0 g/day phytosterol groups were predominantly Caucasian (65% to 86%), whereas those in the 6.0 g/day group were predominantly Black and Hispanic (53%). Mean age was approximately 46 years, and average body weight was 79 kg. The ratio of male to female subjects in each group was approximately evenly distributed ranging from 48% to 61% male across groups.
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Adverse Events
A total of 52 of the 84 (62%) randomized subjects experienced adverse events during the treatment period. Table 3 summarizes the adverse events by body system for each of the groups. The total number of subjects reporting adverse events was not different among groups, nor was the number of reports in each body system category, with the exception of the body as a whole (p = 0.039), a category for those disorders which are general in nature, e.g., hay fever, exhaustion. In this category, the 6.0 and 9.0 g/day groups had a four-to-fivefold greater incidence of adverse events than did the 3.0 g/day and control groups. In pairwise comparisons, there were significant differences between the 3.0 g/day group and the 6.0 (p = 0.042) and 9.0 (p = 0.022) g/day groups, but no significant differences between the control group and the 6.0 and 9.0 g/day groups. Overall, complaints fell predominantly into the gastrointestinal and respiratory disorder categories. Gastrointestinal complaints included primarily dyspepsia, diarrhea and constipation, none of which were severe. A large majority of the respiratory adverse events were cold- and flu-like symptoms and were deemed by the investigator to be unrelated to the study product. There were no study product-related serious adverse events during the study. A total of 20 reported side effects were judged by the investigator to be possibly or probably related to the study product. These included flatulence, discoloration of feces, gastroesophageal reflux, appetite changes, leg cramps, low white blood cells and skin rash.
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- and -tocopherol were also performed, and no significant changes were noted. The cholesterol value utilized in this and all other cholesterol corrections (carotenoids and plant sterols) was from enzymatic analysis.
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-carotene levels differed significantly among groups (p = 0.001 and p = 0.017, respectively). Pairwise comparisons of the trans-ß-carotene responses indicated that the response in the highest level phytosterol ester intake group (-26.3%) differed significantly from responses in both the control group (10.0%) and the lowest level phytosterol ester group (2.9%) (p < 0.05). Pairwise comparisons of -carotene responses indicated that only the highest level phytosterol ester and control groups differed significantly (-25.0% vs. 0%, respectively, p < 0.05). After correction for total cholesterol concentration, the significance of the reduction in the 9.0 g/day group persisted for trans-ß-carotene vs. control (-16.6% vs. 18.6%, p < 0.05), but was of marginal significance for -carotene (-17.5% vs. 9.1%, p = 0.06). All other carotenoid responses, including trans-lycopene, lutein, zeaxanthin and cryptoxanthin were similar among the four groups.
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-tocopherol, dietary intakes were similar between groups at baseline and throughout intervention. Median percent of calories from alcohol was significantly greater in the 3.0 g/day group than in the 9.0 g/day group at baseline (p = 0.044), and at week 8 median alcohol intake was significantly greater in the 0.0 g/day group than in the 6.0 g/day group (p = 0.026). Treatment groups differed significantly in -tocopherol intakes during the intervention period (p = 0.038), but pairwise comparisons showed no significant differences between groups.
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DISCUSSION |
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12% in the control group. Because the changes in the other study groups were of similar magnitude, the laboratory results can likely be attributed to chance. The results of the current study are in agreement with other studies conducted to date which indicate that plant sterol-containing foods are safe and effective for lowering cholesterol [4567]. The safety information available from clinical trials of hypercholesterolemic patients suggests, in general, that phytosterol-enriched margarine is well tolerated with no reported side effects on blood chemistry and hematological variables [5]. Studies conducted in children with severe hypercholesterolemia have also indicated a favorable safety profile for phytosterols [6]. These found no abnormalities present on sonography of the liver or gallbladder after sitosterol administration of 6 g per day. Though ALT, alkaline phosphatase and carotene concentrations fell from baseline, by seven months of therapy, alkaline phosphatase and carotene concentrations had recovered. No changes were seen in leukocyte or platelet counts or hemoglobin, calcium, iron, phosphatase, lipase or CK concentrations with sitosterol administration [6].
Due to their mechanism of action, phytosterol ester-enriched products could potentially affect absorption of fat-soluble vitamins. Though this effect has not yet been fully characterized (due to the lack of investigations using labeled vitamins), there have been no reported clinical problems [14]. Lower serum -tocopherol concentrations following phytostanol consumption have been reported [15]; however, this was not an unexpected finding since most tocopherol is carried in LDL particles, which are also decreased by plant sterol consumption. In fact, the ratio of -tocopherol to cholesterol did not change. In the current study, blood concentrations of all fat-soluble vitamins remained within normal reference ranges, and there were no differences in serum vitamin responses among the four groups.
Since phytosterols could also affect blood carotenoid levels, serum carotenoid levels were monitored. In the current study, there were significant reductions in - and trans-ß-carotene in the 9.0 g/day group vs. control products. This finding is similar to those of previous studies [57] and is expected, given the high lipophilicity of these carotenoids. In addition, though decreased after intervention, carotenoid values remained within normal ranges throughout the study.
All groups receiving phytosterols showed significant increases in serum campesterol levels. Sitosterol levels showed a similar trend; however, these results were not significant vs. control. Serum plant sterol levels increased less in the group consuming phytosterol-enriched dressing only (vs. enriched dressing and spread). The reason for this disparity is unclear as the self-reported compliance was good. However, as serum plant sterols showed a relatively small increase and the serum cholesterol concentration was not reduced to the degree that would be expected from this and other studies, the possibility remains that compliance was suboptimal. Another potential explanation for the response in the group consuming phytosterol-enriched dressing only (vs. enriched dressing and spread) was that the plant sterols in the dressing may not have been effectively incorporated into micelles, which could result in decreased plant sterol absorption and smaller reductions in serum cholesterol.
An important consideration with serum sterol levels is sitosterolemia, a rare inborn error of metabolism. The homozygous state is characterized by tendon and tuberous xanthomas, premature atherosclerotic disease and elevated circulating plant sterol concentrations [161718]. In patients with sitosterolemia, total phytosterols are in the range of 10 to 60 mg/dL, whereas under normal circumstances, total phytosterol levels in the blood are generally reported to be less than 1 mg/L in control patients [17]. Though blood phytosterol levels may also be significantly elevated in persons heterozygous for sitosterolemia relative to normal individuals, phytosterol concentrations are lower than those of homozygotes by ten-to-twentyfold and represent no increased risk for developing premature atherosclerosis [16]. In an examination of three sitosterolemic subjects, mean sitosterol and campesterol levels were 25.9 and 16.1 mg/dL, compared to four daughters of the affected subjects (obligate heterozygotes), who had mean sitosterol and campesterol levels of 1.33 and 1.56 mg/dL, respectively [19]. In the current investigation, the small increases in median serum phytosterol levels in the phytosterol groups at the end of the study, which were less than those seen previously in unaffected heterozygotes, do not suggest increased risk for premature atherosclerosis.
Lipid responses to phytosterol intake were lower than anticipated based on previous studies [457]. The only significant differences in responses among the groups were in triglycerides (-13.3% for 3.0 g/day group vs. 7.8% for control group, p < 0.05) and total:HDL cholesterol (-9.6% for 9.0 g/day group vs. 2.6% for control group, p < 0.05). The minimal changes may have been a result of the low baseline cholesterol levels of the study participants [20]. Although lipid changes were not statistically significant, they were similar in magnitude and direction to those reported previously.
Recently the Food and Drug Administration approved, for food product labels, the health claim that plant sterol ester-enriched foods may reduce the risk of coronary heart disease by lowering blood cholesterol levels [8]. It is anticipated that many individuals will find reduced-fat spreads and dressings easier to incorporate into a low-fat, low-cholesterol diet than other available cholesterol-lowering foods, such as oat bran or psyllium, which require greater dietary adaptation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received January 5, 2001. Revised June 18, 2001. Accepted June 18, 2001.
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REFERENCES |
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