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Fish-oil esters of plant sterols improve the lipid profile of dyslipidemic subjects more than do fish-oil or sunflower oil esters of plant sterols

Isabelle Demonty, Yen-Ming Chan, Dori Pelled and Peter JH Jones

1 From the School of Dietetics and Human Nutrition, McGill University, Sainte-Anne-de-Bellevue, Canada (ID, Y-MC, and PJ), and Enzymotec Ltd, Migdal HaEmeq, Israel (DP)

2 Supported by a Post-Doctoral Industrial Research Fellowship from the Natural Sciences and Engineering Research Council of Canada (to ID) and by Enzymotec Ltd (Migdal HaEmeq, Israel). Margarines containing sunflower oil esters of plant sterols were donated by Unilever Bestfoods NA (Baltimore, MD).

3 Reprints not available. Address correspondence to PJH Jones, School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, PQ H9X 3V9, Canada. E-mail: peter.jones{at}mcgill.ca.


ABSTRACT  
Background: Fish-oil fatty acid esters of plant sterols (FO-PS) were shown to have hypotriglyceridemic and hypocholesterolemic properties in animal models.

Objective: The objective of the study was to evaluate the hypolipidemic effects of FO-PS supplementation in healthy hypercholesterolemic persons fed an olive oil (OO)–based diet.

Design: Twenty-one moderately overweight, hyperlipidemic subjects participated in a semi-randomized, single-blind, 4-period crossover study including 4 experimental isoenergetic diets of 4 wk each and 4-wk intervening washout periods. Diets contained 30% of energy as fat, of which 70% was from extra-virgin OO, and differed only in the supplement oil: OO, fish oil, FO-PS, or sunflower oil esters of plant sterols (SU-PS). Both fish oil and FO-PS provided 5.4 g total eicosapentaenoic and docosahexaenoic acids/d. FO-PS, SU-PS, and OO provided the equivalent of 1.7, 1.7, and 0.02 g free plant sterols/d, respectively.

Results: Fish oil and FO-PS resulted in fasting and postprandial plasma triacylglycerol concentrations that were markedly lower than those observed with OO and SU-PS (P = 0.0001), but to a different extent. LDL cholesterol was significantly lower after supplementation with FO-PS and SU-PS than at the end of the control OO diet (P = 0.0031 and 0.0407, respectively). HDL cholesterol was not affected. FO-PS and SU-PS resulted in a lower ratio of total to HDL cholesterol and lower apolipoprotein (apo) B concentrations than did OO and fish oil. The ratio of apoB to apoA was significantly lower after SU-PS consumption than after consumption of OO (P = 0.0126) and fish oil (P = 0.0292). FO-PS and SU-PS resulted in similar ratios of apoB to apoA. HDL2 and the ratio of HDL2 to HDL3 were significantly higher at the end of the FO-PS treatment than at the end of the OO (P = 0.0006), fish oil (P = 0.0036), and SU-PS (P = 0.0016) treatments.

Conclusion: Supplementation of an OO-based diet with FO-PS may reduce cardiovascular disease risk more than does supplementation with fish oil or SU-PS.

Key Words: Apolipoproteins • cardiovascular risk factors • fish oil • LDL cholesterol • HDL cholesterol • n–3 polyunsaturated fatty acids • olive oil • plant sterols • plasma lipids • triacylglycerols


INTRODUCTION  
Coronary heart disease (CHD) remains one of the leading causes of mortality and morbidity in developed countries (1, 2). Lowering LDL-cholesterol concentrations has been identified by the National Cholesterol Education Program (NCEP) for the detection, evaluation, and treatment of high blood cholesterol in adults as the primary target of therapy for CHD risk (3). However, other risk factors may significantly affect CHD risk, and a growing body of evidence indicates that elevated plasma triacylglycerol concentrations may be considered an independent risk factor (4-6). A recent meta-analysis showed that an increase of 1 mmol triacylglycerol/L was associated with 14% and 37% higher risks of cardiovascular disease in men and women, respectively (7). In addition, high triacylglycerol concentrations are often associated with other atherogenic factors, such as abdominal obesity, low HDL-cholesterol concentrations, small LDL particles, high blood pressure, and insulin resistance (8, 9). This combination of metabolic abnormalities, which is known as the metabolic syndrome, increases CHD risk at any given concentration of LDL cholesterol (3).

During the past 10 y, much effort has gone toward the development of nonpharmacologic approaches to decrease CHD risk. Plant sterols, also called phytosterols, are plant components that are structurally similar to cholesterol (10) and that are thought to interfere with the intestinal absorption of cholesterol by displacing cholesterol from micelles (11, 12). In fact, consumption of 2 g plant sterols or stanols/d is among the recommendations of the NCEP in lowering LDL-cholesterol concentrations (13). However, plant sterols were not shown to have any beneficial effect on circulating triacylglycerol concentrations (11, 14). Because long-chain n–3 polyunsaturated fatty acids (n–3 LC-PUFAs) from fish oil have a potent hypotriglyceridemic effect (15, 16), plant sterols have been recently esterified to fish-oil fatty acids to obtain a final product that would simultaneously reduce plasma LDL-cholesterol and triacylglycerol concentrations. Preliminary studies in animal models suggest that the fish-oil plant sterol esters (FO-PS) may reduce both plasma cholesterol and triacylglycerol concentrations (17-19). However, the efficiency of these novel plant sterol esters has not been shown in humans.

In the current study, we tested the effect of a novel fish-oil supplement containing plant sterols esterified to fish-oil fatty acids via an enzymatic process (ie, FO-PS) on the lipid profile of overweight, hyperlipidemic subjects. Our working hypothesis was that, when supplemented to an olive oil (OO)-based diet, this novel FO-PS supplement would cause an additional improvement in both plasma cholesterol and triacylglycerol concentrations. The effects of the FO-PS esters were compared with those of a nonesterified triacylglycerol fish oil and with sunflower oil of plant sterols (SU-PS).


SUBJECTS AND METHODS  
Subjects
Twenty-four hypercholesterolemic men and postmenopausal women aged 30–65 y were recruited by newspaper advertisement in Montreal, Canada, and the surrounding areas. On the basis of previous research by our group (20), a difference of 0.56 mmol/L in endpoint LDL-cholesterol concentrations could be expected between the phytosterol-containing diets and the control diet. With the use of the SD obtained in that study (ie, 0.84), a sample size of 20 was sufficient to detect a difference of 0.48 mmol/L with 95% CIs and 80% power. Twenty-four subjects were enrolled to compensate for eventual dropouts. Subjects were screened for LDL cholesterol after 12 h of fasting. Criteria for inclusion in the study were plasma LDL-cholesterol concentrations >2.56 mmol/L and body mass index (BMI; in kg/m2) between 24 and 30. Persons who reported having taken lipid-lowering drugs, high-dose dietary supplements, or fish-oil capsules during the previous 3 mo were excluded. Before enrollment, subjects provided a complete medical history and underwent a routine physical examination. Detailed blood chemistry analyses were also performed to rule out any abnormality. Volunteers with diabetes mellitus, kidney disease, or liver disease were excluded. Smokers and volunteers drinking more than the equivalent of 2 glasses (or 284 mL) wine/d were also excluded. One woman taking thyroid hormone therapy was included in the study because she had been shown to be stable in the past few months, and no change was planned in her medication. Women taking hormone replacement therapy were asked to maintain their current regimen for the duration of the study. A physician was on call continually throughout the trial for subjects to contact in case they experienced discomfort.

All subjects provided written informed consent by completing forms that had been approved by the Ethics Review Board (protocol no. REB 808-0403). The experimental protocol was approved by the Faculty of Medicine Ethics Review Board at McGill University (Montreal).

Protocol and diets
Subjects underwent a semirandomized, crossover, single-blind clinical trial at the Mary Emily Clinical Nutrition Research Unit of McGill University. The study consisted of 4 phases of 29 d each, during which subjects were provided with an OO-based, weight-maintaining, North American diet. The composition of the control OO diet is shown in Table 1. In accordance with the Canadian Nutrient Recommended Intakes, the diet provided 55% of energy from carbohydrates, 30% from fat, and 15% from protein. Cholesterol and fiber contents were 80 mg/1000 kcal and 13 g/1000 kcal, respectively. The diet was identical during the 4 dietary phases, except for the treatment oil. The plant sterol and fatty acid compositions of the different treatments are shown in Table 2. All subjects received the control OO diet during the first 1-mo phase. They were then randomly assigned to the 3 remaining dietary treatments with the use of a Latin-square design. During the control OO phase, 70% of energy was provided as extra-virgin OO. During the other 3 phases, a small proportion of OO was replaced by the treatment oils: 1) 7.6 g fish oil providing a total of 1.7 g eicosapentaenoic acid (EPA) and 3.7 g docosahexaenoic acid (DHA), 2) 9.6 g fish oil containing FO-PS and providing the equivalent of 1.7 g free plant sterols as well as a total of 1.7 g eicosapentaenoic acid (EPA) and 3.7 g docosahexaenoic acid (DHA), or 3) 21.4 g low-fat SU-PS margarine (Take Control; Unilever Bestfoods NA, Baltimore, MD) providing the equivalent of 1.7 g free plant sterols. The control OO provided the equivalent of 0.02 g naturally occurring free plant sterols/d. The fish oil was manufactured by Ocean Nutrition Canada Ltd (Halifax, Canada) and contained 51% DHA and 23% EPA (% of total fatty acids). The FO-PS was synthesized by Enzymotec Ltd (Migdal HaEmeq, Israel) by using the same batch of fish oil. Tocopherol mixtures (0.2% by wt) were added to the base stock fish oil used as a control fish-oil treatment and as a source of fatty acids for the esterification of plant sterols. No antioxidant beyond the original formulation was introduced to the tested plant sterol esters of fish oil.


View this table:
TABLE 1. Average composition of the control olive oil–based diet over a period of 3 d1

 

View this table:
TABLE 2. Fatty acid and plant sterol composition of study formulations1

 
The nutrient content of the diet was adjusted by using FOOD PROCESSOR software (version 7.81; Food Processor, Salem, OR). Three isocaloric meals, of similar macronutrient and micronutrient composition, were prepared daily for each subject in the metabolic kitchen of the research unit. Food ingredients were weighted to the nearest 0.5 g. Breakfast, during which treatment oils were ingested, was consumed under supervision at the clinic. The single morning dose of fish oil and FO-PS was stirred into orange juice to neutralize fish-oil organoleptic properties. The control OO was provided in orange juice as well, and the SU-PS margarine was served on French toast, English muffins, or omelets. In both cases, there was no delay between the consumption of the plant sterol esters and the remainder of the meal. Lunch and dinner were packed for consumption at work or home. A 3-d rotating menu was offered to provide a variety of foods. Volunteers were instructed to eat and drink only the food prepared at the clinic, but they were allowed water. Consumption of alcoholic and caffeinated beverages was strictly prohibited during the treatment phases. Subjects were provided with decaffeinated, energy-free beverages to drink between meals. Each treatment phase was followed by a wash-out period of 3 to 4 wk during which subjects consumed their habitual diet and did not visit the clinic. The subjects were regularly asked to maintain their usual level of physical activity throughout the study and to report any symptom, disease onset, medication consumption, or change in their habits.

Basal energy requirements were calculated individually for each subject by using the Mifflin equation (21). Basal energy requirements were then multiplied by 1.7 to supply the additional energy needs for mild-to-moderate activity. Body weight was monitored daily during treatment phases. If subjects gained or lost weight during the first week of the first phase, energy intake was adjusted to maintain constant body weight. The same energy intakes were provided in the next 3 phases.

Fasting blood samples were collected on days 1, 2, 28, and 29 of each treatment phase for measurement of plasma lipid concentrations. On day 28 of each phase, postprandial plasma triacylglycerol concentrations were measured 4 h after breakfast. On day 29 of each phase, an additional blood sample was taken for a complete blood count analysis to ensure that no subject had developed anemia.

Laboratory analyses
Plasma lipids, apolipoproteins and lipoprotein(a)
Blood samples were collected in Vacutainer tubes (Becton Dickinson, Mississauga, Canada) containing polymer gel and silica activator. After 30 min, tubes were centrifuged at 1000 x g for 15 min at 4 °C to isolate plasma. Serum samples were stored at –80 °C until lipids were measured. Serum total cholesterol, HDL-cholesterol, and triacylglycerol concentrations were measured by an enzymatic method using the corresponding Flex reagents on a multianalyzer (Dimension RxL Max; Dade Behring Diagnostics, Marburg, Germany). LDL-cholesterol concentrations were calculated with the equation of Friedewald et al (22) in samples containing <4.5 mmol triacylglycerol/L. When plasma triacylglycerols were >4.5 mmol/L, LDL-cholesterol concentrations were measured directly by using the Flex reagents on the Dimension RxL Max multianalyzer. Subclasses 2 and 3 of HDL cholesterol (HDL2 and HDL3) were obtained by dual precipitation as described previously (23, 24). In brief, plasma apolipoprotein (apo) B–containing lipoproteins were precipitated with manganese chloride-heparin (1.12 mol MnCl2/L, 20 000 USP units heparin/mL; 50:6 by vol) by ultracentrifugation at 1500 x g for 1 h at 4 °C. Part of the supernatant fluid was used to analyze total HDL cholesterol, and the remainder was mixed with dextran sulfate (14.3 mg/mL) to precipitate HDL2 cholesterol. After ultracentrifugation at 1500 x g for 30 min at 4 °C, HDL3 cholesterol was measured in the supernatant fluid. Total HDL and HDL3 cholesterol were measured by using a cholesterol enzymatic kit (Roche Diagnostics, Laval, Canada). Correction factors of 1.1 and 1.21 were used for total HDL and HDL3 cholesterol, respectively, to take into account the dilution by the reagents. HDL2 cholesterol was calculated from the difference between total HDL and HDL3 cholesterol. Apo A-I and apo B were measured by using the N Antisera kit for apo A-I and apo B assays, respectively, on the BN ProSpec Nephelometer (Dade Behring Diagnostics). To reduce day-to-day variation, endpoint lipid concentrations were obtained from the averages of values obtained on days 28 and 29. Lipoprotein(a) [Lp(a)] concentrations were measured in plasma samples collected on day 28 of each phase by using the N Latex Lp(a) assay (Dade Behring Diagnostics) on the BN ProSpec Nephelometer.

Plasma fatty acid profile
Plasma samples from days 1, 2, 28, and 29 were analyzed for fatty acid composition by using gas-liquid chromatography. Total lipids were extracted by a modified Folch extraction (25), and fatty acids were methylated according to the procedure of Morrison and Smith (26). Briefly, an internal standard (heptadecanoic acid, 1 mg/mL) and methanol were added to the serum samples. Total lipids were extracted by using a ratio of chloroform to methanol (4:1, by vol) in the presence of deionized, distilled water. The aqueous phase was separated by centrifugation, and the organic supernatant phase was transferred into another tube. The aqueous phase was then re-extracted by using a ratio of hexane to chloroform (4:1, by vol). The supernatant fluid was added to the first extraction and dried under nitrogen. The methylating reagent (boron fluoride–methanol:hexane:methanol (7:6:7, by vol) was added to the samples, which were heated at 100 °C for 55 min. After the samples were cooled at room temperature, hexane and deionized distilled water were added to the samples. The tubes were centrifuged at 2500 rpm for 5 min at 4 °C (Sorvall model RT 6000B; DuPont Co, Wilmington, DE), and the top layer was transferred to a disposable culture glass tube, dried under nitrogen, redissolved in a smaller volume of hexane containing 50 ppm butylated hydroxytoluene (BHT), and transferred to amber vials.

Fatty acid profiles were determined by using a gas chromatograph (Clarus 500 GC; Perkin-Elmer, Shelton, CT) equipped with a 100-m x 0.2-mm SP-2560 fused silica capillary column (Supelco, Bellefonte, PA) and a flame ionization detector. The column temperature was held at 100 °C for 1 min, increased to 210 °C at a rate of 8 °C/min, and held at 210 °C for the remainder of the run. The injector temperature was set at 250 °C and the detector temperature at 275 °C. Helium was used as the carrier gas at a flow rate of 45 mL/min. Fatty acid methyl esters were identified on the basis of the retention time of known standards (Sigma-Aldrich Canada Ltd, Oakville, Canada). Results were expressed as a percentage of total identified fatty acids by weight.

Plasma plant sterol concentrations
Plasma concentrations of campesterol and ß-sitosterol were determined by using gas-liquid chromatography as reported previously (27). An internal standard of 5-cholestane (Sigma-Aldrich Canada Ltd) was added to each plasma sample. Samples were saponified with 0.5 mol methanolic KOH/L. Nonsaponified materials were then extracted 2 times with petroleum ether and dried under nitrogen flux. The extracts were derivatized by using TMS reagent (pyridine:hexamethyldisilazan:trimethylchlorosilane 9:3:1 by vol) (Sigma-Aldrich Canada Ltd; 28). After evaporation under nitrogen, the samples were dissolved in hexane and injected into a gas-liquid chromatograph (HP 5890 Series II; Hewlett-Packard, Palo Alto, CA) equipped with a flame ionization detector and an auto-injector system. Separation was achieved on a 30-m SAC-5 capillary column with an internal diameter of 0.25 mm and film thickness of 0.25 µm (Supelco). The carrier gas (helium) flow rate was 7.5 psi. Samples were injected at 300 °C. The oven temperature remained at 160 °C for 1 min after injection, was increased to 245 °C at a rate of 15 °C/min, and then was kept constant for 5 min, after which it rose to 285 °C at a rate of 15 °C/min. Then, the temperature was kept constant at 285 °C for 20 min. The detector was set at 310 °C. Plant sterols (ie, campesterol and ß-sitosterol) were identified by using authentic standards (Sigma-Aldrich Canada Ltd).

Plasma retinol and -tocopherol concentrations
Plasma concentrations of the fat-soluble vitamins, ie, retinol and -tocopherol, were measured by using light-protected, reverse-phase HPLC as previously described (29). Briefly, an internal standard solution (10 mg/dL) of retinol acetate (Sigma-Aldrich, St Louis, MO) was added to 250-µL plasma samples. Fat-soluble compounds were extracted with 200 µL methanol and 1000 µL hexane and then were mixed by vortex for 1 min. The organic phase was separated by centrifugation for 10 min at 13 000 x g at room temperature. The hexane layer (750 µL) was then transferred and dried under nitrogen. The extracts were dissolved in 250 µL methanol, mixed by vortex, and injected into an HPLC column (JASCO, Dunmow, United Kingdom) equipped with an ultraviolet detector and an auto-injector system. Separation was achieved on a 150 x 4.6–mm octadecylsilane 3-µm particle column (Supercosil LC-18DB; Sigma-Aldrich), with methanol as a mobile phase, at a flow rate of 1.5 mL/min. Fat-soluble vitamins were identified by using authentic standards (Sigma-Aldrich) and multiwavelength detection (325 and 292 nm for retinol and -tocopherol, respectively) and were quantified by using standard curves.

LDL lipid peroxidation
LDL particles were precipitated according to the method of Gidez et al (23). Briefly, a manganese chloride–heparin solution was added to plasma samples. After centrifugation, the supernatant was discarded and the LDL fraction was resuspended in normal saline. Lipid peroxidation in plasma LDL subfractions was measured by using the thiobarbituric acid–reactive substance (TBARS) assay (Oxitek; ZeptoMetrix Corporation, Buffalo, NY) as described in the manufacturer’s manual.

Statistical analyses
Results are expressed as means ± SEMs, and P < 0.05 was considered significant. Variables that were not normally distributed were log transformed before analysis. Differences in plasma variables were evaluated by using, first, repeated-measures analysis of variance (ANOVA) for carryover effect and, second, repeated-measures ANOVA with the type of dietary matrix in each intervention arm as the within-subject factor and with endpoint values as the between-subject factors. Baseline values, sex, age, BMI, or all of those variables were inserted into the model as covariates if their interactions with dietary matrixes were found to be statistically significant. Subsequently, contrast analyses were used to identify differences between pairs of diets. All statistical analyses were conducted by using SAS software (version 8.2; SAS Institute, Cary, NC).


RESULTS  
Subjects
Twenty-four subjects who fit the study criteria were included in the study. Three subjects withdrew from the study before completion: 2 dropped out for personal reasons, including lack of time and difficulty in reaching the research clinic, and 1 abandoned the study because of gastrointestinal discomfort associated with fish-oil consumption. The remaining 21 subjects completed all 4 treatment phases. The characteristics of the 21 study subjects at the time of selection are shown in Table 3.


View this table:
TABLE 3. Baseline characteristics of the subjects who completed all dietary treatments

 
Most subjects maintained good health throughout the study, and no major adverse events were reported. Approximately one-half of the patients reported burping after ingestion of the fish oil (n = 9) and the FO-PS (n = 10). Some patients complained about a fishy aftertaste (n = 5 and 2 for the fish oil and the FO-PS, respectively). Nausea (n = 1 for each of the fish-oil treatments) and gastrointestinal discomfort (n = 4 and 3 for the fish-oil and FO-PS treatments, respectively) were reported by a few subjects. In most cases, the symptoms were present for 2–4 h after consumption of the supplement, and their intensity decreased from day to day. Chewing sugarless gum helped decrease the intensity of the side effects. One subject had diarrhea for 2–3 d at the beginning of the 2 fish-oil treatments, and another patient had diarrhea for 4 d during the fish-oil phase. In the latter case, it is suspected that the disorder was of infectious origin. Except for a light abdominal discomfort reported by 3 patients at the beginning of the control OO diet, no gastrointestinal symptoms were reported during the control OO or the SU-PS phase.

At endpoints, body weights were 73.3 ± 2.6, 74.4 ± 2.8, 72.4 ± 2.8, and 74.3 ± 2.8 kg for the control OO, control fish-oil, FO-PS, and SU-PS groups, respectively. Because body weights at the end of each treatment phase were significantly correlated with body weights at the beginning of the phases (P = 0.0001), initial BMI values were used as covariates in the model.

Biochemical endpoints
Plasma lipid concentrations at the end of each treatment phase are shown in Table 4. Supplementation of a control OO-based diet with FO-PS and SU-PS resulted in LDL-cholesterol concentrations that were significantly (P = 0.0031 and 0.041, respectively) lower than those observed with the control diet. A strong tendency toward a reduction in total cholesterol concentrations (P = 0.067) was also observed. These observations were associated with significantly lower total:HDL cholesterol after supplementation with FO-PS and SU-PS than after the control OO diet and fish-oil supplementation. Apo B concentrations were 5–6% lower after SU-PS and FO-PS supplementation than after the control OO and fish-oil diet. In addition, SU-PS resulted in apoB:apo A that were significantly lower than those observed with control OO (P = 0.013) or fish oil (P = 0.029). ApoB:apoA at the end of the FO-PS phase did not differ significantly from the ratios observed after consumption of SU-PS and were significantly lower than those obtained with fish oil (P = 0.0049).


View this table:
TABLE 4. Fasting plasma lipid, apolipoprotein, plant sterol, and thiobarbituric acid–reactive substance concentrations in overweight, hyperlipidemic subjects consuming different oil supplements varying in fatty acid and plant sterol content for 4 wk1

 
The effect of the diets on HDL-cholesterol subfractions is also shown in Table 4. HDL2 concentrations were higher at the end of the FO-PS phase than at the end of the other 3 diets. In addition, fish oil and FO-PS resulted in lower HDL3 subfraction concentrations than did OO and SU-PS. These modifications resulted in ratios of HDL2 to HDL3 (HDL2:HDL3) that were 62%, 7%, and 46% higher after supplementation with FO-PS than after consumption of control OO (P = 0.0006), fish oil (P = 0.0036), and SU-PS (P = 0.0016), respectively.

Significant differences were observed between the effects of the dietary treatments on fasting and postprandial plasma triacylglycerol concentrations. Indeed, supplementation of the control OO diet with fish oil and FO-PS resulted in fasting triacylglycerol concentrations that were 40% (P = 0.0004) and 46% (P = 0.0002) lower, respectively, than those observed with OO alone. Moreover, FO-PS and fish oil resulted in plasma triacylglycerol concentrations that were 39% (P < 0.0001) and 32% (P = 0.0001) lower, respectively, than those observed with SU-PS. It is interesting that fasting plasma triacylglycerol concentrations were significantly (P = 0.03) lower after consumption of FO-PS than after fish-oil supplementation. In the postprandial state, plasma triacylglycerol concentrations were 40% and 30% lower at the end of the FO-PS and fish-oil phases, respectively, than at the end of the control OO and SU-PS phases. Similar to the differences in fasting triglycerol concentrations, postprandial triacylglycerol concentrations after the consumption of FO-PS were significantly different from those observed with fish oil (P < 0.0001).

Plasma Lp(a) and TBARS concentrations were not significantly altered by the dietary treatments (Table 4). Consumption of SU-PS resulted in significantly (P = 0.0013 and 0.021) higher plasma campesterol concentrations than did the control OO and fish-oil diets, respectively (Table 4). FO-PS also resulted in high campesterol concentrations, which did not differ from those observed after supplementation with SU-PS. However, plasma ß-sitosterol concentrations were not significantly altered by the different diets. Plasma concentrations of retinol and -tocopherol were not significantly affected by plant sterol ester supplementation. Indeed, retinol concentrations were 5.9% lower after SU-PS supplementation (median: 64.2 µg/dL; range: 48.2–96 µg/dL) than after consumption of the control OO diet (median: 67.2 µg/dL; range: 44.1–85.2 µg/dL), but this difference was not significant (P = 0.43). FO-PS (median: 72.1 µg/dL; range: 46.2–87.4 µg/dL) resulted in retinol concentrations that did not differ from those observed with control OO. SU-PS and FO-PS resulted in -tocopherol concentrations (median: 1660 µg/dL; range: 990–3650 µg/dL for SU-PS; median: 1550 µg/dL; range: 900–3170 µg/dL for FO-PS) that were 10% and 4% higher, respectively, than the concentrations measured at the end of the control OO diet (median: 1490 µg/dL; range: 700–3510 µg/dL); however, this effect was not significant (P = 0.77).

The effect of the different treatments on the plasma fatty acid profile is shown in Table 5. EPA and DHA concentrations were markedly higher after fish-oil and FO-PS supplementation than after the control OO diet (P < 0.0001 and < 0.0001; P < 0.0001 and = 0.0036, respectively) and supplementation with SU-PS (P < 0.0001 and = 0.0034; P = 0.0022 and 0.0466, respectively). Fish-oil supplementation resulted in oleic acid concentrations that were lower than those observed with control OO (P = 0.0334) and SU-PS (P = 0.0019). Both fish-oil diets and the SU-PS resulted in lower arachidonic acid concentrations than did the control OO diet (P = 0.0003, 0.0007, and 0.0303 for fish oil, FO-PS, and SU-PS, respectively); the concentrations with the fish-oil treatments were lower than those with the SU-PS (P = 0.0034 and 0.0215 for fish oil and FO-PS, respectively). Lower linoleic acid concentrations were observed with supplementation of both fish-oil matrixes than with control OO (P = 0.0054 and 0.0365 for fish oil and FO-PS, respectively) and SU-PS (P = 0.0001 and 0.0031 for fish oil and FO-PS, respectively). Supplementation of FO-PS resulted in lower and consumption of SU-PS resulted in higher -linolenic acid concentrations than were observed after consumption of the control OO diet (P = 0.00177 and 0.0054, respectively).


View this table:
TABLE 5. Plasma fatty acid profile in overweight, hyperlipidemic subjects consuming different oil supplements varying in fatty acid and plant sterol content for 4 wk1

 

DISCUSSION  
The current results show that, in overweight, hyperlipidemic subjects consuming an OO-based diet, FO-PS supplementation has a potent hypotriglyceridemic effect that may be even more pronounced than the one observed with regular fish-oil supplementation, and it results in better lipoprotein and apolipoprotein profiles than are seen with fish oil. Moreover, the triacylglycerol-lowering properties of FO-PS, in addition to their increasing effect on HDL2 cholesterol subfractions, make these novel plant sterol esters more beneficial than is SU-PS.

The hypotriglyceridemic effect of fish oil is well documented (15, 16). The 40% reduction in fasting plasma triacylglycerols observed in the current study in patients ingesting 7.6 g fish oil/d is proportional to the 14% decrease in triacylglycerol associated with the average daily consumption of 2.7 g fish oil reported in clinical trials in normotriglyceridemic patients (30). Postprandial triacylglycerol concentrations, which may be a better indicator of CHD than are fasting measurements (31), were also substantially lowered by FO-PS and fish-oil supplementation. The high-dose FO-PS supplement used in this experiment was necessary to provide the equivalent of 1.7 g free plant sterols/d. The lower triacylglycerol concentrations observed with FO-PS than with fish oil suggest a contribution of the plant sterol ester component to the triacylglycerol-lowering effect. Such a hypothesis is supported by data from other studies showing a hypotriglyceridemic effect of ascorbic acid esters of plant sterols in hamsters (32) and of vegetable oil fatty acid esters of plant stanols in hypertriglyceridemic subjects (33). However, the effect on triacylglycerols of SU-PS supplementation did not differ significantly from that of the control OO diet (P = 0.104). Data on the possible hypotriglyceridemic effect of plant sterols are still scarce, and confirmation of such an effect would require further investigations. On the other hand, the current data show that the novel FO-PS are efficient in lowering plasma triacylglycerol concentrations in the context of an OO-based diet, which, on its own, has been reported to induce a 10% triacylglycerol reduction compared with an average American diet (34).

Although n–3 LC-PUFAs from fish are associated with a lower risk of CHD because of their numerous beneficial effects on plasma triacylglycerols (35, 36), inflammation, coagulation, endothelial function (36) and heart rate (36, 37), their effect on plasma cholesterol is usually minor. Indeed, total and LDL-cholesterol concentrations are usually not affected by n–3 LC-PUFA supplementation (38-40). Because plant sterols have a well-established effect of lowering LDL-cholesterol concentrations (14, 41), we expected that plant sterols esterified to fish-oil fatty acids would cause an overall decrease in plasma total and LDL-cholesterol concentrations in hypercholesterolemic subjects. In the current study, LDL-cholesterol concentrations after supplementation with SU-PS and FO-PS were significantly lower than those at the end of the control OO diet phase. However, the reduction in LDL cholesterol was less pronounced (–3% and –6% with FO-PS and SU-PS, respectively) than was expected with the equivalent of 1.7 g free plant sterols [ie, –8.5% according to the meta-analysis of Katan et al (14)].

One explanation for the lower efficacy of plant sterols in the current study than in other trials may be that the plant sterol esters were given in a single dose. Most studies of the hypocholesterolemic effect of plant sterols have used 2–3 doses of plant sterols/d. Our one-dose design was based on the results of Plat et al (42), who observed no difference in LDL-cholesterol reduction between normocholesterolemic and hypercholesterolemic subjects consuming 2.5 g plant sterols/d at lunch or divided over the 3 daily meals (–9.9% and –10.2%, respectively). In another study in which 2.7 g plant sterols was given to hypercholesterolemic subjects in a single dose at lunchtime, LDL cholesterol was 14.6% lower than at baseline (43). It may be hypothesized that the time of administration affects the magnitude of the hypocholesterolemic effect of plant sterols. In the current study, plant sterol esters were given at breakfast. Results of a recent study in mildly hypercholesterolemic subjects showed that the LDL cholesterol–lowering effect of a single dose of 2.8–3.2 g plant sterols/d provided in a yogurt drink >30 min before breakfast was significantly less pronounced than the effect of the same plant sterol drink ingested with lunch (44). The latter results raise questions as whether plant sterol efficacy is affected by postprandial state, diurnal positioning of dose, or both. In the current study, all treatments were ingested at meal time, and no delay occurred between the consumption of the plant sterol esters and the meal. Therefore, differences between treatments could not be attributed to differences in intake occasion. The plant sterol dose itself does not appear to explain the absence of a significant effect on LDL cholesterol in the current study. Although the dose used in our experiment (1.7 g/d) was lower than the doses used in the previous once-a-day studies (1.7 g/d compared with 2.5–3.2 g/d, respectively; 42-44), a previous study showed that increasing the dosage from 1.6 to 3.2 g free sterol equivalent/d did not further increase LDL cholesterol–lowering efficacy (45).

Plasma HDL-cholesterol concentrations usually are inversely correlated with triacylglycerol concentrations (46). However, despite their potent hypotriglyceridemic properties, n–3 LC-PUFAs from fish oil have been reported to have no effect (16) or, when supplemented at high doses, to decrease total HDL-cholesterol concentrations (47). On the other hand, EPA and DHA may alter HDL-cholesterol subclasses. Increases in the HDL2 subfraction have been reported with supplementation of 4 g DHA/d in hyperlipidemic men and patients with type 2 diabetes (48, 49). The effect of EPA on HDL2 subclasses is less clear; a lowering effect on HDL3 concentrations [with no effect on HDL2 (48)] and higher HDL2 measurements (49) have each been observed. When supplemented simultaneously, 1.48 g DHA/d and 1.88 g EPA/d were shown to increase HDL2 concentrations in subjects with familial combined hyperlipidemia, a disorder characterized by low HDL2 concentrations (50). HDL-cholesterol concentrations usually are not significantly affected by plant sterols (10, 14, 51), but a slight increase has been reported in a few studies (52, 53). In the current trial, FO-PS resulted in higher HDL2-cholesterol concentrations and HDL2:HDL3 than did the 3 other diets. This effect may be due to the EPA and DHA components of FO-PS. However, despite a similar fatty acid composition, the fish oil did not significantly improve HDL2 concentrations or HDL2:HDL3 relative to the control diet, which suggests that the esterification to plant sterols may beneficially affect the HDL subclass–modifying properties of n–3 LC-PUFAs from fish oil. HDL2-cholesterol concentrations have been shown to be inversely associated with the risk of established CHD, and this association is stronger than the association with total HDL cholesterol (54). It is therefore possible that the higher HDL2 concentrations observed when FO-PS are supplemented to a monounsaturated fatty acid–rich diet may result in a greater decrease in the risk of myocardial infarction than does supplementation with fish oil.

In our study, apoB:apoA was lower at the end of the SU-PS treatment than after the control OO diet and after supplementation with fish oil. Although apoB:apoA after FO-PS supplementation did not differ significantly from the ratio observed after SU-PS supplementation, the difference between FO-PS and control OO was not significant. However, apoB:apoA at the end of the FO-PS phase was significantly lower than that at the end of the fish-oil phase. In addition, FO-PS resulted in lower total:HDL cholesterol, another risk factor for cardiovascular disease, than did control OO and fish oil. The importance of this variable was shown in the Quebec Cardiovascular Study (55), which concluded that the variation in total:HDL cholesterol may be associated with more substantial alterations in metabolic indexes that are predictive of ischemic heart disease risk and related to the insulin resistance syndrome than is the variation in LDL cholesterol:HDL cholesterol. Whether such a beneficial effect in total:HDL cholesterol could be obtained by concomitant ingestion of fish oil and plant sterols remains to be elucidated.

It is assumed that the active form of plant sterols is the free form. Therefore, the rate of hydrolysis of plant sterol esters of different fatty acids in the intestinal tract could be a factor influencing their efficacy. In the current study, the similar increases in plasma campesterol and sitosterol concentrations observed after the ingestion of FO-PS and SU-PS suggest that the bioavailability of plant sterols is unaltered when they are esterified to fish-oil fatty acids. Reciprocally, n–3 LC-PUFAs from fish oil may be as bioavailable when they are esterified to plant sterols as when they are delivered in the customary fish-oil form. Indeed, the proportions of EPA and DHA were increased similarly at the end of the fish-oil and FO-PS supplementation phases, and both fish-oil treatments had a strong hypotriglyceridemic effect.

In summary, supplementation of an OO-based diet with FO-PS resulted in a marked decrease in plasma triacylglycerol concentrations in overweight, hyperlipidemic subjects. Similarly to the traditional vegetable oil fatty acid esters of plant sterols used in the current study, the novel FO-PS significantly reduced LDL-cholesterol concentrations. However, the LDL cholesterol–lowering effect was less pronounced than expected. This may be due to the time and frequency of plant sterol administration in the current experiment. Nevertheless, for hyperlipidemic persons whose triacylglycerol needs to be lower, FO-PS may provide additional effects by resulting in lower total:HDL cholesterol, lower apo B concentrations and apoB:apoA, and higher HDL2:HDL3 than does fish oil. FO-PS may also present some advantages over SU-PS; in addition to a potent triacylglycerol-lowering effect, FO-PS resulted in higher HDL2 concentrations and HDL2:HDL3. These beneficial effects may result in further reductions in cardiovascular disease risk.


ACKNOWLEDGMENTS  
We thank Joel Lavoie (Montreal Cardiology Institute) for analyzing blood lipid concentrations, William Parsons (School of Dietetics and Human Nutrition, McGill University) for monitoring the participants’ health status during the study, and Marielle Kaplan (Clinical Biochemistry Department, Rambam Medical Center, Haifa, Israel), for analyzing the fat-soluble vitamins. We are indebted to Catherine Vanstone and Maryse Ménard for their participation in patient recruitment and management, Magda Fahmy and Maryse Ménard for their contribution to the development of the study menus, and Linda Lapensée and the staff of the Mary Emily Clinical Nutrition Research Unit for preparing and serving the meals to the study participants. We are grateful to Khatima Khalloufi for her participation in fatty acid extraction and chromatogram analysis. We thank Esther Shabtai (Statistics Services Unit, Sourasky Tel Aviv Medical Center, Tel Aviv, Israel) for assistance with the statistical analyses, Ligia Rujan for venipuncture assistance, and the subjects for their commitment to this study.

PJHJ and DP designed the trial; ID coordinated the study with the participation of Y-MC; ID was responsible for patient recruitment and management, conducted the TBARS and plasma fatty acid laboratory analyses, performed statistical analyses, interpreted the data, and wrote the draft of the manuscript; Y-MC was responsible for the plant sterol and HDL subfraction measurements; ID and Y-MC were responsible for interpretation of the data; and all authors participated in critical review of the manuscript and approved the final version. DP is the Director of Clinical Studies at Enzymotec Ltd. None of the other authors had any personal or financial conflict of interest.


REFERENCES  

Received for publication May 28, 2006. Accepted for publication July 20, 2006.


日期:2008年12月28日 - 来自[2006年84卷第6期]栏目
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Reduced-calorie orange juice beverage with plant sterols lowers C-reactive protein concentrations and improves the lipid profile in human volunteers

Sridevi Devaraj, Bryce C Autret and Ishwarlal Jialal

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.


View this table:
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).


View this table:
TABLE 2. Baseline characteristics of the subjects1

 

View this table:
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).


View this table:
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).


View larger version (12K):
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).


View this table:
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  

Received for publication January 31, 2006. Accepted for publication May 12, 2006.


日期:2008年12月28日 - 来自[2006年84卷第4期]栏目

Plant sterols are efficacious in lowering plasma LDL and non-HDL cholesterol in hypercholesterolemic type 2 diabetic and nondiabetic persons

Vivian WY Lau, Mélanie Journoud and Peter JH Jones

1 From the School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, Canada

2 Presented in part at the Federation of American Societies for Experimental Biology Annual Conference in San Diego, CA (April 11-15, 2003), and at Canadian Society of Clinical Nutrition Annual Conference in Vancouver, Canada (April 24-26, 2003).

3 Supported by the Canadian Diabetes Association.

4 Address reprint requests to PJH Jones, School of Dietetics and Human Nutrition, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9. E-mail: peter.jones{at}mcgill.ca.


ABSTRACT  
Background: Because of hyperglycemia and hyperinsulinemia, diabetic persons have higher cholesterol synthesis and lower cholesterol absorption rates than do nondiabetic persons. Differences in plant sterol efficacy between diabetic and nondiabetic persons have not been examined.

Objective: The objective was to compare the degree of response of plasma lipid concentrations and glycemic control to plant sterol consumption in a controlled diet between hypercholesterolemic type 2 diabetic and nondiabetic subjects.

Design: Fifteen nondiabetic subjects and 14 diabetic subjects participated in a double-blinded, randomized, crossover, placebo-controlled feeding trial. The diet included 1.8 g/d of either plant sterols or cornstarch placebo over 21 d, separated by a 28-d washout period.

Results: Plant sterol consumption significantly reduced (P < 0.05) LDL-cholesterol concentrations from baseline in both nondiabetic and diabetic subjects by 15.1% and 26.8%, respectively. The diabetic subjects had significantly (P < 0.05) lower absolute concentrations of total cholesterol after treatment than did the nondiabetic subjects; however, there was no significant difference in the percentage change from the beginning to the end of the trial. There was also a significant decrease (P < 0.05) in absolute non-HDL-cholesterol concentrations after treatment in both groups.

Conclusions: The results showed that plant sterols are efficacious in lowering LDL cholesterol and non-HDL cholesterol in both diabetic and nondiabetic persons. Plant sterol consumption may exist as a dietary management strategy for hypercholesterolemia in persons with type 2 diabetes.

Key Words: Hypercholesterolemia • type 2 diabetes • plant sterols • cholesterol


INTRODUCTION  
Type 2 diabetes is associated with metabolic disturbances, including hyperinsulinemia, insulin resistance, frequent dyslipidemia, obesity, and impaired ß-cell function (1-3), and is frequently linked with several lipid aberrations, namely, hypertriacylglycerolemia, elevated VLDL cholesterol, and reduced HDL cholesterol (4-7). However, elevated LDL-cholesterol concentrations are not uniformly present in dyslipidemic, type 2 diabetic persons (8). Studies have shown that persons with type 2 diabetes have an increased synthesis and a decreased absorption of cholesterol (9-13). As a result, the risk of developing atherosclerotic vascular diseases, such as cardiovascular disease (CVD), in type 2 diabetic is 2- to 7-fold that in nondiabetic persons (12, 14, 15).

Diabetic patients with no history of heart disease and nondiabetic persons with a history of myocardial infarction are equally at risk of an infarction (15). Thus, the target blood lipid concentrations are more stringent for diabetic than for nondiabetic persons (16) and should be as follows: 2.5 mmol/L for LDL cholesterol, 2.0 mmol/L for triacylglycerol, and 4.0 for the ratio of total cholesterol (TC) to HDL cholesterol (17).

Plant sterols, which chemically resemble cholesterol, have been shown to block the absorption of dietary and endogenously derived cholesterol from the gut (18, 19). Plant sterols are not synthesized by the human body and are minimally absorbed by the human intestine (20). Daily consumption of 0.7–3.2 g plant sterols/d has been shown to reduce plasma TC by 5.0–13.0%, and LDL cholesterol by 5.6–24.4% in both normo- and hypercholesterolemic persons with (10, 21-23) and without (18, 22, 24-35) type 2 diabetes.

The efficacy of plant sterols in diabetic subjects as compared with that in nondiabetic subjects has not been well examined in previous studies. Further investigation is essential to define dietary strategies that can best normalize the risk of CVD and associated complications in type 2 diabetes. Therefore, the objective of this study was to compare the degree of response of plasma lipid concentrations and glycemic control between hypercholesterolemic type 2 diabetic and nondiabetic subjects consuming plant sterols in a precisely controlled dietary setting.


SUBJECTS AND METHODS  
Subjects
Fourteen slightly overweight hypercholesterolemic subjects with type 2 diabetes and 15 hypercholesterolemic nondiabetic subjects were recruited from the surrounding community by advertisements in local newspapers and in private and public medical clinics. The inclusion criteria required all subjects to be ambulatory, to be between the ages of 40 and 80 y, and to have an LDL-cholesterol concentration of 3–5 mmol/L, a triacylglycerol concentration <5 mmol/L, and a body mass index (BMI; in kg/m2) between 23 and 40. The diagnostic criteria for type 2 diabetes included a fasting plasma glucose concentration 7 mmol/L and a glycated hemoglobin (Hb A1c)concentration of 7–8%. Exclusion criteria included the use of ß-blockers or diuretics and a personal history of CVD. Those who reported exercising at a frequency of 5 times/wk, being pregnant, or lactating were also excluded. Subjects were required to have refrained from using drug therapy for hypercholesterolemia for the 8-wk period before the start of the study.

Before acceptance into study, the subjects were required to undergo a complete physical examination. Fasting blood and urine samples were collected for serum biochemistry, hematology, and urinalysis. Subjects were screened for chronic illnesses—including hepatic, renal, thyroid, and cardiac dysfunction—before admission in the study.

The subjects received a thorough explanation of the study protocol and were given opportunities to discuss any concerns with the principal investigator, attending physician, or research coordinators before signing a consent form. The experimental protocol was approved by the Human Ethical Review Committee of the Faculty of Agriculture and Environmental Sciences at McGill University.

Study protocol
The study was a randomized, double-blind, crossover, placebo-controlled clinical trial that consisted of two 21-d dietary feeding periods that were separated by a 28-d washout period. During the washout period, the subjects resumed their habitual diets without restriction.

At baseline (day 0) and at the end (day 21 and day 22) of each dietary phase, fasting blood samples were taken for the measurement of circulating lipid concentrations. In the comparison of endpoints for the 2 dietary phases, the mean of days 21 and 22 was used to account for day-to-day variation in circulating cholesterol concentrations. Day 0 and day 21 blood samples were also used for the measurement of circulating plant sterol, insulin, and Hb A1c concentrations. At the start (day 1) and end (day 21) of each dietary phase, fasting blood samples were taken for the measurement of circulating fatty acids.

Subjects were given routine physical examinations at the beginning and end of each dietary phase by the attending physician. Throughout the trial, a physician familiar with the study protocol and diets was available in case subjects experienced discomfort with the diet.

Experimental diets
The baseline control diet (BCD) was planned based on the Canada Food Guide to Healthy Eating and the Good Healthy Eating Guide (36). The BCD consisted of solid foods typical of those consumed in North America and was provided as 3 meals and 1 snack per day in a 3-d rotating menu. The nutrient content of the BCD was calculated by using FOOD PROCESSOR (Esha Research, Salem, OR), a computerized dietary analysis system. The BCD was designed to meet recommended intakes for all vitamins and minerals. The Mifflin equation was used to estimate individual basal energy requirements (37), which was then multiplied by an activity factor of 1.7 to compensate for the additional energy need of mildly to moderately active healthy adults (38). If subjects gained or lost weight during the first week of each dietary phase, adjustments were made to individual energy requirements to ensure that baseline body weights were maintained (31). Body weight was monitored daily before breakfast during the feeding periods to assess changes.

The BCD contained 55% of energy as carbohydrate, of which 75% was complex carbohydrate; foods with a low glycemic index were selected when possible. The polyunsaturated:monounsaturated:saturated fatty acid ratio of the 30% of energy provided as fat was maintained at 1:1:1. Safflower, canola, and flaxseed oils were used to provide most of the polyunsaturated fatty acids, whereas extra virgin olive oil was selected to provide monounsaturated fatty acids. Saturated fat came from fats in the meat and palm oil. Flaxseed and canola oils were used as sources of essential fatty acids at the level of 2-3%. Protein accounted for 15% of ingested energy.

During each feeding period, a total of 1.8 g/d of either plant sterol (plant sterol phase) powder or placebo phase cornstarch was added to margarine and served on the breakfast toast every morning under supervision. The plant sterol powder was unesterified plant sterols extracted from wood pulp byproducts (40% sitosterol, 30% campesterol, 20% dihydrobrassicasterol, and 10% others; Forbes Medi-Tech Inc, Vancouver, Canada). The placebo powder was cornstarch, because it strongly resembled the white powdery plant sterols (39). To achieve double blinding, plant sterol powder and the cornstarch were portioned in coded containers by an external party so that neither the researchers nor the subjects would know its true identity. The diets were prepared in the metabolic kitchen of the Mary Emily Clinical Nutrition Research Unit of McGill University. All subjects were required to consume breakfast at the Unit under supervision; the other 2 meals and 1 snack were available for takeout. No extra food was allowed between meals, except for decaffeinated, energy-free carbonated beverages and herbal teas, which were obtained from the kitchen’s staff. Alcoholic beverages and coffee were prohibited during the dietary phases.

Plasma lipid concentrations
Blood samples were drawn, after the subjects had fasted overnight for 12 h and had abstained from alcohol for 24 h (day 0), and collected into EDTA-containing Vacutainer (BD, Franklin Lakes, NJ) tubes. Samples were immediately centrifuged (520 x g, 15 min, room temperature), and the resulting plasma and red blood cell (RBC) subfractions were separated within 1 h of collection and stored at –80 °C until analyzed. Plasma total and HDL cholesterol and triacylglycerol were analyzed in quadruplicate with standardized reagents by using a VP Autoanalyser (Abbott Laboratories, North Chicago, IL). Calibration of the machine before each run was performed as per the standardization protocol of the Canadian Reference Laboratory (1996; Vancouver, Canada), which involved direct comparison with fresh specimen samples. Plasma HDL cholesterol was measured after precipitation of apolipoprotein B with dextran sulfate and magnesium chloride (40). LDL-cholesterol concentrations were calculated by using the Friedewald equation (41).

Fatty acid methyl ester composition
RBCs in blood collected on days 1 and 21 were analyzed in duplicate for fatty acid composition by gas-liquid chromatography (GLC) (HP 5890 Series II; Hewlett-Packard, Palo Alto, CA). A modified Folch extraction was used to extract total lipids from the samples (42), and the fatty acids were methylated as per the procedure by Al Makdessi et al (43). Packed RBCs and C17 standard (1 mg/mL) were placed into a culture tube, and MeOH was added to the sample. The culture tube was then heated to 55 °C in a water bath for 15 min. A solution of hexane:chloroform (4:1, by vol) was added and placed in a wrist action shaker for 15 min. Water (Millipore, Nepean, Canada) was added to the sample, and the sample was shaken for an additional 10 min. The sample was centrifuged at 520 x g (1500 rpm) at 4 °C for 15 min, and the organic supernatant fluid was transferred to a culture tube and dried under nitrogen at 45 °C. The aqueous layer was then reextracted by adding hexane:chloroform, and the sample was shaken for 15 min. It was centrifuged at 1500 rpm, and the supernatant fluid was added to the first extraction and dried down. Methylating reagent (7:6:7, BF3MeOH:benzene:MeOH) was added to the sample. The tube was flushed with nitrogen, sealed with polytetrafluoroethylene tape, and mixed by vortex. The tubes were heated at 100 °C for 55 min and allowed to cool in tepid water. Hexane and Millipore water were added to the sample, after which it was vortex mixed. The top layer was transferred to a 1.5-mL crimp seal vial and dried down under nitrogen; chloroform was added and the top layer was transferred to polypropylene vial inserts.

The composition of fatty acid methyl esters in the RBCs was determined by using a Hewlett-Packard 5890 gas-liquid chromatograph equipped with a 30 m x 0.2 mm SP 2330 column (Supelco, Bellefonte, PA), flame ionization detectors, and automated injection (44). Briefly, the oven temperature was held at 100 °C for 1 min and increased to 190 °C at a rate of 3 °C/min, after which it was held at this temperature for the remainder of the run. The injector temperature was set at 210 °C and the detector temperature at 250 °C. Fatty acid methyl esters were identified based on the retention time of known standards (Supelco). All results are expressed as a percentage of total fatty acids by weight (wt/wt%).

Plasma plant sterol concentrations
Plant sterols were measured by GLC (HP 5890 Series II; Hewlett-Packard) facilitated with flame ionization detection and auto-injector system as described (26, 31, 45). A 30-m SAC-5 column (Sigma-Aldrich Canada Ltd, Oakville, Canada) was used. Briefly, an internal standard, 5 -cholestane, was added to each plasma sample. Samples were saponified and sterols were extracted, resuspended in chloroform, and injected into the gas-liquid chromatograph. The column temperature was 285 °C. Isothermal running conditions were maintained for 42 min. The injector and detector were set at 300 and 310 °C, respectively. The carrier gas (helium) flow rate was 1.2 mL/min and inlet splitter set at 100:1. Plant sterols were identified compared with authentic standards (Sigma-Aldrich Canada Ltd). Internal standards were used to calculate detector response factors.

Glycemic control
Insulin concentrations were measured, in duplicate, in the day 0 and day 21 plasma samples with the use of a commercially available radioimmunoassay kits (ICN Pharmaceuticals, Inc, Costa Mesa, CA) with 125I as a tracer. Radioactivity was determined by gamma counting (1282 compugamma CS; LKB Wallac, Fisher Scientific, Montreal, Canada) and collected as counts per min. Plasma values were quantified by using a standard curve and automated data reduction procedures. Insulin values were expressed as µU/mL. Hb A1c was measured in day 0 and day 21 blood samples at a clinical diagnostic laboratory (LDS Laboratories, Montreal, Canada).

Statistics
The results are presented as means ± SEMs. Differences between groups at baseline were analyzed by using an analysis of variance (ANOVA) model. When a significant difference was found, a Tukey’s post hoc test was performed to determine the differences between group means. When baseline differences were noted for a specific variable, an analysis of covariance was performed with the baseline value as a covariate. Differences between group posttreatment values, and the percentage change from the beginning to the end of the trial, were analyzed by using a two-factor ANOVA model that identified diabetic state and plant sterol effects and their interactions. Statistical significance was set at a P value <0.05 in all analyses. Tests for normality were included in the model. Data were analyzed by using SAS software (version 8.0; SAS Institute Inc, Cary, NC).


RESULTS  
Subject compliance and dropout rate
Sixteen hypercholesterolemic nondiabetic and 16 diabetic persons were enrolled in the study. One nondiabetic subject dropped out at the first week of the second feeding cycle because of a myocardial infarction. Two diabetic subjects dropped out at the first week of the first feeding cycle because of personal reasons. Therefore, complete data from 15 nondiabetic and 14 diabetic subjects were collected and analyzed as per the study protocol. The BCD was well tolerated overall. However, some subjects reported minor gastrointestinal discomfort, which did not require medical intervention or lead to the withdrawal of any subject from the study. During the first week of the first feeding trial, some subjects reported that they were given too much food, ie, the meal sizes were larger than those of their habitual diets. However, the subjects consumed all of the food provided.

Subject characteristics
Baseline characteristics of the study subjects are presented in Table 1. Lipid concentrations denoted in the table are based on the values obtained from the initial blood screen. There were no significant differences in age, weight, or TC, LDL, and HDL concentrations. However, BMI, triacylglycerol, fasting blood glucose, and Hb A1c were significantly higher in the diabetic than in the nondiabetic group. There were no significant differences in weight at the endpoint and no changes across either phase for any group (data not shown).


View this table:
TABLE 1. Baseline characteristics of the subjects1

 
Plasma lipid profile in response to treatment
The mean plasma lipid concentrations at baseline (day 0) and at the end (day 21) of each dietary phase are shown in Table 2. There were no significant differences in mean TC concentrations between groups at baseline. When the data were analyzed by using a two-factor ANOVA, diabetic state x sterol interactions were not significant. Additionally, there was no significant main effect of sterol on posttreatment absolute TC values. However, a significant (P < 0.05) main effect of diabetic state was noted on posttreatment absolute TC values.


View this table:
TABLE 2. Plasma lipid concentrations at baseline and after treatment1

 
Mean LDL-cholesterol concentrations were shown to be significantly (P < 0.05) different between groups at baseline. After further analysis, it was shown that the mean baseline LDL-cholesterol concentrations of the diabetic subjects who received placebo were significantly lower (P < 0.05) than those of the nondiabetic subjects. Results of the two-factor ANOVA showed no significant diabetic state x sterol interaction. When LDL-cholesterol concentrations were expressed as the percentage change between pre- and posttreatment values, with baseline as covariate, a significant (P < 0.05) main effect of sterols was noted for nondiabetic and diabetic subjects combined.

Triacylglycerol concentrations were not significantly different between groups at baseline. Results of the two-factor ANOVA showed no significant diabetic state x sterol interactions. Similarly, there were no significant changes in triacylglycerol across groups or treatments.

Mean HDL-cholesterol concentrations at baseline were not significantly different between groups. In addition, diabetic state x sterol interactions were not significant. Moreover, no significant main effects of diabetic state or sterol were noted for absolute HDL-cholesterol concentrations or percent changes posttreatment.

Mean non-HDL-cholesterol concentrations at baseline were not significantly different between groups. In addition, diabetic state x sterol interactions were not significant. However, a significant (P < 0.05) main effect of diabetic state was noted for posttreatment absolute non-HDL-cholesterol values.

Plasma plant sterol concentrations in response to treatment
Plasma plant sterol concentrations and ratios relative to TC are presented in Table 3. When the data were analyzed by using a two-factor ANOVA, diabetic state x sterol interactions were not significant. Absolute plasma campesterol and ß-sitosterol concentrations were not significantly different between groups at baseline or at the end of each dietary phase. Moreover, there were no differences between groups at baseline and there were no changes at the end of each dietary phase for the campesterol:ß-sitosterol, campesterol:TC, or ß-sitosterol:TC ratios. When campesterol concentrations were expressed as the percentage change between pre- and posttreatment values, a significant main effect of both diabetic state (P < 0.04) and sterol (P < 0.02) was observed. When sitosterol concentrations were expressed as the difference between pre- and posttreatment values, a significant (P < 0.02) main effect of sterol was also observed. A significant main effect of sterol (P < 0.02) was also noted for the percentage change in both the campesterol:cholesterol and sitosterol:cholesterol ratios.


View this table:
TABLE 3. Plasma concentrations of plant sterols (PS) at baseline and after treatment1

 
Changes in red blood cell fatty acid composition within each phase
There were no significant differences in the percentage of fatty acids in RBCs between groups at baseline When the data were analyzed by using a two-factor ANOVA, diabetic state x sterol interactions were not significant. Additionally, there was no significant main effect of either diabetic state or sterol treatment on absolute posttreatment concentrations. A significant (P < 0.03) main effect of sterol was noted for the change in oleic acid and the sum of monounsaturated fatty acids, relative to baseline.

Glycemic control in response to treatment
Plasma insulin concentrations at baseline were significantly different (P < 0.01) between diabetic and nondiabetic subjects. When the data were analyzed by using a two-factor ANOVA, diabetic state x sterol interactions were not significant. Absolute concentrations of insulin remained significantly different (P < 0.01) between diabetic and nondiabetic subjects; however, changes in plasma insulin concentrations between the 2 groups were not significantly different with and without plant sterol consumption.

Plasma Hb A1c concentrations at baseline were significantly different (P < 0.001) between diabetic and nondiabetic subjects. When the data were analyzed by using a two-factor ANOVA, diabetic state x sterol interactions were not significant. Absolute concentrations of Hb A1c remained significantly different (P < 0.001) between diabetic and nondiabetic subjects; however, there was no significant change in Hb A1c concentrations for either group after consumption of each diet for 21 d.

Associations between plasma lipid and plasma plant sterol concentrations
Across all subjects, both plasma TC (r = 0.30, P = 0.0017) and LDL-cholesterol (r = 0.38, P = 0.0001) concentrations varied directly with plasma ß-sitosterol concentrations. However, neither plasma TC nor LDL cholesterol was associated with plasma campesterol or the campesterol:ß-sitosterol ratio.


DISCUSSION  
In the current study, overall lipid changes were more favorable in diabetic subjects than in nondiabetic control subjects. There was a significant effect of diabetic state on mean endpoint non-HDL-cholesterol concentrations; the diabetic subjects had lower mean endpoint non-HDL-cholesterol values than did the nondiabetic subjects. Plant sterol consumption also led to significant decreases in LDL cholesterol in diabetics and nondiabetic subjects. Although the 26.8% change in LDL in the diabetic subjects who consumed plant sterols is numerically attractive, it was not statistically different from the value in the control subjects. These effects may have been due to a relatively low absorption efficiency (9), as part of the insulin resistance syndrome (13) in type 2 diabetic subjects. In general, the results of this study agree well with those of earlier studies of various designs using plant sterols, which showed reductions in TC and LDL cholesterol in the range of 5.0–13.0% and 5.6–24.4%, respectively (10, 18, 21-26, 29-32, 34, 35). In the current study, TC decreased from baseline by 14.6% and LDL cholesterol by 26.8% in the diabetic subjects after 21 d. The extent of reduction is generally above that observed in previous studies. On the other hand, the nondiabetic subjects had a 10.4% reduction in TC and a 15.1% reduction in LDL cholesterol; these values are within the previously reported reduction range.

The decrease in TC did not differ significantly between the plant sterol and placebo phases across groups, except for a significant main effect of diabetic state on posttreatment absolute TC values. This result, which was not consistent with the results of most other studies that examined the efficacy of plant sterols, was perhaps due to several features of the study design, including the plasma cholesterol–modifying characteristics of the control diet, subject-specific type of lipid disorder, or plant sterol dose and composition (18). The results of the third National Health and Nutrition Examination Survey showed that moderately high carbohydrate (50–55% of energy) diets, such as the BCD in this study, are associated with low CVD risks and favorable lipid profiles (46). In this study, both the nondiabetic and diabetic subjects had hypercholesterolemia, but the diabetic subjects also had hypertriacylglycerolemia (Table 1).

In the current study, unesterified plant sterol powder (40% sitosterol, 30% campesterol, 20% dihydrobrassicasterol, and 10% others) was mixed in margarine and added to the controlled diet. Although Vanstone et al (39) showed that both plant sterols and stanols, in their unesterified form, significantly and equally reduced both plasma TC and LDL-cholesterol concentrations, the specific composition of unesterified plant sterol powder used in this study differed from that used in previous studies. Moreover, the BCD used in this study was different from that used in previous studies. Thus, the degree of cholesterol lowering observed might also be expected to be different. The use of non-HDL cholesterol to estimate the CVD mortality risk overcomes the limitation of using the Friedewald equation to calculate LDL-cholesterol concentrations. Non-HDL-cholesterol is simple to calculate by subtracting HDL-cholesterol from TC. This eliminates the margin of error obtained when LDL is calculated with the Friedewald equation when the triacylglycerol concentration is >4.5 mmol/L. As a result, their LDL-cholesterol concentrations were not included in this set of results.

In agreement with earlier studies, plant sterols did not affect plasma HDL-cholesterol and triacylglycerol concentrations significantly (24, 26, 27, 29, 31, 47). On the other hand, the current results show for the first time that plant sterol consumption as part of a controlled diet for 21 d substantially lowered non-HDL-cholesterol in a diabetic group (20.2%; P < 0.05). Cui et al (48) indicated that the non-HDL-cholesterol concentration is a stronger and better predictor of CVD mortality than is the LDL-cholesterol concentration in men and women with no clinical evidence of CVD. In addition to the reduction in LDL cholesterol, this study showed that non-HDL-cholesterol was also reduced. Therefore, the risk of CVD mortality might feasibly be reduced in persons with type 2 diabetes consuming plant sterols along with a heart-healthy diet.

RBC fatty acid composition has been shown to reflect dietary habits (49, 50). There were no significant differences in RBC fatty acid composition from day 1 to day 21 within the 2 dietary phases. These data provide indirect evidence that there was good compliance with the dietary modification because the absence of changes in RBC fatty acid composition reflects that there were no changes in the diet within any of the dietary phases. Moreover, given that all diets in this study were isocaloric, there were no statistically significant changes in body weight in either subject group after 2 dietary phases (data not shown). These data provide additional indirect evidence that there was good compliance by the subjects with the diets.

Previous studies have shown that plasma campesterol and sitosterol concentrations increase (31) or remain unchanged (51) with sitosterol feeding. In the current study there was no significant difference between groups in plasma plant sterol concentrations after sterol supplementation. Plasma plant sterol concentrations have been used as an indirect measure of cholesterol absorption. Specifically, serum campesterol concentrations and the campesterol:cholesterol ratio have been shown to correlate positively with intestinal cholesterol absorption. With controlled diets, this association would be expected to reflect cholesterol absorption. However, various plant sterols are absorbed and metabolized differently. Sitosterol made up 40% of the plant sterol regimen used in this study, which has been shown to increase plasma sterol concentrations (31). Thus, it was not appropriate in this study to use plasma campesterol as an indicator of cholesterol absorption.

Another objective of this study was to examine possible effects of plant sterol consumption on glycemic control. There was no improvement in either Hb A1c or insulin with plant sterol feeding. This result disagrees with the recent work of Lee et al (23), in which a significant reduction in Hb A1c occurred in type 2 diabetic subjects, under free-living conditions, after using a plant sterol–enriched spread for 4 wk. However, the duration of the plant sterol diet and the degree of glycemic control of type 2 diabetic subjects were different compared with our study.

In summary, the current study showed that the consumption of plant sterols is efficacious in lowering LDL-cholesterol and non-HDL-cholesterol concentrations in both type 2 diabetic and nondiabetic persons. Plant sterols, therefore, serve as a potential adjunct to dietary management of hypercholesterolemia in patients with type 2 diabetes. The risk of developing CVD is 2- to 7-fold higher in type 2 diabetic than in nondiabetic persons, and this study showed that plant sterol consumption decreases the risk of CVD in this population. In conclusion, incorporation of plant sterols into a low-saturated-fat and low-cholesterol diet for persons at increased risk of CVD mortality could have a positive effect on reducing the mortality rate associated with type 2 diabetes.


ACKNOWLEDGMENTS  
VWYL and MJ recruited the subjects; performed the screening, selection, and randomization; planned the 3-d cycle menu; prepared the daily meals in the kitchen; supervised the subjects at the research unit; and coded the blood samples. VWYL performed the blood lipid and insulin measurements, completed the gas chromatographic analyses of plasma plant sterols and RBCs, and performed the statistical analysis of the data. PJHJ designed the study and acted as the principal investigator.

PJHJ is a consultant to Forbes Medi-Tech Inc, which provided the plant sterol mixture. None of the other authors had a conflict of interest.


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Received for publication February 20, 2004. Accepted for publication May 25, 2005.


日期:2008年12月28日 - 来自[2005年81卷第6期]栏目
循环ads

Plant sterols and endurance training combine to favorably alter plasma lipid profiles in previously sedentary hypercholesterolemic adults after 8 wk

Krista A Varady, Naoyuki Ebine, Catherine A Vanstone, William E Parsons and Peter JH Jones

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.


View this table:
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.


View this table:
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.


View this table:
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.


View this table:
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.


View this table:
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.


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Received for publication December 18, 2003. Accepted for publication June 14, 2004.


日期:2008年12月28日 - 来自[2004年80卷第5期]栏目

Both free and esterified plant sterols reduce cholesterol absorption and the bioavailability of ß-carotene and -tocopherol in normocholesterolemic humans

Myriam Richelle, Marc Enslen, Corinne Hager, Michel Groux, Isabelle Tavazzi, Jean-Philippe Godin, Alvin Berger, Sylviane Métairon, Sylvie Quaile, Christelle Piguet-Welsch, Laurent Sagalowicz, Hilary Green and Laurent Bernard Fay

1 From the Nestlé Research Center, Nestec Ltd, Lausanne, Switzerland (MR, ME, CH, IT, J-PG, AB, SM, SQ, CP-W, LS, HG, and LBF) and Nestlé Product Technology, Centre Konolfingen, Konolfingen, Switzerland (MG)

2 Address reprint requests to M Richelle, Nestec Ltd, Nestlé Research Center, PO Box 44, CH-1000 Lausanne 26, Switzerland. E-mail: myriam.richelle{at}rdls.nestle.com.

See corresponding editorial on page 3.


ABSTRACT  
Background: Plant sterols reduce cholesterol absorption, which leads to a decrease in plasma and LDL-cholesterol concentrations. Plant sterols also lower plasma concentrations of carotenoids and -tocopherol, but the mechanism of action is not yet understood.

Objectives: The aims of this clinical study were to determine whether plant sterols affect the bioavailability of ß-carotene and -tocopherol in normocholesterolemic men and to compare the effects of plant sterol esters and plant free sterols on cholesterol absorption.

Design: Twenty-six normocholesterolemic men completed the double-blind, randomized, crossover study. Subjects consumed daily, for 1 wk, each of the following 3 supplements: a low-fat milk-based beverage alone (control) or the same beverage supplemented with 2.2 g plant sterol equivalents provided as either free sterols or sterol esters. During this 1-wk supplementation period, subjects consumed a standardized diet.

Results: Both of the milks enriched with plant sterols induced a similar (60%) decrease in cholesterol absorption. Plant free sterols and plant sterol esters reduced the bioavailability of ß-carotene by 50% and that of -tocopherol by 20%. The reduction in ß-carotene bioavailability was significantly less with plant free sterols than with plant sterol esters. At the limit of significance (P = 0.054) in the area under the curve, the reduction in -tocopherol bioavailability was also less with plant free sterols than with plant sterol esters.

Conclusions: Both plant sterols reduced ß-carotene and -tocopherol bioavailability and cholesterol absorption in normocholesterolemic men. However, plant sterol esters reduced the bioavailability of ß-carotene and -tocopherol more than did plant free sterols.

Key Words: Plant sterol • cholesterol • absorption • vitamin E • tocopherol • carotenoids


INTRODUCTION  
Plant sterols reduce the absorption of cholesterol in the gut, possibly by competing with cholesterol when they are incorporated into the mixed micelles, by displacing the cholesterol from bile, or by decreasing the hydrolysis of cholesterol esters in the small intestine (1–3). However, the exact cholesterol-lowering mechanism of plant sterols is not yet fully known. Many clinical studies performed in humans show that the administration of plant sterols reduces cholesterol absorption and that, when plant sterols are given over a long period (>3 wk), plasma cholesterol and LDL-cholesterol concentrations are reduced by 5%–15% without major side effects (4–6). The reduction in LDL cholesterol is dose dependent: a measurable reduction of 6% with an intake of 0.9 g plant sterol/d and nearly maximum at 9.6% with an intake of 2 g/d (3, 7–9). Concomitant with cholesterol reduction, plant sterols decrease plasma concentrations of ß-carotene by 25%, -carotene by 10%, and vitamin E by 8%, but plasma vitamin A (retinol) and vitamins D and K are not significantly affected (7–13). Because sterols and stanols reduce the amount of LDL cholesterol and because lipophilic carotenoids and tocopherols are known to be associated with LDL particles, it may be appropriate to adjust the plasma concentrations of these carotenoids and vitamins to reduce LDL cholesterol. With such an adjustment, stanols and sterols do not significantly lower blood concentrations of vitamin E, but concentrations of ß-carotene were reduced by 8%–19% (8). The reason for this decrease in blood concentrations of ß-carotene is not known, but it could be the reduction in its absorption. A reduction in the bioavailability of ß-carotene is of particular concern for persons whose need for vitamin A is greater, such as pregnant and lactating women and young children.

Plant free sterols have been incorporated into a low-fat milk-based beverage. In a previous clinical trial, we showed that midly hypercholesterolemic subjects consuming this low-fat milk-based beverage enriched with 1.8 g plant free sterols/d had a 40% reduction in cholesterol absorption (14). In a longer-term clinical trial performed in midly hypercholesterolemic Danish men and women, daily consumption of 1.2 and 1.6 g plant free sterols in this low-fat milk-based beverage over 4 wk reduced LDL cholesterol by 7.1% and 9.6%, respectively. In addition to cholesterol, both doses of plant sterols decreased the percentage change in plasma - and ß-carotene, lutein, and vitamin E (15).

The objective of the present clinical study was to investigate whether daily consumption of 2.2 g plant sterols (as free equivalent) either as free or ester form would affect the bioavailability of ß-carotene and -tocopherol in humans compared with the same test performed without sterol ingestion (control treatment). In addition, the effect of plant free sterols was compared with that of plant sterol esters. To our knowledge, the effects of nonesters and esters on the bioavailability of these fat-soluble vitamins have not been evaluated in the same study. Esters are presumed to have a greater effect on fat-soluble vitamins because they partition into the oil phase of the intestine, whereas free sterol partitions into the micellar phase (16).


SUBJECTS AND METHODS  
Subjects
Thirty-three healthy men were enrolled in the study. The inclusion criteria were that the subjects be nonvegetarians and nonsmokers and that they not have metabolic disorders such as diabetes; hypertension; renal, hepatic, or pancreatic disease; or ulcers. Subjects were normolipidemic: ie, they had plasma cholesterol concentrations <5.2 mmol/L (or 200 mg/dL), a ratio of plasma cholesterol to HDL cholesterol <5.0 mmol/L, and plasma triacylglycerol concentrations < 2.0 mmol/L. Because of the large amount of blood (827.5 mL) that was drawn during the study, subjects were required to have a blood hemoglobin concentration of >13 g/dL. Subjects were excluded from the study if they used cholesterol-altering medication or vitamin and mineral supplements from 3 mo before the start of the study until the completion of the study; were milk intolerant or had had major gastrointestinal surgery; exercised intensively, such as running marathons; and consumed daily >2 glasses of wine (3 dL), >2 beers (3 dL), or >1 glass (shot glass) of hard liquor.

The protocol was approved by the ethics committee of Nestlé (Lausanne, Switzerland). Subjects received information on the background and design of the study and gave written informed consent before participation. They were free to withdraw from the study at any time.

Study design
This was a placebo-controlled, double-blind, randomized, 3-period, 3-treatment crossover clinical trial. The protocol is presented in Figure 1.


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FIGURE 1.. Scheme of the experimental design of the clinical study.

 
Subjects consumed a standard diet over 6 d that was designed to provide a constant daily intake of 250–260 mg cholesterol. The subjects consumed this standard diet during the three 1-wk treatment periods. Breakfast was a traditional European-style breakfast: ie, bread, butter, and jam. Lunches and dinners consisted of ready-to-eat meals and bread slices. The caloric intake was 2600 kcal. In addition to this standard diet, subjects drank daily, as an intervention, 600 mL of low-fat milk-based beverage supplement—ie, a 300-mL dose at breakfast and a second 300-mL dose at lunch.

On the morning of day 4 after an overnight fast, subjects arrived at the Metabolic Unit of Nestlé Research Center (Vers-chez-les-Blanc, Switzerland) and received a simultaneous intravenous infusion of 45 mg [13C2]cholesterol [incorporated into 15 mL of parenteral emulsion (Interlipid 10%; Pharmacia-Upjohn, Stockholm)] and an oral dose of 15 mg of [2H5]cholesterol (diluted in 1 mL sunflower oil). Both markers were prepared freshly and were administered within 2 h.

On the morning of day 7 after an overnight fast, each subject consumed a standard meal consisting of 15 mg [2H8]ß-carotene and 30 mg [2H6]-tocopherol incorporated in 35 g peanut oil that was mixed with 70 g wheat semolina (cooked with 200 mL tap water). In addition, they consumed 40 g bread, 60 g cooked egg whites, and 600 mL low-fat milk-based beverage supplement. This meal was consumed within 30 min. No other food was allowed over the subsequent 9 h, but subjects were allowed to drink bottled water (Vittel, Vittel, France).

To ensure subject compliance, breakfasts and lunches were consumed in the Metabolic Unit on the weekdays under the supervision of the unit's staff, and packed meals for dinners and the weekend were provided for home consumption. Subjects were repeatedly instructed not to consume any food or beverages other than those provided by the Metabolic Unit.

Milk supplement
Control milk refers to a low-fat, ultrahigh temperature-treated, milk-based beverage (600 mL) containing 0.63% butter oil, 0.56% rapeseed oil, and 0.39% corn oil. The fatty acid profile of the milk comprises 15% palmitic acid, 5% stearic acid, 37% oleic acid, 21% linoleic acid, and 3% -linolenic acid. This milk was packed in a 200-mL sealed container. Control milk (600 mL) enriched with 2.2 g soybean nonhydrogenated, nonesterified plant sterols and 2.2 g sorbitan tristearate (as emulsifier), packed in a 200-mL sealed container, is referred to as plant free sterol milk. Control milk (600 mL) enriched with soybean nonhydrogenated, esterified plant sterols (2.2 g free sterols equivalent), packed in a 200-mL sealed container, is referred to as plant sterol ester milk.

The composition of the 3 milks was adjusted to provide a similar fatty acid profile as well as similar ß-carotene and -tocopherol content. The sterol content in the milk supplement was ascertained after manufacture and was in agreement with the 2.2 g free plant sterol equivalent.

Isotopic markers
The 2, 2, 4, 4, 6-[2H5] cholesterol tracer (95 atom%) (Medical Isotopes, Pelham, NH) was diluted in sunflower oil (15 mg/g) and placed on bread. Ready-to-inject 15-mL syringes containing 45 mg 3-4-[13C2] cholesterol tracer (99 atom%; Medical Isotopes) dissolved in Intralipid 10% were prepared by the Centre Hospitalier Universitaire Vaudois (Lausanne, Switzerland) and checked for sterility.

We purchased 10,10',19,19,19,19',19',19'-[2H8]-ß-carotene (95 atom%) and 2R,4'R,8'R-[2H6]--tocopherol (98 atom%) from Orphachem (Clermont-Ferrand, France). A dose of 15 mg [2H8]-ß-carotene and 30 mg [2H6]--tocopherol was incorporated into 35 g peanut oil.

Collection of blood samples
A fasting blood sample was drawn from an anticubital vein by venipuncture into a potassium EDTA-containing evacuated tube that was immediately placed in an ice-water bath. The tube was protected from light and then centrifuged (10 min, 4 °C, 3000 x g) to separate the plasma, which was isolated and stored at –20 °C until it was analyzed. Samples were analyzed within 6 mo.

For measurement of cholesterol absorption, a fasting blood sample was collected on the morning of day 4 before the administration of the 2 cholesterol markers ([13C2]-cholesterol and [2H5]-cholesterol) and then on the morning of days 6 and 7. For measurement of ß-carotene and -tocopherol bioavailability, a fasting blood sample was collected on the morning of day 7 before the administration of the 2 markers—ie, [2H8]-ß-carotene and [2H6]--tocopherol—as well as at 2.5, 3, 4, 5, 6, 7, 8, and 9 h after the administration of these markers.

Plasma cholesterol enrichment
Plasma lipids were extracted and separated by thin-layer chromatography as described by Pouteau et al (14). The free cholesterol layer of each lipid extract sample was scraped off into a tube and then extracted with 3 mL EtOH:CHCl3 (1:2, by vol). The mixture was mixed by vortex for 10 s and centrifuged for 2 min at 2000 x g at room temperature. Finally, the free cholesterol extract was split into 2 parts: one part of 0.3 mL for measurement of [13C]-cholesterol enrichment by using gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) analysis and another part of 2.7 mL for measurement of [2H]-cholesterol enrichment by high-temperature conversion-elemental analyzer-isotope ratio mass spectrometry (TC-EA/IRMS) on an isotope ratio mass spectrometer (Delta Plus XL; Thermo Finnigan MAT, Bremen, Germany).

The [2H/H] isotope ratio measurement of free cholesterol was performed by using TC-EA/IRMS. The free cholesterol fraction (2.7 mL) was evaporated under N2 at 30 °C. The dry residue was dissolved in 25 µL EtOH/CHCl3 (1:2, by vol), mixed by vortex for 5 s, and then transferred to a silver capsule. The solution was evaporated at 30 °C, and the dried capsule introduced to the solid autosampler of the TC-EA/IRMS system. Analytic conditions of the TC-EA/IRMS system were pyrolysis reactor temperature of 1450 °C, GC column temperature of 90 °C, and helium pressure of 1 bar. We measured the [2H/H] isotopic enrichment of cholesterol by monitoring ions' mass-to-charge ratios of 3 and 2, expressed it in against standard mean ocean water (an international standard for [2H]), and further converted it to molar percent excess.

The [13C:12C] isotope ratio measurement of free cholesterol was performed by using GC/C/IRMS (MAT 252, Thermo Finnigan Mat) according to Pouteau et al (14). The [13C] isotopic enrichment was expressed in against Pee Dee belemnite (an international standard for [13C]) and then converted to molar percent excess.

Triacylglycerol-rich lipoprotein isolation and lipid extraction
Plasma (4 mL) was thawed and introduced in an ultracentrifuge tube. A solution of 1.006 g sodium bromide/mL was deposited on top of the plasma solution without mixing the 2 solutions. The tube was filled with this 1.006 g/mL solution and ultracentrifuged at 100 000 x g and 15 °C for 30 min. The upper phase containing triacylglycerol-rich lipoproteins (TRLs)—mainly chylomicrons with low amounts of VLDL—was collected. TRLs (200–400 µL) were adjusted up to 1 mL with distilled water. Subsequently, 1 mL EtOH, 5 µL deferoxamine mesylate (10 mg/mL water), and 2 mL hexane containing 350 mg BHT/L were added. The tube was mixed by vortex for 20 s, then centrifuged at 2000 x g and room temperature for 10 min in a tabletop centrifuge. The organic phase was collected while the water phase was extracted again with an additional 2 mL hexane-BHT. The hexane layers were combined and evaporated to dryness under N2. The sample was dissolved in 200 µL hexane-BHT for tocopherol analysis and in 120 µL dioxane-ethanol-acentonitrile (1:1:2, by vol) for carotenoid analysis. A volume of 60 or 100 µL was injected onto the HPLC system for tocopherol and carotenoid analysis, respectively. The lipid extract of the biological sample was quite stable for 1 wk at 4 °C and then for 2 d at room temperature before being used for HPLC injection.

Measurement of TRL-[2H8]-ß-carotene
We isolated [2H8]-ß-carotene from ß-carotene by using an HPLC method. The [2H8]-ß-carotene was then quantified with the use of a diode array detector according to the method of Duecker et al (17).

Determination of TRL-D6--tocopherol
We isolated [2H6]--tocopherol from -tocopherol by using an HPLC method. The [2H6]--tocopherol was then quantified with the use of an ultraviolet detector according to the method of Richelle et al (18).

Assessment of bioavailability
Cholesterol
Two cholesterol markers were used: one marker, injected intravenously, gave information on the in vivo metabolism of cholesterol, and the other, administered orally, accounted for intestinal cholesterol absorption and its in vivo metabolism. This method allowed the measurement of the absolute bioavailability of cholesterol in each subject, which was calculated by multiplication of the ratio of the plasma enrichment of cholesterol administered orally and plasma enrichment of cholesterol injected intravenously by the ratio of the intravenous dose of cholesterol and the oral dose administered to the subject, and this total was then multiplied by 100.

ß-Carotene and -tocopherol
The ß-carotene and -tocopherol molecules were administered orally only, and therefore relative bioavailability was ascertained. This assessment was performed by using the pharmacokinetic measurements of the appearance of these labeled molecules in the blood circulation of the subjects, such as area under the curve (AUC), maximum plasma concentration (Cmax), and the time to reach the Cmax (tmax).

Statistical analysis
From time 0 to 9 h, 3 pharmacokinetic parameters were calculated: area under the plasma concentration versus time curve [AUC(0–9 h)], (Cmax), and tmax. The AUC(0–9 h) over baseline was calculated with the use of the trapezoidal rule. These 3 pharmacokinetic values were calculated by using NCSS software (version 2000; NCSS Statistical Software, Kaysville, UT).

For cholesterol, ß-carotene, and -tocopherol, statistical analyses were based on noninferiority tests with a lower equivalence limit of 80% (19). Differences between treatments were analyzed by using a linear mixed-effect model with treatment and period as fixed effects and by using subject as a random effect (SAS software, version 8.2; SAS Corp Inc, Cary, NC). The rejection level in statistical tests was 5%. Data are presented as means ± SEMs.


RESULTS  
Among the 33 subjects enrolled in the study, 7 did not complete the study: 3 for personal reason, 2 because of noncompliance with the treatment, and 2 for medical reasons. Twenty-six subjects, aged 29 ± 1 y, completed the study. Their mean starting body weight was 78 ± 2 kg, and their mean starting body mass index (BMI; in kg/m2) was 24.1 ± 0.4. The values were analyzed only for these 26 subjects who completed the 3 treatment periods. The body weight of the subjects did not change during the course of the study.

Cholesterol absorption
Our normocholesterolemic subjects consuming control milk had cholesterol absorption of 47 ± 2% (Figure 2), which is in agreement with data reported in the literature (20) and our previous study (21). As expected, the administration of plant free sterols and plant ester sterols reduced cholesterol absorption to 23 ± 2% and 20 ± 2%, respectively, of baseline. This reduction in cholesterol absorption by 60% is in agreement with data reported by Lees et al (6) and Ostlund et al (3).


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FIGURE 2.. Mean (±SEM) percentage of cholesterol absorption in subjects who daily consumed a low-fat milk-based beverage (control) or the same beverage supplemented with either 2.2 g plant free sterols or plant sterol esters in quantities equivalent to 2.2 g plant free sterols.

 
TRL production
Dietary lipophilic compounds have to be incorporated into chylomicrons, which are their vehicles in the bloodstream. Chylomicron production is characterized by the triacylglycerol content of the TRLs. Consumption of the standard meal containing 35 g peanut oil led to a marked production of chylomicron particles (Figure 3; Table 1). The pharmacokinetics of the TRL-triacylglycerol concentration consisted of a rapid increase, a Cmax, and a prompt decline. Nine hours after the consumption of the treatments, chylomicrons were totally cleared, which led to TRL-triacylglycerol concentrations that did not differ significantly from the baseline concentration. The AUC, Cmax, and tmax were similar for the 3 treatments, which showed the equivalence of the triacylglycerol absorption (Table 1).


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FIGURE 3.. Mean (±SEM) triacylglycerol-rich lipoprotein (TRL)-triacylglycerol concentrations after the consumption of a standard diet containing 35 g peanut oil and milk. : Control, low-fat milk-based beverage; : free sterols, the same beverage enriched with a daily dose of 2.2 g plant free sterols; : sterol esters, the same beverage enriched with a daily dose of plant sterol esters (2.2 g free sterol equivalent).

 

View this table:
TABLE 1. Pharmacokinetic measurements of lipids of the triacylglycerol-rich lipoproteins after the consumption of the standard meal alone (control) or of the meal enriched with 2.2 g plant free sterols or 2.2 g plant sterol esters (plant free sterol equivalent)1

 
TRL-[2H8]-ß-carotene
A labeled deuterated ß-carotene has been used to distinguish the ß-carotene consumed from endogenous ß-carotene—ie, the ß-carotene already present in the body. Therefore, it is not surprising that, at time 0, there was no [2H8]-ß-carotene in the TRLs (Figure 4). After the consumption of the control milk, TRL-[2H8]-ß-carotene concentrations rose to a Cmax and then declined slowly; detectable concentrations remained 9 h after absorption (Figure 4; Table 1). The 50% reductions in the AUC and the Cmax confirm that both plant sterols decreased the bioavailability of [2H8]-ß-carotene. This reduction was significantly higher with the plant sterol esters (57%) than with the plant free sterols (48%).


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FIGURE 4.. Mean (±SEM) triacylglycerol-rich lipoprotein (TRL)-[2H8]-ß-carotene concentrations after the consumption of a standard diet containing 15 mg [2H8]-ß-carotene and milk. : Control, low-fat milk-based beverage; : free sterols, the same beverage enriched with a daily dose of 2.2 g plant free sterols; : sterol esters, the same beverage enriched with a daily dose of plant sterol esters (2.2 g free plant sterol equivalent).

 
TRL-retinyl palmitate
Within the enterocytes, some of ß-carotene molecules are cleaved by the 15,15' dioxygenase, which leads to retinol that is partly transformed into retinyl palmitate. The TRL-retinyl palmitate pharmacokinetics were quite similar to those described for [2H8]-ß-carotene: a prompt increase to Cmax and a slow decline, leaving concentrations still elevated 9 h after absorption (Figure 5; Table 1). Both plant sterols significantly decreased the concentration of TRL-retinyl palmitate as characterized by reductions in the AUC(0–9 h) and Cmax. Similar to the reduction in [2H8]-ß-carotene, the reduction in TRL-retinyl palmitate was significantly higher with the plant sterol esters (48%) than with the plant free sterols (32%).


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FIGURE 5.. Mean (±SEM) triacylglycerol-rich lipoprotein (TRL)-retinyl palmitate concentrations after the consumption of a standard diet containing 15 mg [2H8]-ß-carotene and milk. : Control, low-fat milk-based beverage; : free sterols, the same beverage enriched with a daily dose of 2.2 g plant free sterols; : sterol esters, the same beverate enriched with a daily dose of plant sterol esters (2.2 g free sterol equivalent).

 
TRL-D6--tocopherol
The pharmacokinetics of [2H6]--tocopherol had a slower increase and decrease than did the pharmakinetics of [2H8]-ß-carotene. Nine hours after the consumption of both treatments, the concentrations of [2H6]--tocopherol remained quite high (Figure 6; Table 1). Plant free sterols had no effect on the bioavailability of [2H6]--tocopherol, whereas plant sterol esters reduced it 27% in comparison to the control treatment. Of the 2 plant sterol treatments, plant sterol esters tended to decrease the bioavailability of [2H6]--tocopherol more than did plant free sterols, as characterized by a significant difference in Cmax whereas the difference in the AUCs was at the limit of significance (P = 0.054).


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FIGURE 6.. Mean (±SEM) triacylglycerol-rich lipoprotein (TRL)-[2H6]--tocopherol concentrations after the consumption of a standard diet containing 30 mg [2H6]--tocopherol and milk. : Control, low-fat milk-based beverage; : free sterols, the same beverage enriched with a daily dose of 2.2 g free plant sterols; : sterol esters, the same beverage enriched with a daily dose of plant sterol esters (2.2 g free plant sterol equivalent).

 

DISCUSSION  
Daily consumption of plant sterols for several weeks has been proven to reduce LDL cholesterol in both normocholesterolemic (11, 20) and hypercholesterolemic (3, 11, 21) persons. Inhibition of the intestinal absorption of exogenous (dietary) and endogenous (biliary) cholesterol was described as the major mechanism of the cholesterol-lowering action of plant sterols, although the exact mechanism of action remains to be investigated in detail. Long-term daily consumption of plant sterols induces a reduction in plasma cholesterol and LDL-cholesterol concentrations. These depletions are related to the dose of plant sterol consumed, the form of the plant sterol, the vehicle the plant sterols are solubilized in, the background diet, genetic factors, and, in some cases, the starting cholesterol concentration (3). The vehicle in which the plant sterols are solubilized can clearly affect the efficacy of plant sterols in lowering LDL cholesterol. Although we have shown in several studies that plant free sterols properly solubilized in low-fat partly vegetable oil-filled milks can lower cholesterol absorption (14) and LDL cholesterol (15), a recent study found that plant sterol did not effectively lower LDL cholesterol in a low-fat liquid matrix (22).

Esters of plant sterols or stanols similar to those of the free forms have been shown to induce a similar effect when provided at the same free sterol equivalent dose (11, 23, 24), but the issue is not completely resolved. In the present study, normocholesterolemic men who daily consumed 2.2 g plant sterols in either free or ester form had similar 60% reductions in cholesterol absorption, which is in agreement with data reported by Lees et al (6) and Ostlund et al (3). Several studies showed that the consumption of stanyl and steryl esters reduces plasma concentrations of fat-soluble antioxidants such as ß-carotene, lycopene, and -tocopherol (7, 9, 11, 25–27). It is hypothesized that, during the absorption processes in the intestine, plant sterols could displace not only cholesterol but also other lipophilic molecules and replace them in incorporation into mixed micelles. It was shown that esters are more likely to partition into the oil phase of the intestine, whereas free sterols are more likely to partition into the mixed micellar phase (16). As a consequence, the absorption of these lipophilic molecules is reduced, and there is a more marked effect with plant sterol esters. Although this assumption is based on lower plasma concentrations when subjects are consuming plant sterols, such reduced absorption has not yet been shown.

Therefore, the purpose of the present study was to test the null hypothesis that consumption of 2.2 g plant sterols either as free or ester form (expressed in free sterol equivalent) would not affect the absorption of ß-carotene and -tocopherol in normocholesterolemic men. The clinical trial was designed as a placebo-controlled, randomized, double-blind, 3-treatment, 3-period study. Subjects consumed a controlled diet that provided similar daily intakes of cholesterol, ß-carotene, and -tocopherol. In addition, they were supplemented daily for 7 consecutive days with a low-fat milk-based beverage enriched with 2.2 g plant sterols (intervention groups) or not enriched (control group). The 1-wk duration of plant sterol administration ensured a sufficient (60%) reduction in cholesterol absorption. In the present study, ß-carotene and -tocopherol bioavailability was assessed at the end of the 1-wk treatment by using a postprandial test. Administered ß-carotene and -tocopherol were both labeled with deuterium to differentiate the dietary intake from the concentration already present in the subject's body. The principle of the postprandial test consists of the administration of a standard meal that allows efficient production of chylomicrons, which will carry dietary lipophilic molecules into the bloodstream of the subject. The pharmacokinetics of TRL-triacylglycerol concentration as measured against time is a measure of bioavailability of lipophilic molecule based on the assumption that the TRL fraction mainly contains intestinally derived lipoproteins (chylomicrons and their remnants) and only some liver-derived lipoproteins (VLDL). To compare the treatments, a reproducible measure of chylomicron production—ie, an intraindividual variability that is relatively low compared with interindividual variability—is required. Van Vliet et al (28) reported an unexpectedly large intraindividual variability (62%) in their population. To minimize the intraindividual variation, they recommended the standardization of ß-carotene bioavailability with chylomicron production, ie, triacylgylcerol responses. In the present study, the 3 treatments led to similar amounts of chylomicron production, as characterized by an equivalent AUC, Cmax, and tmax of TRL-triacylglycerol pharmacokinetics. Thus, ß-carotene and -tocopherol bioavailabilities have not been standardized with the bioavailability of triacylglycerol.

Supplementation with plant sterols, in either free or ester form, reduced the bioavailability of ß-carotene by 50% and that of -tocopherol by 20%. In the case of ß-carotene, the reduction in bioavailability was significantly less with plant free sterols than with plant sterol esters. The reduction in -tocopherol bioavailability was also less with plant free sterols than with plant sterol esters characterized by a lower Cmax and a reduction in AUC at the limit of significance (P = 0.054). These results indicate for the first time that plant sterols reduce not only the absorption of cholesterol but also the bioavailability of ß-carotene and -tocopherol. The results also show that sterol esters provided at the same equivalent dose and in the same vehicle as plant free sterols had a greater effect on ß-carotene and -tocopherol bioavailability than did plant free sterols. It is reasonable to speculate that the absorption of other lipophilic molecules that partition into the intestinal oil phase will be compromised by plant free sterols. The present results contrast with those of Relas et al (29), who showed that a single 1-g dose of dietary stanyl esters incorporated in margarine and combined with fat-soluble vitamins did not detectably interfere with serum concentrations of cholesterol, triacylglycerol, -tocopherol, ß-carotene, retinol, or retinyl palmitate. However, in their study, there was no direct evidence that cholesterol absorption was reduced; they did find a decrease in plasma concentrations of campesterol:cholesterol, which is a marker of cholesterol absorption.

The chronic consumption of plant sterols would be expected to induce a progressive decrease in plasma carotenoid concentrations, although the biological significance of this expected effect is not clear. One way to counterbalance this reduction in absorption is to increase dietary carotenoid intake. Noakes et al (30) showed that daily consumption of 5 servings fruit and vegetables with a minimum of 1 carotenoid-rich serving/d allows the maintenance of lipid-standardized plasma carotenoid concentrations in subjects consuming either 2.5 g plant sterol ester-2 or 2.3 g plant stanol ester-1. An alternative consists of an increase in the intake of carotenoid-rich food such as apricots, cantaloupe, broccoli, and spinach (26) or the consumption of a food supplement (27). In conclusion, plant sterols reduce not only the absorption of cholesterol but also the bioavailability of ß-carotene and -tocopherol.


ACKNOWLEDGMENTS  
We thank Olivier Ballèvre for discussions during the development of this study and Etienne Pouteau for advice on marker selection and dose determination. We greatly thank Patricia Dibling, Sylviane Oguey-Araymon, Annie Blondel-Lubrano, Bernard Decarli, Micheline Chabloz, and Jean-Claude Maire of the Metabolic Unit for their collaboration during this clinical trial. We also thank Isabelle Ré for her participation in the HPLC analyses and Charles Schindler of the Centre Hospitalier Universitaire Vaudois de Lausanne for preparing the emulsions. Finally, we thank the volunteers who participated in this clinical trial.

The study was conceived and designed by MR, ME, CH, AB, and LBF. The design and production of formulations of the phytosterol milk were achieved by MG, AB, and LS. MR coordinated the trial and supervised the analytic aspects. IT, J-PG, SM, SQ, and CP-W contributed to the development and implementation of isotopic analytic methods and to isotope analysis and data collection. ME and CH performed all statistical testing. MR wrote the manuscript, and all authors were involved in interpreting the results and in critical revision of the paper. No authors had any advisory board affiliations.


REFERENCES  

  1. Ikeda I, Tanabe Y, Sugano M. Effects of sitosterol and sitostanol on micellar solubility of cholesterol. J Nutr Sci Vitaminol (Tokyo) 1989;35:361–9.
  2. Ling WH, Jones PJH. Dietary phytosterols: a review of metabolism, benefits and side effects. Life Sci 1995;57:195–206.
  3. Ostlund RE. Phytosterols in human nutrition. Annu Rev Nutr 2002;22:533–49.
  4. Pollack OJ. Reduction of blood cholesterol in man. Circulation 1953;7:702–6.
  5. Farquhar JW, Sokolow M. Response of serum lipids and lipoproteins of man to beta-sitosterol and safflower oil—a long term study. Circulation 1958;17:890–9.
  6. Lees AM, Mok HYI, Lees RS, McCluskey MA, Grundy SM. Plant sterols as cholesterol-lowering agents: clinical trials in patients with hypercholesterolemia and studies of sterol balance. Arteriosclerosis 1977;28:325–8.
  7. Hendriks HFJ, Weststrate JA, van Vliet T, Meijer GW. Spreads enriched with three different concentrations of vegetable oil sterols and the degree of cholesterol lowering in normocholesterolemic and mildly hypercholesterolemic subjects. Eur J Clin Nutr 1999;53:319–27.
  8. Hallikainen MA, Sarkkinen ES, Uusitupa MIJ. Effects of low-fat stanol ester enriched margarines on concentrations of serum carotenoids in subjects with elevated serum cholesterol concentration. Eur J Clin Nutr 1999;53:966–9.
  9. Hallikainen MA, Sarkkinen ES, Gylling H, Erkkila AT, Uusitupa MIJ. Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolaemic subjects on a low-fat diet. Eur J Clin Nutr 2000;54:715–25.
  10. Gylling H, Miettinen TA. Cholesterol reduction by different plant stanol mixtures and with variable fat intake. Metabolism 1999;48:575–80.
  11. Weststrate JA, Meijer GW. Plant sterol enriched margarines and reduction of plasma total and LDL cholesterol concentrations in normocholesterolemic and mildly hypercholesterolaemic subjects. Eur J Clin Nutr 1998;52:334–43.
  12. Plat J, van Onselen ENM, van Heugten MMA, Mensink RP. Effects on serum lipids, lipoproteins and fat soluble antioxidant concentrations of consumption frequency of margarines and shortenings enriched with plant stanol esters. Eur J Clin Nutr 2000;54:671–7.
  13. Moreau RA, Whitaker BD, Hicks KB. Phytosterols, phytostanols and their conjugates in foods: structural diversity, quantitative analysis and health promoting uses. Prog Lipid Res 2002;41:457–500.
  14. Pouteau EB, Monnard IE, Piguet-Welsch C, Groux MJA, Sagalowicz L, Berger A. Non-esterified plant sterols solubilized in low fat milks inhibit cholesterol absorption. Eur J Nutr 2003;42:154–64.
  15. Thomsen AB, Hansen HB, Christiansen C, Green H, Berger A. Effect of free plant sterols in low-fat milk on serum lipid profile in hypercholesterolemic subjects. Eur J Clin Nutr (in press).
  16. Nissinen M, Gylling H, Vuoristo M, Miettinen TA. Micellar distribution of cholesterol and phytosterols after duodenal plant stanol ester infusion. Am J Physiol Gastrointest Liver Physiol 2002;282:G1009–15.
  17. Dueker SR, Jones AD, Smith GM, Clifford AJ. Stable isotope methods for the study of the ß-carotene-d8 metabolism in humans utilizing tandem mass spectrometry and high performance liquid chromatography. Anal Chem 1994;66:4177–85.
  18. Richelle M, Tavazzi I, Fay LB. Simultaneous determination of deuterated and non-deuterated -tocopherol in human plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 2003;794:1–8.
  19. Guidance or statistical procedures for bioequivalence study using a standard two-treatment cross-over design. Rockville, MD: Food and Drug Administration, 1992.
  20. Bosner MS, Lange LG, Stenson WF, Ostlund RE Jr. Percent cholesterol absorption in normal men and women quantified with dual stable isotope tracers and negative ion mass spectrometry, J Lipid Res 1999;40:302–8.
  21. Gremaud G, Piguet C, Baumgartner M, et al. Simultaneous assessment of cholesterol absorption and synthesis in humans, using on-line GC-combustion and GC-pyrolysis isotope ratio mass spectrometry. Rapid Commun Mass Spectrom 2001;15:1207–13.
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  23. Miettinen TA, Vanhannen H. Dietary sitosterol related to absorption, synthesis and serum level of cholesterol in different apolipoprotein E phenotypes. Arterosclerosis 1994;105:217–26.
  24. Jones PJ, Raeni-Sarjaz M, Ntanios FY, Vanstone CA, Feng JY, Parsons WE. Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters. J Lipid Res 2000;41:697–705.
  25. Law M. Plant sterol and stanol margarines and health. BMJ 2000;320:861–4.
  26. Maki KC, Davidson MH, Umporowicz DM, et al. Lipid responses to plant-sterol-enriched reduced-fat spreads incorporated into a National Cholesterol Education Program Step I diet. Am J Clin Nutr 2001;74:33–43.
  27. Neil HAW, Meijer GW, Roe LS. Randomised controlled trial of use by hypercholesterolaemic patients of vegetable oil sterol-enriched fat spread Atherosclerosis 2001;156:329–37.
  28. Van Vliet T, Schreurs WH, van den Berg H. Intestinal ß-carotene absorption and cleavage in men: response of ß-carotene and retinyl esters in the triacylglycerol-rich lipoprotein fraction after a single oral dose of ß-carotene. Am J Clin Nutr 1995;62:110–6.
  29. Relas H, Gylling H, Miettinen TA. Acute effect of dietary ester dose on post-absorptive -tocopherol, ß-carotene, retinol and retinyl palmitate concentrations. Br J Nutr 2001;85:141–7.
  30. Noakes M, Clifton P, Ntanios F, Shrapnel W, Record I, McInerney J. An increase in dietary carotenoids when consuming plant sterols or stanols is effective in maintaining plasma carotenoid concentrations. Am J Clin Nutr 2002;75:79–86.
Received for publication September 25, 2003. Accepted for publication February 9, 2004.


Related articles in AJCN:

The ABCs of vitamin E and ß-carotene absorption
Maret G Traber
AJCN 2004 80: 3-4. [Full Text]  

日期:2008年12月28日 - 来自[2004年80卷第1期]栏目
循环ads

Limitations of plasma plant sterols as indicators of cholesterol absorption

Catherine A Vanstone and Peter JH Jones

Mary Emily Clinical Nutrition Research Unit
School of Dietetics and Human Nutrition
McGill University, Macdonald Campus
21,111 Lakeshore Road
Ste Anne-de-Bellevue, PQ H9X 3V9
Canada
E-mail: jonesp{at}macdonald.mcgill.ca

Dear Sir:

We are writing in reference to the article by Joki et al (1) that was recently published in the Journal. The reported objective of the study was to investigate lipid and sterol metabolism in children with food allergy who were given restricted diets. Outcome measurements included concentrations of serum cholesterol precursors, plant sterols, and lipids. The authors concluded that allergic children have low dietary intakes of cholesterol and low serum cholesterol concentrations. The authors also concluded that serum plant sterol concentrations increased, probably as a result of plant sterols in the rapeseed oil supplement. A careful review of the article by Joki et al identified certain fundamental problems in the study design that severely compromise accurate interpretation of the results.

First, the inclusion of rapeseed oil supplementation confounds the hypothesis of the study. As the authors themselves stated, rapeseed oil is known to contain substantial amounts of plant sterols. Plant sterols are widely known to significantly lower plasma cholesterol concentrations and alter plasma plant sterol concentrations when consumed with the diet. Therefore, what this study actually examined was lipid and sterol metabolism in allergic children supplemented with or without plant sterol-containing rapeseed oil for 3 mo in combination with a restricted diet. This question is very different from the originally intended question about the metabolic effects of a restricted diet alone.

Second, interpretation of the results raises some important questions. The authors report that only 40 of the 52 children were supplemented with the rapeseed oil. It remains unclear to the reader why the supplement was given at all and why only a portion of the children received the supplement. More troubling is the fact that, as the data are presented, we do not know how many children in each group (food allergic and nonallergic) received the supplement or whether those children were the ones contributing the most to the decreased mean cholesterol concentrations. The authors do state that the subjects who received supplementary rapeseed oil had higher plant sterol concentrations than those who did not, but, in the Discussion section, changes in plant sterol concentrations are discussed by comparing allergic subjects with nonallergic subjects. Therefore, it is not clear from the results how many of the allergic and nonallergic subjects were supplemented, which makes it impossible to conclude whether it was the supplementation or the allergic condition that influenced serum plant sterol concentrations.

Third, the authors discuss the importance of cholesterol as an essential lipid molecule for growing children and state that one of the goals of their study was to determine whether the low concentrations of serum cholesterol in their subjects were due to insufficient cholesterol synthesis or absorption or to low intakes of cholesterol and saturated fat. In patients whose cholesterol absorption and intake may be compromised, it seems even more illogical that a supplement with known cholesterol-inhibitory properties would be prescribed. Not only would that predispose the patient to even lower serum cholesterol concentrations, but it also would no longer allow the researcher to accurately assess the hypothesis regarding cholesterol absorption and serum concentrations in allergic children given restricted diets.

Fourth, the authors' rationale for using serum plant sterol concentrations to predict changes in cholesterol absorption is not scientifically sound. Under steady state conditions, plant sterol concentrations in serum are positively related to the efficiency of cholesterol absorption (2-4). However, plant sterol concentrations cannot be used as predictors when the serum concentrations of sterols are in a state of flux as a result of supplementation. When stanols are supplemented in the diet, there is a resultant decrease in plasma campesterol concentrations, which appears to mimic the decrease in cholesterol absorption. Similarily, when sterols are supplemented, cholesterol absorption decreases, but serum concentrations of plant sterols actually increase. Some researchers in the field of phytosterols have inappropriately used the campesterol-to-cholesterol ratio as an indicator of changes in cholesterol absorption efficiency after plant stanol supplementation (5-7). Joki et al also incorrectly refer to this method when assessing changes in cholesterol absorption. In addition, they draw 2 contradictory conclusions regarding cholesterol absorption. First, they conclude that, because the campesterol-to-cholesterol ratio was higher in the allergic subjects than in the nonallergic subjects, cholesterol absorption was not affected, from which they draw the inference that the allergic subjects had no bowel inflammation. Second, they conclude that, because there was an increase in cholesterol synthesis, cholesterol absorption must have been reduced. The authors cannot have it both ways—either cholesterol absorption was altered or it was not.

We feel that the results of the study by Joki et al do not reflect the hypothesis and that this discrepancy is due to flaws in the study design. Moreover, the discussion regarding serum plant sterol concentrations and cholesterol absorption is inconsistent through the article and shows a poor understanding of the metabolic effects of plant sterols. To render this study meaningful to the scientific community, the authors need to restate the hypothesis so that it includes plant sterol supplementation, express the data clearly in terms of allergic and nonallergic subjects and in terms of which subjects from each group were supplemented, and, finally, rework the discussion with a stronger understanding of the effects of plant sterol consumption on metabolism generally and on cholesterol metabolism specifically.

REFERENCES

  1. Joki P, Suomalainen H, Jarvinen KM, et al. Cholesterol precursors and plant sterols in children with food allergy. Am J Clin Nutr 2003;77:51-5.
  2. Tilvis RS, Miettinen TA. Serum plant sterols and their relation to cholesterol absorption. Am J Clin Nutr 1986;43:92-7.
  3. Miettinen TA, Tilvis RS, Kesaniemi YA. Serum cholestanol and plant sterols in relation to cholesterol metabolism in middle-aged men. Metabolism 1989;38:136-40.
  4. Miettinen TA, Tilvis RS, Kesaniemi YA. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. Am J Epidemiol 1990;131:20-31.
  5. Gylling HG, Siimes MA, Miettinen TA. Sitostanol ester margarine in dietary treatment of children with familial hypercholesterolemia. J Lipid Res 1995;36:1807-12.
  6. Gylling HG, Miettinen TA. Cholesterol reduction by different plant stanol mixtures and with variable fat intake. Metabolism 1999;48:575-80.
  7. Miettinen TA, Puska P, Gylling H, Vanhanen H, Vartiainen E. Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholesterolemic population. N Engl J Med 1995;333:1308-12.

日期:2008年12月28日 - 来自[2004年79卷第2期]栏目

Cholesterol precursors and plant sterols in children with food allergy

Päivi Joki, Hanna Suomalainen, Kirsi-Marjut Järvinen, Kaisu Juntunen-Backman, Helena Gylling, Tatu A Miettinen and Marjatta Antikainen

1 From the Skin and Allergy Hospital (PJ, HS, K-MJ, and KJ-B), the Division of Internal Medicine of the Department of Medicine (HG and TAM), and the Hospital for Children and Adolescents (MA), Helsinki University Central Hospital.

2 Address reprint requests to M Antikainen, Hospital for Children and Adolescents, University of Helsinki, FIN-00029 HUS, Helsinki, Finland. E-mail: marjatta.antikainen{at}hus.fi.


ABSTRACT  
Background: The data on lipid metabolism in allergic children is limited.

Objective: We investigated lipid and sterol metabolism in young children whose diets were restricted because of food allergy.

Design: Children in group A [n = 21; mean (± SD) age: 1.78 ± 0.73 y] were allergic to fish, eggs, and either cow milk or cereals; those in group B (n = 31, aged 1.45 ± 0.58 y) were allergic to fish, eggs, and both cow milk and cereals. Cholesterol precursor and plant sterol to cholesterol ratios (102 x µmol/mmol cholesterol) and apolipoprotein E phenotype distributions were analyzed in 36 subjects. The control group for cholesterol precursor and plant sterol measurements consisted of 18 healthy age-matched children.

Results: The mean serum cholesterol concentration was 3.6 ± 0.6 mmol/L, and HDL cholesterol was 1.03 ± 0.3 mmol/L in group A. Corresponding values in group B were 3.4 ± 0.7 and 1.09 ± 0.2 mmol/L. The daily cholesterol intake was low: 61.3 ± 36.0 mg in group A and 50.7 ± 48.5 mg in group B. Cholesterol precursor plant sterol concentrations were significantly higher in allergic subjects than in control subjects.

Conclusions: Allergic children with restricted diets have a low intake of cholesterol and relatively low serum cholesterol concentrations. Dietary intake of plant sterols was obviously increased because of supplementation with rapeseed oil, which is rich in plant sterols, leading to elevated plant sterol concentrations. Plant sterols may have inhibited cholesterol absorption, which in turn stimulated cholesterol synthesis in compensation, also explaining the increased precursor sterol ratios in serum in our subjects.

Key Words: Food allergy • cholesterol • cholesterol precursors • plant sterols • children


INTRODUCTION  
Allergy is a common chronic disease in childhood, and cow-milk allergy is commonly the first manifestation of food allergy in early childhood. Oral ingestion of dietary proteins normally induces clinical tolerance. In allergy, antigenic challenge evokes adverse immune reactions and immunoglobulin secretion. These adverse reactions may impair the barrier function of the intestine. Enhanced absorption of antigens can further increase intestinal permeability and destruction of the intestinal mucosa (1). The dietetic treatment of food allergy consists of elimination of antigens, which preserves the intestinal integrity and reverses the disturbance of the humoral and cell-mediated immune responses (2). However, the restricted dietary regimens may impair the child’s nutritional status and growth (3–5). On the other hand, inadequate elimination maintains intestinal inflammation and decreases nutrient absorption.

Cholesterol is an essential lipid for human cells. Dietary cholesterol esters are digested enzymatically in the small intestine, and the products of lipid digestion are absorbed. Within the intestinal mucosal cells, triacylglycerols, phospholipids, and cholesterol are packaged with specific proteins to form lipoprotein complexes called chylomicrons that carry dietary cholesterol to the liver (6). Enteral cholesterol absorption was shown to be regulated by apolipoprotein E (apo E), a structural protein of lipoproteins (6, 7). Human apo E occurs as 3 genetic isoforms, which can be separated into 3 allelic products: E2, E3, and E4. It was observed that E4 carriers have higher serum cholesterol concentrations than do E3 and E2 carriers. This was also found in infants who were fed either breast milk or low-cholesterol formula (8). One reason is that the E4 allele enhances cholesterol absorption from the gut (6, 7).

Inflammatory bowel diseases affecting the upper part of the gastrointestinal tract may interfere with cholesterol absorption and lead to decreased cholesterol concentrations. This is well documented for patients with celiac disease (9). Serum plant sterol (campesterol and sitosterol) concentrations reflect the effectiveness of cholesterol absorption (10). Endogenous cholesterol synthesis takes place mainly in the liver. Cholesterol synthesis is estimated to require 30 separate reactions involving different enzymatic steps (11). Measurement of the most abundant serum cholesterol precursors—8-cholestenol, lathosterol, and desmosterol—shows the effectiveness of endogenous cholesterol production. Effective cholesterol absorption has been shown to suppress cholesterol synthesis (10). If cholesterol absorption decreases, as in celiac disease, hepatic cholesterol synthesis increases (9).

In this work, we investigated lipid and sterols reflecting cholesterol metabolism in young children kept on a restricted diet because of food allergy by measuring serum total and HDL cholesterol, cholesterol precursor and plant sterol concentrations, and apo E phenotypes.


SUBJECTS AND METHODS  
Subjects
This study comprised 52 children aged 8–39 mo consecutively admitted for oral food challenge at the Skin and Allergy Hospital of Helsinki University Central Hospital and diagnosed to have challenge-proven food allergy to fish, eggs, and cow milk or cereals (rye, wheat, oats, and barley) or both. Food allergy was manifested by skin symptoms such as urticaria and excema (n = 34); gastrointestinal symptoms such as vomiting, loose stools, diarrhea, and abdominal pain (n = 4); and both skin and gastrointestinal symptoms (n = 14). Forty-eight children had atopic heredity (at least one of the first-degree relatives had an atopic disease such as atopic eczema or immunoglobulin E–mediated allergy). The subjects were not on steroids, antihistamines, or any other oral drugs at the time of the study.

Control values for serum cholesterol concentrations and an average cholesterol intake of children in Finland were picked up from the Finnish STRIP Baby Project (12, 13). Control values for cholesterol precursor and plant sterol concentrations were measured with gas chromatography in 18 healthy age-matched children.

The study was approved by the Ethical Committee of the Skin and Allergy Hospital of the Helsinki University Central Hospital. Written informed consent was obtained from each child’s parents.

Diets
When food allergy was diagnosed, the children were put on an elimination diet excluding fish, eggs, and either cow milk or cereals (group A) or fish, eggs, and both cow milk and cereals (group B). The adequacy of the diet was followed and confirmed by a registered dietician. Group A comprised 21 children whose mean (± SD) age was 1.78 ± 0.73 y (range: 0.69–3.0 y); group B had 31 children aged 1.45 ± 0.58 y (range: 0.66–3.28 y).

If cow milk was eliminated from the diet, a tolerated formula [soy (n = 6), protein hydrolysate (n = 16), or amino acid formula (n = 10)] was substituted in infants <2 y of age. For older subjects calcium supplementation was given (n = 12). The 3 youngest babies received breast milk. The mothers of the 3 breast-fed infants were on a cow-milk elimination diet because of the infant’s allergy. During cereal elimination, gluten-containing cereals were excluded from the diet and were replaced with gluten-free rice, corn, millet, and buckwheat, or only millet. Forty subjects received supplementary rapeseed oil.

The food consumption was measured by means of a 3-consecutive-day food record kept by the parents. The portion sizes were estimated in household measures. The data were transferred from the food diaries to a computer, and nutrient intakes were analyzed by using the MICRO-NUTRICA computer program developed at the Research Centre of Social Insurance Institution, Turku, Finland.

Oral food challenge
The children all consumed a milk or cereal (or both) elimination diet for 2–4 wk before the oral food challenge. The challenge was started with a drop of the test antigen on the skin or lips. Thereafter increasing doses of cow milk or cereal were given at 2-h intervals on day 1: 1, 10, 50, and 100 mL cow milk or 1, 2, and 10 g cereal, and on day 2 normal milk or cereal intake appropriate for age was allowed. The challenge was discontinued and the subjects were examined by a pediatrician when any adverse reaction was noted. The time of the clinical reaction to the relevant antigen was defined as the time elapsing from the last given dose eliciting the specific reactions. The subjects were also observed over the 1-wk period of the challenge to detect symptoms developing several days after commencement of the challenge. A period of 2 wk without any symptoms was allowed to elapse before the next challenge.

Cholesterol and apo E measurements
After 3 (geometric : 7.2) months on strict elimination diets, blood samples were taken in the morning after the subjects had fasted for 8 h, and serum total cholesterol and HDL cholesterol concentrations were measured enzymatically (kit no. 236691; Boehringer Diagnostica GmbH, Mannheim, Germany). In a subgroup of 36 subjects, serum sterols and total cholesterol concentration were measured by gas chromatography (14) with a 50-m-long capillary column (Ultra 1; Hewlett Packard, Wilmington, DE). Because plant sterols are mainly transported in cholesterol-containing particles in serum, the absolute concentrations were adjusted for the serum cholesterol concentration (102 x µmol/mmol cholesterol) to eliminate the effect of changes in the serum cholesterol concentration. Apo E phenotypes were separated by isoelectric focusing ( Statistics
One-factor analysis of variance was used to determine differences between the groups in diet records and serum total and HDL-cholesterol concentrations, expressed as means ± SDs. The concentrations of cholesterol precursors and plant sterols were log transformed because the variables were not normally distributed, and the results are expressed as geometric mean values with 95% CIs. Statistical significance is defined as P 0.05. Statistical analyses were carried out with STATVIEW 4.0 software (Abacus Concepts Inc, Berkeley, CA).


RESULTS  
Diet records
Diet records are given in Table 1. The average diet of the children in this study was well within the Nordic Nutrition Recommendations (16). The mean daily total energy intakes in groups A and B were 3826 and 4334 kJ, respectively. The share of total fat and saturated and monounsaturated fatty acids exceeded the recommended amounts. When compared with the average intakes of healthy Finnish children (17, 18), our subjects received lower amounts of proteins and carbohydrates but higher amounts of fats. The quality of the fat, expressed as energy intakes of polyunsaturated fatty acids, monounsaturated fatty acids, and saturated fatty acids was high. The ratio of polyunsaturated to monounsaturated to saturated fatty acids was 0.6:1.2:1.0 in group A and 0.9:1.5:1.0 in group B. No significant difference was found in fat intake or saturated fat intake between groups A and B. Cholesterol intakes were low but comparable with those observed in the Finnish STRIP Baby Project (83.6 ± 36.6 mg/d in the intervention group and 112.8 ± 44.2 mg/d in the control group; 18).


View this table:
TABLE 1 . Fat, fatty acid, carbohydrate, protein, and cholesterol intakes in the 2 study groups  
Serum lipids and sterols
The mean serum cholesterol concentration was 3.6 ± 0.6 mmol/L in group A and 3.4 ± 0.7 mmol/L in group B. The mean HDL-cholesterol concentrations were 1.03 ± 0.3 mmol/L in group A and 1.09 ± 0.2 mmol/L in group B. These values were not significantly different in the 2 groups. Compared with age-matched healthy Finnish children (12), 80% of the subjects in diet group A and 90% of the subjects in group B had low serum total cholesterol concentrations, and 45% and 26%, respectively, had low serum HDL-cholesterol concentrations. The ratios of serum total cholesterol to HDL cholesterol were 3.5 and 3.1 in groups A and B, respectively. The mean serum cholesterol concentration was 3.7 ± 0.61 mmol/L in a small group of 18 healthy children whose total cholesterol concentrations were measured together with the measurements of cholesterol precursor and plant sterol concentrations.

The ratios of cholesterol precursor to cholesterol adjusted for cholesterol concentrations (geometric values with 95% CIs) in groups A and B and in control subjects are given in Table 2
View this table:
TABLE 2 . Serum cholesterol precursor and plant sterol concentrations in the 2 study groups and the control subjects1  
The differences in cholesterol precursor and plant sterol concentrations were also tested in subjects receiving different substitute formulas. No statistically significant differences were observed in these variables between the children fed on the different substitute formulas, although children receiving formulas based on hydrolyzed protein and amino acids tended to have a lower cholesterol intake than did those receiving soy formula or calcium supplementation.

Apo E phenotype distributions were analyzed in a subgroup of 36 children with food allergy. The allele frequencies were as follows: 0.286 for E4, 0.657 for E3, and 0.057 for E2. The phenotype distribution and allelic frequencies were similar to those observed in a large group of adult Finns (15). In Table 3, concentrations of serum cholesterol, its precursors, and plant sterols are given according to the apo E phenotype. Subjects carrying the phenotypes 4/3 and 3/3 had the highest sitosterol concentrations. The lowest campesterol concentrations were seen in subjects carrying phenotype 3/2. There was a tendency, although not significant, for higher serum cholesterol concentrations in subjects carrying the E4 allele than in subjects not carrying E4 allele.


View this table:
TABLE 3 . Serum cholesterol, HDL-cholesterol, cholesterol precursor, and plant sterol concentrations by different apolipoprotein E phenotypes in a subgroup of 36 allergic subjects1  

DISCUSSION  
Cholesterol is an essential lipid molecule for human cells because it is a precursor of steroid hormones, vitamin D metabolites, and bile acids and is also important for neural myelinization and brain growth (11, 19). Cholesterol is therefore especially important for a growing child. Endogenous cholesterol is synthesised in the liver from acetyl-CoA through a complex enzymatic pathway (11). In young infants, the liver enzyme activities needed for cholesterol synthesis are possibly immature. In this study we tried to find out whether the relatively low serum cholesterol concentrations observed in our subjects could be a result of insufficient hepatic cholesterol synthesis. Other possible reasons for low cholesterol concentrations could be diminished absorption of cholesterol or a low amount of cholesterol or saturated fat in the diet.

Within the intestinal lumen, dietary cholesterol is solubilized into mixed micelles. Micelles enter the intestinal mucosal cells and degrade, and the free cholesterol is packed into lipoproteins called chylomicrons. Apo E phenotypes influence cholesterol absorption. The apo E allele frequencies observed in our subjects were comparable with those detected in the adult Finnish population. Thus, abnormal distribution of apo E alleles seemed not to explain low serum cholesterol concentrations in allergic children.

The effects of bowel inflammation on serum lipids and plant sterols were studied previously in celiac patients. In an acute phase of celiac disease (villous atrophy of the jejunal mucosa in biopsy caused by allergic bowel inflammation), cholesterol absorption is low, leading to enhanced cholesterol synthesis in the liver. A gluten-free diet leads to improvement in clinical symptoms and in jejunal villous atrophy. An appropriate diet also improves cholesterol absorption and decreases cholesterol synthesis (9). Cholesterol absorption efficiency also regulates cholesterol synthesis in healthy children receiving plant stanol ester margarine; this margarine lowers the absorption of cholesterol, leading to compensatory activation of cholesterol synthesis (20). In the present study, plant sterol concentrations reflecting cholesterol absorption were higher in our allergic subjects than in the control subjects. The children who received rapeseed oil supplementation had the highest serum plant sterol concentrations. This allows us to assume that, although no intestinal biopsies were taken, our subjects did not have allergic bowel inflammation at the time of the investigation of cholesterol metabolism.

The restricted dietary regimens, including low cholesterol and saturated fat intake, used in a treatment of food allergy contributed to relatively low serum cholesterol concentrations. Cholesterol precursor concentrations were significantly higher in the allergic subjects than in the control subjects. This means that the liver enzyme activities required for cholesterol synthesis are well matured, and there is a compensatory activation of cholesterol synthesis taking place in the liver of the subjects. Dietary intake of plant sterols was obviously increased because of supplementation of rapeseed oil, which is rich in plant sterols, leading to elevated plant sterol concentrations in most of our subjects. Plant sterols may have inhibited cholesterol absorption, which in turn up-regulated cholesterol synthesis in compensation, also explaining the increased precursor sterol ratios in serum. Our earlier plant sterol feeding studies and those with plant sterol ester consumption by Hallikainen et al (21) caused identical changes.

A low-saturated-fat, low-cholesterol diet may reduce not only serum total cholesterol but also HDL-cholesterol concentrations (22). The same observation was made in a large Finnish study of infants with a diet low in saturated fat and cholesterol (18). It is therefore not surprising that our allergic subjects, whose cholesterol intake was very low, had low HDL-cholesterol concentrations. HDL cholesterol is an important lipoprotein in reverse cholesterol transport and is protective against atherosclerosis. One way to assess a risk of atherosclerosis is to calculate the ratio of total cholesterol to HDL cholesterol: the lower the ratio, the lower the risk of coronary artery disease. In our subjects, total cholesterol–HDL-cholesterol ratios were very low compared with the ratios in adult populations, in which the risk of coronary artery disease is evident (23). Thus, our study population does not seem to have an increased risk of this harmful effect of low HDL cholesterol.

In conclusion, allergic children with restricted diets have a low intake of cholesterol and relatively low serum cholesterol concentrations. Dietary intake of plant sterols was obviously increased because of supplementation with rapeseed oil, which is rich in plant sterols, leading to elevated plant sterol concentrations. Plant sterols may have inhibited cholesterol absorption, which in turn stimulated cholesterol synthesis in compensation, also explaining the increased precursor sterol ratios in serum in our subjects.


REFERENCES  

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Received for publication June 27, 2001. Accepted for publication April 1, 2002.


日期:2008年12月28日 - 来自[2003年77卷第1期]栏目
循环ads

Unesterified plant sterols and stanols lower LDL-cholesterol concentrations equivalently in hypercholesterolemic persons

Catherine A Vanstone, Mahmoud Raeini-Sarjaz, William E Parsons and Peter JH Jones

1 From the School of Dietetics and Human Nutrition, McGill University, Ste Anne-de-Bellevue, Quebec.

2 Supported by Dairy Farmers of Canada and Forbes Medi-Tech Inc, Vancouver, Canada.

3 Address reprint requests to PJH Jones, School of Dietetics and Human Nutrition, McGill University, 21,111 Lakeshore Road, Ste Anne-de-Bellevue, Quebec, Canada H9X 3V9. E-mail: jonesp{at}macdonald.mcgill.ca.


ABSTRACT  
Background: Plant sterols, in various forms, have been shown to reduce total and LDL-cholesterol concentrations. Particularly controversial at present is the effect of the degree of hydrogenation of the plant sterols on cholesterol-lowering efficacy and the responsible mechanisms.

Objective: Our goal was to examine the effect of supplementation with unesterified plant sterols and stanols on plasma lipid and phytosterol concentrations and cholesterol absorption, synthesis, and turnover.

Design: Fifteen otherwise healthy hypercholesterolemic subjects consumed each of 4 dietary treatments in a randomized crossover design. Unesterified sterols and stanols were blended into the butter component of the diet at a dosage of 1.8 g/d. The diets contained plant sterols (NS), plant stanols (SS), a 50:50 mixture of sterols and stanols (NSS), or cornstarch (control).

Results: Plasma total cholesterol concentrations were 7.8%, 11.9%, and 13.1% lower (P < 0.01) in the NS, SS, and NSS groups, respectively, than in the control group. LDL-cholesterol concentrations were 11.3%, 13.4%, and 16.0% lower (P < 0.03) in the NS, SS, and NSS groups, respectively, than in the control group. Plasma triacylglycerols and HDL-cholesterol concentrations did not differ significantly across diets. Cholesterol absorption efficiency was 56.0%, 34.4%, and 48.9% lower (P < 0.001) in the NS, SS, and NSS groups, respectively, than in the control group. The fractional synthesis rate was higher by 45.5% (P < 0.003) in the NSS group than in the control group. Plasma campesterol and sitosterol concentrations were higher (P < 0.01) in the NS group and sitosterol concentrations were lower (P < 0.01) in the SS group than in the control group.

Conclusion: These data indicate that, in their free unesterified form, sterols and stanols lower plasma LDL cholesterol equivalently in hypercholesterolemic persons by suppressing cholesterol absorption.

Key Words: Plant sterols • plant stanols • hypercholesterolemia • LDL cholesterol • dietary intervention


INTRODUCTION  
Plant sterols and stanols, structural analogues of cholesterol, have been shown to substantially reduce total and LDL-cholesterol concentrations under a variety of study conditions. Several researchers have claimed that consumption of stanol-containing mixtures is more effective in reducing circulating cholesterol concentrations than is consumption of sterols (1–8). Recently, however, the paradigm has shifted to the position that sterol and stanol esters are comparable plasma cholesterol modulators. It was observed that circulating total and LDL-cholesterol concentrations were equally reduced by 8–13% with both sitosterol-ester and sitostanol-ester margarines at dosages of 1.5–3.3 g/d (9). Similar lowering of total and LDL-cholesterol concentrations was reported with ingestion of sitosterol-esters and sitostanol-esters (10).

This controversy raises another important question: whether unesterified (free) plant sterol and stanol mixtures possess the same cholesterol-lowering efficacy regardless of their degree of hydrogenation, or whether esterification and solubilization of plant sterol mixtures are responsible for their equal effectiveness. Comparison of free sitosterol and sitostanol in pastil form given to children with severe hypercholesterolemia showed that hydrogenation improved the LDL-cholesterol lowering by increasing fecal neutral sterol output to a greater degree than that in the sitosterol-supplemented group (11).

The plasma cholesterol-lowering efficacy of phytosterols varies according to the composition and dose of the phytosterol mix and the vehicle in which they are given. It has been suggested that high intakes of saturated fat and cholesterol may improve the efficacy of phytosterols (12) and phytosterol esters (3). To date, the relative effectiveness of these materials in a dietary context in which saturated fat and cholesterol intakes are at the higher end of the normal physiologic range has not been assessed, nor have the mechanisms of action been fully explored. Therefore, the objective of this study was to examine the effect of supplementation with unesterified sitosterols and sitostanols on plasma lipid and phytosterol concentrations and on cholesterol absorption and synthesis in subjects consuming precisely defined diets.


SUBJECTS AND METHODS  
Subjects
Ten male and 6 female otherwise healthy, free-living volunteers with primary familial hyperlipidemia were recruited. The subjects were aged between 35 and 58 y. Female subjects were either postmenopausal or had undergone a hysterectomy. Subjects were screened for total circulating cholesterol and triacylglycerol concentrations. Inclusion criteria included a plasma total cholesterol concentration in the range of 5.2–9.0 mmol/L and a triacylglycerol concentration <3.5 mmol/L. Before acceptance, subjects were required to provide a medical history and to undergo a complete physical examination. Fasting blood and urine samples were collected for serum biochemistry, hematology, and urinalysis. Subjects were screened for chronic illness, including hepatic, renal, thyroid, and cardiac dysfunction, before admission in the study. Subjects were required to refrain from using drug therapy for hypercholesterolemia during and for 8 wk before the start of the study. Before study commencement, the subjects received a thorough explanation of the study protocol and were given the opportunity to discuss any queries with either the primary investigator, the physician, or the study coordinator before signing the consent form. The experimental protocol was approved by the Human Ethical Review Committee of the Faculty of Agriculture and Environmental Sciences for the School of Dietetics and Human Nutrition at McGill University. The baseline characteristics of the study subjects are presented in Table 1.


View this table:
TABLE 1 . Baseline characteristics of the subjects1  
Experimental design and diets
The study was a randomized, crossover, double-blind clinical trial. Subjects consumed each of 4 dietary treatments. Each dietary treatment phase consisted of 21 feeding days and was separated by a 4-wk washout period during which the subjects consumed their habitual diets. To reduce the error term associated with diet sequencing, subjects were randomly assigned to 1 of 4 predetermined Latin squares, each of which possessed 4 sequenced phases and 4 subjects. In this manner, we ensured that the crossover design was balanced.

Diets were designed on the basis of the recommended nutrient intakes for Canadians to provide 3000 kcal per 70-kg individual daily. The Mifflin equation was used to estimate each subject’s basal energy requirement (13), which was then multiplied by an activity factor of 1.7 to compensate for the additional energy needs of mildly to moderately active healthy adults. If subjects gained or lost weight during the first week of each treatment phase, energy adjustments were made to meet individual requirements and ensure that baseline body weights were maintained.

The diets comprised solid foods, typical of those consumed in North America, and were provided as 3 meals daily in a 3-d rotating menu. The nutrient content of the basal diet was calculated by using FOOD PROCESSOR (Food Processor, Salem, OR), a computerized dietary analysis system with a Canadian database. Dietary carbohydrate, fat, and protein made up 50%, 35%, and 15% of ingested energy, respectively, with 70% of the fat provided as butter.

The diets contained plant sterols (NS), plant stanols (SS), a 50:50 mixture of sterols and stanols (NSS), or cornstarch (control). The NS treatment consisted of purified phytosterols derived from soybeans and contained sitosterol (43%), campesterol (26%), stigmasterol (17%), and other identified phytosterols (14%). For the SS treatment, the same soybean phytosterols were hydrogenated to produce a composition of sitostanol (66%) and campestanol (33%). Equal parts of phytosterols and phytostanols were mixed together to create the NSS treatment. The control product was cornstarch, which strongly resembled the white, powdery phytosterol-containing mixtures. The phytosterol and phytostanol mixtures and the cornstarch control were blended into the butter component of the diet at a dosage of 1.8 g/d; the butter was warmed to 37°C and administered equally across the 3 daily meals. To achieve double blinding, the containers of plant sterols, stanols, and the cornstarch control were coded so that neither the researcher giving the test mixture nor the subject receiving it knew its true identity.

Diets were prepared in the metabolic kitchen of the Mary Emily Clinical Nutrition Research Unit of McGill University. Subjects consumed a minimum of 2 of the 3 daily meals at the unit under supervision. All subjects were required to consume breakfast at the unit and 1 of the other 2 meals was available for take out.

Protocol
At the start (day 1) and end (day 22) of each dietary phase, fasting blood samples were taken for measurement of circulating lipid concentrations. Ninety-six hours before the end of each phase, subjects provided a baseline blood sample before receiving an intravenous injection of 15 mg [25,26,26,26,27,27,27-D7]cholesterol and a 75-mg oral dose of [3,4-13C]cholesterol for cholesterol absorption determination. The ratio of ingested [3,4-13C]cholesterol to injected [25,26,26,26,27,27,27-D7]cholesterol enrichment in serum cholesterol after 24, 48, and 72 h was taken as an indicator of the cholesterol fractional absorption rate. The [D7]cholesterol isotope was prepared for injection by first dissolving it in ethanol at a concentration of 5 mg/mL under sterile conditions at the Royal Victoria Hospital pharmacy. The isotope-ethanol mixture was then added drop-wise to an intravenous fat emulsion (Baxter Corp, Toronto), for a total injectable volume of 9 mL. Cholesterol synthesis was also measured at the end of each diet period by using the deuterium incorporation approach. Seventy-two hours after dosing with [13C]cholesterol and [D7]cholesterol, subjects were dosed with 0.7 g D2O/kg estimated body water (99.8 atom percent excess; CDN Isotopes, Montreal). Body water was estimated to be 60% for calculation of the dose. Deuterium oxide was given immediately after a fasting blood sample was collected at 0800 on day 21 of each diet phase.

Analyses
Plasma lipid concentrations
Blood samples were centrifuged for 15 min at 520 x g and 4°C to separate plasma from red blood cells (RBCs) and were stored at -80°C until analyzed. Plasma total cholesterol, HDL-cholesterol, and triacylglycerol concentrations were analyzed in quadruplicate with standardized reagents in a VP Autoanalyser (Abbott Laboratories, North Chicago, IL). The analyzer was calibrated before each run as per the standardization protocol of the Canadian Reference Laboratory. The Friedewald equation was used to calculate LDL-cholesterol concentrations (14).

Cholesterol absorption
Free cholesterol extracted from RBCs was used to determine [13C]cholesterol and [D7]cholesterol enrichments. Lipid was extracted from the RBCs in duplicate by using a modified Folch extraction procedure (15). Thin-layer chromatography (20 x 20 cm, 250 µ; Scientific Adsorbents Inc, Atlanta) was used to separate free cholesterol from cholesteryl ester. The free cholesterol band was then scraped from the silica gel plate and saponified with 0.5 mol methanolic KOH/L to eliminate any fatty acid contaminants. Free cholesterol extracts were dried under nitrogen and transferred into 18-cm sealed combustion tubes (Vycor; Corning Glass Works, Corning, NY). Cupric oxide (0.6 g) and a 2-cm piece of silver wire were added and the tubes were sealed under a vacuum for 5 min at <20 mTorr. Dual-tracer-labeled cholesterol samples were then combusted to deuterium-enriched water and 13C-enriched carbon dioxide over 4 h at 520°C. The generated carbon dioxide was transferred under vacuum into Vycor tubes for measurement of 13C enrichment, and water was vacuum-distilled into sealed tubes containing 0.06 g Zn (Biogeochemical Laboratories, Indiana University, Bloomington, IN) for deuterium enrichment analysis. Tubes containing the water and zinc were then reduced to deuterium-labeled hydrogen gas at 520°C for 30 min.

Nuclear magnetic resonance was used to verify that the isotopic enrichments of the tracers, [3,4-13C]cholesterol and [D7]cholesterol (CDN Isotopes, Pointe Claire, Canada), were >99 atom percent excess. The 13C enrichments of free cholesterol were measured by differential isotope ratio mass spectrometry (IRMS) with an automated dual-inlet system (SIRA 12; Isomass, Cheshire, United Kingdom). Enrichments were then expressed relative to PeeDee Belemnite (PDB) limestone, which is used as the international reference standard for expressing carbon stable isotopic ratios, from the National Bureau of Standards (NBS). The linearity and gain of response of the SIRA IRMS instrument were assessed by using a carbon dioxide reference tank and NBS standards of known isotopic enrichment. The deuterium enrichments of free cholesterol were measured by differential IRMS with the use of a manually operated dual-inlet system with electrical H3+ compensation (VG Isomass 903D). For deuterium, enrichments were expressed relative to standard mean ocean water (SMOW) and a series of standards of known enrichment from the NBS, which were analyzed concurrently on each day of measurement to correct for any variations in linearity of gain of response of the IRMS.

The average 13C and deuterium enrichments of 48- and 72-h RBC free cholesterol relative to baseline (t = 0) samples were used to calculate the cholesterol absorption coefficient (CAC) by using the ratio of orally ingested [13C]cholesterol to intravenously administered [D7]cholesterol as described by Bosner et al (16):

RESULTS  
Sixteen subjects were enrolled in the study. One male subject dropped out at the end of the first feeding cycle because of difficulties with daily transportation to the unit. Therefore, complete data for 9 men and 6 women were collected and analyzed as per the study protocol. All individuals tolerated the diet without any reported adverse events. Subjects reported no abnormal or atypical smells, tastes, colors, or mouth-feel effects when consuming any of the 4 mixtures and thus were unable to distinguish between dietary treatments. There were no significant mean group weight changes across any of the 3 treatment phases. Blood and urine samples at the beginning and end of each phase for all 15 subjects were sent to LDS Diagnostic Laboratories (Pointe Claire, Canada), where complete blood counts, biochemistry analyses (sequential multiple analysis level C), and urinalyses were carried out. Results from all 4 phases of the feeding trial remained within normal ranges throughout the study period, and the results of regular physical exams showed no suggestion of any clinical irregularities.

Circulating lipids in response to treatment
Concentrations of plasma lipids at the beginning and end of each treatment phase are shown in Table 2. Total cholesterol concentrations and the changes in cholesterol concentrations varied greatly from one subject to another across all phases of the feeding trial. Plasma total cholesterol concentrations were 7.8%, 11.9%, and 13.1% lower at the end of the dietary period (P < 0.01) in the NS, SS, and NSS groups, respectively, than in the control group. LDL-cholesterol concentrations were 11.3%, 13.4%, and 16.0% lower (P < 0.03) in the NS, SS, and NSS groups, respectively, than in the control group. Plasma triacylglycerol and HDL-cholesterol concentrations did not differ significantly across diets. Over the study period, however, HDL cholesterol was lower on day 21 of the NSS treatment period than on day 0 (P < 0.05).


View this table:
TABLE 2 . Plasma lipid concentrations on days 0 and 21 of each dietary period1  
Cholesterol absorption in response to treatment
Cholesterol absorption at the end of each feeding phase was taken as an average of the 48- and 72-h measurements. The mean cholesterol absorption coefficient was lower (P < 0.001) after ingestion of the NS, SS, and NSS diets than after the control diet (Table 3). Therefore, relative to the control period, absorption was 56.0%, 34.4%, and 48.9% lower after the NS, SS, and NSS dietary periods, respectively. Absorption values for the NS and SS dietary periods were significantly (P < 0.05) different from each other; however, the value for the NSS period was not significantly different from that for either the NS or the SS period.


View this table:
TABLE 3 . Cholesterol absorption coefficients and fractional synthesis rates for each dietary period1  
Cholesterol biosynthesis in response to treatment
The FSR in the control group was measured to be 0.044 ± 0.007 pool/d. Ingestion of the NSS diet resulted in a 45.5% higher FSR (0.064 ± 0.007 pool/d; P < 0.003) than did consumption of the control diet. The FSR also tended to be higher after consumption of both the NS and SS diets by 25.0% (0.055 ± 0.008 pool/d) and 34.1% (0.059 ± 0.01 pool/d), respectively (NS). No significant difference in synthesis was observed between groups supplemented with phytosterols or phytostanols.

Cholesterol turnover in response to treatment
Rates of cholesterol turnover were calculated from [D7]cholesterol enrichment values obtained during the 24–72 h period following injection of the isotope at the end of each treatment phase. Turnover rates of free cholesterol, extracted from RBCs, were 0.381 ± 0.05, 0.346 ± 0.06, 0.324 ± 0.04, and 0.364 ± 0.04 pools/d, for the NS, SS, NSS, and control diets, respectively. No significant differences were found between any of the diets.

Plasma plant sterol concentrations in response to treatment
Plasma plant sterol concentrations and ratios relative to total cholesterol are presented in Table 4. Plasma campesterol and sitosterol concentrations were not significantly different between groups at the beginning of each feeding phase. There were, however, differences between groups at the end of phytosterol supplementation. Plasma campesterol and sitosterol concentrations were higher (P < 0.01) in the NS group than in the control and SS groups. Mean plasma campesterol and sitosterol concentrations were 99.3% and 38.6% higher (P < 0.0001), respectively, after the NS diet than after the control diet. Sitosterol concentrations were lower (P < 0.01) by 23.6% after the SS diet than after the control. Campesterol concentrations were also lower with SS feeding, but not significantly so. The NSS diet produced few changes in circulating plant sterol concentrations.


View this table:
TABLE 4 . Plasma plant sterol concentrations on days 0 and 21 of each dietary period1  
Associations between plasma lipid concentrations and kinetic measurements
Across all subjects, both plasma total (r = 0.42, P < 0.001) and LDL cholesterol (r = 0.35, P < 0.006) concentrations varied directly with the cholesterol absorption coefficient. Similarly, ß-sitosterol (r = 0.40, P < 0.002) concentrations varied directly with circulating LDL-cholesterol concentrations. The FSR varied inversely with LDL cholesterol (r = -0.29, P < 0.03) concentrations, further supporting the compensatory relation between cholesterol lowering and increased synthesis rates. The ratio of sitosterol to campesterol correlated directly with plasma LDL cholesterol (r = 0.49, P < 0.0001) and inversely with the FSR (r = -0.42, P < 0.0008). Notably, neither campesterol nor the ratio of campesterol to cholesterol correlated with the cholesterol absorption coefficient.


DISCUSSION  
The major novel finding of the present study is the demonstration that unsaturated, saturated, and an equal mixture of unsaturated and saturated phytosterols, in their unesterified form, significantly and equally reduce both plasma total and LDL-cholesterol concentrations. The degree of cholesterol lowering observed was entirely due to the action of the plant sterols and stanols and not to the basal diet, because plasma total and LDL-cholesterol concentrations marginally increased with the control diet. This reduction in circulating cholesterol concentrations was achieved through inhibition of intestinal cholesterol absorption as evidenced by lower absorption coefficients; however, these reductions were accompanied by a partial compensatory desuppression of cholesterol synthesis, which may be an indication that other mechanisms are also at work.

Despite the relatively high content of saturated fat and cholesterol in the basal diet, sterols and stanols were efficacious in lowering circulating total and LDL-cholesterol concentrations. It has been postulated that elevated intakes of dietary fat and cholesterol (400–450 mg/d) may increase the effectiveness of phytosterols in the intestinal lumen. Unesterified plant sterols blended in butter and supplemented in doses of 0.74 g for 4 wk were shown to decrease total and LDL-cholesterol concentrations by 10% and 15%, respectively, despite a phytosterol dosage of <1 g/d (12). These authors attributed their results to the high cholesterol intake obtained from butter. Several researchers, however, achieved similar degrees of cholesterol suppression when the total fat and cholesterol contents of the diet were much lower (7, 21, 22). These and other studies showed the efficacy of plant sterols and stanols when blended into a fat source such as margarine, butter, mayonnaise, or vegetable oils before supplementation (23). Conversely, when provided as a powder-filled capsule as part of a low-fat diet, plant stanols failed to exert any lipid-modulating effect (24), suggesting that the amount of fat and cholesterol in the diet are not strong modulators of the effectiveness of plant sterols. It is more likely that the effectiveness of plant sterols and stanols depends more on the vehicle in which they are matrixed and added to the diet than on the composition of the diet.

Although structurally similar to cholesterol, plant stanols are believed to be negligibly absorbed by the intestine (1). Therefore, they do not enter the cell and displace cholesterol at the level of the micelle, interrupting absorption (25). Several methods exist to directly measure cholesterol absorption; however, many require fecal collections, radiolabeled cholesterol administration, or both (26, 27). The current study is one of few to use the dual-stable-isotope method. A coefficient of absorption for cholesterol is derived through a time-step comparison of the proportion of an orally administered, labeled bolus of tracer cholesterol appearing in blood relative to the appearance of a bolus labeled with a second tracer administered intravenously (16, 28). The pattern of decay of the intravenous tracer permits correction of the oral tracer response in plasma for loss into routes of excretion or deeper metabolic pools. Previously, selected ion monitoring mass spectrometry was used to measure isotope enrichments (16), whereas this study used more sensitive IRMS to improve precision with lower isotopic dosages. The cholesterol absorption coefficient was determined by calculating the average of the 48 and 72 h time points by using the approach described by Bosner et al (16), who showed that the plasma ratio of oral and intravenous tracers becomes constant between 48 and 72 h after dosing, allowing for accurate assessment of intestinal cholesterol absorption.

The relative effect of unesterified sterols and stanols compared with a control diet on cholesterol absorption has not been previously studied, particularly in the context of a rigidly controlled dietary paradigm. The cholesterol absorption efficiency of 45.4% in the control group is comparable to values reported elsewhere (16, 29, 30). Similarly, the 34–56% lower cholesterol absorption rates after sterol and stanol supplementation agree with data for sterol and stanol esters reported elsewhere for humans (6, 10, 29, 30). The present data indicate that the decrease in circulating cholesterol concentrations in subjects supplemented with unesterified phytosterols or phytostanols is due to this inhibition in the cholesterol absorption efficiency.

Cholesterol absorption varied directly with both total and LDL-cholesterol concentrations, suggesting that circulating cholesterol concentrations are dependent on the uptake of cholesterol in the intestine and that plant sterols and stanols effectively inhibit cholesterol absorption. The group with the lowest absorption coefficient was not, however, the group with the greatest degree of cholesterol lowering. The sterol diet lowered cholesterol absorption by 56% and raised synthesis by 25%; however, a smaller effect on cholesterol lowering was seen than in the other groups. The stanol diet decreased the absorption coefficient by 34.4% and raised synthesis by the same amount (34.1%), and cholesterol concentrations fell more dramatically. Interestingly, the 50:50 mix of sterols and stanols decreased cholesterol absorption by 48.9% and increased synthesis almost 50%, yet lowered cholesterol concentrations to the greatest degree.

Free sitosterol has been shown to more effectively lower cholesterol absorption than does sitosterol ester (29). However, most previous reports concluded that sitostanol more effectively inhibits cholesterol absorption than does sitosterol (1, 6, 7) or results in equal reductions in cholesterol absorption efficiency (10, 30). Although our results are in contrast with those previously reported, the present study enforced a strict dietary regimen, ensuring that all subjects consumed identical foods, in equal proportions, while maintaining a steady weight. This regimen minimized several dietary confounders, making the comparisons between groups more accurate.

Consumption of plant sterols and stanols significantly induces changes in circulating plant sterol and cholesterol concentrations, indicating mutually competitive inhibition between all sterol forms (25). On this basis, plasma plant sterol concentrations have been used as indicators of compliance. Absolute values and percentage changes in campesterol and sitosterol concentrations were similar to those previously reported after phytosterol feeding (8, 10, 23), signaling that the subjects were in fact consuming the treatment.

Plasma plant sterol concentrations have also been used as an indirect measure of cholesterol absorption. Specifically, serum campesterol concentrations and the ratio of campesterol to cholesterol have been shown to correlate positively with intestinal cholesterol absorption. This association would be expected to reflect cholesterol absorption under static dietary conditions. However, different plant sterols are variably absorbed and metabolized; therefore, it is unclear whether the use of campesterol is appropriate for measuring cholesterol absorption under conditions in which plant sterol and stanol intakes are changing. Supplementation with stanols inhibits cholesterol absorption and has consistently produced decreases in sterol concentrations (7, 10, 23). During sitosterol feeding, however, sitosterol and campesterol concentrations have been shown to increase (10) or remain unchanged (11) despite a clear inhibition in the cholesterol absorption efficiency, making this correlation inapplicable as a method of estimating cholesterol absorption in any situation in which phytosterol intakes would be expected to change. In the present study, plasma sterol concentrations decreased with stanols and increased with sterols, whereas concentrations remained similar to those with the control with a 50:50 mix of sterols and stanols. Furthermore, the cholesterol absorption coefficient was not associated with either campesterol concentration or the ratio of campesterol to cholesterol, as would be predicted from investigating stanols alone.

In summary, the results of the present study showed that in free form, sterol and stanol feeding results in eqivalent reductions in total and LDL-cholesterol concentrations. Cholesterol absorption was reduced in response to sterol and stanol feeding and varied directly with reductions in LDL-cholesterol concentration. Cholesterol synthesis was increased, however, but not to an extent that prevented cholesterol lowering. Both unesterified plant sterols and stanols favorably lower LDL cholesterol, independent of their degree of hydrogenation, in hypercholesterolemic individuals.


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Received for publication July 20, 2001. Accepted for publication January 24, 2002.


日期:2008年12月28日 - 来自[2002年76卷第6期]栏目
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