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Nutrient Metabolism

Free Phytosterols Effectively Reduce Plasma and Liver Cholesterol in Gerbils Fed Cholesterol¹

Foster Biomedical Research Laboratory, Brandeis University, Waltham, MA and ^* Novartis Nutrition Research AG, Neuenegg, Switzerland

²To whom correspondence should be addressed. E-mail: kchayes{at}brandeis.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The potential of free phytosterols (including 20% stanols) to lower plasma and liver lipids was assessed in three experiments with gerbils fed diets containing cholesterol. The first explored the ability of phytosterols (0.5%) to block absorption of 0.05, 0.10, and 0.5% cholesterol. The second assessed the importance of consuming phytosterols (0.75%) simultaneously with cholesterol (0.15%). The third compared free phytosterols (0.75%) with similar levels of esterified sterols or stanols using diets containing 0.15% cholesterol. A 5:1 ratio of phytosterols:cholesterol effectively blocked cholesterol absorption when the dietary cholesterol load was moderate. Consuming a 5:1 ratio with every meal was more effective than receiving equal phytosterols in a 10:1 ratio every other day. Finally, free phytosterols dissolved in fat were as effective as esterified sterols and stanols in lowering plasma and liver cholesterol, and all were equally effective at blocking cholesterol absorption as shown by increased fecal cholesterol output. Plant sterol accumulation in the liver was minimal for all test groups.

KEY WORDS: • free phytosterols • esterified phytosterols • plasma cholesterol • liver cholesterol • gerbils

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Phytosterols represent a potentially effective dietary component for lowering plasma total cholesterol (TC)³,especially LDL cholesterol (LDL-C), thereby reducing the risk for coronary heart disease (1 –4 ). These plant sterols are currently available from two major sources, i.e., tall oil (wood pulp by-product) and as a by-product of soybean oil production. The two forms used in food products are the natural sterol or its hydrogenated stanol form. Both forms are generally esterified to make them more soluble in fat because early evidence suggested that poorly soluble free sterols were not very effective at lowering TC (1 ,5 ,6 ). However, recent data suggest that free sterols may be as effective as esters if they are adequately dispersed in the diet (6 –9 ).

Phytosterols are thought to act by displacing dietary cholesterol in gut micelles, thereby excluding free cholesterol (FC) from absorption by the mucosal cells (1 ,2 ). The question of how much phytosterol should be consumed on a daily basis has been studied on numerous occasions, and the answer seems to vary from 1 to 3 g for reasons that are unclear (1 ,2 ). Among possible explanations could be individual variation in the response to dose, whole-body cholesterol dynamics of the host or the dietary cholesterol load itself. A related but distinct question still outstanding is whether phytosterols must always be present when cholesterol is consumed to be maximally effective, or will one large dose last throughout the day?

The following experiments utilized gerbils to address these points on the basis of a preliminary study that revealed that gerbils were slightly superior to hamsters for assessing these effects. Rats and mice were considered inappropriate because their plasma and liver cholesterol is relatively less responsive to dietary cholesterol challenge.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and diets.

Male Mongolian gerbils (Meriones unquiculatus, n = 84; 5–9 wk old; Charles River, Wilmington, MA) were studied in 3 experiments. In each experiment, gerbils were randomly assigned to groups (5–8 per group) and fed for 4–5 wk a purified diet containing 0, 0.05, 0.1, 0.15, or 0.5% (g/100 g) cholesterol and 30% energy(en) from fat. The test diets contained either 0.5 or 0.75% nonesterified (free) phytosterols/stanols derived from tall oil (Reducol, Novartis Consumer Health SA, Nyon, Switzerland), esterified sterols (Take Control, Lipton, Englewood Cliffs, NJ), or esterified stanols (Benecol, McNeil Consumer Healthcare, Port Washington, PA), the latter two as commercial margarines. The free phytosterols were thoroughly dissolved in the fat portion of the mix by heating.

The basal composition of the semipurified gerbil diets was (g/kg dry diet): casein, 200; dextrose, 200; cornstarch, 298; cellulose, 100; fat, 137 (a blend of 59% coconut oil or cholesterol-stripped milk fat + 31% canola oil + 10% soybean oil); mineral mix (Ausman-Hayes), 50; vitamin mix (Hayes-Cathcart), 12; and choline chloride, 3. The composition of the mineral and vitamin mixes was reported previously (10 ). Cholesterol and free phytosterols were added to the diets at the expense of cornstarch, whereas the commercial margarines with sterol/stanol esters were substituted into the fat blend.

Gerbils were housed 2–3 per cage and kept in a controlled environment with a 12-h light:dark cycle with free access to water. Diets were provided daily in predetermined amounts calculated to maintain normal growth. During wk 4, feces were collected in individual hanging cages where indicated. All procedures were approved by the Brandeis animal care and use committee.

After 4–5 wk, gerbils were deprived of food overnight (16 h), blood was collected under O₂/CO₂ anesthesia with an EDTA-wetted syringe by cardiac puncture; after exsanguination, liver, cecum and adipose tissue were excised and weighed. A portion of each liver was stored at -20°C until analyzed. Plasma was separated from EDTA-treated blood by centrifugation at 12,000 x g for 15 min and refrigerated for 1–3 d until analyzed.

Dietary cholesterol load (Experiment 1).

This experiment addressed whether the level of dietary cholesterol influences phytosterol effectiveness when a moderate, but constant intake is consumed as free sterols (80% sterols, 20% stanols). In three studies the phytosterol intake was kept at 0.5% to reflect the concentration consumed at the high end of effectiveness for humans (i.e., equivalent to 2500 mg/d). On the other hand, cholesterol intake was varied (0.05, 0.10 and 0.5%) to exceed the normal range of human dietary cholesterol equivalents (250, 500 and 2500 mg/d, respectively) to identify a reasonably effective paradigm for diet challenge that included final dietary phytosterol:cholesterol ratios of 10:1, 5:1 or 1:1. Data from the three separate studies were combined to evaluate the ratios mentioned. The three studies and diets were similar in nature and each measured body weight gain, plasma lipids and liver cholesterol.

Timing the dose of sterols (Experiment 2).

This experiment was designed to determine whether phytosterols must be consumed with each serving of dietary cholesterol or whether it is possible to elicit a comparable effect with a double dose of phytosterol taken every other day. The implication from Experiment 1 was that phytosterols function by blocking available dietary cholesterol, and that a low dietary cholesterol intake might reduce the opportunity for phytosterols to function. Based on those data and the human literature, the 5:1 ratio used in Experiment 1 still seemed appropriate, but a dose of cholesterol >0.1% and <0.5% was selected because the latter induced extreme liver cholesterol accumulation (Experiment 1). Accordingly, 0.15% diet cholesterol was fed along with five times that amount of phytosterol, i.e., 0.75% (3750 mg/d human equivalent) as a best estimate for enhancing detection of the response in TC. One test group consumed the 5:1 ratio with every bite of food, whereas a second group received a constant intake of cholesterol, but the amount of phytosterol was doubled (10:1 ratio) every other day. In the final analysis, total intake of either phytosterol or cholesterol was the same in the diet groups, but the application of phytosterol differed.

Free vs. esterified sterols (Experiment 3).

This experiment addressed a basic question concerning phytosterol activity and function, i.e., does the form in which it is consumed make a difference? In this case, the free tall oil phytosterols were compared with sterol or stanol esters; the latter two forms were incorporated into the diet as the commercial fat spreads described above. All diets provided 30%en from fat and approximately 0.75% free phytosterol equivalents with 0.15% diet cholesterol, based on the 5:1 ratio of phytosterol to cholesterol identified as most satisfactory in the first two experiments. Phytosterol intake was adjusted for sterol molecular equivalency by assuming that free sterol represented 60% of the ester in the spreads. Gas-liquid chromatography (GLC) analysis of diets confirmed that the dietary total phytosterol content was similar for all test groups and that individual plant sterols/stanols composition varied as expected between the different diets (Table 1 ).

Plasma TC, HDL cholesterol (HDL-C) and triglycerides (TG) were measured by enzymatic assay, with TC and TG using Sigma kits #362 and 336, respectively (Sigma Diagnostics, St. Louis, MO). HDL-C was assayed in the supernatant after sodium phosphtungstate-Mg²⁺ precipitation of apolipoprotein (apo)E- and apoB-containing lipoproteins with reagent #543004 (Boehringer Mannheim Diagnostic, Indianapolis, IN) according to the procedure described by Weingard and Daggy (11 ).

Hepatic cholesterol analysis.

Liver cholesterol was extracted by grinding 0.1 g liver with anhydrous sodium sulfate and extracting three times with 2:1 chloroform/methanol. Liver FC and esterified cholesterol (EC) were determined by HPLC (12 ). Free cholesterol and cholesteryl esters were separated using a Waters Radial-Pack C18 column (8 mm x 10 cm, 10 µ; Milford, MA) eluted isocratically with acetonitrile/isopropanol (50:50, v/v) at 2.0 mL/min. Absorbance of the eluate was measured at 210 nm using a UV detector. Cholesterol (free and individual esters) concentrations were calculated by comparing the peak area of samples with those obtained for the standards (Sigma Chemical). To calculate esterified cholesterol the sum of cholesteryl esters was divided by 1.67, according to the calculation of Witztum et al. (13 ).

Diet, liver and fecal sterol analysis.

Sterol content of diets and feces were determined by GLC after direct saponification with 0.5 mol/L methanolic KOH according to the method of Ntanios and Jones (14 ). For liver samples, sterols were first extracted using 2:1 chloroform/methanol and then saponified. Sterol analysis was performed by GLC equipped with a capillary column (XTI-5, 30 m length, 0.25 mm ID, 0.50 µm df, Baxter, IL) with helium as the carrier gas. Carrier flow was set at 2 mL/min with 20:1 split ratio. The oven temperature was set at 280°C with injector and detector temperature at 300 and 280°C, respectively. Sample sterols were identified by comparison with authentic sterol standards (Sigma, St. Louis, MO).

Statistical analysis.

Statistical comparisons between the various diets were calculated using a one-way ANOVA followed by Scheffé’s F-test utilizing the SuperANOVA statistical software package (Abacus Concepts, Berkeley, CA) Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Dietary cholesterol load (Experiment 1).

Treatments did not affect body weight because all gerbils grew normally (Table 2 ). Increasing dietary cholesterol had two effects on the response to phytosterols. First, the favorable plasma TC– and liver EC–reducing effects of phytosterols became more apparent as dietary cholesterol was increased. Second, a dietary phytosterol:cholesterol ratio > 5:1 did not enhance performance when diet cholesterol intake was modest at 0.05–0.10%. Thus, although the 10:1 phytosterol:cholesterol ratio decreased TC 17% relative to controls (no dietary phytosterols), a lower 5:1 ratio (produced by doubling dietary cholesterol) actually decreased TC by 27%. Both ratios lowered TC to about the basal value (2.1 mmol/L). On the other hand, although the 1:1 ratio (produced by increasing dietary cholesterol fivefold) reduced TC 42%, the resulting absolute TC concentration (5.7 mmol/L) was still markedly elevated (Table 2) . Thus, the enhanced efficacy of free phytosterols in lowering TC as the dietary phytosterol:cholesterol ratio decreased was a function of the absolute increase in dietary cholesterol. A better estimate of efficacy was seen in the absolute liver EC; the EC concentration associated with the1:1 ratio was still extremely high at 158 µmol/g and only 40% lower than in controls (P = 0.10). By contrast, a 70–80% reduction in EC was observed in gerbils fed the diets with 5:1 and 10:1 ratios.

Gerbils that always received phytosterol with cholesterol had reduced plasma TC (-43%) and liver EC (-89%) relative to controls, whereas those that received the equivalent dose of phytosterol every other day had only 22% lower plasma TC (P < 0.05). However, the reduction in liver EC with the every other day treatment was more substantial (-76%) than was predicted from the plasma response (Table 3 .).

All three sources of phytosterols in Experiment 3 equally reduced the plasma TC 50%, with the concentration in the free sterol group tending to be (P 0.33) lower than in the ester-fed groups. The main effect of phytosterols on plasma lipids was to reduce the apoB-rich lipoproteins, thereby lowering the TC/HDL-C ratio 40% relative to the control group. The liver EC was also equally reduced with a tendency (P 0.11) for the lowest level in gerbils fed free sterols. These EC concentrations were essentially normal, representing 10% that of levels in control livers (111 µmol/g) when no dietary phytosterol was present to block cholesterol absorption (Table 4 ).

Accumulation of phytosterols in liver associated with treatments was minimal; the greatest concentrations were in controls (Table 4) . Total hepatic phytosterol concentration in gerbils fed the sterol ester diet did not differ from that of the controls, whereas treatment with the free phytosterols or stanol ester diets reduced concentrations 70–75%. Campesterol was the major liver plant sterol, except in the stanol ester group. Stanols were not detected in livers of any group.

Fecal cholesterol excretion did not differ among the test groups and was fivefold greater than in the control group (Table 5 ). Fecal plant sterols directly reflected dietary sterol composition. Gerbils fed the stanol ester diet produced the most fecal plant sterols, consistent with the nonabsorbability of stanols and their ability to block all sterol absorption (15 ,16 ).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Although previous reports (2 ,6 ,7 ) indicated that free sterols and stanols may be equal to or better than sterol and stanol esters in their ability to lower TC by blocking cholesterol absorption, our data represent the first demonstration of that point in a direct comparison. In addition, we extend this relationship to the stores of body cholesterol represented by the liver. The free sterol is the active form that presumably displaces (outcompetes) free cholesterol in gut micelles; and although esters are typically more readily dispersed in fat, they must first undergo hydrolysis to be active in the gut (1 ,2 ). A critical consideration in this context is to ensure adequate dispersal of the active sterol in the diet and gut. Based on the presumed mechanism of competing for space in the micelle (1 ), the tradeoff between sterol forms reflects their improved fat solubility by esterification countered by their need for hydrolysis once in the gut, vs. the relatively poor solubility of the active free sterols/stanols in dietary fat. Dispersal and incorporation of the free sterols/stanols into micelles apparently was efficient in our experiments because the free sterol preparation proved equivalent, if not superior to the esterified forms provided in the spreads. Nonetheless, our 5:1 ratio of phytosterol to cholesterol may have represented excessive plant sterol, obscuring our ability to discriminate among the three sources of phytosterols. It might be instructive to compare phytosterol function by increasing the cholesterol burden incrementally to determine whether one form of phytosterol becomes less effective as the challenge increases. The surprising effectiveness of our free phytosterols may represent the relatively small particle size that results during their manufacture, a point supported by a recent clinical study that demonstrated the efficacy of microdispersed free sterols (9 ).

It was also noteworthy that none of these preparations caused hepatic plant sterol accumulation relative to the control gerbils. As expected from the literature (15 ,16 ), the stanol ester preparation allowed the least plant sterol absorption and hepatic accumulation; but the free sterol preparation, containing 20% stanols, also prevented hepatic sterol accumulation. The predilection for stored campesterol in the liver of sterol ester–fed gerbils reflected the greater amount in that treatment. The fact that control livers contained the most campesterol indicates that the basal level of this sterol before the study was already high (from nonpurified diet), because none was present in the control purified diet. In fact, lower campesterol in the two treatments providing stanols indicates they must have enhanced campesterol loss during the 4-wk trial.

Dietary cholesterol load.

Our data also suggest that variation in response to phytosterols among human studies may reflect differences in the absolute intake of diet cholesterol relative to that of phytosterols. For example, by specifically reducing the amount of dietary cholesterol present (Experiment 1), the capacity for demonstrating a maximum phytosterol effect was reduced. In other words, extra phytosterol may not lower TC further in situations in which dietary cholesterol is limited, a point recently demonstrated in humans, i.e., 1.1 g/d was as effective as 2.2 g/d (16 ) and 1.5 g as effective as 3.0 g (9 ) when cholesterol intake was only 200 mg/d. However, when dietary cholesterol was high, a dose-dependent reduction in TC by phytosterols could be demonstrated (17 , and this study). In fact, variation in diet cholesterol intake may explain in part the range of 5–15% reduction in LDL-C among human phytosterol experiments (3 ,7 ,8 ,16 –25 ). This conclusion begs the question whether phytosterols can lower TC appreciably when no dietary cholesterol is present, i.e., what effect could be expected from interfering with biliary cholesterol reabsorption in vegetarians with elevated LDL?

A related conclusion is that modeling (i.e., diet designs in animal and human studies) should be realistic in terms of the dietary cholesterol load to avoid misinterpreting results, either to the upside or downside depending on the cholesterol present. For example, from the TC concentrations in Experiment 1, one might conclude that a 1:1 ratio of dietary phytosterol:cholesterol is as good or better than 10:1. However, this ignores the fact that opportunity for success in this model (% decrease in LDL) was much greater at a high cholesterol intake and that hepatic cholesterol concentration was actually a better indicator of efficacy than plasma cholesterol.

Timing phytosterol intake.

In Experiment 2, the plasma TC data (% reduction) suggest that the every other day treatment was exactly half as effective as phytosterol consumed with every meal. The liver data also generally supported this conclusion. Thus, although free phytosterol was partially effective when fed on alternate days, it was most effective when fed in conjunction with each incremental amount of dietary cholesterol. This finding appears to contradict the observation that 1 time/d delivery in humans was as effective as 3 times/d (26 ). However, cholesterol intake was modest (220 mg/d), and if the single daily intake of phytosterol (during lunch) also happened to coincide with the meal providing the most cholesterol, the design may have inadvertently optimized the apparent effectiveness of plant sterols. Had total cholesterol intake been greater, e.g., 400–500 mg/d with most consumed during the evening meal, the result might have been consistent with our gerbil data. Therefore, that design, although not as clear-cut as the present modeling circumstances, does not negate the general consensus that phytosterols are best consumed in meals containing cholesterol. Our data also support the earlier comment that much of the variation in human experiments, and possibly among individuals within experiments, may reflect differences in dietary cholesterol intake, a point not always addressed adequately by the dietary design. These every other day results also support our earlier conclusion that a 10:1 dietary ratio of phytosterol:cholesterol is not necessarily an improvement over 5:1, especially if the mass of cholesterol consumed is not sufficient to warrant the extra phytosterol.

The fecal cholesterol data support the hypothesis that phytosterols function by blocking cholesterol absorption (1 ,2 ,27 ) and that the free sterol form was as effective as esterified sterols and stanols. In summary, free phytosterols/stanols can represent an effective barrier to diet cholesterol absorption. The more cholesterol in the diet, the greater the apparent efficacy of phytosterols to lower liver EC and plasma TC. A free phytosterol:cholesterol ratio of 5:1 appears to be a safe and effective treatment as part of a diet containing modest cholesterol.

	FOOTNOTES

 
¹ Supported in part by a grant from Novartis Consumer Health, Incorporated.

³ Abbreviations used: apo, apolipoprotein; EC, esterified cholesterol; en, energy; FC, free cholesterol; GLC, gas-liquid chromatography; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; TC, total cholesterol; TG, triglycerides.

Manuscript received 10 December 2001. Initial review completed 7 February 2002. Revision accepted 12 April 2002.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hayes, K. C. Articles by Beer, M. Search for Related Content PubMed PubMed Citation Articles by Hayes, K. C. Articles by Beer, M.