Gastrointestinal absorption and plasma kinetics of soy Delta 5-phytosterols and phytostanols in humans

Richard E. Ostlund Jr.1, Janet B. McGill1, Chun-Min Zeng3, Douglas F. Covey3, Jay Stearns5, William F. Stenson2, and Curtis A. Spilburg4

Divisions of 1 Endocrinology, Diabetes and Metabolism, and 2 Gastroenterology, Department of Medicine, Washington University School of Medicine, and 3 Department of Molecular Biology and Pharmacology, Washington University, St. Louis 63110; 4 Lifeline Technologies Inc., Chesterfield, Missouri 63017; and 5 Seres Laboratories, Santa Rosa, California 95403


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our objective was to measure the systemic absorption of lecithin-emulsified Delta 5-phytosterols and phytostanols during test meals by use of dual stable isotopic tracers. Ten healthy subjects underwent two single-meal absorption tests in random order 2 wk apart, one with intravenous dideuterated Delta 5-phytosterols and oral pentadeuterated Delta 5-phytosterols and the other with the corresponding labeled stanols. The oral-to-intravenous tracer ratio in plasma, a reflection of absorption, was measured by a sensitive negative ion mass spectroscopic technique and became constant after 2 days. Absorption from 600 mg of Delta 5-soy sterols given with a standard test breakfast was 0.512 ± 0.038% for sitosterol and 1.89 ± 0.27% for campesterol. The absorption from 600 mg of soy stanols was 0.0441 ± 0.004% for sitostanol and 0.155 ± 0.017% for campestanol. Reduction of the double bond at position 5 decreased absorption by 90%. Plasma t1/2 for stanols was significantly shorter than that for Delta 5-sterols. We conclude that the efficiency of phytosterol absorption is lower than what was reported previously and is critically dependent on the structure of both sterol nucleus and side chain.

diet; sitosterol; campesterol; sitostanol; campestanol; clinical study; mass spectrometry; deuterium; cholesterol


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PLANT-DERIVED delta 5-sterols and stanols (referred to here collectively as phytosterols) are currently consumed in several approved specialty foods in the United States and Europe for reduction of low-density lipoprotein cholesterol and prevention of coronary heart disease. They appear to be safe because they are found in natural foods, but enrichment in the general food supply warrants further work to ensure that no unanticipated adverse effects occur. Phytosterols are thought to act principally within the intestine to block cholesterol absorption while having little systemic absorption themselves (13). However, the exact level of absorption is not settled, and only a few studies have been performed in humans with the use of radioactive tracers (21). Recently, improved formulations of phytosterols employing esterification and solubilization in oil or emulsification with lecithin have enhanced the bioavailability of these hydrophobic compounds and provide a reproducible delivery system, in contrast to previous studies that used crystalline material (17, 18, 24). However, the effects of these procedures on phytosterol absorption are not known. At least some meaningful absorption must take place, because serum Delta 5-phytosterol levels increase moderately after addition to the diet (12). It has been difficult to measure serum phytostanols, and less is known about them. Because the rare disorder phytosterolemia is associated with coronary heart disease (3) and because epidemiological studies relate elevated plasma phytosterol levels to coronary heart disease (6), it is important to understand the absorption and physiology of phytosterols in greater detail.

Our previous studies (4) showed that percent cholesterol absorption can be determined in single-meal tests where differently labeled and distinguishable oral and intravenous deuterated cholesterol tracers are administered simultaneously and plasma enrichments are subsequently measured. Here, we have adapted this technique to soy phytosterols. Tracers either two or five mass units higher than the natural materials were used, and plasma was analyzed by negative ion mass spectrometry with very high sensitivity and specificity (15).

The objective of the present study was to determine the efficiency of absorption of common phytosterols. Most natural phytosterols, like cholesterol, contain a double bond at position 5, as well as a side-chain group (often at position 24) that is not present in cholesterol (7). In some natural phytosterols as well as commercial products (24), the double bond has been reduced to give 5-alpha stanols. Here, we measured the absorption of the major components of both Delta 5-sterols and stanols derived from soybeans and made bioavailable by lecithin emulsification. We found that the absorption of phytosterols was measurable, consistent between subjects, and lower than that previously reported.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. 3,4-2H2-labeled soy sterols (dideuterated) were purchased from MassTrace (Woburn, MA). 2,2,4,4,6-2H5-labeled soy sterols (pentadeuterated) were prepared at Seres Laboratories (Santa Rosa, CA) by a procedure described previously for the deuteration of cholesterol (8). The product was dissolved in boiling hexane, cooled to 45°C, filtered through a 0.2-µm Millex FG solvent-resistant filter (Millipore, Bedford, MA), and allowed to crystalize overnight at 4°C. One-half of these Delta 5-sterol tracers was reduced to the corresponding 5-alpha stanols as described in the next paragraph. Heavy-metal analysis by means of inductively coupled optical emission spectroscopy (Galbraith Laboratories, Knoxville, TN) showed only trace amounts of aluminum and iron and <140 ppm of platinum. Composition of the pentadeuterated Delta 5-soy sterols given orally was 39.4% sitosterol, 26.9% campesterol, and 24.7% stigmasterol, and composition of the pentadeuterated soy phytostanols was 72.6% sitostanol and 23.6% campestanol. Precept 8160 (Central Soya, Fort Wayne, IN) was used as a source of lecithin in all experiments. It is derived from deoiled lecithin by treatment with phospholipase A2 to produce a mixture of lecithin and lysolecithin.

Delta 5-Sterols were reduced to stanols by a modification of a previous method (10). Delta 5-Sterols (5 g) were dissolved in 300 ml of ethyl acetate and 6 ml of acetic acid, and 350 mg of platinum dioxide on carbon were added. The mixture was stirred under a hydrogen atmosphere at 50 psi at room temperature for 18 h. The catalyst was filtered off, the solvent in the filtrate was removed, and the residue was dried and crystalized from ethyl acetate to give the stanols.

Clinical protocol. Ten healthy subjects not taking prescription medications and without active medical or surgical illnesses were recruited (Table 1). Protocols were approved by the Washington University Human Studies Committee, and informed consent was obtained in writing. The study was a randomized, single-blind, crossover design in which each subject received two absorption tests 2 wk apart, a time interval that allowed plasma phytosterol enrichments from the previous test to decay. In one test, the absorption of soy Delta 5-sterols was measured by intravenous injection of dideuterated Delta 5-sterols and oral administration of pentadeuterated Delta 5-sterols, whereas in the other test intravenous dideuterated and oral pentadeuterated soy stanols were used. For each test, fasting subjects consumed a beverage containing 600 mg of soy sterols or stanols and then ate a standard breakfast, prepared by the metabolic kitchen, consisting of 240 ml of orange juice, 240 ml of whole milk, 29 g of corn flakes, and a 60-g bagel. The test meal contained 27 mg of phytosterols and 31 mg of cholesterol exclusive of tracers. Plasma for analysis was collected before the test meal, 8 h after the meal, and then daily for 4 days after an overnight fast.

                              
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Table 1.   Characteristics of the study subjects

Dideuterated phytosterol tracers for intravenous administration were prepared by a modification of a method described previously for labeled cholesterol (4). Tracers were dissolved in ethanol at 6.7 mg/ml at 65°C, cooled to 37°C, and filtered through a 0.2-µm solvent-resistant Millex FG filter. An aliquot was cultured in thioglycollate broth, and another was dried, suspended in water, and analyzed for pyrogens by the limulus assay (BioWhittaker, Walkersville, MD) along with similarly treated sterol samples containing known amounts of added pyrogenic activity. On the day of the experiment, the ethanolic tracer stock solutions were warmed to 55°C, cooled to 37°C, and added dropwise to warm 10% Intralipid intravenous triglyceride emulsion (Fresenius Kabi Nutrition, Clayton, NC) to achieve a concentration of 1.3 mg tracer/ml. After being cooled to room temperature, the tracer-Intralipid emulsion was passed through a 1.2-µm particulate filter (EPS, Feasterville, PA), and a weighed portion of ~7.5 g was administered to each subject over 5 min through an intravenous line. The amount of infused sterol or stanol tracer was 10 mg.

Oral phytosterol tracers were emulsified by dissolving 3.6 g of Delta 5-sterols or stanols and 4.07 g of phospholipid in warm ethyl acetate followed by evaporation of the solvent and removal of trace solvent by lyophilization. On the day before the experiment, 172.5 g of water were added, and after overnight hydration, the mixture was sonicated until homogeneous. Each subject received 30 g of the liquid material diluted into a final volume of 60 ml of Crystal Lite lemonade drink (Kraft Foods, Rye Brook, NY).

Analyses. The general procedures were similar to those described in previous reports (4, 15). Plasma was saponified, and the neutral sterols were extracted and converted to pentafluorobenzoyl esters. Weighed mixtures of the oral and intravenous materials given were treated similarly. Gas chromatography-mass spectrometry was performed with a 15-m × 0.25-mm-ID × 0.5-µm df RTX-200 capillary column (Restek, Bellefonte, PA) and an HP 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA) operating in negative ion chemical ionization mode with methane as reagent gas. Selected ion monitoring of phytosterol molecular anions was performed as follows: sitosterol mass-to-charge ratios (m/z) 608, 610, 613; sitostanol m/z 610, 612, 615; campesterol m/z 594, 596, 599; campestanol m/z 596, 598, 601; cholesterol m/z 581 (M+1) or 582 (M+2). The relative retention times of phytosterols and phytostanols with respect to cholesterol were sitosterol 1.24, sitostanol 1.29, campesterol 1.14, and campestanol 1.18. Delta 5-Sterols and stanols were separated at baseline (0.2 min) so that the contribution of both sterol and stanol at each mass could be measured. Percent absorption of the oral tracer was determined as the quotient of intravenous/oral tracers in plasma averaged over 2-4 days divided by the administered ratio times 100. The interday coefficient of variation of isotope ratios was <2%. The reproducibility of phytosterol absorption measurements was not determined here, but previous work showed that repeated single-meal cholesterol absorption tests in the same subjects had a coefficient of variation of 5% of the measured value (5).

Statistics. Results are expressed as means ± SE. Differences between phytosterols were determined by the paired t-test. Half-times for labeled intravenous phytosterols were calculated by regression with the use of a single-compartment model.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Figures 1 and 2 show the percent absorption of lecithin-formulated soy Delta 5-sterols or soy stanols calculated from plasma oral/intravenous isotope ratios over 4 days. For both Delta 5-sterols and stanols, the isotope ratios became constant 2 days after the test meal, and mean values for days 2-4 are presented in Table 2. Absorption of all phytosterols was low, with campesterol showing the highest value of 1.89% and sitostanol having the lowest at 0.044%. These are in marked contrast to cholesterol absorption, which was 56.2%, measured by the same technique as that employed here (4). Under the conditions of our studies, certain trends are apparent. A double bond at position 5 in the sterol nucleus that is present in cholesterol and Delta 5-sterols, but not stanols, enhances absorption. Increasing the length of the side-chain group at position 24 (hydrogen for cholesterol, methyl for campesterol and campestanol, and ethyl for sitosterol and sitostanol) progressively diminishes absorption.


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Fig. 1.   Percent absorption of soy sterols. Bars represent SE.



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Fig. 2.   Percent absorption of soy stanol. Bars represent SE.


                              
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Table 2.   Phytosterol absorption

The absorption of stanols was an order of magnitude less than that of Delta 5-sterols and was consistent across the two major structural groups, differing in the side chain. Sitostanol absorption was 8.6% of sitosterol absorption (P < 0.0001), and campestanol absorption was 8.2% of campesterol (P = 0.0001). Within the stanols, campestanol absorption was 3.5 times that of sitostanol (P < 0.0001), whereas within the Delta 5-sterols campesterol absorption was 3.7 times that of sitosterol (P = 0.0003). Thus the effects of side-chain structure and the presence of a double bond at position 5 had consistent and independent effects on cholesterol absorption.

Plasma kinetics of the phytosterols were calculated from the decay of intravenously injected dideuterated tracers (Figs. 3 and 4). The decay is log linear, suggesting that over this period a single-compartment model can be used to calculate the data. The half-life (t1/2) of stanols was consistently shorter than that for Delta 5-sterols (Table 3). Campestanol had a t1/2 of 1.69 days compared with 4.06 days for campesterol (P < 0.0001) whereas the t1/2 of sitostanol was 1.84 days compared with 2.94 for sitosterol (P = 0.0005). The campesterol t1/2 of 4.06 days was significantly longer than the value of 2.94 days for sitosterol (P = 0.004), but there was no difference in t1/2 between campestanol (1.69 days) and sitostanol (1.84 days) (P = 0.55).


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Fig. 3.   Turnover of sitosterol and sitostanol. Bars represent SE.



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Fig. 4.   Turnover of campesterol and campestanol. Bars represent SE.


                              
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Table 3.   Phytosterol turnover


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Despite their widespread therapeutic use and occurrence in natural foods, there is little quantitative information about the absorption of phytosterols in humans. Formal absorption measurements are important in assessing safety, since plasma phytosterol levels alone may give an incomplete picture of their metabolism. Normal subjects are known to have only small increases (less than a doubling) in plasma phytosterol levels with doses several times the usual dietary intake (20, 25). This resistance may be due, in part, to increased excretion of absorbed phytosterols, as demonstrated by heterozygotes for sitosterolemia, who are able to maintain normal plasma levels of sitosterol despite increased absorption because of an offsetting increase in sitosterol excretion (22).

For many years, phytosterols have been used as nonabsorbable stool markers in cholesterol balance studies (9), but formal measurements to define the exact residual level of absorption have been performed in only a few individuals. In four subjects consuming a normal amount of sitosterol (330 mg/day) under metabolic ward conditions, sitosterol absorption was 2.8 ± 0.8%, calculated from dietary intake and plasma radiolabeled sitosterol turnover (20). However, a repeat study was performed in a single subject at a much higher sitosterol dose (1,909 mg/day), and the percent absorption fell from 2.2 to 0.6%, very similar to the value of 0.512% reported here. In another study, with an intake of 105 mg sitosterol/day, five subjects absorbed 7.5 ± 2.2% of sitosterol by plasma turnover, whereas another absorbed 5% with the use of the radioactive dual-isotope ratio method (22). In a different study, a single subject had sitosterol absorption of 4% by the dual radioactive isotope procedure while consuming 300 mg sitosterol/day (21). Finally, three subjects consuming 145 mg sitosterol/day had sitosterol absorption of 5 ± 1% in a well validated fecal recovery study (14). Taken together with our work, the data suggest that the absolute amount of sitosterol absorbed is very small, even when the material is made bioavailable by emulsification with lecithin. Absorption does not increase linearly when the phytosterol dose is increased, indicating that the absorption of plant sterols may be a saturable process.

Our results for phytosterol percent absorption are low compared with those from other reports, a difference that may be due, in part, to the choice of analytical methods. The mass spectroscopic procedure used here has the advantage that tracer material appearing in plasma can be characterized definitively during quantitation, even when many sterol species are present. In contrast, previous work with radioactive isotopes assumed that the activity in plasma was structurally identical to the major material present in the original tracer. However, radioactive tracers are seldom pure, either before or after labeling, and when only a small fraction of the orally administered counts appears in plasma there is an inherent question about identity, even when methods such as thin-layer chromatography are used for characterization.

Compared with the percent absorption for cholesterol of 56.2 ± 12.1% obtained in normal subjects under the same test conditions (4), the absorbability of soy sterols and stanols reported here ranges from a high of 1.89% for campesterol to 0.04% for sitostanol. It is known that campesterol is more avidly absorbed than sitosterol, and in three normal subjects consuming 27 mg/day, the percent campesterol absorption was 16 ± 1% or 4.3 mg (14). From our data, an intake of 161 mg of campesterol emulsified with lecithin and 439 mg of other Delta 5-soy sterols resulted in an absorption of 3.0 mg. Thus the absolute absorption of campesterol found here (3.0 mg) was similar to that in the previous work (4.3 mg), even though the percentage absorbed was much less (16.1% vs. 1.89%).

Little is known about the absorption of campestanol. During intestinal intubation and perfusion studies, campestanol absorption was found to be 12.5%, the highest of any Delta 5-sterol or stanol measured and higher even than that for campesterol, which was 9.6% absorbed (11). The infused campestanol occurred naturally in a commercial feeding solution, and the infusion rate was rather low at 0.5 mg/h. This relatively high level of absorption has led to concern about the potential safety of campestanol-containing products. However, our results show that, at least when administered with sitostanol in a preparation of mixed soy stanols, the absorption of campestanol is only 0.16%. There are two possible explanations for these different results. First, the larger value for absorption observed previously may reflect the small amounts of campestanol used, but it is also possible that differences in experimental design may be important. The intubation experiment measured disappearance of campestanol from the intestinal lumen, whereas the dual-isotope procedure measures the appearance of campestanol in the systemic circulation. The two processes are not identical, because phytosterols can be taken up in large amounts by enterocytes but not absorbed at the same level into lymph (2). Hence, it is possible that campestanol is rapidly taken up into the enterocyte but not ultimately absorbed. Although it is possible that our deuterated tracers might differ in transport behavior from natural phytosterols, previous work with cholesterol tracers enriched by five to six mass units has not been associated with significant isotope effects (4, 16).

Sitostanol has been found to be very poorly absorbed in humans (14) and animals (23). Our results confirm that the levels absorbed are indeed low at 0.04%. The absorption of both campestanol and sitostanol was only ~10% of that of the corresponding Delta 5-sterols, indicating a dominant effect of double-bond saturation on absorption.

In addition to the reduced absorption of soy stanols, our results indicate that their turnover is more rapid than that of Delta 5-sterols, a process that further attenuates increases in serum stanols during treatment. Indeed, serum levels of sitostanol and campestanol change little with sitostanol treatment in patients with sitosterolemia (14). Because we measured decay of intravenously injected tracer for only 4 days, complete kinetic analysis cannot be performed. However, our data fit well the first exponential decay of plasma phytosterols and give results similar to those reported after measuring plasma radioactive isotope decay for many weeks. For example, we found that sitosterol has a t1/2 of 2.94 days compared with a previously reported first exponential decay of 3.8 days (20). Because the more slowly mixing second sitosterol pool comprises only ~35% of sitosterol mass in the body, it is likely that our results reflect accurately the bulk of Delta 5-sterol and stanol turnover.

Finally, the marked differences between Delta 5-sterols and stanols in absorption and turnover are not consistent with simple physical/chemical processes, since these compounds are difficult to separate, even with sophisticated analytical techniques. Recent work has identified a membrane cholesterol transporter mutation in sitosterolemia that appears to mediate the clinical disorder (1, 19). It is likely that differential interaction of Delta 5-sterols and stanols with this and similar transporters in the enterocyte causes the discrimination between cholesterol and other sterols that is one of the hallmarks of mammalian intestinal cholesterol absorption. Exploitation of these pathways may provide potent drug candidates for hypercholesterolemia and coronary heart disease.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health (NIH) Grant R43 HL-62780 to Lifeline Technologies, NIH Grants R01 HL-50420 to R. E. Ostlund, RR-00036 to the Washington University General Clinical Research Center, RR-00954 to the Washington University Mass Spectrometry Resource, and P30 DK-56341 to the Washington University Clinical Nutrition Research Unit.


    FOOTNOTES

Address for reprint requests and other correspondence: R. E. Ostlund, Washington Univ. School of Medicine, Division of Diabetes, Endocrinology and Metabolism, Box 8127, 660 South Euclid Ave., St. Louis, MO 63110 (E-mail: Rostlund{at}im.wustl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00328.2001

Received 19 July 2001; accepted in final form 25 November 2001.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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