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
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ABSTRACT |
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Our
objective was to measure the systemic absorption of lecithin-emulsified
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
5-phytosterols and oral
pentadeuterated
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
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
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
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INTRODUCTION |
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PLANT-DERIVED
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
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- stanols. Here, we measured the absorption of the major components of both
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.
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METHODS |
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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 5-sterol tracers was reduced to
the corresponding 5-
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
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.
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 5-sterols was measured by intravenous injection of
dideuterated
5-sterols and oral administration of
pentadeuterated
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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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. 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.
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RESULTS |
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Figures 1 and
2 show the percent absorption of
lecithin-formulated soy 5-sterols or soy stanols
calculated from plasma oral/intravenous isotope ratios over 4 days. For
both
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
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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The absorption of stanols was an order of magnitude less than that of
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
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 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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DISCUSSION |
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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 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 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 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 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
5-sterol and stanol turnover.
Finally, the marked differences between 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
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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