Am J Physiol Gastrointest Liver Physiol 282: G1009–G1015, 2002;
10.1152/ajpgi.00446.2001.
Micellar distribution of cholesterol and phytosterols
after duodenal plant stanol ester infusion
MARKKU NISSINEN, HELENA GYLLING, MATTI VUORISTO, AND TATU A. MIETTINEN
Department of Medicine, Division of Internal Medicine,
University of Helsinki, Helsinki FI-00029 HUCH, Finland
Received 16 October 2001; accepted in final form 23 January 2002
Nissinen, Markku, Helena Gylling, Matti Vuoristo,
and Tatu A. Miettinen. Micellar distribution of cholesterol
and phytosterols after duodenal plant stanol ester infusion.
Am J Physiol Gastrointest Liver Physiol 282: G1009–G1015,
2002; 10.1152/ajpgi.00446.2001.—Properties of the intestinal
digestion of the dietary phytosterols, cholesterol and choles-
tanol, and the mechanisms by which phytosterols inhibit the
intestinal absorption of cholesterol in healthy human sub-
jects are poorly known. We have studied the hydrolysis of
dietary plant sterol and stanol esters and their subsequent
micellar solubilization by determining their concentrations
in micellar and oil phases of the jejunal contents. Two liquid
formulas with low (formula 1) and high (formula 2) plant
stanol concentrations were infused via a nasogastric tube to
the descending duodenum of 8 healthy human subjects, and
intestinal contents were sampled for gas-liquid chromato-
graphic sterol analysis 60 cm more distally. During the
duodenal transit, phytosterol esters were hydrolyzed. This
was especially profound for sitostanol, as its esterified frac-
tion per milligram of sitosterol decreased 80% (P ⬍ 0.001) in
formula 1 and 61% (P ⬍ 0.001) in formula 2. Contrary to that,
esterified fraction of cholesterol per milligram of sitosterol
was increased fourfold (P ⬍ 0.001) in formula 1 and almost
sixfold (P ⬍ 0.001) in formula 2, whereas that of cholestanol
remained unchanged. Percentages of esterified sterols and
stanols in total intestinal fluid samples were higher after the
administration of formula 2 than of formula 1. Esterified
cholesterol and stanols accumulated in the oil phase, and
free stanols replaced cholesterol in the micellar phase. At
high intestinal plant stanol concentrations, cholesterol
looses its micellar solubility possibly by replacement of its
free fraction in the micellar phase by hydrolyzed plant
stanols, which leads to a decreased intestinal absorption of
cholesterol.
intestinal absorption of cholesterol; phytosterol esters; mi-
celle formation
MICELLE FORMATION OF DIETARY lipids and their digestion
products in the presence of bile acids and phospholip-
ids is crucial for the intestinal absorption of cholesterol
from the gut lumen to the intestinal epithelial cells (30,
31). Absorption of both free and esterified cholesterol
has been suggested (3, 17, 29, 34) to be protein-medi-
ated. Scavenger receptor class B type I in the small-
intestine brush-border membrane was suggested to
Address for reprint requests and other correspondence: T. A.
Miettinen, Dept. of Medicine, University of Helsinki, PO Box 340,
Helsinki FI-00029 HUCH, Finland.
http://www.ajpgi.org 0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society
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facilitate the uptake of dietary free and esterified cho-
lesterol, triglycerides, and phospholipids (12). The per-
centage of absorbed dietary free cholesterol is higher
than that of the esterified form because of its better
micellar solubility (3). Thus to improve the cholesterol
absorption, dietary esterified cholesterol should be hy-
drolyzed by bile acid-activated pancreatic cholesterol
hydrolase. Contrary to cholesterol, dietary plant ste-
rols (phytosterols), including sitosterol, stigmasterol,
and campesterol, occur in diet in free, esterified, and
glycosidic forms and are only weakly absorbed from the
gut. Compared with cholesterol, these sterols have
different intraluminal solubilization, different uptake,
or intracellular processing by the enterocytes (3, 13,
28). Absorption of cholestanol, campesterol, and sitos-
terol is directly related to cholesterol absorption effi-
ciency (24, 35). That dietary plant sterols lead to a
reduction in serum cholesterol was first shown by Pol-
lak (27), and their inhibitory effect on cholesterol ab-
sorption was evidenced by Grundy et al. (6). The plant
stanols sitostanol and campestanol are 5␣-saturated
derivatives of sitosterol and campesterol. These stanols
appeared to be almost unabsorbable, and they dimin-
ished both intestinal absorption and the serum level of
cholesterol more effectively than their unsaturated
parent plant sterols both in experimental animal stud-
ies (16, 32, 33) and human studies (2, 14, 15). Poorly
soluble plant stanols were converted to highly fat sol-
uble by their esterification with rapeseed oil fatty acids
(39). This resulted in development of plant stanol ester
margarine for serum cholesterol lowering. Daily con-
sumption of rapeseed oil margarine (spread) with and
without plant stanol esters revealed that the plant
stanols inhibited cholesterol (and plant sterol) absorp-
tion, cholesterol elimination as neutral sterols, and,
despite a compensatory increase in cholesterol synthe-
sis, lowered serum total and low density lipoprotein
cholesterol (7, 10, 11, 21). Effective impairment of
cholesterol and plant sterol absorption by plant stanol
esters apparently provides that the esters are hydro-
lyzed by intestinal sterol ester hydrolase and that
released free plant stanols interfere subsequently with
micellar solubilization of cholesterol and plant sterols.
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.
G1009
G1010 CHOLESTEROL AND PHYTOSTEROLS IN DUODENAL DIGESTION

In addition, excessive amounts of plant stanols may
interfere also with hydrolysis of dietary cholesterol and
plant sterol esters during food digestion. However,
although plant stanols are believed to interfere with
micellar solubilization of cholesterol and other sterols,
no studies are available on intestinal distribution of
sterols in oil and micellar phases during ingestion of
normal amounts of plant sterols or especially after
consumption of large doses of stanol ester margarine.
Therefore, we carried out an intestinal plant stanol
ester perfusion study to clarify the hydrolysis of cho-
lesterol, cholestanol, plant sterol and stanol esters, and
their distribution to the micellar and oil phases in the
upper part of the small intestine in healthy subjects.
The studies were performed with stanol esters, but
sterol esters might give similar results.
METHODS
Subjects. Eight healthy medical students (4 females, 4
males) with normal liver and thyroid function and with no
evidence of diabetes or gastrointestinal disease or hypolipi-
demic medication volunteered for the study. Their body mass
indexes were within normal limits (19.3–23.5 kg/m2). The
study protocol followed principles of the Helsinki Declara-
tion. Subjects were informed of the nature and purpose of the
investigations and the study, which was approved by the
Ethics Committee of the Department of Medicine, Helsinki
University Central Hospital.
Experimental design. A triple-lumen radiopaque tube
(model AN 20; H. W. Andersen Products) was used for the
infusion of the test formulas and for the collection of the
samples from the proximal jejunum for the sterol analysis.
The study subjects fasted overnight. The tube was placed
under X-ray control such that the proximal (infusion) outlet
was in the middle of the second part of duodenum, i.e.,
adjacent to the ampulla of Vater. The second and third
outlets were located 10 and 60 cm below the first one. Two
kinds of liquid formulas were used (Table 1). The liquid
formula 1 was prepared by sonication of 10 g of rapeseed oil
margarine, one egg yolk, 4.5 g of glycerol-L-mono-oleate
(Fluka), and 200 ml of water in a final volume of 218 ml. The
liquid formula 2 was prepared by sonication of 0.9 g of plant
stanols (sitostanol/campestanol ⫽ 3.5) as their esters (Raisio
Group, Raisio, Finland) in 10 g of the rapeseed oil margarine,
one egg yolk, 4.5 g of glycerol-L-mono-oleate, and 200 ml of
water in a final volume of 218 ml. These formulas were
infused at a constant rate of 50 ml/h through the proximal
outlet of the tube by using an infusion pump (model 871–102;
B. Braun, Melsungen, Germany). The liquid formula 1 was
administered first for a period of 1 h. After an equilibration
period of 40 min, followed by infusion of formula 2, infusion
was started via the same route at the same speed for an
additional period of 2.5 h. During both infusions, samples
were aspirated at intervals of 15 min via the third lumen.
Jejunal samples (5–10 ml) were placed in a water bath at
80°C for 5 min to destroy the lipase and hydrolase activities
and then were stored at ⫺20°C until analyzed.
Chemical analysis. Separation of the oil and micellar
phases and the sediment of the formulas and total intestinal
fluid were obtained by ultracentrifugation of samples at a
speed of 15,000 rpm for 1 h at 37°C. Samples from both the
total intestinal fluid and, after ultracentrifugation, from the
oil droplet on the surface of the ultracentrifuged sample and
from the micellar phase were collected. After removal of the
oil phase and most of the micellar phase, sediment in a
measured amount of micellar phase was also sampled. The
samples and those of the two formulas were extracted with
chloroform-methanol, evaporated, and subjected to thin-
layer chromatography in a diethyl ether/heptane (50:50) so-
lution to separate free and esterified sterols, which were then
eluated. Ester phases were saponified with 2 M KOH in 90%
ethanol and nonsaponifiable lipids were extracted from the
alkaline alcoholic-water medium with hexane. After the ad-
dition of the internal standard 5␣-cholestane, the sterol frac-
tions were silylated, and the sterols were quantified by gas-
liquid chromatography on a 50-m long SE-30 capillary
column (Ultra 2 column; Hewlett-Packard) with an auto-
mated electronic integrator (Sigma 10; Hewlett-Packard,
Palo Alto, CA.) for measurement of peak areas (18–21).
Calculations. The esterification percentages of cholesterol,
cholestanol, plant sterols (sitosterol and campesterol),
stanols (sitostanol and campestanol), and their free, esteri-
fied, and total concentrations were calculated in formulas,
total intestinal fluids, and in micellar, oil, and sediment
fractions of aspirated samples from the proximal jejunum
(third outlet). Because sitosterol was the largest plant sterol
fraction in formula 1 and was probably negligibly absorbed
during passage of the short upper intestinal segment, it was
used as the internal marker to correct dilutions. Use of
sitosterol as an unabsorbable marker in intestinal perfusion
studies has been thoroughly examined and validated (4, 7, 8,
23). Thus the absolute values of sterols and their esterified
forms were calculated as ratios to sitosterol to evaluate
effects of the different intestinal plant stanol contents and

Table 1. Absolute values of total sterols and their ratios to sitosterol in test meal formulas
and proximal jejunum
Formula 1 Formula 2

mg/dl mg/mg of sitosterol mg/dl mg/mg of sitosterol

Proximal Proximal Proximal Proximal

Sterol Infusate Jejunum Infusate Jejunum Infusate Jejunum Infusate Jejunum

Cholesterol 79.3 ⫾ 4.4 58.5 ⫾ 11.3 10.38 ⫾ 0.97 22.5 ⫾ 4.3a 86.0 ⫾ 1.8 57.1 ⫾ 6.4c 6.37 ⫾ 0.05e 9.21 ⫾ 0.4a,e
Cholestanol 0.6 ⫾ 0.0 0.6 ⫾ 0.1 0.08 ⫾ 0.01 0.23 ⫾ 0.0a 1.9 ⫾ 0.4d 0.9 ⫾ 0.1 0.14 ⫾ 0.04 0.15 ⫾ 0.0d
Campesterol 5.4 ⫾ 0.1 1.7 ⫾ 0.3c 0.71 ⫾ 0.01 0.65 ⫾ 0.1 8.8 ⫾ 0.2f 3.8 ⫾ 0.5c,e 0.65 ⫾ 0.00 0.61 ⫾ 0.1
Campestanol 0.5 ⫾ 0.1 0.1 ⫾ 0.0c 0.7 ⫾ 0.00 0.04 ⫾ 0.0 75.9 ⫾ 1.3f 26.8 ⫾ 3.3c,f 5.62 ⫾ 0.05f 4.32 ⫾ 0.3b,f
Sitosterol 7.6 ⫾ 0.3 2.6 ⫾ 0.3c 1.00 1.00 13.5 ⫾ 0.3f 6.2 ⫾ 0.6c,f 1.00 1.00
Sitostanol 1.6 ⫾ 0.1 0.3 ⫾ 0.0c 0.21 ⫾ 0.01 0.12 ⫾ 0.0 266.7 ⫾ 4.8f 83.7 ⫾ 13.8c,f 19.76 ⫾ 0.22f 13.5 ⫾ 1.1c,f
Values are means ⫾ SE; n ⫽ 5 analyses in the infusate groups and 8 analyses in the proximal jejunum groups. a P ⬍ 0.05, b P ⬍ 0.01,
c
P ⬍ 0.001, compared with corresponding value of infusate by unpaired t-test; d P ⬍ 0.05, e P ⬍ 0.01, f P ⬍ 0.001, compared with
corresponding value of formula 1 by paired t-test.

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 CHOLESTEROL AND PHYTOSTEROLS IN DUODENAL DIGESTION G1011

Table 2. Esterified sterols per sitosterol in test meal RESULTS

formulas and proximal jejunum
Test meal formulas. The liquid formulas 1 and 2
Formula 1 Formula 2 contained similar contents of cholesterol, whereas
Proximal Proximal
those of other sterols, especially that of campestanol
Infusate Jejunum Infusate Jejunum and sitostanol, were higher in formula 2 than in for-
mula 1 (Table 1). Contrary to the high esterification
Cholesterol 0.55 ⫾ 0.03 2.19 ⫾ 0.30c 0.42 ⫾ 0.07 2.45 ⫾ 0.18c
Cholestanol 0.01 ⫾ 0.00 0.04 ⫾ 0.02 0.08 ⫾ 0.01 0.05 ⫾ 0.01 percentages of the phytosterols (64–92%) those of cho-
Campesterol 0.46 ⫾ 0.01 0.08 ⫾ 0.04c 0.50 ⫾ 0.00 0.27 ⫾ 0.06b,e lesterol (5.3%) and cholestanol (10.6%) were lower in
Campestanol 0.05 ⫾ 0.04 0.02 ⫾ 0.00 5.38 ⫾ 0.00 2.44 ⫾ 0.33c,f formula 1 and the esterification percentage of stanols
Sitosterol 0.65 ⫾ 0.02 0.27 ⫾ 0.05c 0.76 ⫾ 0.00 0.47 ⫾ 0.03c,d was higher than that of sterols in both formulas (Table
Sitostanol 0.20 ⫾ 0.01 0.04 ⫾ 0.00c 18.90 ⫾ 0.02 7.31 ⫾ 0.76c,f
3). Owing to transesterification of the stanol prepara-
Values are means ⫾ SE in mg/mg of total sitosterol calculated by tion, esterification percentage of cholestanol (55.5%)
dividing the ester concentrations of Table 3 by the respective sitos- and phytosterols (76–96%) were higher in formula 2
terol concentrations in Table 1. Since the sitosterol level was 1.77
higher in formula 2 than in formula 1, the jejunal concentration after than in formula 1. In formula 1, absolute concentra-
formula 2 was administered was 2.39 times higher than after for- tions of cholesterol, campesterol, and sitosterol esters
mula 1 was administered; n ⫽ 5 analyses in the infusate group and were similar (4–5 mg/dl), but the sum of phytosterol
8 analyses in the proximal jejunum group. a P ⬍ 0.05, b P ⬍ 0.01, esters (10.4 mg/dl) was more than twice the content of
c
P ⬍ 0.001, compared with corresponding value of infusate by
unpaired t-test; d P ⬍ 0.05, e P ⬍ 0.01, f P ⬍ 0.001, compared with cholesterol esters (Table 3). Ester contents of plant
corresponding value of formula 1 by paired t-test. sterols, especially of campestanol and particularly of
sitostanol, exceeded that of cholesterol in the formula 2
(Table 3). Cholesterol (90–94%), cholestanol (90–98%)
the duodenal transit (Tables 1 and 2). The free sterols in and plant sterols and stanols (over 99%) were in the oil
micellar fractions of the upper jejunal samples were calcu- phase in both formulas 1 and 2, respectively.
lated as %portion of the total amount of the corresponding
sterol (Fig. 1). The sterol composition of the intestinal micel-
Sterol levels in proximal jejunal contents. Comparing
lar and oil phases was also calculated on a molar percent phytosterol contents in the intestinal fluid with those
(M%) basis. Because the sterol concentrations in the sedi- in formula 1 showed that the concentrations were
ment fractions were inconsistently different from those in the diluted three to five times the highest dilutions (five-
respective micellar phase, the difference between the total fold) being found for the plant stanols (for sitosterol
and micellar phases was considered to represent the oil 2.92 ⫾ 0.66) (Table 1). Use of sitosterol as an unabsorb-
phase. The term phytosterol in this text will mean saturated able marker showed that cholesterol per milligram of
(5␣) or unsaturated (⌬5) sterols or their mixture. Statistical sitosterol was increased from 10.4 to 22.5 mg and that
analyses were carried out using Student’s unpaired (Tables
1–3) and paired t-test (Tables 1–4 and Fig. 1). Logarithmic
of cholestanol was about threefold in the proximal
transformations were used with skewed distributions. The jejunum compared with those in formula 1, probably
differences between the means were considered statistically due to biliary secretion (Table 1). The respective phy-
significant if the P value was ⬍ 0.05. Mean ⫾ SE values are tosterol values tended to decrease, although the use of
given in the text, Tables 1–4, and Fig. 1. campesterol as the unabsorbable marker showed an

Fig. 1. Columns show percentage

amounts of micellar and oil-free and
esterified sterols in proximal jejunal
aspirates of test meal formulas (F) 1
and 2. Concentrations (mg/dl) of sterols
in total intestinal fluid samples are on
the tops of the columns (mean ⫾ SE).
Free micellar sterols, percentage of to-
tal intestinal sterols, are marked
within the columns (mean ⫾ SE). As-
terisks on the right hand side of the
columns indicate statistical signifi-
cances of esterification percentages of
micellar and oil phase sterols between
formulas 1 and 2, and those below the
columns indicate total esters, percent-
age of total intestinal sterols. *P ⬍
0.05, **P ⬍ 0.01, ***P ⬍ 0.001 from the
respective value of formula 1 by paired
t-test.

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G1012 CHOLESTEROL AND PHYTOSTEROLS IN DUODENAL DIGESTION

Table 3. Sterol esters and esterification in test meal formulas and proximal jejunum
Formula 1 Formula 2

Esters, mg/dl Ester% Esters, mg/dl Ester%

Proximal Proximal Proximal Proximal

Sterols Infusate Jejunum Infusate Jejunum Infusate Jejunum Infusate Jejunum

Cholesterol 4.2 ⫾ 0.2 5.7 ⫾ 1.2 5.3 ⫾ 0.3 9.7 ⫾ 2.1 5.7 ⫾ 0.9 15.1 ⫾ 1.1c,f 6.6 ⫾ 1.2 26.4 ⫾ 1.9c,e
Cholestanol 0.1 ⫾ 0.0 0.1 ⫾ 0.0 10.6 ⫾ 1.9 17.6 ⫾ 5.6 1.1 ⫾ 0.0f 0.3 ⫾ 0.1c,e 55.5 ⫾ 0.5f 37.4 ⫾ 6.0a,d
Campesterol 3.5 ⫾ 0.1 0.2 ⫾ 0.1c 65.0 ⫾ 1.7 12.9 ⫾ 3.8c 6.7 ⫾ 0.0f 1.6 ⫾ 0.2c,f 75.7 ⫾ 0.5f 43.9 ⫾ 6.4b,e
Campestanol 0.4 ⫾ 0.1 0.04 ⫾ 0.00c 76.8 ⫾ 12.9 35.8 ⫾ 3.6a 72.6 ⫾ 0.1f 15.0 ⫾ 0.4c,f 95.7 ⫾ 0.2 56.0 ⫾ 6.0c,d
Sitosterol 4.9 ⫾ 0.1 0.7 ⫾ 0.1c 63.8 ⫾ 1.7 26.8 ⫾ 4.6c 10.3 ⫾ 0.0f 2.9 ⫾ 0.2c,f 76.4 ⫾ 0.3f 46.8 ⫾ 3.3c,e
Sitostanol 1.5 ⫾ 0.0 0.1 ⫾ 0.0b 91.7 ⫾ 2.8 38.6 ⫾ 3.4c 255.2 ⫾ 0.3f 45.0 ⫾ 5.0c,f 95.7 ⫾ 0.2 53.8 ⫾ 6.0c,d
Values are means ⫾ SE; n ⫽ 5 analyses in infusate group and 8 analyses in the proximal jejunum group. a P ⬍ 0.05, b P ⬍ 0.01, c P ⬍
0.001, compared with corresponding value of infusate by unpaired t-test; d P ⬍ 0.05, e P ⬍ 0.01, f P ⬍ 0.001, compared with corresponding
value of formula 1 by paired t-test.

opposite trend. After formula 2, the dilution of the
phytosterols was two to three times, again the highest
(threefold) for plant stanols (for sitosterol 2.18 ⫾ 0.27;
P ⫽ 0.06 vs. formula 1). Despite the slightly higher
dilution after formula 1 infusion, jejunal cholesterol
and cholestanol concentrations were similar in the two
studies, whereas phytosterol levels were higher after
formula 2 than formula 1 as in the infusates. Choles-
terol standardized by infused sitosterol was increased,
cholestanol and campesterol were unchanged, and
plant stanols tended to decrease compared with the
ratios in formula 2 (Table 1).
Sterol esters and their hydrolysis during duodenal
transit. Comparison of the infused sterols with those in
the intestinal fluids of the proximal jejunum (Table 3)
revealed that jejunal levels of cholesterol and choles-
tanol esters were similar to the infused ones, whereas
those of phytosterols were markedly decreased and the
percentages of esterified cholesterol and cholestanol
tended to increase and those of other esterified sterols
were markedly decreased after infusion of formula 1.
Thus 80% of campesterol and 60% of sitosterol esters
had been hydrolyzed, respective values being 54% for
campestanol and 58% for sitostanol. After formula 2,
the concentration of jejunal esterified cholesterol was
almost threefold higher than that of the infusate, those
of the other sterols being markedly diluted. All the
jejunal sterol ester concentrations were clearly higher
after formula 2 than formula 1. The %ester of choles-
terol was increased from 6.6% in the infusate to 26.4%
in the intestinal contents, whereas those of other ste-
rols were markedly decreased, indicating that 39–44%
of the sterol esters, including the two stanol esters, had
been hydrolyzed during the passage of the infusate
through the first 60 cm of the upper small intestine.
Owing to markedly high campestanol and sitostanol
ester contents of the infusate, their mean absolute
hydrolysis was highest from among the noncholesterol
sterols.
The absolute amount of esterified cholesterol in re-
lation to total sitosterol was increased in the proximal
jejunum by a factor of almost four (P ⬍ 0.001) of that in
formula 1 and by fivefold (P ⬍ 0.001) after formula 2,
the actual increase being probably over fivefold higher
by a similar sitosterol intake of formulas 1 and 2 (Table
2). For the four phytosterols, the esterification values
were reduced by 58–82% after the formula 1 and less
so by 39–61% after formula 2 (Table 2).
Sterols in oil and micellar phases of intestinal con-
tents. Relative distributions of free and esterified phy-
tosterols are shown for the oil and micellar phases in
Fig. 1. Total concentration of each sterol in the intes-

Table 4. Concentration of jejunal free and esterified sterols in oil and micellar phases after infusion
of formulas 1 and 2 test meals
Oil Phase Micellar Phase

Sterols F Free Ester Free Ester

Cholesterol 1 4.5 ⫾ 1.0 1.5 ⫾ 0.5 48.3 ⫾ 12.2 4.2 ⫾ 0.6

2 9.4 ⫾ 1.8 10.1 ⫾ 1.2** 32.1 ⫾ 4.9 5.0 ⫾ 0.6
Cholestanol 1 0.08 ⫾ 0.01 0.03 ⫾ 0.00 0.43 ⫾ 0.09 0.08 ⫾ 0.02
2 0.15 ⫾ 0.09 0.20 ⫾ 0.13** 0.39 ⫾ 0.05 0.12 ⫾ 0.02
Campesterol 1 0.14 ⫾ 0.04 0.08 ⫾ 0.02 1.34 ⫾ 0.31 0.14 ⫾ 0.03
2 0.61 ⫾ 0.10** 1.26 ⫾ 0.34*** 1.50 ⫾ 0.27 0.39 ⫾ 0.07*
Campestanol 1 0.01 ⫾ 0.00 0.00 ⫾ 0.00 0.07 ⫾ 0.01 0.04 ⫾ 0.01
2 3.2 ⫾ 0.4*** 11.9 ⫾ 0.4*** 8.2 ⫾ 1.8*** 3.1 ⫾ 0.4***
Sitosterol 1 0.4 ⫾ 0.1 0.1 ⫾ 0.0 1.6 ⫾ 0.3 0.6 ⫾ 0.1
2 0.9 ⫾ 0.2 2.1 ⫾ 0.1*** 2.3 ⫾ 0.3 0.8 ⫾ 0.1
Sitostanol 1 0.02 ⫾ 0.01 0.04 ⫾ 0.02 0.15 ⫾ 0.03 0.11 ⫾ 0.03
2 8.7 ⫾ 1.7*** 33.9 ⫾ 1.1*** 30.0 ⫾ 6.5*** 11.1 ⫾ 1.1***
Values are means ⫾ SE in mg/dl; n ⫽ 8 analyses. F, formula. * P ⬍ 0.05, ** P ⬍ 0.01, *** P ⬍ 0.001, compared with the liquid formula
1 by paired t-test.

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 CHOLESTEROL AND PHYTOSTEROLS IN DUODENAL DIGESTION
tinal fluid is shown on the top of each column. Respec-
tive concentrations of the free and esterified sterols in
the oil and micellar phases are shown in Table 4.
Respective concentrations of intestinal cholesterol
and cholestanol (about 1% cholesterol) were similar
after infusion of the two formulas (Fig. 1). The relative
distributions of their free and esterified forms were
similar in the respective oil and micellar phases after
infusion of formula 1, over two-thirds of sterols being
found as free sterols in the micellar phase. Respective
distributions of the two sterols were also similar after
formula 2, but proportions of esterified sterols had
markedly increased in the oil phase, and the respective
amounts of free, but not esterified, micellar sterols had
decreased. Micellar free cholesterol concentration was
decreased by 16.2 ⫾ 5.8 mg/dl (P ⫽ 0.06) (Table 4).
However, micellar-free cholesterol comprised 82.7 ⫾
3.6% of total intestinal cholesterol in the formula 1
study and 56.2 ⫾ 6.4% (P ⬍ 0.001) in the sitostanol
ester-enriched formula 2 study. The respective values
for cholestanol were 69.7 ⫾ 7.0 vs. 45.5 ⫾ 3.7% (P ⬍
0.05) (Fig. 1). Distribution of the two plant stanols
differed from that of the respective sterols mainly by
surprisingly large micellar ester fractions after for-
mula 1.
Infusion of formula 2 increased intestinal plant sta-
nol concentrations ⬎200 times and those of plant ste-
rols 2–3 times compared with those after infusion of
formula 1 (Tables 1 and 4, Fig. 1). As in the cases of
cholesterol and cholestanol, the infusion of plant stanol
ester-enriched formula markedly increased the oil
phase fractions due to enlargement of all esterified
phytosterols, especially of stanols, whereas the respec-
tive proportions of micellar fractions were decreased
most consistently for free sterols but also for esters of
phytosterols, except campesterol (Fig. 1). Despite the
relative reduction of micellar sterols, the concentra-
tions of micellar-free and esterified phytosterols were
increased compared with the respective values after
infusion of formula 1 (Table 4). The micellar concen-
tration of esterified campesterol increased almost three
times, the oil phase free campesterol increased four
times, and the esterified one increased 16 times. The
plant stanol levels increased up to hundreds of times in
both phases by formula 2 vs. formula 1.
M% of intestinal sterols calculated for the sterols of
Table 4 showed that M% of cholesterol was decreased
in both the oil (from 87 to 24) and micellar phases (from
92 to 39) by formula 2. The respective values increased
markedly for campestanol, both in the oil (from 0.1 to
18) and micellar (from 0.2 to 12) phases and especially
for sitostanol, also in both the oil (0.9 to 52) and
micellar (0.5 to 43) phases.
DISCUSSION
The major difference between the two test meal
formulas was the 200 times higher concentrations of
esterified plant stanols in formula 2 than formula 1.
We found that the esterification percentage of choles-
terol only tended to increase after infusion of formula
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G1013
1, but it was significantly increased after the formula 2
infusion during the transit of the infusate from the
proximal duodenum to the proximal jejunum (Table 3).
Effective absorption of unesterified cholesterol in the
upper small intestine could have increased the esteri-
fied percentage of cholesterol. However, calculation of
absolute amounts of sterol esters in milligram per
milligram of total sitosterol (plant stanols could have
been better unabsorbable markers, but their baseline
contents were low in formula 1) in the formulas and
proximal jejunal contents showed, as in the earlier
study (23) that the amount of esterified cholesterol was
four times increased in the jejunal contents compared
with that in infused formula. In the previous study
(23), in contrast to the present one, infused cholesterol
was mainly esterified (98%) and sitosterol mainly un-
esterified (95%); the respective increase of jejunal es-
terified cholesterol was only ⬃35%, whereas that of
sitosterol was fourfold. Secretion of bile into the intes-
tinal contents increased unesterified cholesterol frac-
tion, because biliary cholesterol comprises ⬃97% of
gallbladder bile sterols of, which virtually all are un-
esterified (20). Contrary to cholesterol, esters of the
phytosterols of both formulas were significantly hydro-
lyzed in the upper intestine, whereas that of cholesta-
nol tended to increase after infusion of formula 1.
Absolute amount of sitosterol esters was reduced by
⬃60%, and those of other phytosterols were reduced by
up to 80%, indicating that the plant sterol esters of our
normal food are rapidly hydrolyzed after food intake
already in the upper part of the small intestine. Anal-
ogous to cholesterol, cholestanol exhibited percentage
of low ester in formula 1 but its relationship to sitos-
terol was increased fourfold in the intestinal contents.
The method of analysis used here allows assessment of
the relative contributions of different sterols to the
total sterol composition, but it does not allow assess-
ment of the absolute amounts of different sterols and
stanols in the intestinal contents.
Infusion of stanol esters increased the absolute
amount of cholesterol esters significantly by a factor of
five, in contrast to fourfold after formula 1, but the
increased sitosterol infusion also indicates a higher
absolute increase of cholesterol esters. Infusion of for-
mula 2 reduced esters of cholestanol and phytosterols
somewhat less than after formula 1. Thus even in the
presence of excessively large amounts of intestinal
stanol esters, hydrolysis of minor sterol esters is effec-
tive, and stanol ester hydrolysis occurs rapidly already
in the short, upper intestinal segment (Fig. 1, Table 3).
The significant increase of cholesterol esters in rela-
tionship to sitosterol in the small intestine by the
addition of a large stanol ester amount is a surprising
finding suggesting an inverted reaction in ester hydro-
lysis. However, hydrolysis may also occur in the lower
part at the small intestine, because esterified percent-
age of cholesterol is low in excreta of colectomized
patients (25).
Esterified amounts of sterols in the oil fractions were
markedly increased in the proximal jejunal contents
after infusion of the plant stanol esters with concomi-
282 • JUNE 2002 • www.ajpgi.org
G1014 CHOLESTEROL AND PHYTOSTEROLS IN DUODENAL DIGESTION
tant marked absolute increase of free plant stanols and
decrease of cholesterol in the micellar fractions. A
decrease of the cholesterol content of the intestinal
micelles after formula 2 infusion was also evident as
calculated on an M% basis. Incorporation of free
stanols into the duodenal and jejunal micelles may
thus prevent entry of free cholesterol into micelles,
resulting also in accumulation of unesterified choles-
terol and other sterols in the oil phase. The increase of
free sterols and fatty acids may retard hydrolysis of all
sterol esters and could even contribute to increased
formation of cholesterol esters, possibly by competition
with cholesterol as a substrate of the pancreatic cho-
lesterol esterase. Accordingly, sitostanol may enhance
hydrolysis of various plant sterols over that of choles-
terol, resulting in accumulation of esterified choles-
terol. Replacement of cholesterol in the micellar phase
by stanols is crucial in preventing cholesterol absorp-
tion because that occurs from the intestinal micellar
phase (5). Solubilization of stanols, particularly in lec-
ithin micelles, reduced cholesterol absorption by 37%
in humans (26). Although Heinemann et al. (13) have
shown that esterified cholesterol also is taken up by the
enterocyte, the proportion of cholesterol absorbed in
esterified form may be much less compared with that of
free cholesterol because the esterified form has a poor
solubility in the bile acid micelle (13, 15). In general,
esterified cholesterol has been believed to be hydro-
lyzed before absorption (36, 38). Our findings on the
increasing esterification percentage of cholesterol dur-
ing the duodenal transit already at low, but especially
at high, plant stanol concentrations suggest that the
esters are only weakly solubilized into the micellar
phase and that they accumulate in the oil phase and
thus are less effectively absorbed into the enterocytes.
Furthermore, the length of the side chain and hydrog-
enization of the unsaturated phytosterol ⌬5-double
bond in the stanol molecule led to increased hydropho-
bicity and solubility into the micelle (1, 13) and, hence,
enable the replacement of cholesterol from the micelle.
Cholestanol and plant stanols have a lower intesti-
nal absorbability than cholesterol due to the saturated
⌬5-double bond of 5␣-stanols (1, 37). Our results
showed a higher esterification percentage for cholesta-
nol in formula 2 than in formula 1, whereas that of
cholesterol was the same in the two formulas. How-
ever, in the proximal jejunum, the relative distribu-
tions in oil and micellar-free and esterified fractions
were similar to cholesterol. Additionally, the micellar-
free sterol proportion of both cholesterol and cholesta-
nol was lower with formula 2 than with formula 1.
In conclusion, our results suggest that the sitostanol
ester is effectively hydrolyzed and entered into the bile
acid micelles already during the duodenal transit. At a
high sitostanol ester concentration, it is owing to its
rapid hydrolysis that plant stanols replace cholesterol
in the micelle and increase accumulation of cholesterol
and its esters in the oil phase. These events may be
essential for the reduction of cholesterol absorption
caused by sitostanol ester.
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The authors thank Leena Kaipiainen, Orvokki Ahlroos, Pia Hoff-
ström, Anne Honkonen, and Ritva Nissilä for expert technical assis-
tance.
The study was supported with grants from the Helsinki Univer-
sity Central Hospital.
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