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Prebiotic oligosaccharides and the enterohepatic circulation of bile salts
in rats
Hester van Meer,
1
Gunther Boehm,
2,3
Frans Stellaard,
1
Aldwin Vriesema,
4
Jan Knol,
4
Rick Havinga,
1
Pieter J. Sauer,
1
and Henkjan J. Verkade
1
1
Pediatric Gastroenterology, Department of Pediatrics, University Medical Center Groningen, University of Groningen,
Groningen, The Netherlands;
2
Sophia Children’s Hospital, Erasmus Medical Center, University of Rotterdam, Rotterdam,
The Netherlands;
3
Numico Research, Friederichsdorf, Germany;
4
Biomedical Research Department, Numico Research BV,
Wageningen, The Netherlands
Submitted 30 August 2007; accepted in final form 10 December 2007
Van Meer H, Boehm G, Stellaard F, Vriesema A, Knol J,
Havinga R, Sauer PJ, Verkade HJ. Prebiotic oligosaccharides and
the enterohepatic circulation of bile salts in rats. Am J Physiol
Gastrointest Liver Physiol 294: G540–G547, 2008. First published
December 13, 2007; doi:10.1152/ajpgi.00396.2007.—Human milk
contains prebiotic oligosaccharides, which stimulate the growth of
intestinal bifidobacteria and lactobacilli. It is unclear whether the
prebiotic capacity of human milk contributes to the larger bile salt
pool size and the more efficient fat absorption in infants fed human
milk compared with formula. We determined the effect of prebiotic
oligosaccharides on bile salt metabolism in rats. Rats were fed a
control diet or an isocaloric diet containing a mixture of galactooli-
gosaccharides (GOS), long-chain fructooligosaccharides (lcFOS), and
acidified oligosaccharides (AOS) for 3 wk. We determined synthesis
rate, pool size, and fractional turnover rate (FTR) of the primary bile
salt cholate by using stable isotope dilution methodology. We quan-
tified bile flow and biliary bile salt secretion rates through bile
cannulation. Prebiotic intervention resulted in significant changes in
fecal and colonic flora: the proportion of lactobacilli increased 344%
(P⬍0.01) in colon content and 139% (P⬍0.01) in feces compared
with the control group. The number of bifidobacteria also increased
366% (P⬍0.01) in colon content and 282% in feces after the
prebiotic treatment. Furthermore, pH in both colon and feces de-
creased significantly with 1.0 and 0.5 pH point, respectively. How-
ever, despite this alteration of intestinal bacterial flora, no significant
effect on relevant parameters of bile salt metabolism and cholate
kinetics was found. The present data in rats do not support the
hypothesis that prebiotics naturally present in human milk contribute
to a larger bile salt pool size or altered bile salt pool kinetics.
bile salt kinetics; infant nutrition; cholate
THE ENTEROHEPATIC CIRCULATION of bile salts, the major constit-
uents of bile, serves two important functions in the human
body. Bile salts enhance the absorption of long-chain saturated
fatty acids and fat-soluble vitamins from the intestine. Further-
more, the enterohepatic circulation of bile salts promotes the
excretion of lipophilic molecules via the bile into the feces
(e.g., cholesterol and bilirubin) and is critically important for
cholesterol homeostasis in the body. The dietary fat intake in
infants is relatively high compared with the dietary fat intake in
adults, whereas the bile salt pool size of the former is lower
(33). Efficient absorption of dietary fat is essential for optimal
growth and development during infancy.
Human milk is the gold standard in infant feeding. One of
the beneficial qualities of human milk involves the more
efficient absorption of dietary fat compared with that from
formula (34). Interestingly, the bile salt pool is larger in
premature infants fed human milk compared with formula, but
it has not been clarified whether this contributes to the mech-
anism underlying the more efficient fat absorption (34). Previ-
ously, we reported that the maturation of fat absorption in
human neonates is functionally related to an increased capacity
to absorb long-chain fatty acids from the intestine, possibly due
to developmental changes in bile salt composition and bile salt
pool (28).
Prebiotics are nondigestible food ingredients that stimulate
the growth and activity of specific bacteria in the colon (i.e.,
bifidobacteria and lactobacilli) (14). Oligosaccharides are a
major constituent of human milk and have been demonstrated
to increase the proportion of bifidobacteria and lactobacilli in
the infant’s colon (4, 9). The prebiotic galactooligosaccharides
(GOS) and fructooligosaccharides (FOS) are known to increase
the number of bifidobacteria and lactobacilli in the gut in both
human and animal studies (3, 26, 32). Previous studies in rats,
using the prebiotic substrate FOS, have shown an increase in
the amount of intestinal bifidobacteria and lactobacillus
(25, 31).
It is not known whether oligosaccharides, the prebiotic
constituent of human milk, influence the bile salt metabolism
and thereby play a role in the more efficient fat absorption of
breast milk. Interactions between intestinal flora and bile salts
are well known. The bacterial metabolism of bile salts in the
intestinal lumen can involve deconjugation and dehydroxyla-
tion, converting primary bile salts such as cholate and
chenodeoxycholate into secondary bile salts (i.e., deoxycholate
and lithocholate). Bacterial metabolism of bile salts is partly
responsible for the fractional turnover rate (FTR) of primary
bile salts, i.e., the portion of the pool that is newly synthesized
per day. Bacterial metabolism may also influence the physio-
logical activity of bile salts since secondary bile salts are more
hydrophobic than primary bile salts and therefore have a
greater capacity to interact with dietary fat.
Recently we developed a method to quantify cholate fluxes
in the enterohepatic circulation of experimental animals (19).
Address for reprint requests and other correspondence: H. J. Verkade,
Center for Liver, Digestive and Metabolic Diseases, Pediatric Gastroenterol-
ogy, Dept. of Pediatrics, CMC IV, Rm. Y4.107a, Univ. Medical Center
Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands (e-mail:
h.j.verkade@med.umcg.nl).
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.
Am J Physiol Gastrointest Liver Physiol 294: G540–G547, 2008.
First published December 13, 2007; doi:10.1152/ajpgi.00396.2007.
0193-1857/08 $8.00 Copyright ©2008 the American Physiological Society http://www.ajpgi.orgG540
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This method is based on a stable isotope dilution methodology
used in humans that was successfully downscaled to allow
measurement of cholate fluxes in experimental animals (19).
The enterohepatic circulation of bile salts can be quantitatively
characterized by specific kinetic parameters: the pool size (the
amount of bile salts in the body), the FTR (the fraction of the
pool that is newly synthesized each day), the synthesis rate,
and, finally, the cycling time (the time it takes the cholate pool
to undergo one full cycle in the enterohepatic circulation). In
the present study, we determined the effect of dietary prebiotic
oligosaccharides on the enterohepatic circulation of cholate,
the major primary bile salt in rats.
MATERIALS AND METHODS
Animals and materials. Six-week-old male rats (Harlan Laborato-
ries, Zeist, The Netherlands) were housed in a light- and temperature-
controlled facility with free access to tap water and to either the
prebiotic diet or the control diet. Experimental protocols were ap-
proved by the Ethical Committee for Animal Experiments, Faculty of
Medical Sciences (University Medical Center Groningen, The Neth-
erlands). 2,2,4,4-Tetradeuterated cholic acid ([
2
H
4
]-CA; isotopic pu-
rity, 98%) was obtained from Isotec (Miamisburg, OH). All other
chemicals and solvents used were of the highest purity commercially
available. Animals received semi-purified AIN-93-based diets pressed
into pellets (Research Diet Services, Wijk bij Duurstede, The Neth-
erlands). In the prebiotic diet, the supplemented oligosaccharides were
exchanged for the same amount of carbohydrates. The experimental
diet was supplemented with GOS, lcFOS, and AOS in a dose of 7.65,
0.85, and 0.90 wt%, respectively (Table 1).
Experimental procedures. Male Wistar rats (253 ⫾11 g body wt)
were fed the control diet for 3 wk as a run in, after which they were
randomly assigned to the prebiotic or the control diet for another 3 wk
(n⫽8 per group). Body weight was measured weekly; food intake
and fecal production were measured during 72 h in the third week
after randomization. Three weeks after randomization, relevant
parameters of synthesis and enterohepatic circulation of cholate were
determined by using the previously mentioned stable isotope dilution
technique (19). In short, 3.0 mg of [
2
H
4
]-CA in a solution of 0.5%
NaHCO
3
in phosphate-buffered saline was slowly injected via the
penile vein during isoflurane anesthesia. Blood samples were taken
before injection and at 12, 24, 36, 48, 60, and 72 h after injection.
Blood samples (300 l) were collected by tailbleeding under isoflu-
rane anesthesia. Blood was collected in EDTA tubes and centrifuged
to obtain plasma. After centrifugion (3,000 rpm for 10 min at 4°C),
plasma was stored at ⫺20°C until analysis. At the last day of the
experiment, rats were equipped with a catheter in the bile duct, and
bile was collected for 30 min. Animals were euthanized by heart
puncture, after which the liver and colon contents were collected and
stored at ⫺80°C until analysis.
Analytical procedures. Plasma alanine transaminase, aspartate
transaminase, alkaline phosphatase, cholesterol, and triglyceride con-
centrations were determined by routine laboratory techniques. Con-
centrations of biliary cholesterol and phospholipids were measured as
described (13, 16), as were bile salt concentrations in feces and in bile
(24). Fecal bile salt composition and fecal neutral sterols were
analyzed as follows: 50 mg of dried feces were boiled in 1 ml of
alkaline methanol (1 M NaOH-methanol, 1:3 vol/vol) at 80°C for 2 h
after addition of 50 nmol 5␣-cholestane and 14 nmol 7␣,12␣-dihy-
droxy-5-cholanic acid as internal standard for neutral sterols and bile
salts, respectively. After cooling down to room temperature, neutral
sterols were extracted by using 3 ⫻3 ml of petroleum ether, boiling
range 60 – 80
°
C. The residual sample was diluted 1:9 with distilled
water. A sample (100 l) of the solution was subjected to an
enzymatic total bile salt measurement (24). The remaining solution
was used for bile salt isolation by reversed-phase solid-phase (C18)
extraction (24, 30). The eluate was evaporated to dryness, and bile
salts were derivatized to the methyl ester-trimethylsilyl derivatives for
gas chromatography analysis. The extracted neutral sterols were
derivatized to the trimethylsilyl derivatives by applying the same
procedure that was used for bile salts. Bile salt composition of
prepared bile samples, fecal samples, and neutral sterol composition
of prepared feces samples were determined by capillary gas chroma-
tography on an Agilent gas chromatograph (HP 6890), equipped with
a25m⫻0.25 mm CP-Sil-19-fused silica column (Varian, Middel-
burg, The Netherlands) and a flame ionization detector. The condi-
tions were as follows: injector temperature 280°C; pressure 16.0 psi;
column flow constant at 0.8 ml/min; oven temperature program:
240°C (4 min), 10°C/min to 280°C (27 min); detector temperature
300°C. Hepatic concentrations of triglycerides and cholesterol were
measured by using commercial kits (Wako Chemicals, Neuss, Ger-
many and Roche Diagnostics, Mannheim, Germany) after livers were
homogenized. Pooled plasma samples were used for lipoprotein sep-
aration by fast-protein liquid chromatography (FPLC) on a Superose
6B 10/30 column (Amersham Biosciences). The concentration of
triglycerides in the various fractions was determined by using a
commercial kit (Wako Chemicals).
Gas-liquid chromatography electron capture negative chemical
ionization mass spectrometry and calculations. Plasma samples were
prepared for isotopic analysis of bile salts by gas chromatography-
mass spectrometry (GC-MS) as described (19, 27). Analyses were
performed at the pentafluoro-TMS derivative by using a Finnigan
SSQ7000 Quadrupole GC-MS (Finnigan MAT, San Jose´, CA). GC
separation was performed on a 15 m ⫻0.25 mm column, 0.25-m
film thickness (AT-5MS; Alltech Associates, Deerfield, IL). The area
ratio M4/M0 is calculated after computerized integration of peak areas
of M4 CA and M0 CA in the mass chromatograms for mass-to-charge
ratio 627.3 and 623.3, by using LCQuan software (Finnigan MAT).
Enrichment is defined as the increase of M4/M0 after administration
of [
2
H
4
]-CA and is expressed as the natural logarithm of the atom
percent excess (ln APE) value. The decay of ln APE over time was
described by linear regression analysis. From this linear decay curve,
the FTR and pool size of CA were calculated. The FTR (day
⫺1
)
equals the slope of the regression line. The pool size (mol/100 g
body wt) is determined according to the formula: pool size ⫽(D ⫻
b⫻100/e
a
)⫺D, where D is the administered amount of label, b is
the isotopic purity, and a is the intercept on the y-axis of the ln
APE-vs.-time curve. The CA synthesis rate (mol䡠100 g body
wt
⫺1
䡠day
⫺1
) is determined by multiplying pool size and FTR.
Enterohepatic cycling time. The cycling time of the enterohepatic
circulation is the time it takes for a bile salt to undergo one full cycle.
The cycling time for cholate can be estimated by dividing the cholate
pool size by the biliary secretion rate of cholate (which was calculated
by using bile salt composition in bile and bile flow rates after bile
cannulation). The fraction of cholate lost per cycle was calculated by
Table 1. Composition of the prebiotic diet and the
control diet
Ingredients, g/kg
Control Diet
(AIN-93G)
Prebiotic Diet
(GOS-1cFOS-AOS supplemented)
Cornstarch 397.5 397.5
Dextrinized
cornstarch 132.0 65.9
Sucrose 100.0 72.3
Cellulose 50.0 50.0
Pure carbohydrate
mix Numico* 93.8
Soybean oil 70.0 70.0
*85 g galactooligosaccharides (GOS), long-chain fructooligosacharides (lcFOS)
(9:1), and 8.8 g acidified oligosaccharides (AOS).
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dividing fractional cholate synthesis rate by cholate cycling fre-
quency.
Fecal flora composition. Frozen fecal or colon samples were
thawed by adding 1 ml of MilliQ water and heating at 90°C. The
suspensions were subsequently homogenized and frozen overnight at
⫺20°C. The homogenized samples were thawed at room temperature,
followed by DNA isolation by using the NucliSense Isolation Extrac-
tion Kit (BioMerieux, Boxtel, The Netherlands). For the relative
quantification of the genera Bifidobacterium and Lactobacillus in
relation to the total bacterial load, a duplex 5⬘nuclease quantitative
real-time PCR assay was used (15).
Briefly, different primers and probes for the genus Bifidobacterium
or Lactobacillus in combination with primers and probes for total
bacteria were used in a temperature profile consisting of 2 min at
50°C, 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 60°C
for 1 min, run on ABI Prism 7700 PCR equipment (Applied Biosys-
tems, Nieuwerkerk a/d IJssel, The Netherlands). The relative amounts
of the genus Bifidobacterium or Lactobacillus in the samples were
calculated with respect to the total bacterial load according to Liu
et al. (23) and expressed in percentages. All samples were analyzed in
triplicate.
Statistics. Values represent means ⫾SD for the indicated number
of animals per group. Differences between the two groups were
determined by Student’s t-test for normally distributed values, and
Mann-Whitney exact two-tailed U-test was used for nonnormally
distributed data. P⬍0.05 was considered significant. Analysis was
performed using SPSS 12.0 for Windows software (SPSS, Chi-
cago, IL).
RESULTS
Animal characteristics. Animals fed the prebiotic diet or the
control diet were comparable in body weight, growth, fecal
production, and food intake (Table 2). The prebiotic diet
significantly elevated serum lathosterol, an intermediate in the
cholesterol synthesis pathway, whereas plasma concentrations
of cholesterol, triglycerides, alanine transaminase, aspartate
transaminase, and alkaline phosphatase were not altered upon
treatment (Table 2).
Effect of prebiotic treatment on the composition of intestinal
flora. Figure 1 shows that the prebiotic diet had a prominent
bifidogenic effect. After 3 wk the relative numbers of bi-
fidobacteria and of lactobacilli were significantly higher in
Table 2. Animal characteristics, plasma and hepatic
parameters of lipid metabolism, and liver function after
feeding rats a prebiotc or a control diet
Control Diet Prebiotic Diet
Animal characteristics
Body weight 252⫾12 254⫾11
Body weight at termination 456⫾35 476⫾28
Feces (wet), g 䡠day
⫺1
䡠100 g body wt
⫺1
0.4⫾0.1 0.4⫾0.1
Food intake, g/24 h 27.9⫾1.7 26.7⫾1.6
Hepatic parameters
Liver weight
-absolute, g 13.8⫾1.4 14.8⫾1.7
-relative, % of body wt 3.0⫾0.2 3.1⫾0.2
Triglycerides, nmol/mg liver 14.3⫾5.4 14.7⫾5.6
Cholesterol, nmol/mg liver 5.4⫾0.7 5.7⫾1.7
Plasma parameters
Alanine transaminase, units/l 46⫾10 45⫾7
Aspartate transaminase, units/l 153⫾75 155⫾49
Alkaline phosphatase, units/l 6⫾72⫾1
Cholesterol, mmol/l 1.8⫾0.3 1.9⫾0.5
Triglycerides, mmol/l 1.4⫾0.5 1.9⫾1.0
Lathosterol, mol/l 0.7 ⫾0.1 1.3 ⫾0.5*
*P⬍0.05.
Fig. 1. Effect of prebiotic treatment (solid bars) on percentage of total
bacterial load of bifidobacteria (A) and lactobacilli (B) in feces and colon
content compared with control rats (open bars). Effect of prebiotic treatment on
pH in feces and colon content (C). Prebiotic-treated rats are significantly
different from controls. Data are expressed in means ⫾SD of n⫽8 per group;
*P⬍0.05; **P⬍0.01.
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colon content and in feces in rats fed the prebiotic diet
compared with controls. The proportion of lactobacilli in-
creased 344% (P⬍0.01) in colon content and 139% (P⬍
0.05) in feces compared with rats on control diet (Fig. 1).
Prebiotic treatment also increased the contribution of bi-
fidobacteria 366% (P⬍0.01) in colon content and 282% (P⬍
0.01) in feces compared with the control group (Fig. 1). The
prebiotic diet significantly lowered pH in both colon content
and feces with 1.0 and 0.5 pH point, respectively (Fig. 1).
Effect of prebiotic intervention on parameters of the entero-
hepatic circulation of bile salts. Bile flow and biliary secre-
tion rates of bile salts, phospholipids, and cholesterol were
similar between the groups (Fig. 2), as were biliary bile salt
composition (Fig. 3), fecal neutral sterol composition, and
fecal bile salt excretion (Fig. 4). Prebiotic treatment did not
affect the enterohepatic circulation of bile salts. Kinetic
parameters of cholate, the main bile salt in rodents, were not
significantly altered by the prebiotic diet. Cholate synthesis
rate, pool size, and FTR, as well as fecal excretion, were
similar in both groups (Table 3 and Fig. 5).
Effect of prebiotic treatment on hepatic and plasma lipids.
The prebiotic treatment did not affect hepatic and plasma
concentrations of cholesterol and triglycerides (Table 2). Upon
FPLC separation of plasma lipoproteins, the distribution of
triglycerides in the different fractions was comparable in pre-
biotic-treated rats and controls (Fig. 6).
DISCUSSION
We investigated whether prebiotic treatment and subsequent
alteration of the intestinal bacterial flora affects the enterohe-
patic circulation and composition of bile salts in rats. The
results show that prebiotic treatment significantly increased the
relative contributions of both lactobacilli and bifidobacteria in
the colon and the feces of rats. Furthermore, pH in both colon
and feces significantly decreased after feeding rats a prebiotic
diet. This prebiotic effect, however, did not significantly affect
bile flow or bile salt composition or the synthesis and entero-
hepatic circulation of cholate.
The liver parenchymal cells synthesize the primary bile salts
cholate and chenodeoxycholate. Primary bile salts are conju-
gated before their secretion into bile (in rats predominantly to
taurine). Upon entering the proximal small intestine, conju-
gated bile salts stimulate the emulsification and absorption of
dietary fat. Conjugated bile salts are efficiently reabsorbed in
the terminal ileum, mediated by the apical sodium-dependent
bile salt transporter (Asbt) and the basolateral transporter Ost
␣/. Primary bile salts that escape absorption in the terminal
Fig. 2. Effect of prebiotic diet (solid bars) on bile flow (A), total bile salts (B), phospholipids (C), and cholesterol (D) in bile compared with the control rats (open
bars). Data are expressed in means ⫾SD of n⫽7 or 8 per group. No significant differences between prebiotic-treated rats compared with controls in any of
the parameters. BW, body weight.
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ileum may be deconjugated by the bacterial flora in the colon
and then undergo (7-alpha-) dehydroxylation resulting in the
formation of secondary bile salts such as deoxycholate and
lithocholate. Various intestinal bacterial species, including lac-
tobacilli and bifidobacteria, have the capacity to metabolize
bile salts (10). The secondary bile salts are partly reabsorbed
by passive diffusion or are lost via the feces. Intestinal reab-
sorption of secondary bile salts, dependent on passive diffu-
sion, is less efficient than the Asbt-mediated transport of
primary bile salts.
Under steady-state conditions, fecal bile salt loss is compen-
sated by synthesis of primary bile salts in the liver. In theory,
modification of intestinal bacterial flora and subsequent altered
distribution of bile salts between primary and secondary type
could influence intestinal absorption and fecal excretion of bile
salts and therefore affect kinetic parameters of bile salt
homeostasis. In preterm infants fed human milk, the bile salt
pool size is larger compared with formula-fed infants, indicat-
ing that dietary factors affect the pool size (34). Also, fecal bile
salt concentrations are higher in infants fed human milk and
contain a smaller fraction of secondary bile salts compared
with those fed formula (16). Breast-fed infants have an intes-
tinal bacterial flora that is characterized by relatively high
amounts of bifidobacteria and lactobacilli (4, 17, 20, 29).
Because of the fermenting ability of these bacteria, a lower
stool pH is found in infants fed human milk compared with
formula-fed infants. The prebiotic capacity of human milk
induces a different intestinal environment, which could, in
theory, alter the enterohepatic circulation of bile salts. Our
present data show that a prebiotic treatment altered the intes-
tinal bacterial flora in adult rats, indicated by a significant
induction of colonic and fecal bifidobacteria and lactobacilli
and a significant decrease in fecal pH. Interestingly, however,
the prebiotic diet did not affect either the bile salt composition
or the kinetic parameters of the enterohepatic bile salt circula-
tion (Fig. 5).
Theoretically, these observations could be due to a different
response to a prebiotic diet in rats compared with other species.
The present prebiotic treatment resulted in lactobacilli, ac-
counting for 2.0 and 1.8% of total bacterial load in colon and
feces, respectively. The same diet (AIN-93G) supplemented
with 10 wt% GOS-lcFOS (ratio 9:1) resulted in 7.5% lactoba-
cilli in feces of mice (32). Haarman et al. (15) found fecal flora
to contain 4.4% lactobacilli in infants receiving a formula
supplemented with 0.8 g/100 ml GOS-lcFOS (ratio 9:1) for 6
wk, similar to values observed in breast-fed infants. Despite the
still limited absolute contribution of lactobacilli, the deceased
colonic and fecal pH indicates that a physiological response is
achieved.
However, the bacterial flora of infants fed human milk
contains ⬎60% bifidobacteria within 1 wk after birth (17). The
addition of a GOS-FOS mixture to infant formula resulted in an
increase of fecal bifidobacteria of almost 60% in healthy
infants (20). Vos et al. (32) used an identical prebiotic diet (10
wt% GOS-lcFOS; ratio 9:1) in a murine model, resulting in
40% bifidobacteria of total bacterial load in fecal samples.
Fig. 3. Effect of prebiotic treatment (solid bars) on biliary bile salt composi-
tion compared with control rats (open bars). Data are expressed in means ⫾SD
of n⫽7 or 8 per group. No significant differences between prebiotic-treated
rats compared with controls. C, cholate; DC, deoxycholate; CDC, chenode-
oxycholate; ␣-M, ␣-muricholate; -M, -muricholate; ⌬22-M, ⌬22-muri-
cholate; HDC, hyodeoxycholate.
Fig. 4. Effect of prebiotic treatment on fecal bile salt excretion (A) and fecal
neutral sterol excretion (B). Prebiotic diet (solid bars), control diet (open bars).
Values are expressed in means ⫾SD, and n⫽8 per group. No significant
differences between prebiotic-treated animals compared with controls in any of
the parameters. Copr, coprostanol; epiCopr, epicoprostonol; Chol, cholesterol;
Dih-Chol, dihydrocholesterol.
Table 3. Pool size, fractional turnover rate, and synthesis
rates of cholate
Prebiotic Diet Control Diet
Fractional turnover rate, day
⫺1
0.28⫾0.04 0.26⫾0.07
Pool size, mol/100 g body wt 8.11⫾1.82 8.80⫾2.57
Synthesis rate, mol 䡠100 g
⫺1
䡠day
⫺1
2.25⫾0.55 2.23⫾0.72
Values are obtained by [
2
H
4
]-cholic acid isotope enrichment measurements
in plasma of rats fed the prebiotic diet or the control diet, as detailed in
MATERIALS AND METHODS.
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Surprisingly, the present study showed a contribution of
bifidobacteria of 1.3 and 1.5% in feces and colon content,
respectively, after prebiotic treatment. Thus, although the pres-
ently used diet resulted in an increase of lactobacillus in the
same range as mice and humans and a significant decrease in
fecal and colonic pH, the less pronounced bifidogenic response
could theoretically contribute to the observed lack of effect on
the enterohepatic circulation of bile salts.
It should be realized that the situation studied in the present
rat model differs in several aspects from the physiology in
human infants. Besides the less pronounced prebiotic response,
it cannot be excluded that the absence of a gallbladder in rats
influences the dynamics of the enterohepatic circulation of bile
salts. Furthermore, immaturity of intestinal absorption of nu-
trients (28) and bile salts (6) intervene with the enterohepatic
circulation of bile salts, which may render the dynamics of bile
salt metabolism in human infants not completely comparable to
the presently studied rat model. Finally, only in rats, the
primary bile salt chenodeoxycholate is further metabolized to
␣-muricholic acid and -muricholic acid, rendering the bile
salt pool more hydrophilic compared with the human situation.
In the present experiment, however, the amount of muricholic
bile salts measured in bile did not exceed 25%. Nevertheless,
we are aware of the possibility that one or more of the
aforementioned differences may limit the extrapolation of the
present results to the human situation.
Besides the addressed differences in bile salt physiology
between human infants and the presently used rat model,
human milk contains high concentrations of cholesterol (2.6 –
3.9 mmol/l) compared with formulas (0.3– 0.9 mmol/l) (5). The
colonic flora of human infants develops its dehydroxylating
capacity over the first months of life (2), whereas in the adult
rat the dehydroxylating bacteria are well established. It is
tempting to speculate that the larger bile salt pool size found in
breast-fed infants could be related to the substantially higher
dietary intake of cholesterol and not, based on our present
observations, on differences in dietary oligosaccharides.
Enhanced conjugation of bile salts by bile hydrolase-pro-
ducing bacteria such as lactobacilli could, in theory, be ex-
pected to increase the amount of deconjugated bile salts and the
subsequent fecal excretion hereof. In the present study, both
amount and composition of fecal bile salts were unaffected
upon prebiotic treatment. We hypothesize that this can be due
to the relative low contribution of lactobacillus and bifidobac-
teria to the total intestinal deconjugating capacity. Bile salt
hydrolase is apart from lactobacillus and bifidobacteria, also
detected in other bacterial species (12). Furthermore, efficient
passive absorption of deconjugated bile salts may have masked
a deconjugating, enhancing effect of prebiotic treatment.
The presently applied methodology allowed determination
of bile salt kinetic parameters in a physiologically uncompro-
mised animal model. The parameters of cholate kinetics deter-
mined in this experiment were in line with previous experi-
ments performed in rats by using the same method (19). Under
steady-state conditions, the bile salt pool size is regulated by
hepatic de novo synthesis of bile salts and by the efficiency by
Fig. 6. Distribution of triglycerides in plasma lipoprotein fractions in prebi-
otic-treated rats (closed symbols) and controls (open symbols). Lipoproteins
were separated by using fast-protein liquid chromatography (FPLC). Plasma
from all individual rats per group (n⫽8 per group) was pooled and subjected
to gel filtration by using Superose 6 columns. Triglyceride concentration in
each fraction was measured as described in Analytical procedures. The amount
of triglycerides in the separated fractions is comparable in the prebiotic-treated
rats and controls.
Fig. 5. Cholate synthesis rate, pool size, and fecal
excretion in rats fed a prebiotic diet (A)ora
control diet (B); n⫽7 or 8 per group. The cycling
time of the enterohepatic circulation is the time it
takes for a bile salt to undergo one full cycle. The
cycling time for cholate can be estimated by
dividing the cholate pool size by the biliary se-
cretion rate of cholate. The biliary secretion rate
of cholate was similar in both groups (data not
shown). The fraction of cholate lost per cycle was
calculated by dividing fractional cholate synthesis
rate by cholate cycling frequency. No significant
differences between prebiotic-treated rats com-
pared with controls in any of the parameters.
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which bile salts are reabsorbed in the intestine. Under steady-
state conditions, the amount lost via the feces is equal to the
amount of bile salts newly synthesized by the liver. A differ-
ence neither in cholate synthesis rate nor in fecal bile salt
excretion was observed, indicating that prebiotic treatment
does not affect the two important rate-limiting parameters of
the enterohepatic circulation. We also observed that the fecal
excretion of chenodeoxycholate-derived metabolites (muri-
cholate and hyodeoxycholate) was not altered either, suggest-
ing no effect of prebiotic treatment on the synthesis of the other
primary bile salt, chenodeoxycholate.
Apart from an important role in absorption of dietary lipids,
bile salts are critically involved in cholesterol homeostasis in
the body. Prebiotic substances are suggested to have a plasma
lipid-lowering effect in both animal and human studies. Serum
cholesterol concentrations in bottle-fed infants decreased as the
number of lactobacilli in their stools increased (18). Possible
mechanisms could be modulation of the intestinal bacterial
flora with production of short-chain fatty acids inhibiting
hepatic fatty acid or cholesterol synthesis or alteration of the
bacterial bile acid deconjugating capacity and a subsequent
altered fecal bile salt excretion (8). Studies in rats have con-
sistently shown a decrease in plasma triglycerides after a diet
supplemented with inulin and oligofructose (reviewed in Ref.
7). Data on plasma cholesterol, however, are less straightfor-
ward. A 10 wt% oligofructose-supplemented diet decreased
serum cholesterol concentrations in rats in one study (11), but
the addition of oligofructose to the diet of lean rats did not
induce a decrease in plasma cholesterol levels in another study
(21). Human studies investigating the effect of oligofructans on
lipid metabolism have shown variable results (reviewed in Ref.
8). Our prebiotic diet did not significantly affect plasma con-
centrations of cholesterol in rats, whereas it increased the
serum lathosterol concentration. Lathosterol is an intermediate
in the cholesterol synthesis pathway, which could suggest that
cholesterol synthesis was enhanced during prebiotic treatment.
However, cholesterol synthesis was not determined in this
experiment, and no other indications of increased cholesterol
synthesis were obtained. For example, the hepatic mRNA
expression of HMG-CoA reductase, encoding for the rate-
limiting enzyme of cholesterol synthesis, was not upregulated
by prebiotic treatment (data not shown). The presently used
diet did not affect serum triglycerides or hepatic triglycerides.
Also, the amount of triglycerides in the VLDL fraction of
lipoproteins was comparable in prebiotic-treated and control
rats, indicating that the present treatment did not affect hepatic
VLDL composition or triglyceride distribution over the various
plasma lipoproteins (Fig. 6). Rat studies on lipid metabolism or
bile salt metabolism using diets supplemented with the pres-
ently used GOS-lcFOS, have, to the best of our knowledge, not
been performed. In accordance with the present results, a study
recently performed in human infants showed unaffected
plasma levels of cholesterol and of triglycerides in infants fed
a formula supplemented with identical prebiotic substances in
the same ratio used in the present study (1).
In summary, we conclude that feeding rats a prebiotic diet
induces modification of the intestinal flora and decreases the
intestinal pH in the colon and the feces. However, the prebiotic
diet does not influence the metabolism of bile salts in rats. The
present data in rats do not support the hypothesis that prebiotics
naturally present in human milk contribute to a larger bile salt
pool size or altered pool kinetics.
ACKNOWLEDGMENTS
The authors thank M. Haarman for the quantitative real-time PCR analysis
of Bifidobacterium and Lactobaccilus.
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