1Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, and 2Division of Gastroenterology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School and Harvard Digestive Diseases Center, Boston, Massachusetts 02215; 3First Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan; and 4Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
Submitted 2 April 2003 ; accepted in final form 7 May 2003
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ABSTRACT |
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ATP-binding cassette transporters; bile flow; bile salt pool size; biliary secretion; mixed micelles
Nonetheless, several studies in the literature suggest that effects of different bile acids on cholesterol absorption may not be related simply to their solubilization capacity. In two animal studies (42, 49), the investigators noted that feeding CA promotes intestinal cholesterol absorption more efficiently than CDCA, yet in vitro the conjugates of the former solubilize less cholesterol at equilibrium than do the latter (40). Furthermore, other workers (11, 27) have suggested that neither CA nor its taurine conjugate (taurocholate; TC) added to chow or to cholesterol-enriched diets influence cholesterol absorption in the mouse. Together, these divergent studies indicate that the influence of individual bile acids on cholesterol absorption require systematic evaluation in a well-characterized animal model.
In the current study, we used gallstone-susceptible C57L mice carrying
several Lith alleles
(44) to investigate how
variations in the hydrophilic-hydrophobic balance of bile acids influence
intestinal cholesterol absorption. Our results demonstrate that the overall
hydrophilic-hydrophobic balance of the bile salt pool, and not the fed bile
acids per se, is the critical determinant of cholesterol absorption from the
small intestine. As inferred from gene expression studies, the intestinal
sterol efflux transporters Abcg5 and Abcg8 were upregulated
by feeding CA but not by the more hydrophilic -muricholic acid
(
-MCA) nor by the more hydrophobic deoxycholic acid (DCA). Most likely
this finding is related to the augmented mass of cholesterol and oxysterols
that were absorbed by being solubilized in a bile salt pool composed
principally of TC. Overall, our study establishes that natural hydrophilic
bile acids are powerful inhibitors of intestinal cholesterol absorption in the
mouse.
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MATERIALS AND METHODS |
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Animals and diets. Inbred C57L/J male mice, 6-8 wk old, were
purchased from Jackson Laboratory (Bar Harbor, ME) and were maintained in a
temperature-controlled room (22 ± 1°C) with a 12:12-h light-dark
cycle (lights on 6 AM-6 PM). Mice were allowed to adapt to the environment for
at least 2 wk before bile acid feeding and were provided free access to water
and rodent chow (Harlan Teklad Laboratory, Madison, WI) containing trace
(<0.02%) cholesterol (42).
Once mice reached 10 wk of age, they were fed chow or chow supplemented with
0.5% (by weight) each of CA, CDCA, DHCA, DCA, HCA, HDCA, -MCA,
-MCA,
-MCA, UDCA, or UCA. Bile acids were added in ethanol to the
powdered chow and dried for 48 h on trays under reduced pressure in a 60°C
oven. All experiments were executed according to accepted criteria for the
care and experimental use of laboratory animals, and euthanasia was consistent
with recommendations of the American Veterinary Medical Association. Protocols
were approved by the Institutional Animal Care and Use Committees of Harvard
University.
Cannulation of the common bile duct and collection of hepatic bile. For biliary lipid secretion studies (44), 12 groups of mice (n = 5/group) fed chow or chow supplemented with 0.5% of each bile acid (see Animals and diets) for 7 days were used. In brief, nonfasted mice were anesthetized with intraperitoneal injections of 35 mg/kg pentobarbital. The cystic duct was doubly ligated, and a cholecystectomy was performed. The common bile duct was then cannulated via a PE-10 polyethylene catheter. Hepatic bile was collected by gravity during the first hour of acute fistulation for measurement of biliary lipid secretion rates and molecular compositions of the bile salt pool. The circulating bile salt pool sizes were determined by employing 8-h biliary "washout" techniques (44). During surgery and bile collection, mouse body temperature was maintained at 37 ± 0.5°C with a heating lamp and monitored with a thermometer. After hepatic bile volumes were determined by weighing and employing a specific density of 1, bile samples were frozen and stored at -20°C for later lipid analyses (see Lipid analyses).
Determination of intestinal cholesterol absorption. For measurement of intestinal cholesterol absorption, 12 groups of mice (n = 10-22/group) were fed either chow or chow supplemented with 0.5% bile acid (see Animals and diets) for 7 days. We validated earlier that this feeding period was sufficient to reach a steady state of bile salt compositions in bile (44). Cholesterol absorption was determined by a plasma dual-isotope ratio method (41, 42, 46). In brief, nonfasted mice were anesthetized with pentobarbital. Exactly 2.5 µCi of [3H]cholesterol in 100 µl of Intralipid was injected intravenously via the jugular vein. Following this procedure, each animal was given, by gavage, an intragastric dose of 1 µCi of [14C]cholesterol in 150 µl of medium-chain triglyceride. After being dosed, mice were transferred to individual cages with wire-mesh bottoms, where they were free to eat chow or the appropriate bile acid and diet for an additional 3 days. Mice were then anesthetized and bled from the heart into heparinized microtubes. Ten milliliters of EcoLite were mixed with 100 µl of plasma and 100 µl of the original dosing mixture. Plasma ratios of the two radiolabels were determined by liquid scintillation spectrometry, and the percent cholesterol absorption was calculated (41, 42, 46).
Measurement of small intestinal transit times. Small intestinal
transit was measured according to previous methods
(46). In brief, mice
(n = 7/group) were housed individually in cages with wire-mesh
bottoms and fed chow or chow supplemented with 0.5% CA or -MCA until
being fasted 18 h before study. Exactly 2 µCi of [3H]sitostanol
in 100 µl of medium-chain triglyceride was instilled into the duodenum via
a transabdominal catheter implanted surgically 24 h earlier. Exactly 30 min
later, the mice were anesthetized with pentobarbital, the abdomen was opened
quickly, and the stomach, small and large intestines, and cecum were removed.
The small intestine was frozen with liquid nitrogen and cut into 20 equal
segments with a scalpel blade. The radioisotope was isolated from individual
intestinal segments, and radioactivity was determined by liquid scintillation
counting. Samples of stomach, cecum, and large intestine were also analyzed,
but none showed appreciable radioactivity. Intestinal transit time was
evaluated by a geometric center method as described elsewhere
(46).
Quantitative real-time PCR assays of Abcg5/g8, the sterol efflux transporters. Freshly harvested small intestines were flushed with ice-cold saline and cut into three segments with duodenum/jejunum/ileum length ratios of 1:3:2. In the middle of each segment, 1.5 cm of intestinal tissue was removed, and tissues from four mice per strain were pooled. Total RNA was extracted using RNeasy Midi (Qiagen, Valencia, CA), and reverse-transcription reaction was performed using the SuperScript II first-strand synthesis system (Invitrogen, Carlsbad, CA) with 5 µg of total RNA and random hexamers to generate cDNA. Primer Express software (Applied Biosystems, Foster City, CA) was used to design the following primers: mouse Abcg5 (AF312713 [GenBank] ), forward 5'-TTGAAATCTCTCGGATACAATCGA-3', reverse 5'-AAGCCCATTGTTTGCCCAT-3', probe 5'-CGTCTGACCTTTGGGATCCTCACCA-3'; and mouse Abcg8 (AF324495 [GenBank] ), forward 5'-TGGATAGTGCCTGCATGGATC-3', reverse 5'-AATTGAATCTGCATCAGCCCC-3', probe 5'-CAAGCTGTCGTTCCTCCGGTGGTG-3'. Real-time PCR assays were performed in triplicate on a GeneAmp 5700 sequence detection system (Applied Biosystems). The real-time PCR reaction contained, in a final volume of 50 µl, 1 µl of cDNA, optimized concentration (100-300 µM) of each primer, 200 µM of probe, and 25 µl of 2x TagMan Universal PCR Master Mix (Applied Biosystems). Relative mRNA levels were calculated by using the threshold cycle of an unknown sample against a standard curve with known copy numbers. To obtain a normalized target value, the target amount was divided by the endogenous reference quantity of rodent Gapdh as control (Applied Biosystems).
Lipid analyses. Total and individual bile salt concentrations were measured by HPLC according to the method of Rossi et al. (28). Biliary phospholipids were analyzed as inorganic phosphorus by the method of Bartlett (2). Cholesterol content in chow and biliary cholesterol levels were determined by HPLC (42). Hydrophobicity indices of individual bile acids as well as bile salts in hepatic biles were calculated according to Heuman's method (18).
Statistical methods. All data are expressed as means ± SD. Statistically significant differences among groups of mice fed chow or different bile acids were assessed by Student's t-test or by Mann-Whitney U-test. Analyses were performed with SuperANOVA software (Abacus Concepts, Berkeley, CA). Employing linear regression analyses, parameters associated significantly with the hydrophobicity index and percent cholesterol absorption were further assessed by a stepwise multiple regression analysis to identify independence of the associations. Statistical significance was defined as a two-tailed probability of <0.05.
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RESULTS |
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HPLC analyses were carried out during the first hour of biliary secretion
in individual hepatic biles of C57L mice fed bile acids for 7 days. This
revealed that all bile salts were taurine conjugated. The predominant bile
salt species in chow-fed mice were TC (49.7 ± 6.7%) and T--MC
(42.3 ± 5.7), whereas T-
-MC (1.4 ± 0.6), TUDC (2.6
± 1.6%), TCDC (0.6 ± 0.3%), and TDC (3.4 ± 1.3%) were
present in small concentrations. In mice fed UDCA, UCA, CA,
-MCA,
-MCA,
-MCA, HCA, or HDCA, the conjugates of the fed bile acids
became the predominant (50-86%) bile salts in bile
(Table 1). This indicates that
the fed bile acids were absorbed from the intestine, delivered in portal blood
to the liver, conjugated with taurine, and incorporated efficiently into the
bile salt pool. Furthermore, feeding UDCA, UCA,
-MCA,
-MCA, HCA,
and HDCA reduced TC and T-
-MC levels in bile significantly
(Table 1). Mice fed CA display
a marked increase in TC (83.3 ± 4.7%) and a strong decrease in
T-
-MC concentrations (3.9 ± 1.6%). In certain instances, bile
acid biotransformation occurred consequent to bile acid feeding: for example,
during CDCA administration, T-
-MC (64.2 ± 1.8%) became the major
constituent of the bile salt pool, followed by TCDC (24.8 ± 8.3%), with
TC showing a significant decrease (5.2 ± 1.5%). When mice were fed DCA,
the formation of TC (66.2 ± 8.8%) from rodent hepatic
7
-hydroxylation was appreciably larger than that of TDC (26.5 ±
7.9%), and both were accompanied by a significant decrease in T-
-MC (4.7
± 2.2%). Feeding DHCA significantly increased TDC (18.0 ± 3.4%),
TCDC (9.1 ± 2.3%), and TUDC (7.1 ± 3.7%), but TC remained
unchanged and T-
-MC (12.2 ± 3.0%) decreased. These
biotransformations occurred as a result of the rich capacity of the mouse
liver to hydroxylate the steroid nucleus of both endogenous and exogenous bile
acids in the 6
and 7
positions. Because of hepatic metabolism of
specific bile acids, hydrophobicity indices of biliary bile salt pools
(Table 1) differed markedly
from the individually fed bile acids (Fig.
1).
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Influence of bile salt hydrophobicity on bile flow, biliary lipid
secretion, and bile salt pool sizes.
Table 2 lists mean bile flow
rates at 7 days during acute (first-hour) interruption of the enterohepatic
circulation in mice fed chow or chow supplemented with 0.5% bile acid. Bile
flow rates demonstrated a great degree of variability (87-119 µl ·
min-1 · kg-1) but
approximated the results observed with chow [P = not significant
(NS)]. Table 2 also summarizes
biliary cholesterol, phospholipid, and bile salt secretion rates. Compared
with lipid secretion rates of chow-fed mice (cholesterol = 10.2 ± 3.5
and phospholipid = 25.6 ± 4.5 µmol ·
h-1 · kg-1), biliary
cholesterol outputs were markedly decreased (8.1-8.9 µmol ·
h-1 · kg-1) with unaltered
phospholipid outputs (26.2-31.9
µmol·h-1·kg-1) in
mice fed hydrophilic bile acids (UDCA, UCA, -,
-, and
-MCA, and HCA). In contrast, administration of more hydrophobic bile
acids (CA and DCA) increased both biliary cholesterol (11.2-12.6 µmol
· h-1 · kg-1) and
phospholipid outputs (29.1-31.2 µmol · h-1
· kg-1). We noted that dietary supplementation
with the more hydrophilic DHCA markedly augmented biliary cholesterol
secretion by 40% (14.2 ± 5.6 µmol · h-1
· kg-1) but decreased biliary phospholipid
secretion by 10% (23.4 ± 4.9 µmol · h-1
· kg-1). Because its biotransformation produced
T-
-MC, CDCA reduced biliary cholesterol secretion (9.0 ± 3.3
µmol · h-1 ·
kg-1) without altering biliary phospholipid output (25.6
± 4.5 µmol · h-1 ·
kg-1). Figure
2A shows that there are significant and positive linear
correlations between the hydrophobicity indices of the biliary bile salt pools
and biliary cholesterol outputs (P < 0.0001, r = 0.91).
Figure 2B shows a
similar positive correlation for cholesterol/phospholipid molar ratios in
hepatic bile and hydrophobicity indices (P < 0.001, r =
0.85). This suggests that decreasing hydrophobicity of the bile salt pool
reduces biliary cholesterol secretion significantly in mice
(5). No correlations were found
between the hydrophobicity indices of the bile salt pools and the biliary
outputs of either phospholipids or bile salts. The range of bile salt outputs
(Table 2) in mice fed various
bile acids (159.3-200.7 µmol · h-1 ·
kg-1) was comparable with those on chow (157.9 ±
56.8 µmol · h-1 ·
kg-1) and showed no statistically significant
differences. Furthermore, compared with chow, we did not find any significant
differences in sizes of circulating (3.2-3.9 µmol) or total (4.7-5.3
µmol) bile salt pools in mice fed bile acids.
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Influence of bile acid species on intestinal cholesterol
absorption. Figure 3
displays percent cholesterol absorption for each of the dietary bile acids
tested as well as the chow control. Compared with cholesterol absorption on
the chow diet (37 ± 5%), percent cholesterol absorption was inhibited
significantly in mice fed the seven hydrophilic bile acids
(Fig. 3) increasing in the rank
order: -MCA (11 ± 2%),
-MCA (12 ± 2%), HCA (14
± 3%),
-MCA (15 ± 3%), UCA (17 ± 4%), UDCA (19
± 5%), and HDCA (30 ± 4%). In contrast, feeding the hydrophobic
bile acids (DCA and CA) led to significant increases in intestinal cholesterol
absorption (51 ± 6 and 63 ± 7%, respectively). Because of
hepatic biotransformations with resulting alterations in the hydrophobicity
indices of the bile salt pools (Table
2), feeding CDCA, a more hydrophobic bile acid, significantly
reduced cholesterol absorption to 25 ± 3% because bile salt pool became
more hydrophilic from increases in T-
-MC
(Table 1). In contrast, feeding
DHCA, the more hydrophilic bile acid, significantly increased cholesterol
absorption to 45 ± 6% because of increasing hydrophobicity of the bile
salt pool (Table 1).
Figure 4 displays a significant
linear (P < 0.0001), positive (r = 0.95) correlation
between the percent cholesterol absorption and hydrophobicity indices of the
bile salt pool. As a corollary, the efficiency of intestinal cholesterol
absorption is curtailed significantly by decreasing hydrophobicity of the bile
salt pool.
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Small intestinal transit times. We found that small intestinal
transit, as inferred from the distributions of radioactivity in the small
intestine, were essentially unchanged in mice fed the relatively hydrophobic
CA or hydrophilic -MCA compared with chow (data not shown). Mean values
for the geometric centers of the profiles were 10.5 ± 1.1 for chow,
11.0 ± 0.9 for CA, and 10.7 ± 1.0 for
-MCA (P =
NS). These results demonstrate that feeding neither hydrophobic nor
hydrophilic bile acids alters small intestinal transit time in the mouse.
Gene expression of intestinal sterol efflux transporters.
Figure 5 shows relative mRNA
levels of Abcg5 and Abcg8, two genes that encode the
half-transporters for efflux of sterols from enterocytes in duodenum, jejunum,
and ileum of C57L mice fed chow and chow supplemented with 0.5% -MCA,
CA, or DCA for 7 days. Gene expression levels of Abcg5 and
Abcg8 in the duodenum were lower than in jejunum or ileum but were
essentially identical among the four groups of mice. In the jejunum and ileum,
gene expression levels were similar in mice fed
-MCA and DCA compared
with chow feeding but were increased significantly by CA feeding.
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DISCUSSION |
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When an individual bile acid is fully ionized, its hydrophobicity index
(18) is determined by the
number, position, and orientation of hydroxyl groups, which are crucial
factors in determining their physical-chemical and physiological functions.
Bile salts, together with ionized and nonionized fatty acids,
monoacylglycerides, lysophospholipids, and unesterified cholesterol, form
intestinal mixed micelles (17,
34). These micelles function
as a concentrated reservoir and transport vehicle for cholesterol across the
unstirred water layer toward the brush border of the small intestine to
facilitate uptake of monomeric cholesterol by the enterocyte. As shown in the
present study, the efficiency of intestinal cholesterol absorption was
inhibited significantly by feeding hydrophilic bile acids (Figs.
3 and
4). Within the intestinal
lumen, the presence of hydrophilic bile salts may reduce solubility of
cholesterol by inducing phase separation of the sterol from mixed micelles to
a coexisting liquid crystalline vesicle phase. Most likely, hydrophilic bile
salts facilitate incorporation of cholesterol molecules into a stable liquid
crystalline/vesicle phase (17,
34) from which they are poorly
absorbed by small intestinal enterocytes
(8). In contrast, feeding CA
increased hydrophobicity of bile salts in bile, which markedly increased
micellar cholesterol solubility
(40) and thereby augmented
cholesterol absorption (Ref.
42 and
Fig. 3). This suggests that
hydrophobic bile acids are more effective promoters of cholesterol absorption
than hydrophilic bile acids. However, CDCA, a more hydrophobic bile acid than
CA, paradoxically caused a reduction of intestinal cholesterol absorption
since it was extensively biotransformed in the liver to the hydrophilic
T--MC (Table 1). In
contrast, the very hydrophilic DHCA augmented cholesterol absorption markedly
because of its biotransformation into TDC and TCDC, both hydrophobic partners
in the bile salt pool. Because of their novel biotransformations in this
rodent model, the hydrophobicity indices of individual bile acids fed cannot
invariably forecast their cholesterol absorption efficiency. It is clear that
a knowledge of the hydrophobicity index of the bile salt pool is necessary to
predict the influence of individual bile acids on intestinal cholesterol
absorption.
Biliary secretion rates of bile salts can also demonstrate marked effects on intestinal cholesterol absorption (29, 39, 43, 47), most likely related to the increased micellar solubilization capacity of an augmented flow of bile salts within the upper small intestine. We (44, 46) have observed that gallstone-susceptible C57L mice display significantly higher biliary bile salt outputs and intestinal cholesterol absorption compared with gallstone-resistant AKR mice. However, we found in this study that feeding various bile acids did not alter bile salt pool sizes or biliary outputs significantly (Table 2). This is consistent with an adaptive response to alterations in the bile salt pool sizes and outputs at the level of the ileal apical sodium-dependent bile acid transporter (1). In addition, we observed a significant positive correlation between hydrophobicity of the biliary bile salts and biliary outputs of cholesterol (Fig. 2A), suggesting that, compared with hydrophobic bile acids, not only do hydrophilic bile acids curtail intestinal cholesterol absorption, but they are also less efficient in promoting biliary cholesterol secretion (Table 2). These hydrophilic bile acids, as might be expected, are significantly associated with decreases in cholesterol/phospholipid ratios in bile (Fig. 2B) and consequently in the upper small intestine, where during digestion they may reduce transfer of cholesterol molecules from intermediate liquid crystalline/vesicle phases to mixed micellar phases (9, 17, 34). Overall, enrichment of bile with hydrophilic bile acids may reduce both concentration and bioavailability of intraluminal cholesterol molecules for absorption by enterocytes.
The newly identified intestinal ATP-binding cassette transporters ABCG5 and
ABCG8 (3,
21) in humans are apical
cholesterol export pumps that efflux cholesterol (and most phytosterols) from
enterocytes back to the intestinal lumen and reduce their fractional
absorption (50). We observed
that feeding the hydrophilic -MCA did not influence expression of the
genes for these two transporters. Nonetheless, feeding CA, and to a lesser
extent DCA (Fig. 5), did so
significantly by upregulating mRNA expression of the jejunal and ileal
Abcg5 and Abcg8. This response is most likely an indirect
effect because expression of these ABC transporters is highly sensitive to the
mass of absorbed cholesterol
(3,
10), which is promoted
principally by fed CA (Fig. 3).
Another possible explanation is that CA feeding may markedly increase
intestinal absorption of biliary (and dietary) oxysterols
(15) that bind and activate
the oxysterol receptor LXR, a transcriptional regulator of these transporter
genes (26). Consistent with
our results, Watt and Simmonds
(48) observed many years ago
that TC infused intraduodenally in bile-diverted rats produced significantly
higher uptake and transport of intestinal cholesterol compared with TDC or
TUDC. This may explain why intestinal Abcg5 and Abcg8
expression increased significantly only during CA feeding as a result of
increased sterol uptake. Our data do not exclude the possibility that CA per
se may have a specific enhancing effect on the expression of the genes for
these sterol efflux transporters. We took note that in the chow-fed mouse, the
mRNA levels of Abcg5/g8 are slightly higher in the jejunum and
duodenum compared with the ileum as assayed by Northern blot analysis
(3,
26,
50). In the present study, the
expression levels of Abcg5/g8 are somewhat greater in the jejunum and
ileum than in the duodenum as measured by our highly precise quantitative
real-time PCR techniques. A possible reason for these differences is that we
cut the small intestines into three segments with length ratios of 1:3:2
(duodenum/jejunum/ileum). Total RNA was extracted from the middle 1.5 cm of
intestinal tissue in each segment. Other investigators
(3,
26,
50) extracted total RNA from
the entire intestinal mucosa of three segments with length ratios of 1:1:1
(duodenum/jejunum/ileum). Another possible explanation is suggested by the
different genetic backgrounds of the murine strains employed in previous and
present studies.
The importance of small intestinal transit rates as a determining factor of cholesterol absorption has been validated in human (25) and animal studies (Ref. 36 and Wang DQ-H, Kopin AS, and Carey MC, unpublished observations). Reynier et al. (27) concluded that intestinal transit time is similar in mice whether CA, CDCA, or UDCA is fed, as evidenced by the rates of fecal excretion of unabsorbed cholesterol and its derivatives together with a carmine red marker. In the present study, our new and validated methodology (46) adapted to luminal lipid transit in the mouse allowed us to observe that feeding bile acids with different hydrophobicities did not influence small intestinal transit times in C57L mice.
In summary, the overall results of our study establish that modulating the
hydrophilic-hydrophobic balance of the bile salt pool profoundly influences
intestinal cholesterol absorption. We speculate that the mechanisms whereby
hydrophilic bile acids inhibit intestinal cholesterol absorption is via the
uptake step by curtailing micellar cholesterol solubilization intraluminally.
Hence, decreasing the hydrophobicity index of the biliary bile salt pool
reduces cholesterol's bioavailability for absorption by enterocytes. Although
the size and composition of the intestinal bile salt pool exert major
influences on the amount of chylomicron cholesterol reaching the liver from
the intestine, the pharmacological inhibition of various proteins involved in
other steps in the absorption process, e.g., the putative sterol transporter
in the brush-border membrane, acyl-CoA:cholesterol acyltransferase isoform 2,
or microsomal triglyceride transfer protein in the enterocyte, can also effect
a dramatic change in cholesterol absorption without there being any change in
the amount or species of bile salts in the intestinal pool. Nonetheless, our
current study suggests that natural hydrophilic bile acids efficiently
suppress cholesterol absorption and may act as potent plasma and biliary
cholesterol-lowering agents, even more so than UDCA, for prevention of
cholesterol deposition diseases in humans. An example of the latter is our
recent study (47) showing that
-MCA prevents cholesterol gallstone formation in gallstone-susceptible
C57L mice by chronically inhibiting intestinal cholesterol absorption.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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D. Q.-H. Wang is a recipient of a New Scholar Award from the Ellison Medical Foundation (1999-2003).
This paper was presented in part at the Annual Meeting of the American Association of the Study for Liver Diseases, Dallas, TX, in 1999 and published as an abstract in Hepatology (30: 395A, 1999).
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FOOTNOTES |
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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.
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REFERENCES |
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