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• W1992305758 abstract "Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1α and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation. Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1α and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation. Despite decades of research, gallstone disease remains a significant health problem worldwide, particularly in the female adult population. In the United States and European countries, 10–20% of adults develop gallstones, mostly cholesterol-rich stones (1Dowling R.H. Review. Pathogenesis of gallstones.Aliment. Pharmacol. Ther. 2000; 2: 39-47Google Scholar). Even though cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect (2LaMont J.T. Carey M.C. Cholesterol gallstone formation. II. Pathobiology and pathomechanics.Prog. Liver Dis. 1992; 10: 165-191Google Scholar), the molecular pathogenesis of cholesterol gallstone formation remains poorly understood. Disorders contributing to the cholesterol supersaturation of bile could result from a) uncoupling of phospholipid and/or cholesterol secretion from bile acid secretion or b) augmentation of hepatic cholesterol synthesis or uptake. The source of the excess cholesterol is unclear, but it is probably derived from lipoprotein (3Fuchs M. Ivandic B. Muller O. Schalla C. Scheibner J. Bartsch P. Stange E.F. Biliary cholesterol hypersecretion in gallstone-susceptible mice is associated with hepatic up-regulation of the high-density lipoprotein receptor SRBI.Hepatology. 2001; 33: 1451-1459Google Scholar) rather than from synthesis (4Empen K. Lange K. Stange E.F. Scheibner J. Newly synthesized cholesterol in human bile and plasma: quantification by mass isotopomer distribution analysis.Am. J. Physiol. 1997; 272: G367-G373Google Scholar). Furthermore, evidence is available that c) alterations of intestinal bile acid recycling (5Shoda J. He B.F. Tanaka N. Matsuzaki Y. Osuga T. Yamamori S. Miyazaki H. Sjovall J. Increase of deoxycholate in supersaturated bile of patients with cholesterol gallstone disease and its correlation with de novo syntheses of cholesterol and bile acids in liver, gallbladder emptying, and small intestinal transit.Hepatology. 1995; 21: 1291-1302Google Scholar), d) prolonged intestinal transit (5Shoda J. He B.F. Tanaka N. Matsuzaki Y. Osuga T. Yamamori S. Miyazaki H. Sjovall J. Increase of deoxycholate in supersaturated bile of patients with cholesterol gallstone disease and its correlation with de novo syntheses of cholesterol and bile acids in liver, gallbladder emptying, and small intestinal transit.Hepatology. 1995; 21: 1291-1302Google Scholar), e) altered bile salt synthesis, and f) gallbladder motility defects are important in human gallstone formation and biliary pain (6Brand B. Lerche L. Stange E.F. Symptomatic or asymptomatic gallstone disease: is the gallbladder motility the clue?.Hepatogastroenterology. 2002; 49: 1208-1212Google Scholar). Accordingly, the pools of cholic and chenodeoxycholic acid have been found to be reduced in most normal weight gallstone patients, whereas that of deoxycholic acid is often increased (7Berr F. Pratschke E. Fischer S. Paumgartner G. Disorders of bile acid metabolism in cholesterol gallstone disease.J. Clin. Invest. 1992; 90: 859-868Google Scholar). Cholic acid is almost completely 7-α-dehydroxylated to deoxycholic acid by anaerobic bacteria in the colon (8Morris J.S. Low-Beer T.S. Heaton K.W. Bile salt metabolism and the colon.Scand. J. Gastroenterol. 1973; 8: 425-431Google Scholar), and ∼30–40% of this deoxycholic acid is absorbed from the intestinal lumen (7Berr F. Pratschke E. Fischer S. Paumgartner G. Disorders of bile acid metabolism in cholesterol gallstone disease.J. Clin. Invest. 1992; 90: 859-868Google Scholar). The expansion of the deoxycholic acid pool observed in gallstone patients (7Berr F. Pratschke E. Fischer S. Paumgartner G. Disorders of bile acid metabolism in cholesterol gallstone disease.J. Clin. Invest. 1992; 90: 859-868Google Scholar) could possibly result from increased cholic acid synthesis, small intestinal spill of cholic acid into the colon, bacterial overgrowth, or a change in bacterial flora favoring cholic acid deconjugation.Bile acids synthesized in the liver undergo a very efficient cycling between the liver and the intestine. A key component of the enterohepatic circulation of bile salts is the intestinal reclamation of bile acids (9Shneider B.L. Intestinal bile acid transport: biology, physiology, and pathophysiology.J. Pediatr. Gastroenterol. Nutr. 2001; 32: 407-417Google Scholar). Both conjugated and unconjugated bile acids are passively recovered along the entire axis of the intestine. In the terminal ileum, the bulk of conjugated bile acids are reabsorbed by an active sodium-dependent dimeric transport system [the ileal apical sodium bile acid transporter (ASBT)] (10Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter.J. Biol. Chem. 1994; 269: 1340-1347Google Scholar). Bile acids escaping active reabsorption in the distal ileum are 7-α-dehydroxylated and deconjugated by colonic bacteria; these secondary bile acids may then reach the portal circulation by passive diffusion along the colon. Decreased bile acid uptake as a result of genetic disruption of ASBT activity (11Heubi J.E. Balistreri W.F. Fondacaro J.D. Partin J.C. Schubert W.K. Primary bile acid malabsorption: defective in vitro ileal active bile acid transport.Gastroenterology. 1982; 83: 804-811Google Scholar, 12Wong M.H. Oelkers P. Dawson P.A. Identification of a mutation in the ileal sodium-dependent bile acid transporter gene that abolishes transport activity.J. Biol. Chem. 1995; 270: 27228-27234Google Scholar), ileal diseases, ileal resection, or congenital primary bile acid malabsorption may lead to bile acid pool depletion and the subsequent development of cholesterol gallstones (13Tougaard L. Giese B. Pedersen B.H. Binder V. Bile acid metabolism in patients with Crohn's disease in terminal ileum.Scand. J. Gastroenterol. 1986; 21: 627-633Google Scholar, 14Heuman R. Sjodahl R. Tobiasson P. Tagesson C. Postprandial serum bile acids in resected and non-resected patients with Crohn's disease.Scand. J. Gastroenterol. 1982; 17: 137-140Google Scholar, 15Nyhlin H. Brydon G. Danielsson A. Westman S. Clinical application of a selenium (75Se)-labelled bile acid for the investigation of terminal ileal function.Hepatogastroenterology. 1984; 31: 187-191Google Scholar).After being actively reabsorbed from the ileal lumen by the integral brush border membrane glycoprotein ASBT, bile acids are presumed to be associated with the 14 kDa ileal lipid binding protein (ILBP) for cytosolic transport (16Kramer W. Girbig F. Gutjahr U. Kowalewski S. Jouvenal K. Müller G. Tripier D. Wess G. Intestinal bile acid absorption.J. Biol. Chem. 1993; 268: 18035-18046Google Scholar, 17Gong Y.Z. Everett E.T. Schwartz D.A. Norris J.S. Wilson F.A. Molecular cloning, tissue distribution, and expression of a 14-kDa bile acid-binding protein from rat ileal cytosol.Proc. Natl. Acad. Sci. USA. 1994; 91: 4741-4745Google Scholar). The secretion of bile salts from the basolateral surface of enterocytes into the splanchnic circulation has not yet been fully clarified. Recently, it was shown that besides an alternatively spliced truncated form of ASBT (18Lazaridis K.N. Pham L. Tietz P. Marinelli R.A. deGroen P.C. Levine S. Dawson P.A. LaRusso N.F. Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter.J. Clin. Invest. 1997; 100: 2714-2721Google Scholar) and/or multidrug resistance protein 3 (19Hirohashi T. Suzuki H. Takikawa H. Sugiyama Y. ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3).J. Biol. Chem. 2000; 275: 2905-2910Google Scholar), the organic solute transporter α-β (20Dawson P.A. Hubbert M. Haywood J. Craddock A.L. Zerangue N. Christian W.V. Ballatori N. The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter.J. Biol. Chem. 2005; 280: 6960-6968Google Scholar) seems to be involved in the efflux of bile acids in the intestine.Binding sites of several nuclear receptors [e.g., hepatic nuclear factor 1α (HNF1α) and peroxisome proliferator-activated receptor α (PPARα)] have been identified in the promoter of ASBT (21Shih D.Q. Bussen M. Sehayek E. Ananthanarayanan M. Shneider B.L. Suchy F.J. Shefer S. Bollileni J.S. Gonzalez F.J. Breslow J.L. et al.Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism.Nat. Genet. 2001; 27: 375-382Google Scholar, 22Jung D. Fried M. Kullak-Ublick G.A. Human apical sodium-dependent bile salt transporter (SLC10A2) gene is regulated by the peroxisome proliferator-activated receptor alpha 4.J. Biol. Chem. 2002; 277: 30559-30566Google Scholar, 23Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Google Scholar). HNF1α seems to be of particular importance. For example, the minimal ASBT promoter construct that confers full transcriptional activity contains three functional HNF1α recognition sites (22Jung D. Fried M. Kullak-Ublick G.A. Human apical sodium-dependent bile salt transporter (SLC10A2) gene is regulated by the peroxisome proliferator-activated receptor alpha 4.J. Biol. Chem. 2002; 277: 30559-30566Google Scholar). Furthermore, site-directed mutagenesis of HNF1α binding sites in the ASBT promoter abrogates transcription activity, and HNF1α knockout mice are characterized by the absence of ileal ASBT expression along with marked fecal bile acid wasting (21Shih D.Q. Bussen M. Sehayek E. Ananthanarayanan M. Shneider B.L. Suchy F.J. Shefer S. Bollileni J.S. Gonzalez F.J. Breslow J.L. et al.Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism.Nat. Genet. 2001; 27: 375-382Google Scholar). In addition, results of in vitro studies suggest a PPARα-dependant induction of human ASBT gene expression (22Jung D. Fried M. Kullak-Ublick G.A. Human apical sodium-dependent bile salt transporter (SLC10A2) gene is regulated by the peroxisome proliferator-activated receptor alpha 4.J. Biol. Chem. 2002; 277: 30559-30566Google Scholar). A transcriptional activation of ILBP gene expression has been assigned to the direct effect of a complex of bile acids and the farnesoid X-receptor (FXR) (24Grober J. Zaghini I. Fujii H. Jones S.A. Kliewer S.A. Willson T.M. Ono T. Besnard P. Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer.J. Biol. Chem. 1999; 274: 29749-29754Google Scholar). In addition, binding sites of other nuclear transcription factors, such as the liver X-receptor and the sterol element regulator protein 1 (24Grober J. Zaghini I. Fujii H. Jones S.A. Kliewer S.A. Willson T.M. Ono T. Besnard P. Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer.J. Biol. Chem. 1999; 274: 29749-29754Google Scholar, 25Zaghini I. Landrier J.F. Grober J. Krief S. Jones S.A. Monnot M.C. Lefrere I. Watson M.A. Collins J.L. Fujii H. et al.Sterol regulatory element-binding protein-1c is responsible for cholesterol regulation of ileal bile acid-binding protein gene in vivo. Possible involvement of liver-X-receptor.J. Biol. Chem. 2002; 277: 1324-1331Google Scholar), have been identified in the ILBP promoter.Because little is known about the expression of these bile acid transporters in human cholelithiasis, the main objective of this study was to determine the expression of ASBT and ILBP in the ileum of patients with gallstones and controls. In addition, protein levels of the nuclear transcription factors HNF1α, FXR, and PPARα were measured.MATERIALS AND METHODSSubjectsThe study protocol was approved by the Ethics Committee of the University of Tuebingen (Tuebingen, Germany). Informed consent was obtained from all subjects. Subjects included had a) no history of taking lipid-lowering drugs or drugs interfering with bile acid uptake, b) no known medical conditions affecting lipid metabolism (e.g., diabetes), c) normal liver function tests and absence of signs of hemolysis or other conditions associated with pigment stones, d) no medical records indicating findings affecting bile acid uptake (e.g., inflammatory bowel diseases) or colonic surgery, and e) no clinical indication of impaired nutritional status. A total of 41 female subjects, all of whom were undergoing colonoscopy for medical reasons, were included in the study. Seventeen subjects had gallstones, and 24 gallstone-free subjects served as controls. None of the subjects had symptomatic gallstone disease. Six of the 17 gallstone carriers had a known history of gallstone disease that was first diagnosed by routinely performed abdominal ultrasound; however, in addition, the presence or absence of gallstones was confirmed in all patients and controls by ultrasound on the day of colonoscopy. None of the gallstone carriers suffered from a disease associated with pigment gallstones (e.g., hemolytic syndromes), so most likely patients has cholesterol gallstones in this cohort. The characteristics of patients and controls did not differ between groups and are summarized in Table 1. None of the patients or controls displayed any histological signs of inflammation in the ileum. Using standard pinch forceps, eight biopsies were obtained from the ileum within 10 cm of the ileocecal valve and either placed immediately in liquid nitrogen and stored at −80°C until use or fixed in 10% buffered formalin.TABLE 1.Characteristics of study participantsCharacteristicAll womenOverweight womenNormal weight womenControlGallstone carriersControlGallstone carriersControlGallstone carriersNumber241789168Age (years)57 ± 264 ± 355 ± 462 ± 559 ± 266 ± 4Body mass index (kg/m2)24.0 ± 0.825.6 ± 0.827.5 ± 0.828.6 ± 0.722.0 ± 0.722.8 ± 0.4Triglyceride (mg/dl) (1–200)aNormal range.151 ± 14122 ± 13167 ± 21122 ± 19128 ± 10130 ± 18Cholesterol (mg/dl) (140–240)aNormal range.187 ± 12197 ± 7187 ± 18204 ± 12190 ± 15197 ± 9Bilirubin (mg/dl) (0.2–1.4)aNormal range.0.53 ± 0.050.73 ± 0.110.49 ± 0.050.68 ± 0.150.59 ± 0.060.73 ± 0.11Values are given as means ± SEM.a Normal range. Open table in a new tab Isolation of RNA and proteinTotal RNA and protein were isolated using Trizol (Invitrogen), based on the single-step method described by Chomczynski and Sacchi (26Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Google Scholar).Real-time reverse transcription-polymerase chain reactionThe integrity, quality, and quantity of RNA were analyzed by subjecting samples to gel electrophoresis (1.2% agarose gel) and measuring absorption at 260 and 280 nm. First-strand cDNA was synthesized from 400 ng of total RNA by the random primer method using an avian myeloblastosis virus (AMV)-reverse transcriptase system (Promega). Using real-time PCR, amplification of ASBT (sense primer, 5′-ATGCAGAACACGCAGCTATG-3′; antisense primer, 5′-GCTCCGTTCCATTTTCTTG-3′) and ILBP (sense primer, 5′-CCTCAGCAACTGGGAGAGTTTAT-3′; antisense primer, 5′-TTTTATTGGTGGGTTTGTAGCTC-3′) was performed with the LightCycler sequence detection system (Roche Molecular Biochemicals). Because SYBR Green was used for measurements of amplification-associated fluorescence, RT-PCR products were also analyzed on ethidium bromide-stained agarose gels to ensure that a single amplicon of the expected size was obtained. Villin amplification was used to account for variability in the initial quantities of cDNA and to account for the varying numbers of enterocytes in biopsy specimens (sense primer, 5′-AGCCAGATCACTGCTGAGGT-3′; antisense primer, 5′-TGGACAGGTGTTCCTCCTTC-3′). The relative quantity for any given transcript was calculated using the second derivative maximum method (LightCycler software 3.5) according to the manufacturer's instructions. Individual quantities of each sample were determined in triplicate.Western blot analysisAntibodies used for the detection of human ASBT and human ILBP were kind gifts of P. Dawson and W. Kramer, respectively. Primary antibodies for the detection of FXR, HNF1α, and PPARα were purchased from Santa Cruz Biotechnology. Protein concentration was determined using a commercial kit (Bio-Rad). Protein extracted from biopsies obtained from 10 different controls was pooled and used as a standard on each blot (see supplementary Fig. 1). Samples of 6–12 μg of total protein and serial dilutions of standard protein (5, 10, and 20 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk in TBS-Tween 20 and probed with dilutions of primary antibody in 5% nonfat milk, TBS-Tween 20. After being washed three times, immunoblots were incubated with peroxidase-conjugated anti-rabbit IgG and anti-chicken IgG (both Dianova) and exposed to a chemiluminescent reagent (SuperSignal® West Dura; Pierce). Bands were photographed (Camera LAS 1000; Fuji), and immunoquantitation was accomplished by densitometric analysis using the software AIDA (Raytest). Furthermore, to account for variability in the amounts of enterocytes in biopsy specimens, villin contents of all samples were determined using a primary antibody against human villin (Sigma) and a secondary peroxidase-conjugated anti-mouse IgG (Oncogene). All measurements were carried out in duplicate.Fig. 1.Apical sodium bile acid transporter (ASBT) protein levels and mRNA expression in ileal mucosa biopsies of women. A: Quantitative analysis of ASBT protein levels of gallstone carriers (G) and controls (C). Protein levels of villin were determined to normalize to the amount of enterocytes in biopsies. Data are means ± SEM. * P < 0.05 compared with controls. B, C: Representative Western blot (B) and immunostaining (C) of ASBT in terminal ileum biopsy specimens of female normal weight gallstone carriers and controls. D: Relative ASBT mRNA expression, normalized to villin. Expression of ASBT and villin was measured by real-time RT-PCR. Data are means ± SEM. * P < 0.05 compared with controls.View Large Image Figure ViewerDownload (PPT)Fig. 1.Apical sodium bile acid transporter (ASBT) protein levels and mRNA expression in ileal mucosa biopsies of women. A: Quantitative analysis of ASBT protein levels of gallstone carriers (G) and controls (C). Protein levels of villin were determined to normalize to the amount of enterocytes in biopsies. Data are means ± SEM. * P < 0.05 compared with controls. B, C: Representative Western blot (B) and immunostaining (C) of ASBT in terminal ileum biopsy specimens of female normal weight gallstone carriers and controls. D: Relative ASBT mRNA expression, normalized to villin. Expression of ASBT and villin was measured by real-time RT-PCR. Data are means ± SEM. * P < 0.05 compared with controls.View Large Image Figure ViewerDownload (PPT)ImmunhistochemistryFormalin-fixed biopsies were embedded in paraffin and cut into 3 μm sections. The immunohistochemical localization the ASBT was performed using the EnVision technique (EnVision™ Detection Kit; DAKO), according to the manufacturer's instructions. Briefly, tissue sections were deparaffinized in xylene and rehydrated in a series of ethanol solutions (100–70%). Endogenous peroxidase activity was blocked by the addition of 0.9% H2O2 in methanol for 30 min, and sections were washed in Tris saline (pH 7.6), microwaved, and again washed with Tris saline (0.15 M). Slides were then incubated with the polyclonal rabbit anti-ASBT primary antibody (overnight), followed by a 30 min incubation with a horseradish peroxidase-labeled polymer secondary antibody (dextran backbone coupled with peroxidase and a multifunctional secondary antibody; EnVision™). To stop the reaction, sections were rinsed with tap water. Slides were then counterstained with hemalaun for 15 s.For the immunochemical detection of ILBP, the EnVision technique described above was modified slightly, as the primary antibody used for the detection of ILBP was raised in chicken. Therefore, before incubating with EnVision solution and the secondary antibody, dextran-enzyme complex sections were incubated with an unconjungated secondary rabbit anti-chicken antibody.To ensure the specificity of staining of ASBT, the following controls were used: 1) omission of primary antibody; 2) omission of horseradish peroxidase-labeled secondary antibody; 3) omission of EnVision complex; 4) omission of primary and secondary antibody as well as EnVision complex; 5) omission of primary and secondary antibody; 6) omission of secondary antibody and EnVision complex; and 7) positive controls using tissue sections of the terminal ileum and antibodies against CK20. All of these controls were used to ensure the specificity of staining of ILBP as well.Statistical analysisResults are presented as means ± SEM. The statistical comparison between groups was performed using the Mann-Whitney U-test. Correlation was tested by calculating Spearman's rank-order correlation coefficient. P < 0.05 was considered statistically significant.RESULTSASBT: protein levels and mRNA expression in terminal ileumFigure 1 and supplementary Fig. 2 summarize the results of protein and mRNA measurements performed in biopsy specimens obtained from gallstone carriers and controls. Western blot analyses demonstrated that ASBT was present as a major immunoreactive band representing the 48 kDa monomer (Fig. 1B, lane 1 depicts a representative Western blot for the control). Infrequently, a 93 kDa band representing the dimeric form of the protein was visible but too faint to be included in the densitometic estimations. ASBT protein levels of women with gallstones and controls did not differ; however, ASBT differed considerably among subjects. Therefore, study participants were further subgrouped by weight into normal weight (body mass index < 25) and overweight (body mass index > 25). Protein levels of ASBT did not differ between overweight gallstone carriers and controls. However, when ASBT protein levels of normal weight female gallstone carriers (n = 8) and controls (n = 16) were compared, ASBT levels were found to be significantly lower in patients with gallstones than in the controls. Specifically, mean ASBT protein levels of normal weight gallstone carriers were ∼48% lower than those of controls (Fig. 1A, B). In addition, ASBT was analyzed by immunohistochemical methods in four normal weight gallstone carriers and five controls. Staining was restricted to the apical membrane of enterocytes. Figure 1C shows representative photomicrographs of ASBT protein staining in paraffin-embedded tissue of normal weight gallstone carriers and controls.Fig. 2.Ileal lipid binding protein (ILBP) protein levels and mRNA expression in ileal mucosa biopsies of women. A: Quantitative analysis of ILBP protein levels of gallstone carriers (G) and controls (C). Protein levels of villin were determined to normalize to the amount of enterocytes in biopsies. Data are means ± SEM. * P < 0.05 compared with controls. B, C: Representative Western blot (B) and immunostaining (C) of ILBP in female normal weight gallstone carriers and controls. D: Relative mRNA expression of ILBP in gallstone carriers and controls. Expression of ILBP was measured by real-time RT-PCR and normalized to villin. Data are means ± SEM. * P < 0.05 compared with controls.View Large Image Figure ViewerDownload (PPT)Fig. 2.Ileal lipid binding protein (ILBP) protein levels and mRNA expression in ileal mucosa biopsies of women. A: Quantitative analysis of ILBP protein levels of gallstone carriers (G) and controls (C). Protein levels of villin were determined to normalize to the amount of enterocytes in biopsies. Data are means ± SEM. * P < 0.05 compared with controls. B, C: Representative Western blot (B) and immunostaining (C) of ILBP in female normal weight gallstone carriers and controls. D: Relative mRNA expression of ILBP in gallstone carriers and controls. Expression of ILBP was measured by real-time RT-PCR and normalized to villin. Data are means ± SEM. * P < 0.05 compared with controls.View Large Image Figure ViewerDownload (PPT)Fig. 2.Ileal lipid binding protein (ILBP) protein levels and mRNA expression in ileal mucosa biopsies of women. A: Quantitative analysis of ILBP protein levels of gallstone carriers (G) and controls (C). Protein levels of villin were determined to normalize to the amount of enterocytes in biopsies. Data are means ± SEM. * P < 0.05 compared with controls. B, C: Representative Western blot (B) and immunostaining (C) of ILBP in female normal weight gallstone carriers and controls. D: Relative mRNA expression of ILBP in gallstone carriers and controls. Expression of ILBP was measured by real-time RT-PCR and normalized to villin. Data are means ± SEM. * P < 0.05 compared with controls.View Large Image Figure ViewerDownload (PPT)Fig. 2.Ileal lipid binding protein (ILBP) protein levels and mRNA expression in ileal mucosa biopsies of women. A: Quantitative analysis of ILBP protein levels of gallstone carriers (G) and controls (C). Protein levels of villin were determined to normalize to the amount of enterocytes in biopsies. Data are means ± SEM. * P < 0.05 compared with controls. B, C: Representative Western blot (B) and immunostaining (C) of ILBP in female normal weight gallstone carriers and controls. D: Relative mRNA expression of ILBP in gallstone carriers and controls. Expression of ILBP was measured by real-time RT-PCR and normalized to villin. Data are means ± SEM. * P < 0.05 compared with controls.View Large Image Figure ViewerDownload (PPT)Real-time RT-PCR measurements of ASBT expression also revealed significant differences between groups (Fig. 1D). Specifically, ASBT mRNA levels of gallstone carriers were ∼45% lower than those of controls when comparing all patients and controls, regardless of body weight. Subjects were again subgrouped by weight as described above. ASBT mRNA levels did not differ significantly between overweight gallstone carriers and controls. However, mRNA expression of ASBT was ∼65% lower in normal weight gallstone carriers than in controls.Furthermore, ASBT protein and mRNA levels were correlated. Regardless of body weight, ASBT protein and mRNA levels were correlated significantly in a positive manner. Specifically, when correlating ASBT mRNA and protein levels of all study participants, R = 0.41, with a level of st" @default.
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• W1992305758 title "Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers" @default.
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