Search Article Keyword:  

PubMed Submission Abstract PDF   Click Count: 2392 DownLoad Count: 699 

ISSN 1007-9327 CN 14-1219/R  World J Gastroenterol  2006 December 21; 12(47): 7710-7714


Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption


Marco Montagnani, Anna Abrahamsson, Cecilia Gälman, Gösta Eggertsen, Hanns-Ulrich Marschall, Elisa Ravaioli, Curt Einarsson, Paul A Dawson

Marco Montagnani, Elisa Ravaioli, Dipartimento di Medicina Interna e Gastroenterologia, Università di Bologna, Bologna, Italy

Marco Montagnani, Elisa Ravaioli, Center for Applied Biomedi-cal Research, Università di Bologna, Bologna, Italy

Anna Abrahamsson, Hanns-Ulrich Marschall, Curt Einarsson, Center for Gastroenterology and Hepatology, Karolinska University Hospital Huddinge, Stockholm, Sweden

Cecilia Gälman, Center for Metabolism and Endocrinology, Karolinska University Hospital Huddinge, Stockholm, Sweden

Gösta Eggertsen, Department of Laboratory Medicine, Division of Clinical Chemistry, Karolinska University Hospital Huddinge, Stockholm, Sweden

Paul A Dawson, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157,

United States

Supported by grants from the Swedish Research Council, the Karolinska Institutet and the Swedish Society of Medicine (to CE) and National Institutes of Health grants DK-47987 (to PAD)

Correspondence to: Paul A Dawson, PhD, Department of Internal Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, United States.

Telephone: +1-336-7164633  Fax: +1-336-7166279

Received: 2006-09-19             Accepted: 2006-11-20



The etiology of most cases of idiopathic bile acid malabsorption (IBAM) is unknown. In this study, a Swedish family with bile acid malabsorption in three consecutive generations was screened for mutations in the ileal apical sodium-bile acid cotransporter gene (ASBT; gene symbol, SLC10A2) and in the genes for several of the nuclear receptors known to be important for ASBT expression: the farnesoid X receptor (FXR) and peroxisome proliferator activated receptor alpha (PPARa). The patients presented with a clinical history of idiopathic chronic watery diarrhea, which was responsive to cholestyramine treatment and consistent with IBAM. Bile acid absorption was determined using 75Se-homocholic acid taurine (SeHCAT); bile acid synthesis was estimated by measuring the plasma levels of 7α-hydroxy-4-cholesten-3-one (C4). The ASBT, FXR, and PPARα genes in the affected and unaffected family members were analyzed using single stranded conformation polymorphism (SSCP), denaturing HPLC, and direct sequencing. No ASBT mutations were identified and the ASBT gene did not segregate with the bile acid malabsorption phenotype. Similarly, no mutations or polymorphisms were identified in the FXR or PPARa genes associated with the bile acid malabsorption phenotype. These studies indicate that the intestinal bile acid malabsorption in these patients cannot be attributed to defects in ASBT. In the absence of apparent ileal disease, alternative explanations such as accelerated transit through the small intestine may be responsible for the IBAM.


© 2006 The WJG Press. All rights reserved.


Key words: Bile acid malabsorption; Diarrhea; Genetics; 75Se-homocholic acid taurine test; Nuclear receptors


Montagnani M, Abrahamsson A, Gälman C, Eggertsen G, Marschall HU, Ravaioli E, Einarsson C, Dawson PA. Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption. World J Gastroenterol 2006; 12(47): 7710-7714



Bile acids are synthesized from cholesterol in the liver and secreted into the small intestine, where they facilitate absorption of fat, fat-soluble vitamins and cholesterol[1]. The bile acids are then reabsorbed from the intestine and returned to the liver via the portal venous circulation. The enterohepatic cycling of bile acids is an extremely efficient process, and less than 5% of the intestinal bile acids escape reabsorption and are eliminated in the feces. The ileal apical sodium/bile acid cotransporter (ASBT)[1] mediates the first step in the active uptake of bile acids from the intestine, and defects in ileal ASBT function may be responsible for bile acid malabsorption associated with watery diarrhea. Impaired ileal uptake of bile acids has been documented in several patients[2] and inherited ASBT mutations were demonstrated in congenital primary bile acid malabsorption (PBAM)[3]. However, ASBT mutations are not found in most patients with adult-onset bile acid malabsorption, chronic diarrhea, and a morphological and functionally normal ileum[4], a more common condition termed idiopathic bile acid malabsorption (IBAM)[5-7]. In this study we examined the association between IBAM and inherited mutations affecting the ASBT and several of the nuclear receptors known to be important for ASBT expression, the farnesoid X receptor (FXR)[8] and peroxisome proliferator activated receptor A (PPARα)[9], in a Swedish family with three generations of bile acid malabsorption.



Three family members (subjects 1, 10, and 11) reported chronic diarrhea, occurring especially after meals (Figure 1). Fasting blood samples were obtained from each family member. Informed consent to participate in the study was obtained from each subject and the protocol was approved by the Ethics Committee of Karolinska University Hospital Huddinge. Bile acid absorption was determined using 75Se­homocholic acid taurine (SeHCAT), a synthetic analog of taurocholic acid, as previously described[10]. Briefly, a capsule containing 10 μCi of 75SeHCAT was given orally and retained activity was measured after 3 h and 7 d using an uncollimated gamma counter. Retention of less than 10% of the administered radiolabeled bile acid was considered abnormal. The plasma level of 7α-hydroxy-4-cholesten-3-one (C4) (normal < 19 ng/mL), an intermediate product in the synthesis of bile acids, was measured as described[11]. C4 is a reliable marker for the activity of hepatic cholesterol 7α-hydroxylase, the rate-determining enzyme in bile acid synthesis[12,13].


Figure 1  Family pedigree and genetic analysis. Individuals with bile acid malabsorption are indicated by the shaded symbols. The haplotype for each subject is provided below the symbol in the pedigree. The affected subjects’ number, age, BMI, SeHCAT results, and C4 results are indicated below the pedigree.


Patient 11 had a history of diarrhea since adulthood with 15 to 20 watery bowel movements per day over the past 10 years. Clinical history was unremarkable except for a cholecystectomy at age 24. Patient 10 had a history of frequent watery diarrhea since her teenage years. Patient 1 reported frequent bowel movements following a meal. In all three patients, celiac disease was excluded; lactose tolerance tests, vitamin B12 absorption, and routine laboratory blood tests including hemoglobin, sedimentation rate and liver function tests were normal. Barium contrast gastrointestinal exams and ileocolonoscopy with mucosal biopsies (patients 10 and 11) were normal. SeHCAT tests (patients 10 and 11) and plasma levels of C4 (patients 1, 10 and 11) were markedly abnormal (Figure 1). Treatment of patients 10 and 11 with cholestyramine (Questran, Bristol-Myers) reduced the stool frequency and improved the stool consistency.

Dysfunctional mutations in the ASBT gene were previously identified in a subject with PBAM[3]. To determine if similar mutations in ASBT are associated with bile acid malabsorption in this family, we employed simple sequence length polymorphism (SSLP) analysis using a dinucleotide repeat linked to the ASBT gene and SSCP analysis to screen for mutations in the ASBT coding and proximal promoter regions. The SSCP primers designed for ASBT intron or exon sequences and PCR amplification conditions have been described previously[3,14]. PCR amplification products were resolved using three different gel electrophoresis conditions, gels contained 10% glycerol, 1 × TBE buffer, and 6% acrylamide, 10% acrylamide (acrylamide: N, N’-methylenebisacrylamide ratio 50:1), or 0.4 × MDE (Mutation Detection Enhancement acrylamide; FMC Bioproducts, Rockland, Maine), in order to increase the assay sensitivity[15,16], and the nucleotide sequence changes responsible for the SSCP band shifts were subsequently identified by PCR amplification and sequencing. No ASBT mutations or polymorphisms were found in patients 1 or 10, whereas patient 11 was heterozygous for two common polymorphisms that do not affect ASBT function[14], a G-to-T transversion in exon 3 that causes an alanine to serine substitution at position 171 (A171S) and an intronic A-to-G transition located 20 bp upstream of exon 6 (int 5). Following SSCP analysis, the two ASBT alleles could then be distinguished in the original proband (patient 11) using a combination of the linked dinucleotide repeat marker and the single nucleotide polymorphisms (A171S and int 5). The affected individuals shared only one ASBT allele, and four unaffected individuals (subjects 2, 3, 6 and 8) also inherited this allele.

Since the nuclear receptor, FXR, is an important regulator of ASBT expression and bile acid metabolism, the coding region of the FXR gene, exons 3 to 11[17], was also analyzed in patients 1, 10, and 11 using SSCP conditions described by Lind et al[18]. Sequences for SSCP primers designed for FXR intron and exon sequences are shown in Table 1. Briefly, exons 3-11 were amplified by PCR from genomic DNA, generating fragments varying from 200 to 250 bp in length, except exon 10 (315 bp) and exon 11 (385 bp). Due to its larger size, exon 4 was PCR-amplified using two sets of primers that yielded products of 240 and 300 bp. The fragments were separated on precast polyacrylamide gels visualized by silver staining (GenePhor DNA Separation System, Amersham Bioscience, Uppsala, Sweden). This analysis detected no mutations or polymorphisms in the human FXR gene of these patients.


Table 1  Sequences for the primers for SSCP on human FXR


Polymorphisms in the intronic and exonic regions of the PPARa gene (PPARA) have been previously described[19,20], and PPARα is a known regulator of ASBT gene expression[21]. We analyzed two well-characterized polymorphic regions of PPARA, exon 5 and intron 7, in the PBAM family in order to determine if a mutation in this gene could be associated with the disease. Specific primers were employed for PCR amplification of exon 5 (forward: 5’-AGTAAAGCAAGTGCGCTGGT-3’; reverse: 5’-AAGGAAGGGGAACTGAGGAA-3’) and intron 7 (forward: 5’-CCTCCCGAGTATCTGGGATT-3’; reverse: 5’-TGAGCTGCCTTTAGATATTGTCA-3’). The PCR products were analyzed for polymorphisms or mutations by denaturing HPLC (D-HPLC) (Transgenomic Wave, Transgenomic, Omaha, Nebraska) and automatic sequencing (automatic sequencer CEQTM8000 XL, Beckman Coulter Inc., Fullerton. CA). PPARA gene analysis did not show any new mutations. Analysis of the L162V polymorphism of exon 5 and G > C polymorphism of intron 7 revealed that PPARA alleles did not segregate with the bile acid malabsorption symptoms (Figure 1).



The enterohepatic circulation efficiently conserves bile acids, thereby maintaining bile flow and adequate intraluminal bile acid concentrations for micellular solubilization and absorption of lipids[22]. Defective small intestinal absorption leads to increased concentrations of dihydroxy bile acids reaching the colon, where they alter water and electrolyte movement leading to secretory diarrhea[23,24]. Three types of intestinal bile acid malabsorption are generally recognized[25]. Typebile acid malabsorption is the most common form and is caused by ileal resection, ileal disease such as Crohn’s disease, ileal bypass, and radiation enteritis[26,27]. Type bile acid malabsorption is associated with conditions such as cholecystectomy, peptic ulcer surgery, chronic pancreatitis, celiac disease, diabetes mellitus, cystic fibrosis, and the use of various drugs[28].

In contrast to typesand , type bile acid malabsorption (also called primary or idiopathic bile acid malabsorption) is not associated with obvious ileal disease. A very rare congenital form of typebile acid malabsorption (primary bile acid malabsorption) exhibiting refractory infantile diarrhea, steatorrhea, and growth failure[2,25] was found to be associated with inherited mutations in the ASBT gene[3]. However, most patients with adult-onset idiopathic bile acid malabsorption appear to have a normal ASBT gene[4] and the etiology is still obscure. The identification of a family with idiopathic bile acid malabsorption in three consecutive generations offered a rare occasion to further evaluate association of this syndrome with inherited mutations affecting the ASBT. These patients were diagnosed with IBAM on the basis of clinical presentation, low SeHCAT test values, increased bile acid synthesis, and response to cholestyramine treatment. Analysis of these individuals and unaffected family members conclusively demonstrated that the intestinal bile acid malabsorption in these subjects is not due to inherited defects in the ASBT gene. In addition, we also looked for polymorphisms of PPARα and FXR, two nuclear receptors known to be important for the regulation of the ASBT. To our knowledge, polymorphism analysis of the human FXR has not been described previously and no mutations of the FXR gene were found in the present study. Likewise, no association between PPARA and IBAM was found in this family.

There is increasing evidence emerging in support of IBAM etiologies other than defective ileal uptake of bile acids. Earlier studies had provided evidence for an increased ileal uptake of bile acids[29] as well as an expanded bile acid pool in some patients with type bile acid malabsorption[28]. Very recently, Bajor et al[30] demonstrated elevated in vitro bile acid uptake and ASBT protein expression in ileal biopsies from patients with bile acid malabsorption, abnormal SeHCAT-retention values, and elevated plasma C4 levels. This apparent increase in ASBT activity and expression could be explained by accelerated small bowel transit in IBAM patients, thereby reducing the contact time between the luminal contents and the mucosa. In support of this hypothesis, a more rapid small bowel transit has been reported for patients with IBAM[31]. The etiology of the postulated accelerated small bowel transit in these patients is not clear. However, more rapid small bowel transit has been noted in subjects with elevated BMI[31,32] and in subjects consuming high fat diets[33], suggesting dysregulation of gut motility under these conditions. The rapid small bowel transit is predicted to reduce the opportunity for ileal absorption, leading to decreased levels of bile acids in the ileal enterocytes and increased ASBT expression. Previous in vitro studies have shown that the human ASBT promoter is negatively regulated by bile acids through an FXR dependent mechanism[8]. The decreased enterocyte levels of bile acids are also predicted to reduce the FXR-dependent induction of FGF19 expression, thereby increasing hepatic bile acid synthesis and plasma C4 levels. FGF19 is an ileal enterocyte derived factor that mediates repression of the hepatic cholesterol 7α-hydroxylase gene and bile acid synthesis[34,35].

In conclusion, the present findings further argue against defective ileal uptake of bile acids as the direct cause of IBAM and support the exploration of alternative explanations such as reduced contact time with the ileal mucosa due to changes in small intestinal motility.



1    Hofmann AF. The continuing importance of bile acids in liver and intestinal disease. Arch Intern Med 1999; 159: 2647-2658  PubMed

2    Heubi JE, Balistreri WF, Fondacaro JD, Partin JC, Schubert WK. Primary bile acid malabsorption: defective in vitro ileal active bile acid transport. Gastroenterology 1982; 83: 804-811  PubMed

3    Oelkers P, Kirby LC, Heubi JE, Dawson PA. Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2). J Clin Invest 1997; 99: 1880-1887  PubMed

4    Montagnani M, Love MW, Rossel P, Dawson PA, Qvist P. Absence of dysfunctional ileal sodium-bile acid cotransporter gene mutations in patients with adult-onset idiopathic bile acid malabsorption. Scand J Gastroenterol 2001; 36: 1077-1080  PubMed

5    Wildt S, Norby Rasmussen S, Lysgard Madsen J, Rumessen JJ. Bile acid malabsorption in patients with chronic diarrhoea: clinical value of SeHCAT test. Scand J Gastroenterol 2003; 38: 826-830  PubMed

6    Rossel P, Sortsoe Jensen H, Qvist P, Arveschoug A. Prognosis of adult-onset idiopathic bile acid malabsorption. Scand J Gastroenterol 1999; 34: 587-590  PubMed

7    Thaysen EH, Pedersen L. Idiopathic bile acid catharsis. Gut 1976; 17: 965-­970  PubMed

8    Neimark E, Chen F, Li X, Shneider BL. Bile acid-induced negative feedback regulation of the human ileal bile acid transporter. Hepatology 2004; 40: 149-156  PubMed

9    Jung D, Fried M, Kullak-Ublick GA. Human apical sodium-dependent bile salt transporter gene (SLC10A2) is regulated by the peroxisome proliferator-activated receptor alpha. J Biol Chem 2002; 277: 30559-30566  PubMed

10  Eusufzai S, Ericsson S, Cederlund T, Einarsson K, Angelin B. Effect of ursodeoxycholic acid treatment on ileal absorption of bile acids in man as determined by the SeHCAT test. Gut 1991; 32: 1044-1048  PubMed

11  Galman C, Arvidsson I, Angelin B, Rudling M. Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alpha-hydroxy-4-cholesten-3-one in peripheral blood. J Lipid Res 2003; 44: 859-866  PubMed

12  Axelson M, Bjorkhem I, Reihner E, Einarsson K. The plasma level of 7 alpha-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7 alpha-hydroxylase in man. FEBS Lett 1991; 284: 216-218  PubMed

13  Sauter G, Berr F, Beuers U, Fischer S, Paumgartner G. Serum concentrations of 7alpha-hydroxy-4-cholesten-3-one reflect bile acid synthesis in humans. Hepatology 1996; 24: 123-126  PubMed

14  Love MW, Craddock AL, Angelin B, Brunzell JD, Duane WC, Dawson PA. Analysis of the ileal bile acid transporter gene, SLC10A2, in subjects with familial hypertriglyceridemia. Arterioscler Thromb Vasc Biol 2001; 21: 2039-2045  PubMed

15  Ravnik-Glavac M, Glavac D, Dean M. Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum Mol Genet 1994; 3: 801-807  PubMed

16  Highsmith WE Jr, Nataraj AJ, Jin Q, O’Connor JM, El-Nabi SH, Kusukawa N, Garner MM. Use of DNA toolbox for the characterization of mutation scanning methods. II: evaluation of single-strand conformation polymorphism analysis. Electrophoresis 1999; 20: 1195-1203  PubMed

17  Huber RM, Murphy K, Miao B, Link JR, Cunningham MR, Rupar MJ, Gunyuzlu PL, Haws TF, Kassam A, Powell F, Hollis GF, Young PR, Mukherjee R, Burn TC. Generation of multiple farnesoid-X-receptor isoforms through the use of alternative promoters. Gene 2002; 290: 35-43  PubMed

18  Lind S, Rystedt E, Eriksson M, Wiklund O, Angelin B, Eggertsen G. Genetic characterization of Swedish patients with familial hypercholesterolemia: a heterogeneous pattern of mutations in the LDL receptor gene. Atherosclerosis 2002; 163: 399-407  PubMed

19  Flavell DM, Jamshidi Y, Hawe E, Pineda Torra I, Taskinen MR, Frick MH, Nieminen MS, Kesaniemi YA, Pasternack A, Staels B, Miller G, Humphries SE, Talmud PJ, Syvanne M. Peroxisome proliferator-activated receptor alpha gene variants influence progression of coronary atherosclerosis and risk of coronary artery disease. Circulation 2002; 105: 1440-1445  PubMed

20  Jamshidi Y, Montgomery HE, Hense HW, Myerson SG, Torra IP, Staels B, World MJ, Doering A, Erdmann J, Hengstenberg C, Humphries SE, Schunkert H, Flavell DM. Peroxisome proliferator--activated receptor alpha gene regulates left ventricular growth in response to exercise and hypertension. Circulation 2002; 105: 950-955  PubMed

21  Eloranta JJ, Kullak-Ublick GA. Coordinate transcriptional regulation of bile acid homeostasis and drug metabolism. Arch Biochem Biophys 2005; 433: 397-412  PubMed

22  Hofmann AF, Schteingart CD, Lillienau J. Biological and medical aspects of active ileal transport of bile acids. Ann Med 1991; 23: 169-175  PubMed

23  Mekhjian HS, Phillips SF. Perfusion of the canine colon with unconjugated bile acids. Effect on water and electrolyte transport, morphology, and bile acid absorption. Gastroenterology 1970; 59: 120-129  PubMed

24  McJunkin B, Fromm H, Sarva RP, Amin P. Factors in the mechanism of diarrhea in bile acid malabsorption: fecal pH--a key determinant. Gastroenterology 1981; 80: 1454-1464  PubMed

25  Balistreri WF, Heubi JE, Suchy FJ. Bile acid metabolism: relationship of bile acid malabsorption and diarrhea. J Pediatr Gastroenterol Nutr 1983; 2: 105­-121  PubMed

26  Aldini R, Roda A, Festi D, Sama C, Mazzella G, Bazzoli F, Morselli AM, Roda E, Barbara L. Bile acid malabsorption and bile acid diarrhea in intestinal resection. Dig Dis Sci 1982; 27: 495-502  PubMed

27  Hofmann AF. Bile acid malabsorption caused by ileal resection. Arch Intern Med 1972; 130: 597-605  PubMed

28  van Tilburg AJ, de Rooij FW, van den Berg JW, van Blankenstein M. Primary bile acid malabsorption: a pathophysiologic and clinical entity? Scand J Gastroenterol Suppl 1992; 194: 66-70  PubMed

29  van Tilburg AJ, de Rooij FW, van den Berg JW, van Blankenstein M. Primary bile acid diarrhoea without an ileal carrier defect: quantification of active bile acid transport across the ileal brush border membrane. Gut 1991; 32: 500-503  PubMed

30  Bajor A, Kilander A, Fae A, Galman C, Jonsson O, Ohman L, Rudling M, Sjovall H, Stotzer PO, Ung KA. Normal or increased bile acid uptake in isolated mucosa from patients with bile acid malabsorption. Eur J Gastroenterol Hepatol 2006; 18: 397-403  PubMed

31  Sadik R, Abrahamsson H, Ung KA, Stotzer PO. Accelerated regional bowel transit and overweight shown in idiopathic bile acid malabsorption. Am J Gastroenterol 2004; 99: 711-718  PubMed

32  Sadik R, Abrahamsson H, Stotzer PO. Gender differences in gut transit shown with a newly developed radiological procedure. Scand J Gastroenterol 2003; 38: 36-42  PubMed

33  Cunningham KM, Daly J, Horowitz M, Read NW. Gastrointestinal adaptation to diets of differing fat composition in human volunteers. Gut 1991; 32: 483-486  PubMed

34  Angelin B. Telling the liver (not) to make bile acids: a new voice from the gut? Cell Metab 2005; 2: 209-210  PubMed

35  Inagaki T, Choi M, Moschetta A, Peng L, Cummins CL, McDonald JG, Luo G, Jones SA, Goodwin B, Richardson JA, Gerard RD, Repa JJ, Mangelsdorf DJ, Kliewer SA. Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis. Cell Metab 2005; 2: 217-225  PubMed


                                                                                       S- Editor  Wang GP   L- Editor  Zhu LH    E- Editor  Bai SH








Reviews Add

Related Articles:
Physiology of bile secretion
Intestinal bile acid physiology and pathophysiology
Endocrine and paracrine role of bile acids
Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption
Bile acid interactions with cholangiocytes