1. L IVER B ILE S ALTS Anson Lowe October 2, 2015 Medicine

2. METHODS IN ENZYMOLOGY, VOL. 400

3. Understand the physiologic function of bile salts The determinants and regulation of bile salts synthesis Enterohepatic recycling Bile salts; enterohepatic recycling; cholesterol

4. Liver Functions Bilirubin metabolism Protein Synthesis –Albumin –Coagulation factors (II, V, VII, IX, X) Bile Salt Metabolism Lipid Metabolism Glycogen storage and gluconeogenesis Drug metabolism/Xenobiotic transformation

5. Bile H2084% Bile Salts11.5% Phosphatidyl Choline (lecithin) 3.0% Bile pigments, protein, inorganic ions 1.0%

7. Bile Salt Function Lipid digestion and absorption Essential for the absorption of cholesterol and the fat soluble vitamins; A, D, E, and K. Major route for cholesterol degradation Influences cholesterol synthesis and degradation

8. Bile Salts Synthesized in the liver and excreted into the biliary system Stored in the gallbladder Participate in lipid digestion and absorption in the intestine. Recycled through absorption in the terminal ileum and transport back to the liver

9. Undergraduate Teaching Project ©AGA, Unit 27, slide 54, produced by Milner-Fenwick, Timonium, MD.

10. Bile Salt Synthesis Cholesterol is the starting material ◦ hydrophobic Through a series of hydroxylation steps, cholesterol is turned into either cholic acid or chenodeoxycholic acid

12. Classical Pathway CYP7A1 - this enzyme marks the entry point into the classical pathway. ◦ Hydroxylates the 7  carbon of cholesterol ◦ Expressed only in the liver ◦ Can be regulated  Negative feedback based on bile salt concentration

13. CYP7A1 Regulation FXR - a transcription factor characterized by a DNA binding domain and a ligand binding domain that binds chenodeoxycholic and cholic acid.

14. CYP7A1 Regulation Bile salts will bind to FXR, which promotes an association with other transcription factors and results in negative feedback on CYP7A1. ◦ Thus the negative feedback on CYP7A1 inhibits cholesterol breakdown and potentially contributes to higher levels serum cholesterol and increased cardiovascular risk.

16. CYP7A1 Regulation FXR responds to the the level of oxysterols (derived from cholesterol). ◦ Reduces CYP7A1 activity ◦ Promotes cholesterol efflux from the enterocyte

17. CYP7A1 Regulation Approaches for decreasing cholesterol levels and cardiovascular risk. ◦ Lowering the bile salt pool size  Resection of the terminal ileum to reduce bile salt recycling  Use of bile sequestrants, which bind and removes bile salts in the feces. ◦ Develop agents that inhibit FXR binding to the CYP7A1 DNA regulatory domain.

18. Tu et al. TCB 10:30 (2000)

19. FXR Regulation Positively regulates IBABP (also known as ILLBP) in the enterocyte where bile salts are absorbed and recycled in the terminal ileum.

21. CYP7A1 Deficiency First identified in 2 brothers with a frameshift mutation that disrupts CYP7A1 expression ◦ Early gallstone disease ◦ Hypercholesterolemia  Hypothesis: decrease degradation of cholesterol > down regulates LDL receptor > serum cholesterol levels rise ◦ Hypertriglyceridemia

23. Fuchs, Am J Physiol (2003) 284:G551

24. Abdelmalek et al., J Am Acad Dermatol, 46:161 (2002)

26. CYP27A1 Deficiency Decrease in total bile acid synthesis, but have normal cholesterol levels Cerebrotendinous xanthomatosis ◦ Tendon xanthomatosis ◦ Accumulation of cholesterol in all tissues ◦ Progressive neurologic dysfunction ◦ Premature atherosclerosis Patients respond to administration of chenodeoxycholic acid ◦ ? mechanism

28. Bile Acid Conjugation

29. pKa Cholic Acid5.0 Glycocholate Acid3.9 Taurocholate Acid2.0

30. Bile Acid Conjugation Bile acid CoA:Amino Acid N-acyltransferase (BAAT) ◦ Conjugation with either glycine or taurine is mediated by BAAT

31. Familial Hypercholanemia (FHC) Mutations in BAAT resulting in high serum bile acid concentrations ◦ Low transport out of the hepatocyte Fat malabsorption ◦ Coagulation defects because of vitamin K malabsorption ◦ steatorrhea Itching - deposition of bile acids in the skin

32. Familial Hypercholanemia (FHC) Treatment ◦ Administration of ursodeoxycholic acid  Source of conjugated bile acids for lipid absorption  Provides feedback inhibition of bile acid synthesis

33. Undergraduate Teaching Project ©AGA, Unit 27, slide 54, produced by Milner-Fenwick, Timonium, MD.

35. Bacterial Affects on Bile Acid 7  dehydroxylation ◦ Chenodeoxycholic acid > lithocholic acid ◦ Cholic acid > deoxycholic acid Deconjugation > increases hydrophobicity

37. Bile Acid Absorption Conjugated bile acids are reabsorbed by specific uptake mechanisms Unconjugated bile acids are passively absorbed

38. Bile Acid Uptake Conjugated bile acids are taken up in the terminal ileum by a specific sodium dependent transporter, SLC10A2 (IBAT, ileal sodium/bile salt transporter). ◦ SLC10A2 is specifically expressed in the ileum

39. Primary Bile Acid Malabsorption (PBAM) Features ◦ Congenital diarrhea, steatorrhea, reduced plasma cholesterol levels, loss of enterohepatic recirculation of bile acids Mutations in SLC10A2

40. Oelkers et al., J Clin Invest.,(1997) 99:1880 Primary Bile Acid Malabsorption - SLC10A2

42. Ileal Lipid-Binding Protein Also known as IBABP, ILLBP, ILBP. Mediates cytoplasmic transport of bile acids Specifically expressed in the ileum Expression is regulated by FXR, which enhances bile acid uptake

44. Wolkoff, A.w. and Cohen, D.E., Am J Physiol 284:G175 (2003)

45. Undergraduate Teaching Project ©AGA, Unit 27, slide 54, produced by Milner-Fenwick, Timonium, MD.

46. Trauner et al., NEJM (1998) 339:1217

47. Bile Salt Export ATP-dependent transport by the Bile Salt Export Pump (BSEP). Also called SPGP. ◦ Hepatocyte specific expression

48. Progressive Familial Intrahepatic Cholestasis, type 2 (PFIC2) Autosomal recessive characterized by severe cholestasis that results in liver failure and death Mutations in BSEP were found by positional cloning

49. Bile Salt: Hepatocyte > Bile Duct > Gallbladder > Duct

50. Bile Formation Bile Acids Phospholipids Cholesterol Bilirubin Water and bicarbonate

51. Phospholipid (Lecithin) Secretion MDR3 - ATP dependent transporter for phospholipids.

52. Progressive Familial Intrahepatic Cholestasis, type 3 (PFIC3) Most mutations have resulted in a prematurely truncated MDR3 protein Presents with childhood jaundice, icterus, diarrhea, hepatosplenomegaly Liver histology: bile duct proliferation and fibrosis, portal inflammation Therapy: liver transplantation

53. Intrahepatic Cholestasis of Pregnancy (ICP) Usually presents in the 3rd trimester with jaundice Exhibits a higher rate of fetal death Maternal cholestasis resolves after delivery Patients are heterozygotes for a missense mutation in MDR3

54. Cholesterol Secretion Cholesterol secretion is influenced to an extent by phospholipid secretion ◦ Mice in which only one MDR3 gene was disrupted displayed half the normal phospholipid secretion but normal cholesterol secretion ◦ Homozygous knockouts had no cholesterol secretion Recently defined transporters for cholesterol have been discovered and are likely to play a role

56. Copyright ©2002 American Society for Clinical Investigation Lecithin = phospholipid

57. Feldman: Sleisenger & Fordtran’s Gastrointestinal and Liver Disease, 7th ed.

58. Undergraduate Teaching Project ©AGA, Unit 27, slide 54, produced by Milner-Fenwick, Timonium, MD.