Bile acids: Trying to understand their chemistry and biology with the hope of helping patients

Authors

  • Alan F. Hofmann

    Corresponding author
    1. Division of Gastroenterology, Department of Medicine, University of California, San Diego, San Diego, CA
    • Division of Gastroenterology, Department of Medicine, University of California, San Diego 92093-0063
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    • fax: (858) 459-1754.


  • Potential conflict of interest: Dr. Hofmann received grants from and holds intellectual property rights for Falk Pharma. He is a consultant for Daiichi Sankyo, Sorbent Technologies, Relypsa, and Chiasma.

Abstract

An informal review of the author's five decades of research on the chemistry and biology of bile acids in health and disease is presented. The review begins with a discussion of bile acid structure and its remarkable diversity in vertebrates. Methods for tagging bile acids with tritium for metabolic or transport studies are summarized. Bile acids solubilize polar lipids in mixed micelles; progress in elucidating the structure of the mixed micelle is discussed. Extensive studies on bile acid metabolism in humans have permitted the development of physiological pharmacokinetic models that can be used to simulate bile acid metabolism. Consequences of defective bile acid biosynthesis and transport have been clarified, and therapy has been developed. Methods for measuring bile acids have been improved. The rise and fall of medical and contact dissolution of cholesterol gallstones is chronicled. Finally, principles of therapy with bile acid agonists and antagonists are given. Advances in understanding bile acid biology and chemistry have helped to improve the lives of patients with hepatobiliary or digestive disease. (HEPATOLOGY 2009.)

In contrast to others in this series, I am not a master hepatologist. Rather, I have spent the last 50 years trying to understand the biology and chemistry of bile acids in health and disease, with the hope that such understanding would help the human condition. I feel that I have been extraordinarily fortunate to be able to pursue almost full-time research in the departments of medicine at the Rockefeller University, the Mayo Clinic, and the University of California, San Diego (UCSD). In this pursuit, I have been blessed by the association with several scores of talented fellows as well as with many collaborators. In this brief retrospective, I will summarize some successes, some failures, and some remaining challenges. The topics discussed involve both the intestine and the liver, because I have followed the enterohepatic circulation—from intestine to liver and back to the intestine. Space limitations preclude a discussion of many adventures. They also preclude a complete bibliography, as well as historical details or personal anecdotes; the last would have made this essay much more fun to read. Seldom is the circuitous path of discovery described in full!

Getting Started

In 1959, while serving as a physician to the National Symphony during its pan-American tour, I received a cable stating that the National Foundation would support me for 2 years to work in the laboratory of Bengt Borgström at the University of Lund, Sweden, on the mechanism of fat absorption. I had chosen to work on this problem because at the time lipid absorption was poorly understood, and Borgström was writing about it with clarity and simplicity. The decision to go to Lund was a wise one. Under Borgström's gentle tutelage, I was able to create model systems simulating small intestinal content during triglyceride absorption and elucidate principles of solubilization of lipolysis products in mixed micelles.1

I intubated myself and my laboratory colleagues (without benefit of informed consent); collected small intestinal content during digestion of a test meal; isolated the clear, micellar phase by ultracentrifugation; and determined its chemical composition.2 Its contents (partly ionized fatty acids and monoglycerides) were exactly as predicted from our in vitro studies. It was an exciting time as we realized the mixed micelle was the final common path for all dietary lipids that were absorbed. Borgström also taught me the power of chromatography3 and the utility of adding a radioactive tag to follow the fate of a molecule under study.4

Abbreviations

ABC, adenosine triphosphate binding cassette; BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; FDA, U.S. Food and Drug Administration; FGF-15, fibroblast growth factor-15; FXR, farnesoid X receptor; HPLC, high performance liquid chromatography; LC-MS-MS, liquid chromatography-mass spectrometry-mass spectrometry; MDR2, multidrug resistance protein 2; NCGS, National Cooperative Gallstone Study; NIH, U.S. National Institutes of Health; NTCP, sodium taurocholate cotransporting peptide; PSC, primary sclerosing cholangitis; Tmax, transport maximum; UCSD, University of California, San Diego; UDCA, ursodeoxycholic acid.

Bile Acid Chemistry

To study model systems simulating small intestinal content during digestion, one needed the taurine and glycine conjugates of the three major bile acids in human bile. The proportions of these major bile acids—cholic, deoxycholic, and chenodeoxycholic—had just been clarified by paper chromatography by Jan Sjövall, who had done his doctoral work in Lund before moving to Stockholm with his mentor, the late Sune Bergström. Cholic acid and deoxycholic acid were readily available from commercial sources, because of the long tradition of isolating them from ox bile. There are only traces of chenodeoxycholic acid (CDCA) in bovid bile, so if one wanted CDCA in gram quantities, it had to be synthesized from cholic acid. The only method available had been reported in 1948 by Louis Fieser, a legendary Harvard organic chemist, who was exploring ways of converting bile acids into cortisone. The method was tedious and gave a product full of impurities. I bravely wrote to Fieser asking if there were not a simpler way to make CDCA. He wrote back about a new method5 in which ethanedithiol was used to functionalize the hydroxy group on the 12th carbon atom that had to be eliminated when cholic acid was converted to CDCA. But ethanedithiol had such a terrible stench (it is a molecule that smells at both ends) that the whole floor stunk and the laboratory personnel complained bitterly. I was forced to give up the method and return to the method originally described by Fieser. My final product—beautiful white crystals—had the wrong melting point, and I was thrust abruptly into learning about polymorphism of organic molecules.6 Years later, when CDCA had been shown to induce gallstone dissolution, I could visit chemical plants and see large drums containing kilograms of pure CDCA waiting to be encapsulated.

When I returned to the United States in 1962 and continued postdoctoral work at Rockefeller University (in the laboratory of E. H. Ahrens), I developed an outside collaboration with the late Erwin Mosbach who had fed rabbits cholestanol (a saturated derivative of cholesterol) and observed that the animals developed gallstones. Together, we showed that the stones were composed of the calcium salt of the glycine conjugate of allodeoxycholic acid, a steric isomer of deoxycholic acid7 in which the juncture of the A and B rings is trans and not cis as it is in most common, natural bile acids. This was a wonderfully complex disease of the enterohepatic circulation involving both the synthesis of an unusual bile acid by the liver and its modification by the colonic flora.

Two decades later, at UCSD, working with the late Karol Mysels (a master colloid chemist), we (first Craig Jones, then Jing-Jing Gu) measured the solubility products of the calcium salts of common, natural bile acids and learned why humans rarely precipitate the calcium salts of bile acids in their biliary tract.8, 9, 10 With Susan Cummings, we explored the determinants of biliary calcium secretion in the dog.11 As a result, we could link the physiology to the physical chemistry. The principle that emerged from these experiments was that the concentration of ionized Ca2+ in bile was constant, irrespective of the rate of bile flow because of the high permeability of the biliary tract to Ca2+ ions. Consequently the formation of calcium-containing gallstones resulted from an increased concentration of a calcium-sensitive ion such as unconjugated bilirubin, carbonate, ionized fatty acid, or sulindac.

At the Mayo Clinic, the major chemical effort was to develop bile acids tagged with tritium to be used for metabolic studies. We first introduced tritium into the 2 and 4 positions12 and later into the 11 and 12 positions.13 We learned in a study led with great ability by Nick LaRusso that the 2,2′,4,4′ label was not completely stable in vivo.14 At UCSD, we finally solved the problem of inserting a stable tritium label by putting a double bond into the side chain and reducing it with carrier-free tritium gas.15 With William Duane, we showed this label was stable in vivo during enterohepatic cycling.16 We have used this method to prepare tritium-conjugated bile acids with high specific activity. Such labeled bile acids have been highly useful for characterizing bile acid transport.17, 18

At UCSD, whose faculty I joined in 1977, we began a whole new line of bile acid chemistry. I was most fortunate to have Lee Hagey join the laboratory, initially as a technician, then a graduate student pursuing his doctorate, and finally as a faculty member. Lee is an extraordinary combination of instrument engineer, analytical biochemist, and evolutionary biologist. Lee arranged to receive bile from every animal that died at the San Diego Zoo. He then analyzed the biliary bile acids by high-performance liquid chromatography (HPLC), gas chromatography–mass spectroscopy (MS), and electrospray ionization tandem MS.

In this program, we have made a number of discoveries, not all of which have been published. We showed in a collaborative study with the laboratories of Takahikio Hoshita in Hiroshima, Japan, and the late Erwin Mosbach that the manatee does not have bile acids, but instead has C27 bile alcohols in bile.19 We developed the idea that CDCA is the root C24 bile acid and that the majority of species add one more hydroxy group. We identified 3,7,16-trihydroxy bile acids as being extremely common in birds.20 Indeed, because of the large number of avian species, this bile acid (avicholic acid) is present in more species than any other bile acid, yet is not mentioned in a single textbook of biochemistry. Swans and some related geese have 3,7,15-trihydroxy bile acids.21 The remarkable diversity of chemical structures in the three great bile salt classes—C27 bile alcohols, C27 bile acids, and C24 bile acids—is illustrated in Fig. 1. Table 1 summarizes the types of bile acid classes that are present in different vertebrate orders.

Figure 1.

Chemical structures of bile salts identified to date in vertebrates. Cholesterol, the precursor, is shown in (A). The three major classes of bile salts are shown in (B-D), with their default structures that contain hydroxy groups at C-3 and C-7. (B) C27 bile alcohols. (C) C27 bile acids. (D) C24 bile acids. In bile, C27 bile alcohols are esterified with sulfate at C-27. C27 bile acids are amidated with taurine. C24 bile acids are amidated with glycine or taurine. Sites of hydroxylation that occur in many species are indicated by the broad arrows. Sites of hydroxylation that occur in only one or a few species are indicated by the small arrows. Since this figure was made, C27 bile acids conjugated at C-1 have been identified in the bile of the red-winged tinamou, an early-evolving wingless bird. Additional sites of hydroxylation are likely to be discovered in all three classes. Probably >95% of bile salts are shown in the figure. Nevertheless, there are species with C23-nor bile acids, as well as species with C28 bile acids. The figure has been published previously.141

Table 1. Occurrence of Major Bile Acid Classes in Vertebrates
Vertebrate OrderC27 Bile AlcoholsC27 Bile AcidsC24 Bile Acids
Fish   
 Ancientx  
 Cartilaginousx  
 Cyprinidsx  
 Transitionalx x
 Bony  x
Amphibiaxxx
Reptiles   
 Skinks x 
 Lizards  x
 Turtles x 
 Crocodiles xx
 Snakes  x
Birds   
 Early evolving xx
 Later evolving  x
Mammals   
 Early evolvingx x
 Later evolving  x

Proof of structure of any new bile acid isolated from bile requires nuclear magnetic resonance spectroscopy and if possible, confirmation by chemical synthesis. We have been fortunate to have established collaboration with Takashi Iida, a Professor of Chemistry at Nihon University in Japan. Takashi has had a long career synthesizing novel bile acids. In our collaborative studies, he has synthesized the C-15 and C-16 trihydroxy bile acids.22 We are now collaborating with Matt Krasowski, University of Pittsburgh, PA, to attempt to relate phylogenetic relationships to bile acid structure,23 continuing the work of the late Geoffrey Haslewood, Professor of Biochemistry at Guy's Hospital Medical School, London, UK.24

Bile Acid Physical Chemistry: The Molecular Arrangement of the Mixed Micelle

Bile acids are planar amphipaths. They are rigid molecules with a hydrophilic side and a hydrophobic side, and a negative charge at the end of the molecule. A typical bile acid is shown in Fig. 2. In aqueous solution, bile acid anions self-associate to form simple micelles. The most remarkable quality of bile acids is their ability to solubilize lipid bilayers that form mixed micelles.

Figure 2.

Depiction of a space-filling model of the taurine conjugate of cholic acid (viewed from the side) showing its planar amphipathic structure. So far as is known, all natural bile acids and bile alcohols have the same planar amphipathic character. The greater the hydrophobic area, the lower the critical micellization concentration. The figure has been published previously.141

The mixed micelle of bile contains mostly phosphatidylcholine. The mixed micelle of intestinal content contains mostly monoglyceride and partly ionized fatty acid. Rex Hjelm, a talented biophysicist, had used small angle neutron scattering to deduce the molecular arrangement of the biliary micelle. The lipid aggregates (containing bile acids and phosphatidylcholine) were spherical or worm-like; the bile acids were on the surface, pushing their hydrophobic back between the polar heads of the phospholipid. I persuaded Rex to look at bile acid–monoglyceride micelles by small angle neutron scattering. He found they had exactly the same pattern as the mixed micelles of bile.25

Freeze fracture microphotographs of the canaliculus show hemivesicles, presumably composed mostly of phosphatidylcholine, budding from the canalicular membrane. These are transformed into mixed micelles by bile acid anions. Exactly the same process occurs at the surface of the triglyceride droplets in the small intestinal lumen as pancreatic lipase generates bilayers composed of monoglyceride and partly ionized fatty acid. These adsorb bile acid anions and are transformed into mixed micelles. Thus, the physicochemical processes in both biliary lipid secretion and fat digestion are quite similar. The micelles in bile are export micelles, transporting cholesterol into the intestine from which it is inefficiently absorbed. The micelles in intestinal content are import micelles, transporting insoluble fatty acids, monoglycerides, and fat-soluble vitamins to the hungry enterocyte. The conversion of bilayers to mixed micelles, as occurs in the biliary canaliculus and at the aqueous interface of lipid droplets in the small intestine, is illustrated in Fig. 3.

Figure 3.

Conversion by bile acid anions of lipid vesicles or lipid bilayers to mixed cylindrical micelles. Micelles may also be spherical depending on the ratio of solubilized lipids to bile acids. Vesicles containing mostly phosphatidylcholine bud out from the outer face of the apical membrane of the biliary canaliculus. Bilayers of fatty acid (partly ionized) and 2-monoacyl glycerides are generated by the action of pancreatic lipase on the surface of triglyceride droplets during fat digestion in small intestinal content. The figure has been published previously.141

One question that caused much controversy in the 1980s was the ionization behavior of bile acids. The subject is important, because ionized molecules do not cross cell membranes in the absence of a transporter, whereas nonionized bile acid molecules traverse membranes by passive flip-flop. To straighten this matter out, I (with advice of others) convened a small workshop of 12 chemically oriented physicians and 12 clinically interested chemists, luxuriously supported by the Kroc Foundation. It was an exciting meeting and we did straighten the matter out!26

Bile Acid Metabolism

The laboratory at Rockefeller University was doing tedious balance studies, looking at the effect of different diets on bile acid and cholesterol biosynthesis. I thought we should try to learn what was happening in the small intestine. We, including the late Wilfred Simmonds and Emanuel Theodor, perfused a micellar solution of bile acid, monoglyceride, and cholesterol into the small intestine of healthy volunteers and observed net plant sterol secretion into the perfused loop.27 This was a novel observation, because no one had dreamed there were sterol extruders. Our study was also one of the first uses of the triple lumen tube technique to measure both secretion and absorption. The technique was later modified at the Mayo Clinic to study pancreatic (and biliary) secretion,28 starting Bill Go on his very successful career as a pancreatologist.

The Mayo Clinic was an ideal place to perform clinical investigation because of helpful colleagues and cooperative patients. We labeled the steroid moiety and amino acid moieties of conjugated bile acids separately, and used such molecules to define the metabolism of the major bile acids occurring in humans.29-31 Steady-state models were developed with Neville Hoffman.32, 33 Figure 4 illustrates our attempt to show the metabolism of cholic acid and its conversion to deoxycholic acid.

Figure 4.

The metabolism and enterohepatic cycling of cholic acid and its metabolic product, deoxycholic acid. The lower arc denotes unconjugated bile acids returning to the liver to undergo reconjugation. The input of newly formed deoxycholic acid averages about one-third of the rate of cholic acid synthesis in the adult.40

Then, in consultation with Bill Go, Wilf Simmonds and Solko Schalm and others developed radioimmunoassays for the conjugated forms of the primary bile acids, permitting “high-throughput” analyses of plasma bile acids.34, 35 The results disclosed the rhythm of the enterohepatic circulation36 as illustrated in Fig. 5. Nick LaRusso showed that the only source of bile acids in plasma in health was intestinal absorption.37

Figure 5.

Plasma levels of immunoreactive cholyl conjugates (taurocholate and glycocholate) in response to three equicaloric meals. Data from healthy volunteers, cholecystectomized patients, and patients with bile acid malabsorption due to ileal resection are shown. If first-pass extraction by the liver is assumed to be the same in all three groups and uninfluenced by meals, the levels indicate the rate of bile acid absorption from the intestine.

Thanks to the generosity of the National Institutes of Health (NIH), I was able to spend some months at the Politecnico di Torino in Torino, Italy. Here, working with Gianpaolo Molino, Mario Milanese, Gustavo Belforte, and their colleagues, I was able to develop physiological pharmacokinetic models of bile acid metabolism, and use these models to simulate bile acid metabolism in the healthy human38–40 as well as in some disease conditions.41 I was happy with this work, but it seems to have had little impact in the modeling field and has hardly been noticed by pharmacokineticists.

To gain insight as to why bile acids were conjugated only with taurine or glycine, we prepared bile acids conjugated with other amino acids, in work performed with Suzie Huijghebaert. She showed that all of these unnatural conjugates were cleaved by pancreatic carboxypeptidases.42 We could now declare conjugated bile acids to be indigestible and unabsorbable—properties contributing to their high intraluminal concentration.

The enterohepatic circulation of bile acids results from efficient bile acid transport by the hepatocyte and the terminal ileal enterocyte. We could not have dreamed 30 years ago that the four transporters—two apical and two basolateral—of the ileal enterocyte and the hepatocyte would be cloned and characterized. At the International Physiological Congress in 1989, I heard Ernie Wright describe his expression cloning of glut1,43 and thought we could use the same technique to clone the ileal apical bile acid transporter. We were getting started15 when Paul Dawson, a most talented molecular biologist and physiologist, succeeded.44

The disease phenotypes associated with defects in hepatic transporters are now well understood. Defective bile acid uptake by the hepatocyte should be associated with pruritus and fat malabsorption. Defective canalicular transport leads to bile acid retention in hepatocytes and cell death.45 Defective bile acid uptake by the ileal enterocyte leads to diarrhea.46 The clinical consequences of defective basolateral transport by the ileal enterocyte are not yet clarified, but the mouse knockout has bile acid malabsorption without the expected compensatory increase in bile acid biosynthesis.47 The failure to increase bile acid synthesis is attributed to inappropriate and excessive release of fibroblast growth factor-15 (FGF-15) from the ileal enterocyte. FGF-15 (in the mouse) and FGF-19, its human analog, are peptides that appear essential for bile acid–mediated suppression of bile acid biosynthesis in the hepatocyte.48

Bile Acid Functions

Besides their role in lipid digestion, bile acids also denature dietary proteins, enhancing their rate of cleavage by pancreatic proteolytic enzymes.49 Bile acids also have direct and indirect antimicrobial effects that are poorly understood50; Bacterial overgrowth and translocation to lymph nodes can be prevented in cirrhotic rats by oral bile acid administration51 or by activation of farnesoid X receptor (FXR), the nuclear receptor for bile acids.52

The multiple functions of bile acids—in the liver, biliary tract, and small and large intestine—are summarized in Table 2. Whether bile acids in plasma will have a function remains to be seen. Of course, the most obvious function of bile acids is to promote the excretion of cholesterol and thereby contribute to cholesterol homeostasis. Cholesterol metabolism is summarized succinctly in Fig. 6.

Table 2. Functions (Micellar and Nonmicellar) of Bile Acids in Mammals
Whole Organism
 Elimination of cholesterol
Liver
 Hepatocyte
  Insertion of canalicular bile acid and phospholipid transporters
  Induction of bile flow and biliary lipid secretion
  Promotion of mitosis during hepatic regeneration
  Regulation of gene expression by activation of FXR
 Endothelial cells
  Regulation of hepatic blood flow via activation of TGR5
Biliary Tract
 Ductular lumen
  Solubilization and transport of cholesterol and organic anions
  Solubilization and transport of heavy metal cations
 Cholangiocyte
  Stimulation of bicarbonate secretion via CFTR and AE2
  Promotion of proliferation when obstruction to bile flow
 Gallbladder lumen
  Solubilization of lipids and heavy metal cations
 Gallbladder epithelium
  Modulation of cAMP-mediated secretion
  Promotion of mucin secretion
Small Intestine
 Lumen
  Micellar solubilization of dietary lipids
  Cofactor for bile salt dependent lipase
  Antimicrobial effects
  Denaturation of dietary protein resulting in accelerated proteolysis
 Ileal enterocyte
  Regulation of gene expression via nuclear receptors
  Release of FGF-15, a peptide down-regulating bile acid biosynthesis
 Ileal epithelium
  Secretion of antimicrobial factors (FXR mediated)
Large Intestine
 Colonic epithelium
  Promotion of fluid absorption at low concentrations
  Induction of secretion at high concentrations
 Colonic muscular layer
  Promotion of defecation by increasing propulsive motility
Brown Adipose Tissue
 Adipocytes
  Promotion of thermogenesis via TGR5
Figure 6.

Global view of cholesterol and bile acid metabolism. In humans, sterol balance studies suggest that cholesterol is eliminated more in the form of cholesterol than by conversion to bile acids. In most animals, as inferred from biliary lipid composition, most cholesterol is eliminated by conversion to bile acids.144 Statins, widely used to treat hypercholesterolemia, have little effect on the values shown.

Bile Acids and the Intestine

At the Mayo Clinic, in collaboration with Sidney Phillips (and executed by Hagop Mekhjian), we perfused the human colon and showed that deoxycholic acid and CDCA, irrespective of the state of conjugation, induce colonic secretion,53 an observation confirmed recently in polarized monolayers of T84 cells, a colonic cell line.54 Discovery that bile acids were secretagogues suggested that diarrhea caused by bile acid malabsorption should respond to cholestyramine. Results testing the efficacy of cholestyramine in our first patient are shown in Fig. 7. Then, with Rainer Poley, we extended this observation by testing the efficacy of cholestyramine in a single blind study in multiple patients who had diarrhea caused by bile acid malabsorption because of ileal resection.55 These outpatient studies were complemented by inpatient studies in ileal resection patients that helped to clarify the role of bile acid malabsorption versus fat malabsorption in the pathogenesis of diarrhea.56 Colesevelam, a more potent bile acid sequestrant that has recently been developed, is likely to be even more effective than cholestyramine in treating bile acid–induced diarrhea.

Figure 7.

A balance study in a patient with a distal ileal resection, bile acid malabsorption, and increased bile acid biosynthesis, showing the striking reduction in fecal mass and Na+ during 6-day periods when cholestyramine was ingested. Despite binding of bile acids in the proximal small intestine, cholestyramine administration did not induce steatorrhea.

Bile acid malabsorption is also common in diarrhea-predominant irritable bowel syndrome.57 The mechanism has been unknown, but recently Julian Walters and his colleagues have shown that some of such patients have extremely low levels of FGF-19 in plasma, presumably because of defective formation and/or release from the ileal enterocyte. Such a deficiency of FGF-19 should lead in turn to inappropriate up-regulation of bile acid biosynthesis.58 I have argued unsuccessfully that every patient with chronic diarrhea should have a therapeutic trial of a bile acid sequestrant. Bile acid malabsorption can now be diagnosed by measuring plasma levels of C4 (an intermediate in bile acid biosynthesis); plasma C4 increases in direct proportion to the rate of bile acid biosynthesis.59 It can also be measured by showing decreased colonic retention (or rapid loss from the gallbladder) of 75Se–homocholic acid–taurine (75SeHCAT), the gamma-emitting radionuclide that is metabolized much like taurocholate.60 Unfortunately, this valuable compound has never been available in the United States and even its commercial availability outside the United States in the future is uncertain.

One topic that merits further study is whether in patients with increased intestinal permeability, bile acids are absorbed via the paracellular route. Were this to occur, bile acids might activate mast cells in the mucosa.

Bile Acid Metabolism in Cholestatic Liver Disease

In San Diego, with Jan Lillienau and Claudio Schteingart, we showed in guinea pigs that ileal transport of bile acids is regulated in a negative feedback fashion.61 These experimental results are shown in Fig. 8. This observation explains why in humans there is only a modest increase in bile acid secretion when cholic acid or CDCA is fed.

Figure 8.

Evidence for feedback inhibition of ileal bile acid transport in the guinea pig. In these studies, the rate of ileal transport of taurovrsodexycholate was measured by “clamping” luminal bile acid, i.e. bile acid was infused at such a rapid rate, that its luminal concentration was kept constant. Bile acid absorption was measured by recovery in bile from a biliary fistula. Animals had been fed conjugated bile acids or cholestyramine for a week. Abbreviations: CDCG, glycochenodeoxycholate; CT, taurocholate; CS, cholylsarcosine, a bile acid resistant to deconjugation-dehydroxylation.

This work suggested that ileal bile acid absorption should increase in cholestatic liver disease,62 an observation confirmed by Lanzini et al. who reported a greatly prolonged half-life of bile acids (examined by the use of 75SeHCAT in patients with cholestasis).63 Such inappropriate conservation of bile acids could be prevented, at least in principle, by sequestrant administration or by inhibiting ileal transport. A potent nonabsorbable inhibitor of the ileal apical transporter was developed by Monsanto-Searle,64 but has been shelved by Pfizer who took over Monsanto-Searle, presumably because the market for such an agent is tiny.

Presently, children with cholestatic pruritus are treated by ileal bypass or partial biliary diversion, both of which prevent the postulated inappropriate conservation of bile acids by the ileal bile acid transport system. My own view is that bile acid retention in plasma induces pruritus directly or indirectly because increased plasma bile acid levels fulfill most of the requirements of cause and effect.65, 66

Bile Acid Metabolism in Short Bowel Syndrome

Most patients with short bowel syndrome have profound bile acid malabsorption because the resected gut includes the terminal ileum. Maximum bile acid synthesis (5–10 mmol/day) is less than daily bile acid secretion in health (24 mmol/day).67 With Adrian Schmassmann68, 69 and later Sally Longmire-Cook and Jan Lillienau,70, 71 we showed that the sarcosine (N-methylglycine) conjugate of cholic acid was resistant to bacterial deconjugation and dehydroxylation, was not cathartic, and enhanced lipid absorption in the rat. In patients with ileal resections and steatorrhea but with intact colons, cholylsarcosine increased fat absorption and patients gained weight.72 However, our use patent was not licensed, and the compound is now in the public domain, precluding its commercial development. The inability to bring cholylsarcosine to the market is one of my failures. Because bile acids often do not undergo 7-dehydroxylation in the colon of patients with short bowel syndrome, the possibility remains that taurocholate would also be efficacious and not induce diarrhea.

Quantifying Bile Acids

The lab was forced to develop analytical methods to solve biological problems. Bengt Borgström had purchased the first thin-layer chromatography device in Scandinavia, and it was thrilling to see how cleanly individual bile acids could be separated.3 I also reported the use of hydroxyapatite layers to separate 1-monoglycerides and 2-monoglycerides (without isomerization) and confirmed the positional specificity of pancreatic lipase in humans.73

The enzymatic measurement of total 3α-hydroxy bile acids was a Japanese import and was based on the pioneering studies on 3α-hydroxysteroid dehydrogenase by Paul Talalay at the Johns Hopkins University, my alma mater. With Aldo Roda, Juergen Schoelmerich, and the late Marlene DeLuca, we converted the enzymatic measurement of bile acids into a bioluminescence technique for measuring 3α-hydroxy74 and 7α-hydroxy75 bile acids. We used this highly sensitive technique to show that CDCA was well absorbed,76 as well as to examine changes in the postprandial elevation of bile acids during bile acid sequestrant administration.77 Later, with Jim Converse and Steve Rossi, we developed an HPLC method that resolved the 12 conjugated bile acids in human bile.78 We used this technique to show (with Rosemarie Fisher) that hepatotoxicity of CDCA in the National Gallstone Study appeared to be due to the intrinsic toxicity of CDCA and not to defective sulfation of lithocholate.79 In our most recent work on bile acid composition of human cecal content80 and bile acid metabolism in childhood functional constipation,81 we used LC-MS-MS (executed by Terry Griffin). This method seems to me to be the ultimate analytical method, especially if combined with prior class separations.82

In all of this work, we were fortunate to be able to fund a chemistry lab. I was most fortunate to have had two great chemists in my lab: Rick DiPietro and Claudio Schteingart. Rick now directs the pilot plant of IBM in San Jose, CA. He developed a group of photoresist molecules, based on bile acids, that are widely used in microcircuit manufacture.83 This was not a case of bench discovery to bedside application, but rather an uncommon transfer of technology from a Department of Medicine to the electronic industry.

Dissolution of Cholesterol Gallstones by Oral Bile Acid Administration

The idea of feeding bile acids to dissolve cholesterol gallstones is a very old one. Nonetheless, J. L. W. Thudichum in his master work on gallstone disease published in 186384 does not mention bile acid feeding. A decade later, the great physiologist Moritz Schiff proposed the use of oral bile acids for gallstone dissolution.85

In 1957, Charles Johnston, a surgeon, showed together with Fumio Nakayama, a visiting Japanese surgeon, that cholic acid administration did not improve the ability of bile to dissolve cholesterol.86 He contemplated the feeding of CDCA but did not have a source of the material. In 1970, the late Reno Vlahcevic and his colleagues using the isotope dilution technique developed by Sven Lindstedt in Lund, Sweden, reported that the exchangeable pool size of bile acids was decreased in patients with cholesterol gallstones.87 This report provided a clear-cut rationale for feeding bile acids to restore the bile acid pool to normal.

The late Leslie Schoenfield returned to the Mayo Clinic in 1966 from the Karolinska Institute in Stockholm where he had learned to measure bile acids by gas chromatography under the tutelage of Jan Sjövall. Stimulated by the report of Vlahcevic and his colleagues, Leslie Schoenfield decided to feed bile acids in an attempt to restore the bile acid pool and to desaturate bile in cholesterol. He had planned to feed cholic acid and hyodeoxycholic acid, two bile acids that were commercially available. While at Rockefeller University, I had fed cholic acid to a patient with hypercholesterolemia and observed that it decreased cholesterol biosynthesis by the sterol balance technique.88 In 1965, I learned of a British company producing CDCA and later was able to purchase 1 kg with the hope of initiating clinical studies at the Mayo Clinic. I suggested to Les that his study would be improved if he fed not only cholic acid, but also CDCA, because it was the other primary bile acid in humans. The actual study was done by Johnson Thistle. Their finding that CDCA, but not cholic acid, decreased the cholesterol saturation of gallbladder bile was presented to the Central Society for Clinical Research in 1970. I had thought that the presentation would cause great excitement. Indeed, before the presentation, I telephoned New York to order another kilogram of CDCA, thinking that the American supply would soon be exhausted. But no one in the audience other than Les, John, and myself saw the significance of the finding.

Leslie Schoenfield accepted a new position in Los Angeles, and the work on gallstone dissolution continued in a collaboration between Johnson Thistle, Rudy Danzinger, and myself. Later that year, gallstone dissolution in patients receiving CDCA was first observed, leading to our report in 1972.89 Suddenly, there was general interest in bile acids! In the next few years, I worked closely with Herbert Falk of Falk Pharma in Germany as well as the Gipharmex company in Italy as these firms endeavored to bring the compound to market.

While our clinical studies with CDCA were proceeding in Rochester, MN, toxicity studies were being performed in Europe. In 1973, I received a telephone call that CDCA was highly toxic in monkeys and that all European studies had been stopped.90 Our patients seemed to be fine. I convened a Data Safety Monitoring Group to follow our studies closely and we continued our therapeutic trial. We (Alastair Cowen, Mel Korman, Bob Allan, and Paul Thomas) pursued humans studies to show that lithocholate was eliminated solely in bile and was in part sulfated.91, 92 At the same time, in a project executed by Tom Gadacz, we collaborated with Eberhard Mack, a surgeon at the University of Wisconsin, to show that lithocholic acid, the major bacterial metabolite of CDCA, was not sulfated in the rhesus monkey.93 Later, with Michael Schwenk at the University of Tübingen, we could show that the chimpanzee, like humans, also sulfates lithocholate.94 I think that CDCA is the only compound ever to have been approved by the U.S. Food and Drug Administration (FDA) that kills rhesus monkeys!

The pharmaceutical and pharmacological properties of CDCA were nicely elucidated by Gerard van Berge Henegouwen,95, 96 who went on to a distinguished career as Department of Medicine Chief at the University of Utrecht, Netherlands. With the late Tim Northfield, we measured secretion rates of biliary lipids and found (to our astonishment) that CDCA acted by decreasing cholesterol secretion rather than increasing bile acid secretion.97 Curt Einarsson and his colleagues in Sweden, who in parallel to our own group were also studying bile acid metabolism, found that most patients with cholesterol gallstones hypersecreted cholesterol.98 Thus, CDCA corrected the pathophysiological defect. Very recently, cholesterol hypersecretion appears attributable to gain-of-function mutations in ABC5/ABC8, the sterol extruders responsible for a major fraction of biliary cholesterol secretion.99

The NIH, following the recommendation of Bob Gordon, made the decision to support a multicenter double-blind placebo-controlled study, thinking that industry was unlikely to do so. Leslie Schoenfield organized and directed the National Cooperative Gallstone Study (NCGS) with great energy and skill. The study showed that CDCA at a dose of 15 mg/kg body weight induced gradual gallstone dissolution in the majority of treated patients and that CDCA appeared to be reasonably safe.100, 101 After the study was started, it was realized that the dose was suboptimal, but changing the design during the study was impossible.102 During the study, ursodeoxycholic acid (UDCA) had been shown in Japan to induce cholesterol gallstone dissolution, but UDCA could not be easily added to the ongoing NCGS study. There were hopes of a NCGSII which would examine the efficacy and safety of UDCA for radiolucent gallstone dissolution, but the application to NIH for this study was not funded.

There were problems with medical dissolution even with UDCA, which appeared to be almost as effective as CDCA and was totally devoid of hepatotoxicity. First, medical dissolution was only recommended for patients whose gallbladders visualized during oral cholecystography. This technique was being replaced by ultrasonography of the gallbladder. Second, gallstone dissolution by oral bile acid therapy was too slow, often requiring 2 years for complete dissolution; long-term compliance became a problem. Third, it did not always work. Research by the late Karlheinrich Wolpers in Germany103 suggested that when acute cholecystitis occurred, a noncholesterol layer was deposited on the cholesterol gallstone, precluding or at least slowing dissolution. Furthermore, analyses of the natural history of gallstone disease indicated that the asymptomatic gallstone should not be treated.104 Thus, either the patient did not need treatment if asymptomatic, or if biliary colic had occurred, it was too late for treatment. There was no obvious window of opportunity. Finally, there was recurrence after dissolution. In the history of gallstone disease, it was recurrence that led cholecystotomy to be replaced by cholecystectomy.105

Extracorporeal shockwave lithotripsy, a German development, was shown to fragment stones into small pieces, exposing their cholesterol surface; fragmentation speeded dissolution when UDCA was administered. Nonetheless, dissolution was slow, there was pain as stone fragments passed from the gallbladder, and recurrence was not infrequent.106

In contrast, surgery was curative and in the hands of most surgeons was totally safe. The death knell for medical dissolution came with the rapid adaptation of laparoscopic cholecystectomy.

Contact Dissolution of Cholesterol Gallstones

While at the Mayo Clinic, I had shown (with Johnson Thistle and Gerry Carlson) that mono-octanoin (mono-octanoyl glycerol), a kind of salad oil, could dissolve cholesterol stones107 based on a collaborative study with Gordon Flynn at the University of Michigan who examined cholesterol solubility in organic solvents.108 Mono-octanoin had a brief usage for dissolution of retained common duct stones after cholecystectomy,109, 110 but the agent worked very slowly, and endoscopic management became preferred.

Some years later, the Mayo Clinic group led by Johnson Thistle returned to contact dissolution of gallstones. They described dissolution of gallbladder stones using methyl tert-butyl ether (MTBE), a noxious gasoline additive, via a percutaneous transhepatic catheter.111 The Mayo group also developed a spark-free oscillating pump to move MTBE into and out of the gallbladder. Dissolution occurred in several days. Studies in pigs conducted by Oliver Esch in our laboratory indicated that the gallbladder epithelium was completely destroyed by MTBE, but quickly regenerated.112

In our laboratory in San Diego, Salam Zakko, a fellow with remarkable talents in electronics, developed a highly intelligent pump capable of inducing turbulent flow around the gallstone; a pressure sensor that fed back on the inflow and outflow pumps kept the solvent in the gallbladder even if there was gallbladder contraction.113, 114 We found that ethyl propionate (a C5 ester) dissolved gallstones in vitro as rapidly as MTBE (a C5 ether), and was much better tolerated by the patient because of its lower volatility and rapid metabolism.115 We successfully treated six patients.116

Nonetheless, contact dissolution failed for multiple reasons. First, placing the catheter in the gallbladder via the transhepatic route or retrograde via the common bile duct and then the cystic duct was difficult, requiring a skilled interventional radiologist or endoscopist. Second, dissolution was labor intensive and required several days. Third, it was never clear who would do the dissolution: the internist, the interventional radiologist, the endoscopist, or the surgeon. Fourth, if MTBE inadvertently entered the bloodstream, it was highly toxic, causing severe renal damage. Finally, the FDA was unhappy with the atypical route of development and with the inherent toxicity of organic solvents. Venture capital was never obtained, and the procedure is probably relegated to medical history. On a lighter note, I thought that the intelligent, pressure-sensing pump of Salam Zakko could be used to dissolve fecal impactions, but I was not able to convince him of this potential use.

Bile Acids and Biliary Ductules

We decided to synthesize 24-nor bile acids, i.e. C23 bile acids (four carbons instead of five in the side chain) and study their physiological properties. The motivation was to extend a study (with Devorah Gurantz) on the relationship between the physicochemical properties and bile acid–induced biliary lipid secretion.117 Lindstedt and Tryding, working under Sune Bergstrom in Lund, had shown that norcholate was excreted into bile in unchanged form, i.e., without being conjugated with glycine or taurine.118 With Eamonn O'Maille, Devorah Gurantz, and Steve Kozmary, we confirmed this finding.119 We then turned to norCDCA, a project led by Kel Palmer,120 who has since gone on to a distinguished career in endoscopy. In the biliary fistula rat, norCDCA induced a bicarbonate-rich choleresis. We (with Yong Bum Yoon) showed that a similar bicarbonate-rich choleresis was induced by norUDCA. We characterized the complex metabolism of norUDCA, observing that it was secreted in part intact, in part as glucuronides, and in part as a trihydroxy metabolite which turned out to be the 5-hydroxy derivative of norUDCA.121

To explain the remarkable bicarbonate-rich choleresis, we proposed cholehepatic shunting of the unchanged norUDCA, as illustrated in Fig. 9. This idea was greeted with considerable skepticism at its initial presentation, but I think is now widely accepted. The cholehepatic circulation concept also provided an explanation for the bicarbonate-rich choleresis induced by UDCA when it was infused into animals at a rate exceeding the conjugation capacity of the liver122 The choleresis was of canalicular, not ductular, origin based on measurements of mannitol clearance, a technique introduced into biliary physiology by the late Henry Wheeler.123 We were also able to show that a large dose (6 moles) in humans induces a bicarbonate-rich hypercholeresis and possibly set a world's record for the greatest bile flow ever induced in an experimental subject.124

Figure 9.

Cholehepatic shunting of nordihydroxy bile acids or any lipophilic weak acid that is secreted into bile in membrane permeable form (when protonated). The source of the proton is suggested to be carbonic acid. The molecule that is absorbed is replaced by a bicarbonate anion in bile. The absorbed molecule exits the cholangiocyte (shown with hatching) and returns via the periductular capillary plexus to sinusoidal blood. The compound is cleared by the hepatocyte and resecreted into bile, generating osmotic bile flow. The results of multiple such cycles is a bicarbonate rich choleresis of canalicular origin, as evidenced by increased mannitol clearance.

We (Ulrich Bolder, Nanh Trang, and Claudio Schteingart) extended the idea of cholehepatic shunting to sulindac, thus indicating that cholehepatic shunting should occur for any weak, lipophilic acid that is secreted into canalicular bile in membrane permeable form.125 The idea thus arose of “ductular targeting”. The Tmax for biliary secretion of sulindac was so high that we thought that it was transported by the bile salt export pump (BSEP). We speculated that some drug hepatotoxicity might be attributable to interaction of the drug with the BSEP, and that idea has now been confirmed in multiple studies.126

The discovery by Ronald Oude Elferink, Piet Borst, and their colleagues that the multidrug resistance protein 2 (MDR2) (encoded by the gene ABCB4) is a canalicular phosphatidylcholine flippase was a major advance in our understanding of biliary lipid secretion. The ABCB4 knockout mouse has no phospholipid in bile.127 In the absence of biliary phospholipid, the simple bile acid micelles attack cholangiocyte apical membranes, leading ultimately to a peribiliary fibrosis resembling the lesion of primary sclerosing cholangitis (PSC). The group of Michael Trauner and Peter Fickert has been characterizing the pathology of these animals.128 I suggested to the group that they test norUDCA in their animals, and the result was truly remarkable.129 Ductular proliferation decreased, fibrosis cleared, and leukocyte infiltration decreased. The mechanisms of these striking events are under active study by the Graz group. Preclinical studies of norUDCA, sponsored by Falk Pharma, are in progress, with the eventual aim of conducting a clinical trial in PSC. Of course, in human PSC, there is no abnormality of phospholipid secretion and it may be that the compound has no effect whatsoever on this disease. Nonetheless, patients with an MDR3 deficiency are now being recognized with increasing frequency,130 and it is conceivable that treatment with norUDCA will be of therapeutic value in such patients.

Therapy with Bile Acid Agonists and Antagonists

Therapy with exogenous bile acids is still a small market. Medical dissolution of gallstones has been largely displaced by laparoscopic cholecystectomy which can even be performed on an ambulatory basis. UDCA is widely used in treating primary biliary cirrhosis, but it is an uncommon disease. Those clinicians with vast clinical experience are convinced that UDCA slows disease progression,131, 132 even though a meta-analysis failed to show efficacy.133 In PSC, emerging data suggest that UDCA is not helpful. Both of these conditions are considered to be autoimmune diseases, and it is doubtful whether UDCA has any immunomodulatory properties. UDCA certainly decreases the cytotoxicity of circulating bile acids and has antioxidant properties, but it does not attack the etiology of the disease. UDCA is clearly useful in the syndrome of cholestatic disease of pregnancy134 which is likely to be a caused by a defect in canalicular transport.

Conjugated bile acid replacement in short bowel syndrome has been shown to be efficacious, but there appears to be little commercial interest in this application. Primary bile acid feeding is life-saving in some inborn errors of bile acid biosynthesis,135 because exogenous primary bile acids restore the circulating bile acids and also down-regulate bile acid biosynthesis by activating FXR, the transcription factor activated by bile acids. Such conditions are extremely rare.

New synthetic FXR agonists, far more potent than CDCA, are now being reported.136, 137 The impetus for targeting FXR in cholestatic liver disease is that FXR up-regulates BSEP and down-regulates NTCP, the basolateral bile acid uptake transporter. This result should lower the intrahepatocyte concentration of bile acids, thereby decreasing their cytotoxic effects. FXR is also considered to have potent effects on carbohydrate and lipid metabolism.138 In addition, bile acids have been shown to activate TGR5, a G protein–coupled receptor in brown adipose tissue139 that in turn leads to thyroxin production, suggesting a role for bile acids in thermogenesis and possibly weight loss. Animal studies with bile acid derivatives that have a high affinity for the TGR5 receptor are in progress. A potent FXR agonist, 6-ethylCDCA,140 is in clinical trials for the treatment of primary biliary cirrhosis.

The above discussion indicates there are currently five rationales for administering bile acids or bile acid derivatives.141 The first is bile acid displacement in which the composition but not the secretion rate of the circulating bile acids is altered, usually by enrichment in the administered bile acid. This is achieved by UDCA administration in cholestatic liver disease and cholesterol gallstone disease. The second is bile acid replacement, where bile acids are administered to correct a deficiency of bile acids caused by defective biosynthesis (inborn errors of metabolism) or defective intestinal conservation (short bowel syndrome). The third is the administration of compounds that may or may not be bile acids aimed at activating the nuclear receptor FXR. The aim of such therapy is discussed above. The fourth is the administration of bile acids or bile acid derivatives that are potent TGR5 agonists for weight loss. The last is the administration of bile acids with modified side chains that undergo cholehepatic shunting and may possibly benefit cholangiopathies.

Bile acid sequestrants may be considered bile acid antagonists. A more potent sequestrant, colesevelam, has been claimed in anecdotal reports to be superior to cholestyramine in treating bile acid diarrhea as well as cholestatic pruritus. Still more potent bile acid sequestrants are likely to be developed in the future. Bile acid sequestrant administration increases insulin sensitivity modestly but consistently,142 possibly by moving the site of fatty acid absorption to the ileum and thereby stimulating the release of glucagon-like peptide-1.143 The FDA has now approved the use of colesevelam in the treatment of type II diabetes.

Epilogue: Leon Schiff, who was a master hepatologist and who helped to found the American Association for the Study of the Liver, convened the first American meeting on bile acids just 40 years ago. Were he with us today, I think he would be thrilled by the progress that has been made in unraveling the complex biology and chemistry of bile acids. Progress is continuing as the power of molecular biology is being applied to disclose new functions of bile acids in regulating carbohydrate and lipid metabolism. The bile acid community has always emphasized translational research. I think it can take some satisfaction in knowing its discoveries have not only dispelled ignorance but have also have helped patients suffering from intestinal and hepatobiliary disease.

Acknowledgements

During much of my time at the Mayo Clinic and at UCSD, I was most fortunate to have Vicky Huebner as a secretary, administrative assistant, and then editor, Her secretarial, administrative, and editing skills let me work in peace and contributed to a decent acceptance rate for the many manuscripts produced by the laboratory. Joseph Steinbach, Ph.D., provided invaluable advice for statistical analysis, and solved all problems in graphics. I also thank the anonymous reviewers of my submitted manuscripts whose criticisms improved them greatly.

Work described above had many sources of support, among them the Mayo Foundation, the Falk Foundation, many generous pharmaceutical companies, and above all, the National Institutes of Health. I also acknowledge the support of the Alexander von Humboldt Foundation that permitted a brief sabbatical on two occasions. I also acknowledge the continuing loving support of my wife, Heli, who has often been neglected by my deep involvement in my work.

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