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Bile Acid Chemistry, Biology, and Therapeutics
During the Last 80 Years: Historical Aspects
Alan F. Hofmann and Lee R. Hagey
Running footline: History of bile acid research
Department of Medicine, University of California, San Diego 92093-063
Correspondence to Alan F. Hofmann ([email protected]) or Lee R. Hagey ([email protected])
Alan F. Hofmann, M.D., Telephone and fax: (858) 459-1754
Lee R. Hagey, Ph.D. Telephone (619) 543-2281
Abbreviations: ASBT, apical sodium co-dependent bile acid transporter; AUC, area under the curve;
BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; CMC, critical micellization concentration;
CMT, critical micellization temperature; CMpH, critical micellization pH; DCA, deoxycholic acid; FATP,
fatty acid transport protein; FGF, fibroblast growth factor; FGFR4, fibroblast growth factor receptor 4;
FRET, Forster resonance energy transfer; FXR, farnesoid X-Receptor; GLP-1, glucagon-like peptide 1;
MDR, multidrug resistance (protein); MRP4, multidrug resistance protein 4; MTBE, methyl tertbutyl
ether; NTCP, Na+ taurocholate co-transporting polypeptide; OATP, organic anion transporting
polypeptide; OST, organic solute transporter; PC, phosphatidylcholine; PFIC2, progressive familial
intrahepatic cholestasis, type 2; SLCO, solute carrier organic anion; TGR5, transmembrane G protein-
coupled receptor 4; UDCA, ursodeoxycholic acid.
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Abstract
During the last 80 years there have been extraordinary advances in our knowledge of the chemistry and
biology of bile acids. We present here a brief history of the major achievements as we perceive them.
Bernal, a physicist, determined the x-ray structure of cholesterol crystals, and his data together with the
vast chemical studies of Wieland and Windaus enabled the correct structure of the steroid nucleus to be
deduced. Today, C24 and C27 bile acids together with C27 bile alcohols constitute most of the bile acid
“family”. Patterns of bile acid hydroxylation and conjugation are summarized. Bile acid measurement
encompasses the techniques of gas chromatography, HPLC, mass spectrometry, as well as enzymatic,
bioluminescent, and competitive binding methods. The enterohepatic circulation of bile acids results from
vectorial transport of bile acids by the ileal enterocyte and hepatocyte; the key transporters have been
cloned. Bile acids are amphipathic, self-associate in solution and form mixed micelles with polar lipids --
phosphatidylcholine in bile, and fatty acids in intestinal content during triglyceride digestion. The rise and
decline of dissolution of cholesterol gallstones by the ingestion of 3,7 dihydroxy bile acids is chronicled.
Scientists from throughout the world have contributed to these achievements.
Key words: Bile acid transport, enterohepatic circulation, bile acid physical chemistry; bile acid analysis;
bile acid metabolism
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Table of contents
I. The ebb and flow of medical interest in bile acids 5
II. Bile acid structure: history and current concepts
A. The carbon skeleton 13
B. the A/B ring junction 16
C. Hydroxylation sites 18
D. Conjugation 25
E. Structure-function relationships 28
III. Measurement
A. Isolation and chromatographic separation 30
B. Mass spectrometry 33
C. Enzymatic and competitive binding techniques 34
D. Diagnostic utility of plasma bile acids 35
E. Determinants of bile acid profiles in body fluids 36
IV. Bile acid metabolism: transport and the enterohepatic circulation
A. Bile acid transport 37
B. Enterohepatic circulation in health 48
C. Perturbations of the enterohepatic circulation 57
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V. Physicochemical aspects of bile acids
A. Overview 58
B. Bile acids in aqueous solution 59
C. Solubilization of polar lipids by bile acid micelles 66
D. Physical chemistry of bile and cholesterol gallstone disease 70
E. Physical chemistry of fat digestion 73
VI. Dissolution of cholesterol gallstones
A. Gallstone dissolution by oral bile acids 75
B. Acceleration of dissolution by shock wave lithotripsy 79
C. Topical dissolution with organic solvents 80
D. Concurrent advances in gallstone management 81
VII. Epilogue: omissions and apologies 82
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This article contains aspects of the history of bile acid research during the last eight decades. The
authors, now in the twilight of their careers, are not trained historians, and there will be omissions,
misconceptions, and provocative judgments. Let us hope the omissions are minor, misconceptions are
rare, and provocative judgments stand the test of time. Let us also hope that this effort will be both useful
and entertaining. Each of the topics discussed merits at least a full length article, so there will be of
necessity in many instances consideration of only what we perceive to be the highlights.
I. The Ebb and Flow of Medical Interest in Bile Acids
After the elucidation of the true chemical structure of bile acids in 1932 (see below), there was little
interest in bile acids in the Western world. One exception to this statement was the laboratory of
Siegfried Thannhauser, who wrote the first textbook of metabolic biochemistry in Germany. He studied
cholesterol and bile acid balance in the biliary fistula dog (1). During this time, bile acids were sold as
liver tonics and laxatives, but there were no placebo-controlled studies showing efficacy. Indeed, bile
acids were considered by the medical profession to have no useful therapeutic properties. The tri-oxo
derivative of cholic acid (called “dehydrocholic acid”) was known to induce bile flow in animals (2), and
was occasionally used to stimulate bile flow in patients; but again there were no controlled studies
showing efficacy in hepatobiliary disease.
Albert Boehringer founded a pharmaceutical company in Ingelheim, Germany. His wife was a cousin
of Heinrich Wieland, then Professor in Munich. Wieland was successful in persuading the company to
begin the isolation and marketing of cholic acid from ox bile in 1917. Thus, cholic acid of high purity
soon became available to the scientific community. During this time, Japanese students came to
laboratories such as that of Wieland to pursue their doctoral studies. When they returned to Japan, they
continued their studies on bile acid and bile alcohol structure. Their work, as well as that occurring in
Europe is summarized in the book “Ueber die Chemie and Physiologie der Gallensäuren” authored by
Shimizu in 1935 (3).
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During the 1930’s and 1940’s, an enormous international effort went into defining the structure of
the major hormonal steroids. When it was recognized that cortisone had a C-11 oxygen atom, it was
logical to use deoxycholic acid (possessing a C-12 hydroxyl group) as a chemical precursor for the
synthesis of corticosteroids. Deoxycholic acid (DCA) was easily isolated from bovine bile or synthesized
from cholic acid, and it was not too difficult for a talented chemist to move the oxygen from C-12 to C-
11. In 1946, H. Sarett (at the Merck Company) reported a complex synthesis (37 steps!) of cortisone
from DCA (4), and this led to its commercial production on a small scale. Seven years later, Hench, a
rheumatologist at the Mayo Clinic who worked closely with his colleague Kendall, an able steroid
chemist, obtained a small supply of cortisone supplied by Merck (5). Hench showed that this compound
caused a striking symptomatic improvement in patients with rheumatoid arthritis. The translational
research of Hench and Kendall resulted in their being awarded the Nobel Prize in 1950 (6).
As a result of the truly exciting advent of cortisone in the treatment of rheumatoid arthritis, there was
an enormous effort to develop a simple, efficient synthesis of this hormone from DCA. Soon, there was
concern that the world’s supply of DCA (derived from cow and sheep bile) would be insufficient to meet
the medical demand. Then, workers at Upjohn discovered that hydroxylation at C-11 could be achieved
using a fungus. Other workers found a plant saponin that could be used as a substrate for the fungal
hydroxylation at C-11, after which the side chain was easily altered to that of cortisone. (For details, see
Fieser and Fieser) (7). DCA was no longer needed, and chemical interest in bile acids collapsed.
After the Second World War, a few laboratories pursued the search for new bile acids, as well as
defining the metabolism of bile acids in mammals. The availability of 14C and 3H which could be
incorporated into the bile acid molecule, together with the development of automatic liquid scintillation
counters and chromatography enabled biotransformations to be identified and measured. Sune
Bergström, then working in Lund, Sweden recruited a highly talented group of doctoral students -- Jan
Sjövall, Henry Danielsson, Arne Norman, Sven Lindstedt, Bengt Samuelsson, Sven Eriksson, Bengt
Borgström, among others -- who carried out fundamental studies of bile acid metabolism in a variety of
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species including man (8). (It is said that Bergström chose his graduate students on the golf course. It is
also said that the lights never went off at night in the Bergström laboratory).
Sjövall developed gas chromatography and then later, after he had moved to the Karolinska Institute in
Stockholm, developed mass spectrometry for the measurement of bile acids (9). He used these techniques
and others to define important aspects of bile acid metabolism over the next four decades (10). Norman
developed a simple synthesis of conjugated bile acids, prepared the conjugates of all the major bile acids
known at that time (11), and with Sjövall performed metabolic studies in animals which distinguished
primary bile acids (made in the liver from cholesterol) from secondary bile acids (made from primary bile
acids by intestinal bacteria). Their work proved that DCA was a secondary bile acid (12, 13). Lindstedt
measured bile acid kinetics (pool size, turnover rate, and synthesis) in man (14). Danielsson identified
key intermediates in bile acid biosynthesis (15). Samuelsson synthesized ursodeoxycholic acid (UDCA)
and defined its metabolism (16). Eriksson showed that bile acid synthesis in the rat was under negative
feedback control (17). Borgström with his colleagues Sjövall, Lundh, and Dahlquist, intubated healthy
volunteers and defined the site at which individual nutrients are absorbed (18). This study proposed that
the bile acid pool circulates about twice per meal. Borgström became director of the Department of
Physiological Chemistry in Lund in 1958, replacing Bergström who had moved earlier that year to the
Karolinska Institute in Stockholm with the bile acid group. Bergström and his pupil Samuelsson then left
the bile acid field and turned their attention to prostaglandins. Their outstanding work in this field led to
their receiving the Nobel Prize in 1982. However, at the Karolinska Hospital, Swedish scientists
continued to work on bile acids, clinical and biochemical studies being conducted by Kurt Einarsson, Bo
Angelin, Ingemar Björkhem, and Kjell Wikvall, among others.
In 1961, news of the work of Lack and Weiner (19) which showed unequivocally that the ileum was
the site of conjugated bile acid absorption reached Sweden. Borgström directed the senior author, to
perform studies showing that in man, conjugated bile acids are absorbed mainly in the ileum (20).
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In London, Geoffrey Haslewood pursued the structure of bile acids in different species, based on his
belief that bile acid composition was a powerful phenotype that would aid in the identification of
evolutionary relationships (21). Edward Doisy, Chairman of the Department of Biochemistry at St. Louis
University, having already won the Nobel prize for his work on vitamin K, pursued the identity of the
3,6,7-trihydroxy bile acids in rats and identified the α- β- and ω-muricholic acids (22) His student,
William Elliott synthesized numerous bile acids and described their chemical and chromatographic
properties (23).
In Japan, a school of bile acid studies was established by Tayei Shimizu (24), who returned to his
native country after having spent three years in the laboratory of Heinrich Wieland. Japanese
investigators pursued the structure of natural bile acids, the work continuing with Taro Kazuno and his
pupil Takahiko Hoshita (25) and, in turn, with Hoshita’s student Mizuho Une (25, 26). Japanese workers
also attempted to define the early steps in bile acid biosynthesis from cholesterol (27).
In the United States, James Carey, working at the University of Minnesota, was directed by his mentor,
Cecil Watson, to pursue bile acid metabolism in man. He identified chenodeoxycholic acid (CDCA) as a
major biliary bile acid (28) and proposed that lithocholic acid, its bacterial metabolite, caused liver injury
in man (29). Edward (“Pete”) Ahrens, working with Lyman Craig at the Rockefeller University, showed
that countercurrent distribution could be used to separate conjugated and unconjugated bile acids (30).
Later, two of his postdoctoral fellows, Scott Grundy and Tatu Miettinen, developed a gas
chromatographic method for measurement of fecal bile acids, whose output, in the steady state, is
equivalent to bile acid synthesis (31). Grundy went on to a distinguished career in human nutrition (32).
Miettinen continued to do sterol balance studies for much of his very productive career (33).
The first symposium devoted solely to bile acids was organized by Leon Schiff, a clinical hepatologist,
who was one of the founders of the American Association for the Study of Liver Diseases. This
symposium held in 1967 was quite exciting for its participants who are shown in Figure 1.
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However, it is safe to say that the study of bile acids was pursued by only a small number of
laboratories, some in Departments of Biochemistry and some in Departments of Medicine. Erwin
Mosbach, one of the early workers in bile acid metabolism, once stated to his wife, “Whenever I go to the
podium to give a paper on bile acids, everyone leaves the room”.
In 1965, the senior author, working in the laboratory of E.H. Ahrens, began feeding studies with cholic
acid in a patient with severe hypercholesterolemia, and showed that cholic acid feeding was a potent
suppressor of bile acid and cholesterol biosynthesis (34), based on measurement of fecal bile acids and
sterols, using the newly developed gas chromatographic method for fecal bile acids that had been
developed in this laboratory (32). It was logical to test CDCA, the other primary bile acid, but at that
time, the world’s supply of pure CDCA was thought to be less than 10 grams, and the synthesis from
cholic acid was difficult. However, in the 1960’s, a small English pharmaceutical company (Weddell
Pharmaceuticals) began the manufacture of CDCA for unknown reasons. A kilogram was purchased for
the senior author by the Mayo Clinic in 1967. Leslie Schoenfield returned to the Mayo Clinic in 1966
after having spent a year in the laboratory of Sjövall, and initiated a clinical trial with his fellow Johnson
Thistle to test whether oral cholic acid or hyodeoxycholic acid would lower cholesterol in bile and
ultimately induce cholesterol gallstone dissolution. The senior author persuaded Schoenfield to add
CDCA to his protocol, and this study of Thistle and Schoenfield showed that CDCA feeding decreased
biliary cholesterol saturation, whereas neither cholic acid nor hyodeoxycholic acid had any effect (35). In
1972, the first gallstone dissolution induced by the ingestion of CDCA was observed, initially at the Mayo
Clinic (36), and later in London by a group led by Hermon Dowling (37). (This was not the first time that
the efficacy of oral bile acids had been tested at the Mayo Clinic. In 1938, Philip Hench had fed a
mixture of conjugated bile salts in an unsuccessful attempt to treat rheumatoid arthritis) (38).
The discovery that CDCA would induce gradual dissolution of cholesterol gallstones led to the next
resurgence of interest in bile acids. For the very first time, CDCA was made in kilogram quantities by
several manufacturers, and became the third bile acid available as a fine chemical.
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In Japan, ursodeoxycholic acid (UDCA) had been brought to market in the 1950’s by Tokyo Tanabe as
a liver tonic, based on the legendary therapeutic effects of bear bile. It was packaged as 10 mg tablets,
and the recommended dose is unlikely to have had any pharmacodynamic effects. Two decades later in
Japan, UDCA, in much larger doses, was observed to induce cholesterol gallstone dissolution (39, 40).
UDCA soon replaced CDCA for gallstone dissolution throughout the world because UDCA showed an
efficacy similar to that of CDCA, but in contrast, had virtually no hepatotoxicity. To provide UDCA for
therapeutic trials performed around the world, UDCA was synthesized in kilogram quantities and became
the fourth bile acid to become available as a fine chemical. A Japanese perspective on the discovery of
UDCA as a therapeutic agent is available (41).
As discussed later, dissolution of cholesterol gallstones by ingestion of UDCA began to decline in the
1990’s because of the development of laparoscopic cholecystectomy which promised a rapid cure for the
problems of gallstone disease, irrespective of gallstone type. Once again, bile acids seemed to have little
medical value, and interest in their biology and chemistry declined.
Nonetheless, the ready availability of UDCA meant that it continued to be used for gallstone patients
who were poor operative candidates. Ulrich Leuschner, a hepatologist based in Frankfurt, Germany, noted
that UDCA ingestion improved biochemical parameters in patients with primary biliary cirrhosis (42).
UDCA was subsequently shown to slow disease progression in primary biliary cirrhosis, initially by
Raoul Poupon in Paris (43), and later by Keith Lindor and his colleagues at the Mayo Clinic (44). UDCA
treatment of primary biliary cirrhosis is now standard of care. UDCA was also shown to be effective in
cholestasis of pregnancy (45). UDCA has been widely used for other cholestatic diseases and to restore
bile flow following liver transplantation, but proof of efficacy in these conditions based on placebo-
controlled trials has been uncommon.
At present, it is estimated that about 1000 metric tons or about 1 million kilograms -- of cholic acid are
produced globally from bovine bile, the majority of which is used for the production of UDCA (S. Yorke,
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New Zealand Pharmaceuticals, Ltd.; personal communication). Bile acids are also used as precursors for
the synthesis of nuclear receptor agonists such as obeticholic acid (the 6α-ethyl derivative of CDCA), as
building blocks for other molecules (46), and as key constituents of some bacteria media. Bile acids have
even used in the electronic industry as precursors of photoresist molecules in integrated circuits (47).
(This unlikely technology transfer from a biological laboratory to the electronic world resulted from one
of the early chemists in our laboratory, Rick DiPietro, taking a position in the electronics industry and
envisioning a truly novel use for bile acids).
In 1984, the results of the Coronary Primary Prevention Trial showed that the ingestion of
cholestyramine, the first bile acid sequestrant to be marketed, lowered plasma cholesterol levels and
reduced cardiovascular events. This was of considerable interest to the cardiovascular community, as the
study provided the first convincing evidence for the validity of the cholesterol hypothesis (48).
Nonetheless, the demonstrated efficacy of treating hypercholesterolemia with a bile acid sequestrant had a
rather modest effect on clinical practice because of the poor tolerability of cholestyramine and the
emergence of the statins which were still more potent and usually well tolerated.
The current rebirth of interest in bile acids arises from the astonishing discovery of their signaling
properties. Bile acids were found to be the ligand for the orphan nuclear receptor FXR (farnesoid X-
receptor) (49, 50, 51), meaning that bile acids regulate the activity of many genes (52, 53, 54). Bile acid
derivatives that are potent FXR agonists have been shown to induce the synthesis and basolateral release
of the protein fibroblast growth factor 15/19 (FGF19) from the ileal enterocyte (55). FGF19 travels in
portal venous blood to the liver and down-regulates bile acid biosynthesis (55, 56). FGF19 promotes
hepatocyte growth (57), and FXR agonism also has antidiabetic effects mediated by multiple mechanisms
(58), as well as anti-inflammatory activities (59). Several potent FXR agonists have been synthesized (53,
60) and one of these, obeticholic acid (61), is now being tested for its efficacy in primary biliary cirrhosis
and non-alcoholic steatohepatitis (62).
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Two Japanese groups discovered a transmembrane G-coupled protein receptor (TGR5) activated by
bile acids (63, 64). TGR5 has a wide tissue distribution, being most heavily expressed in the enteric
nervous system (65); it is also present in spinal cord neurons (66) and the primary cilia of cholangiocytes
(67). The consequences of this activation include release of the hormone glucagon-like peptide 1 (GLP-
1), which has appetite-suppressing and antidiabetic effects (68). TGR5 also has effects on hepatic blood
flow (69), distal intestinal motility (70), and in addition, has anti-inflammatory effects (71) that are under
active investigation.
Discovery of the signaling properties of bile acids is certainly a new paradigm in the bile acid field.
Bile acids have become hormones. Now highly talented molecular biologists in both academe and the
biotechnology industry are pursuing bile acid biology. Bile acid signaling has been discussed only
briefly in this review, as its aim is to focus on events in the past.
As bile acids have been the subject of scientific study for more than 150 years, the bile acid
literature is vast. The early history of bile acid discovery is available in the two monographs by Harry
Sobotka (72, 73), as well as the masterly text on steroids by the Fiesers published in 1955 (7). A small
pharmaceutical company, Dr. Falk Pharma, based in Freiburg, Germany was the first European company
to bring CDCA to market for gallstone dissolution. Since 1972, the Falk Foundation, funded by Dr. Falk
Pharma, has hosted biennial meetings on bile acid biology, chemistry, and therapeutics. The proceedings
of these meetings, usually published in book form, serve to document the advances that have occurred in
the last four decades. G.A.D. Haslewood’s slim monograph summarizes current knowledge in 1966 (74)
and Haslewood later authored a monograph on the biological importance of bile salts (75). The Elsevier
Encyclopedia of Organic Chemistry (76) contains detailed information on the chemistry of bile acids and
their derivatives. A four volume series “The Bile Acids” edited by Nair and Kritchevsky (77) treats many
of the topics discussed in this article in much greater detail. A book entitled “Sterols and Bile Acids” is
Volume 12 of the series New Comprehensive Biochemistry. This volume, published in 1985, has 6
chapters dealing with varying aspects of bile acids and bile alcohols (78). A multiauthor book describing
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many aspects of bile acid research with particular emphasis on Italian contributions was published in
2000 (79). The senior author (80) and Hermon Dowling (81) have both written overviews of the field
from a medical standpoint in 1985. The Journal of Lipid Research published a number of excellent
reviews on the biology and chemistry of bile acids a few years ago.
The remainder of this historical review will discuss selected topics in more detail with an emphasis on
structure-activity relationships. The review will not summarize our extensive work on bile acids in
different vertebrates, as our findings have been published elsewhere (82). Other topics that have been
omitted are discussed in the epilogue.
II. Bile acid structure: History and current concepts
A. The carbon skeleton
1. The steroid nucleus
In December, 1929, a unique event occurred in Stockholm. Two Nobel prizes in Chemistry were
awarded. In 1927, no prize in Chemistry had been awarded because the Nobel Prize Selection Committee
could not agree on a suitable candidate. During the following year, the committee decided to award the
1927 prize to Heinrich Wieland and the 1928 prize to Adolf Windaus. In his Nobel lecture, Wieland
reviewed his laborious attempts to define the structure of bile acids and presented his current vision of the
structure of cholic acid (83). Windaus presented his proposed structure of cholesterol as well as his
brilliant studies on the formation of vitamin D2 from cholesterol by radiation (84). These two
extraordinarily talented chemists, using the most primitive of chemical techniques, had slowly developed
the idea that both cholesterol and bile acids share a four ring structure with a side chain of five carbon
atoms for bile acids and eight carbons for cholesterol. However, the structures of the steroid nucleus
shown in their Nobel lectures were not correct, and each of these giants described their structures as
“probable”.
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The path to the correct structure came from an unlikely origin. The polymath Desmond Bernal,
working in the Mineralogical Museum in Cambridge, England decided together with his colleague
William Astbury to undertake X-ray diffraction studies of organic molecules. Bernal, nominally a
physicist, began his studies in 1931. He studied ergosterol, cholesterol, and vitamin D and for all three of
these compounds obtained a probable structure of a flat molecule. He published his findings as a note in
Nature in 1932 (85). This report caught the attention of Rosenheim and King in London, and Wieland
and Dane in Munich. Later, in that same year, Wieland and Dane followed by Rosenheim and King
published the application of Bernal’s pioneering studies to the extensive literature on bile acid structure
(86). Each group proposed the cyclopentanoperhydrophenanthrene structure for the steroid nucleus.
Their final structure has stood the test of time with confirmation by both X-ray diffraction (87) and a total
synthesis of CDCA (88). Figure 2 shows the structure of CDCA and the numbering system for the steroid
nucleus and side chain that was proposed in the 1930’s and has been used ever since.
Bernal’s X-ray diffraction studies were the first biological application of this revolutionary technique.
Bernal later used X-ray diffraction to deduce the structure of other organic molecules, and his pupils such
as Dorothy Crowfoot Hodgkin extended the work, its ultimate triumph being the helical structure of DNA
and RNA by Watson and Crick. Bernal’s left wing political views and his Bohemian life style probably
kept him from being awarded a Nobel Prize (89,90).
2. The side chain
The structure of the C5 side chain of the common natural C24 bile acids – isopentanoic acid or more
specifically γ-methylbutanoic acid – was elucidated by Wieland, and like the structure of the steroid
nucleus, has also been confirmed by X-ray diffraction (87) and total synthesis (88).
The structure of the C8 side chain in C27 bile alcohols and bile acids was more problematic. It was
not clear initially that these molecules possessed the side chain of cholesterol because it was quite
uncertain whether these compounds and even the common natural C24 bile acids were derived from
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cholesterol. The uncertainty arose from the failure of a dietary cholesterol load to increase bile acid
output in the biliary fistula dog. George Whipple, a leading physiologist, wrote in his 1922 review on the
constituents of bile (91). “Cholesterol....has often been suspected as the precursor of cholic acid, ... but
careful experiments negative [sic] this interesting suggestion”. Nonetheless, Shimizu in his book “Ueber
die Chemie und Physiologie der Gallensäuren”, published in 1935 (3) noted that, “Interesting
intermediates in the biological catabolism of cholesterol to bile acids can be found in scymnol and
tetraoxybufostan [both C27 bile alcohols]”. Even earlier, in 1923, Ruzicka had proposed that bile acids are
formed from cholesterol (92).
Proof that cholesterol was converted to bile acids had to await the introduction of isotopes into
biochemistry by Rudolph Schoenheimer (93). Bloch, Berg, and Rittenberg prepared cholesterol labeled
with deuterium, gave it to a dog with a surgical connection between the biliary tract and the renal pelvis;
in this chronic preparation bile is excreted in the urine. Cholic acid was isolated from urine and shown to
have incorporated deuterium into its structure, thus establishing the conversion of cholesterol to C24 bile
acids (94). Figure 3 shows the structure of cholesterol and the changes that occur as it is converted to
CDCA. (Although Konrad Bloch received the Nobel Prize in 1964 for his extraordinary work on fatty
acid and sterol synthesis, his Nobel lecture does not even mention this key experiment).
The first bile acid with a longer side chain than the common natural C24 bile acids was isolated
from the bile of the toad Bufo vulgaris formosus by Shimizu and Oda in 1934 (95). The structure of this
bile acid was established by Takahiko Hoshita et al as a trihydroxy bile acid with a double bond at C-22
and a carboxyl group at C-24 (25). Thus this bile acid is a C28 bile acid having one more carbon than its
precursor cholesterol. Other C28 bile acids with a methyl group on the side chain have been reported (26),
but are quite uncommon in nature.
C27 bile acids are much more common (82). They were isolated from the bile of a number of
early evolving vertebrates. In particular, bile from the alligator, which was readily available in large
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amounts (studies at that time began with up to 5 liters of bile), became the object of intense study as it
contains a great variety of C27 bile acids (96, 97).
The carbon skeleton of the side-chain of C27 bile acids was assumed to have remained unchanged
from that of the parent compound, cholesterol, but the location of the carboxyl group was unclear. In
1952, Bridgwater and Haslewood synthesized a defined side-chain starting from the steroid nucleus.
They then showed that their product was identical to 3α,7α,12α-trihydroxy-5β-cholestan-27-oic acid, the
major bile acid isolated from the bile of crocodiles and alligators, and they confirmed its structure by X-
ray diffraction (98). Subsequently, the laboratory of Takashi Iida reported the synthesis of the 25R and
25S stereoisomers of this compound (99).
The first C27 bile alcohol to be isolated was scymnol, obtained from the bile of the Greenland
shark (Scymnus borealis) by Olof Hammarsten, working in Sweden in 1898 (100). The finding that the
molecule had 27 carbon atoms is a tribute to the precision and accuracy of the combustion techniques that
were used at that time to determine elemental analysis. A half century later, another C27 bile alcohol was
isolated from the bile of the American bullfrog (Rana catesbeiana) by Kazuno et al (101). Subsequently,
Haslewood (102) found the same bile alcohol in the European common frog (Rana temporia), and gave it
the common name “ranol”. Our extensive survey of vertebrate bile acids (82) has established that most
bile alcohols have an iso-octanoic acid (α,ε-dimethyl-hexanoic acid) side chain. The exceptions identified
to date are C26 and C25 alcohols that are present in the bile of some frog species (25, 26), as well as a C24
bile alcohol, petromyzonol that occurs in the lamprey as a disulfate (103,104). Trace proportions of C24
bile alcohols with the nuclear structure of cholic acid and CDCA are found in the bile of many species
(105).
B. The A/B ring junction
In most natural bile acids whether C24 or C27, the junction of the A and B rings is cis. This steric
conformation is indicated by depicting the bond connecting the C-5 hydrogen atom to the A/B ring
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juncture as a solid line or wedge. The much rarer “allo” bile acids (A/B ring junction is trans) are usually
denoted by a dashed line coming from the ring junction to the hydrogen atom. For the non-steroid
chemist, it is not easy to grasp that the orientation of the C-5 hydrogen atom indicates the type of A/B ring
junction, and thus determines the shape of the two markedly different bile acid epimers. The structures of
cis and trans decalin, model compounds for the A and B rings of bile acids are shown in Figure 4. Every
bile acid and bile alcohol has, in principle, a 5α and a 5β epimer.
Both Wieland and Windaus were fully aware of the problem of bile acid geometric isomerism at the
time of their Nobel Prize lectures. A detailed review of some of the experimental work elucidating the
nature of the A/B ring junction in various bile acids is available in Fieser and Fieser’s comprehensive text
on steroids (7). Probably, it was Windaus who introduced the prefix allo to denote 5α (A/B trans) bile
acids.
Elliott has summarized the chemical and biological properties of allo bile acids in his chapter in The
Bile Acids, volume I (106). One way to distinguish 5α bile acids from 5β bile acids is to prepare their
per-oxo derivatives, which are readily distinguished using optical rotatory dispersion (107).
C24 allo bile acids are the dominant primary bile acids only in lizards, although they are present in trace
proportions in the bile of many species (82). If cholestanol (the saturated 5α-derivative of cholesterol) is
administered to rabbits, it is converted to 5α- bile acids (107). Allocholic acid, the biological precursor of
allodeoxycholic acid, has been prepared synthetically and some its biological properties explored (108).
Allo bile acids have also been identified in fecal bile acids (109), presumably generated by bacterial
enzymes acting on the 3-oxo-Δ4,6 intermediate that is formed during the process of C-7 dehydroxylation
(110). Enantiomeric bile acids, in which the steric configuration of all ring junctions is reversed, have
recently been synthesized (111).
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Bile alcohols (C27) with a 5α configuration are not uncommon in nature. The simplest
biotransformation of cholesterol (Δ5) into a bile alcohol results in a 5α nucleus, and many early evolving
vertebrates form A/B trans bile alcohols (25, 26, 82).
The preceding discussion thus indicates that there are only four carbon skeletons in most natural bile
acids and alcohols – the C27 cholestane skeleton (with either A/B cis or A/B trans structure) and the C24
cholane skeleton (with either A/B cis or A/B trans A/B structure). The side chain of each of the four
skeletons may end in either a primary alcohol group or a carboxyl group. As noted above, C24 bile
alcohols are very rare in nature. Thus, to simplify bile acid and alcohol nomenclature, it is useful to divide
all “cholanoids” and “cholestanoids” into only three great classes – C27 bile alcohols, C27 bile acids and
C24 bile acids. In each of these classes, there are 5α and 5β epimers. Within these three great classes, and
for a given A/B ring junction, the pattern of hydroxylation – on the steroid nucleus and side chain or both
-- defines each individual bile alcohol or bile acid.
C. Hydroxylation sites
1. The steroid nucleus
a. C24 primary bile acids
Bile acid nomenclature, i.e. the trivial names of bile acids, is awkward because it is based on cholic
acid. Ox bile from the slaughter house was readily available to chemists, and Strecker is given credit for
isolating cholic acid and obtaining the correct empirical formula of C24H40O5 by combustion analysis in
1848 (112). Mylius isolated DCA in 1886 and the term “deoxy” was attached as a prefix because DCA
had one less oxygen atom by combustion analysis (113). When CDCA was isolated from goose bile, it
was named Chenosäure (cheno acid) for its animal source of origin (cheno denotes goose in Greek)
(114), and some years later the adjective “deoxy” was added, based on its elemental composition, giving
rise to the polysyllabic name of chenodeoxycholic (acid). Other trihydroxy bile acids were named for
their animal source: muricholic acid (from rodents), and hyocholic acid (from pigs).
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We have proposed (82) that the root C24 bile acid should be CDCA, as every bile acid must have a
hydroxyl group at C-3 (from the C-3 hydroxyl group of cholesterol) and a hydroxyl group at C-7, as 7α-
hydroxylation of cholesterol is the rate limiting step in the major pathway of bile acid biosynthesis. Thus,
all natural primary C24 trihydroxy bile acids can be considered as derivatives of CDCA. The only
exception to this useful convention occurs in those few mammals (nutria, beavers, bears, and some
cavimorphs) where UDCA, the 7β-hydroxy epimer of CDCA, is a primary bile acid. The route of
formation of this 7β-hydroxy epimer of CDCA is unknown. One can speculate on the existence of a
cholesterol 7β-hydroxylase or a pathway that results in the epimerization of the C-7 hydroxy group.
The most frequent site (in terms of the number of vertebrate species) of additional hydroxylation of
CDCA is at C-12 in cholic acid. The second most common site of hydroxylation is probably at C-16,
because so many avian species have the 3α,7α,16α-trihydroxy bile acid as a major biliary bile acid (82).
Another common site of hydroxylation is at C-6, occurring in hyocholic acid (pigs) and the three
muricholic acids (rats and mice). Other “third site” (nuclear) hydroxylations occurring in biliary bile
acids of the healthy animal are at C-1 (α or β), C-2 (β), C-4(�), C-5(β), C-9(β), C-11(α), C-15(α), and C-
19 (β). Table 1 summarizes the species in which these rare biliary bile acids occur, and the appropriate
citation.
C24 bile acids with four hydroxyl groups on the steroid nucleus occur rarely. Some snakes have
3α,7α,12α,16α-tetrahydroxy bile acids (82). Tetrahydroxy bile acids of unknown structure are found in
trace proportions in the bile of many vertebrate species. After experimental ligation of the common bile
duct, tetrahydroxy- and even penta-hydroxy C24 bile acids occur in the plasma and urine of mice (115).
Such bile acids also occur in mice when the canalicular bile salt export pump has been deleted genetically
(116).
An ad hoc committee of bile acid workers met in Freiburg in 1980 under the auspices of the Falk
Foundation. The group proposed working rules for bile acid nomenclature and bile acid abbreviations
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that were published and noted by the IUPAC nomenclature committee. At the suggestion of Gerhart Kurz
of the University of Freiburg, it was agreed that there would be no more trivial names, except for
conversational purposes (117).
b. C24 secondary bile acids
As noted before, bile acids are modified by bacterial enzymes in the distal intestine. Modification
of the hydroxyl groups forms new bile acids that are termed “secondary”. This distinction has limitations,
because bile acids such as UDCA or certain of the hydroxy-oxo bile acids can be both primary and
secondary. A consideration of secondary bile acids is important because they can be major biliary bile
acids. After their formation in the distal intestine, they are absorbed in part. They return to the liver in
portal venous blood, and traverse the hepatocyte where they may or may not undergo further metabolism
before joining the pool of primary bile acids (formed de novo from cholesterol) in the enterohepatic
circulation.
The major bacterial biotransformations of conjugated bile acids are deconjugation, 7-
dehydroxylation, oxido-reduction, epimerization, and side chain desaturation (detailed in 110).
Deconjugation, i.e. hydrolysis of the amide bond between the terminal carboxylic acid group of the bile
acid and the amino group of taurine or glycine, is mediated by many bacterial species and does not
require anaerobic conditions. Unconjugated bile acids formed by bacterial deconjugation are still
considered primary bile acids.
In the large intestine, anaerobic modifications of the hydroxyl groups occur. The most striking change is
7-dehydroxylation that occurs via a 3-oxo-Δ4,6 nucleotide intermediate (110). The 7-dehydroxylation
product of cholic acid is DCA; that of CDCA or UDCA is lithocholic acid. The 7-deoxy derivative of
hyocholic acid and ω-muricholic acid is termed hyodeoxycholic acid and that of α- and β-muricholic acid
is termed murideoxycholic acid. To date, the C-7 deoxy derivative of “avicholic” (3α,7α,16α-trihydroxy)
has not been identified, but bacterial 16-dehydroxylation has been reported for some steroids (118), and is
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likely to occur in some birds. Such 16α-dehydroxylation would result in the formation of CDCA, that in
turn would likely undergo 7α-dehydroxylation to form lithocholic acid.
During transit through the large intestine, any hydroxyl group can be oxidized and then reduced. If the
reduction is to the other possible epimer, a new epimer will be formed. 3β-Hydroxy bile acids (“iso” bile
acids) are major fecal bile acids in man (119). 7β-Hydroxy bile acids and even 12β-hydroxy bile acids
are also formed, but to a far lesser extent. Elimination of the 3-hydroxy group with formation of a Δ2 or
Δ3 bile acid may also occur (120).
Secondary bile acids may undergo additional hydroxylation during hepatocyte transport. Such
rehydroxylation at C-7 generates the original primary bile acid that had undergone bacterial 7-
dehydroxylation in the intestine. This rehydroxylation does not occur in man and the cow, occurs to a
limited extent in the rat and hamster, and is complete in the prairie dog (121). Alternatively, secondary
bile acids may undergo hydroxylation at a new site, generating a novel “tertiary” bile acid. One example
is 16α-hydroxylation of DCA to form “pythocholic” acid (3α,12α,16α- trihydroxy) that occurs in the
python (75). In the wombat, lithocholic acid undergoes hydroxylation at C-15 (122). “Iso” (3β-hydroxy)
bile acids are epimerized to 3α-hydroxy bile acids during transport through the hepatocyte (123,124).
Biotransformation of the 3β-epimers of the muricholic and hyocholic acid families has not been reported,
but is likely to occur. In man, the 7β-hydroxy group of UDCA does not undergo epimerization during
transport through the hepatocyte, and the small proportion of UDCA in the biliary bile acids is likely to be
formed from CDCA in the distal intestine by bacteria. Bacterial modification of bile acids and subsequent
hepatic biotransformation has been termed by us, “damage and repair”.
The percent of secondary bile acids in biliary bile acids is usually less than 50%, but there are
exceptions. The most remarkable example is in the rabbit, in which DCA constitutes 80% of the
circulating bile acids (82). In some snakes, the 3α,12α,16α-trihdroxy bile acid, which can be formed by
16α-hydroxylation of DCA, exceeds 50% (82).
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c. C27 bile acids
Nuclear hydroxylation in C27 bile acids occurs at C-3 and C-7 (the default structure), and also at C-12 in
most species. In the Andean condor, hydroxylation at C-16 occurs (82). We have identified a 1β,3α,7α-
trihydroxy C27 bile acid in the bile of the tinamou, an ancient flightless bird (125). A tetrahydroxy bile
acid with hydroxyl groups at C-3, C-7, and C-12 plus a 2β-hydroxy group has been isolated from the bile
of the large freshwater fish Arapaima gigas (126). It seems likely that there are additional sites of nuclear
hydroxylation in C27 bile acids yet to be discovered.
C27 bile acids undergo 7-dehydroxylation in the alligator (97), but the extent of 7-dehydroxylation of
C27 bile acids in other species has received little attention.
d. C27 bile alcohols
In C27 bile alcohols, default nuclear hydroxylation occurs at C-3 and C-7; additional hydroxylation
occurs at C-12, and rarely at C-6 or C-16 (25,26, 127). The C-3 hydroxyl group has a β-configuration in
latimerol and myxinol, meaning that it remains unchanged from that of cholesterol. Hydroxylation at C-
16 occurs in myxinol. The bile of Arapaima gigas contains a bile alcohol with a β-hydroxy group at C-2
in addition to hydroxyl groups at C-3, C-7, and C-12, so that C27 bile alcohols with four hydroxyl groups
on the nucleus do occur (126).
No C27 bile alcohols are recognized as “secondary”, i.e. with hydroxyl groups modified by bacterial
enzymes. However, the presence of 7-deoxyscymnol in the Megamouth shark, as reported by Ishida et al
(128), suggests that bacterial dehydroxylation at C-7 may also occur for C27 bile alcohols.
. 2. The side chain
a. C24 bile acids
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With only four available carbons in the side chain, C24 bile acids are likely to undergo side chain
hydroxylation only at C-23 or C-22, each of which has potential R and S epimers. The most common C-
23 hydroxy bile acid is termed “phocaecholic acid” and has the nucleus of CDCA. (The name “phoca” is
given to the marine mammal family from which Hammarsten (129) isolated the bile acid). The structure
of phocaecholic acid was later established as the 23-hydroxy derivative of CDCA by Windaus and van
Schoor (130). The R and S epimers have been synthesized (131,132) and the R form identified as the
naturally occurring epimer (131). In addition to marine mammals, phocaecholic acid also occurs in
wading birds and some song birds (82). The 12-hydroxy derivative of phocaecholic acid also occurs in
marine mammals and is therefore a natural tetrahydroxy- bile acid.
A 3α,7α-dihydroxy bile acid with a hydroxy group at C-22 was shown to occur in the fish
Parapristipoma trilineatum by the Hoshita laboratory (133) and given the awkward name of
haemulcholic acid. The compound was synthesized by both the Hoshita laboratory (134) and the
Pellicciari laboratory (135), and the S epimer was shown to occur in nature. Some of its physicochemical
and biological properties were characterized by Roda et al (135). Haemulcholic acid was also identified
in freshwater fish by Haslewood during his Zaire River expedition (75).
Side chain desaturation forming Δ22 bile acids is a major pathway in rats (136,137) and cavimorphs
(82). Such bile acids could arise from further β-oxidation of the side chain in the liver and/or by bacterial
action in the distal intestine.
b. C27 bile acids
Elucidation of the structure of the C27 bile acids with hydroxyl group substituents on the C8 side chain
was not simple, as side products such as lactones may be generated during alkaline hydrolysis.
Unfortunately, an enzyme preparation that hydrolyzes the amide bond of C27 taurine conjugated bile acids
is not available at present (In contrast, cholylglycyl hydrolase, which rapidly cleaves the amide bond in
C24 conjugated bile acids, is available commercially).
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So far, all reported C27 bile acids have only a single hydroxyl group in the side chain. It occurs at C-22
in turtles, at C-24 in lizards, and at C-26 in bullfrogs (25, 26); each of these bile acids has four potential
epimers. The four cholestanoic acid epimers with a hydroxyl group at C-24 have each been synthesized
by Une et al. (138). The most common diastereoismer is varanic acid, which has the 24R, 25R
configuration. In collaboration with the Iida and Une groups, we have found that the 24R,25S epimer is
present in the bile of several varanids (139).
c. C27 bile alcohols
As noted earlier, Hammarsten determined that he had isolated a C27 bile alcohol from the bile of the
Greenland shark (100). The challenge of determining the structure of this compound would occupy
chemists for many decades. Windaus et al. (140) initially took up the problem of determining the
structure of Hammarsten’s compound (named scymnol) and proposed that it contained one primary and
three secondary hydroxyl groups, with an epoxy ring. The three secondary hydroxyl groups were found
to be located in the sterol nucleus (at positions 3α, 7α, and 12α) as scymnol could be converted into cholic
acid by partial oxidation of the side-chain (141,142). Since the side-chain of cholesterol has a C-27-
methyl group, different paired combinations of carbons 23, 24, and 25 were proposed to contain the
epoxy group, with the primary alcohol group being on C-27. Bridgwater and Haslewood showed (using
Hammarsten’s original sample) that the epoxy group was an artifact of alkaline hydrolysis, and that the
side-chain of scymnol actually contained two secondary hydroxyl groups and a primary hydroxyl group
bonded to sulfate (143,144). In 1961, Cross used 1H-NMR to elucidate the structure of both scymnol and
anhydroscymnol, the epoxide formed during alkaline hydrolysis (145). He found that the side chain
hydroxyl group of scymnol was unexpectedly on C-26 (the only carbon with a single proton), consistent
with a side-chain structure having hydroxyl groups at C-24 and C-26, and a primary hydroxyl group at C-
27. The structure of scymnol was ultimately established as 3α,7α,12α,24,26,27-hexahydroxy-5β-
cholestane by comparison with a synthetic standard (143). Carbon-24 is chiral, and the configuration of
the 24-hydroxyl group was determined to be 24R, in the crystallographic study of Ishida et.al. (146). This
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group has also found that whereas most shark species have the (24R,25R)-epimer of scymnol, a few
species also form the (24R,25S)-epimer (147). Other named C27 bile alcohols are tabulated in our
previous review (82). Additional information is available in the cogent review of Une and Hoshita (26).
Bile alcohols, just as bile acids, have been named for the species in which they occur, and the name
gives no clue whatsoever to the structure of the bile alcohol. It is convenient to think of the trihydroxy
bile alcohol with α-hydroxyl groups at C-3, C-7, and a primary alcohol function at C-27 as the “root” C27
bile alcohol, and to look on other bile alcohols as derivatives of that simplest bile alcohol. At present, this
“root” bile alcohol has no trivial name, but has been synthesized by the Mosbach group (148).
D. Conjugation
1. N-acyl amidation with taurine or glycine.
In virtually all species, bile acids, whether C24 or C27, are found in bile in N-acyl amidated form, i.e.
conjugated in amide linkage with taurine (or a taurine derivative) or far less commonly with glycine.
Taurine conjugation is the rule in fish, amphibians, reptiles, and most avian species. It also occurs to a
variable extent in mammals (82). In some angelfish species, N-methyltaurine conjugates are present
(149), the N-methyltaurine thought to be of dietary origin. Similarly, in some marine fish, bile acids are
conjugated with cysteinolic acid, a hydroxy-ethyl derivative of taurine that is of dietary origin (150).
Glycine conjugation occurs in a few avian species, some bovids, cavimorphs, and primates, including
humans (82). Sarcosine (N-methylglycine) conjugates have been synthesized (151,152,153) and shown to
be more resistant to bacterial deconjugation during enterohepatic cycling (154). Some workers have
considered bile acid amidation to be the final step in bile acid biosynthesis (155). It seems more logical to
us to define the bile acid biosynthesis pathway as ending with a mature bile acid or bile alcohol before it
undergoes a phase II biotransformation step such as N-acylamidation, sulfation, or glucuronidation.
The process of conjugation occurs both in hepatocytes and in cholangiocytes (156). However,
conjugation in the hepatocyte greatly exceeds that occurring in cholangiocytes. The conjugation process
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begins with the formation of a bile acid adenylate (157) followed by formation of the coenzyme A thio-
ester. A bile acid aminotransferase then transfers the CoA bile acid to the amino group of glycine or
taurine; the human enzyme has been cloned and shown to mediate conjugation with both glycine and
taurine (158). Conjugation of newly synthesized bile acids occurs in peroxisomes (159), and recent work
suggests that “reconjugation" of unconjugated bile acids returning from the intestine also occurs in these
organelles (160). Other than their pKa value, which in turn is responsible for the pH-solubility profile (see
later), there is little difference in the biological properties of glycine and taurine conjugated bile acids,
except that taurine conjugates are deconjugated by bacteria more slowly than glycine conjugates
(161,162).
That bile acids are present in bile in “conjugated” form was recognized in the 19th century. At that
time, neither the structure of the bile acid nor the site of linkage was known. This resulted in bile acid
conjugates having a misleading name, in that “taurocholate” implies that the cholic acid moiety is capable
of ionization. We and others have suggested that the name “cholyltaurine” (analogous to glycylglycine)
would be more appropriate (117), but few investigators have followed this proposal. The extremely low
proportion of unconjugated bile acids in biliary bile acids has led some investigators to conclude that the
conjugation process is highly efficient. However, unconjugated bile acids, especially dihydroxy bile
acids, if secreted into bile are rapidly absorbed by the biliary ductules (163,164), resulting in their
removal from ductal bile. Thus, the state of conjugation in canalicular bile can be considerably less than
that of ductal bile.
2. Other types of bile acid conjugation
When the term “conjugated” was applied to bile acids, it denoted N-acyl amidation with taurine or
glycine. When bile acid sulfation and glucuronidation were discovered to be important pathways under
some circumstances, it was necessary to distinguish different modes of “conjugation” for bile acids. In
principle, there are six modes of bile acid conjugation: N-acyl amidation (using glycine, taurine, or
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possibly other amino acids), sulfation, glucuronidation, conjugation with glutathione, and conjugation
with N-acetylglucosamine or glucose, and bile acids may have more than one mode of conjugation. In
vertebrates, the only bile acid sulfates that occur in appreciable proportions in biliary bile acids are the 3-
sulfate of lithocholylglycine and lithocholyltaurine amidates that occur in man (165) and probably great
apes (166). In patients with severe cholestasis, bile acid sulfation (mostly at C-3) followed by urinary
excretion becomes a major pathway (167,168). In the bile duct ligated hamster, esterification with sulfate
occurs at C-7 (169).
In the Mayo Clinic studies of Thistle and Schoenfield, in which bile acids were administered to
gallstone patients (35), one group received hyodeoxycholic acid (3α,6α-dihydroxy-. Since
hyodeoxycholic acid should be absorbed efficiently from the small intestine by passive mechanisms, its
absence in biliary bile acids was completely mystifying. In 1978, B. Almè and J. Sjövall reported a
patient with malabsorption who excreted in urine 6-hydroxy bile acids that were present as O-linked
(ethereal) glucuronides. (170). Five years later, Sacquet et al, working in the laboratory of Reca Infante in
Paris, reported that when hyodeoxycholic acid was administered to human subjects, it was largely
excreted in urine as its glucuronide (171), explaining its absence in biliary bile acids. This group did not
know the site of glucuronidation but proposed that it was at C-3.
Four years later, Anna Radominska-Pyrek and colleagues showed that human microsomes rapidly
glucuronidate hyodeoxycholic acid at C-6 (172). Thus the curious fate of hyodeoxycholic acid in humans
was finally settled. It presumably results from defective N-acylamidation, and hyodeoxycholic acid is
consequently metabolized in the same manner as a C24–nor (C23) dihydroxy bile acid (see below), i.e. by
glucuronidation in the endoplasmic reticulum. To date, this pathway has not been identified in any other
animal. When hyodeoxycholic acid is given to rats, it is probably conjugated with taurine and not
glucuronidated (173).
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When C23 dihydroxy-bile acids, are administered to animals including man, little amidation occurs, as
both the CoA ligase and the glycine/taurine amino acid transferase act to only a very limited extent on bile
acids with a shortened (C4) side-chain (174). Such bile acids undergo glucuronidation both at C-3 and C-
23 (163,164). In man, when norUDCA is given, it forms the C-23 ester glucuronide which is excreted in
both bile and urine (175). In rats, N-acyl amidation is also much less efficient for α-hydroxy bile acids
(131,132) and hydrophilic epimers of cholic acid, for example 3α,7β,12β-trihydroxy-cholanoate (176).
In patients ingesting UDCA, UDCA also undergoes conjugation with N-acetylglucosamine at C-7, in
addition to amidation with glycine or taurine at C-24 (177). In Niemann-Pick disease type C, a novel Δ5
3β,7β-dihydroxy cholestanoic acid is formed (178); this compound may be considered an acidic 7β-
hydroxy derivative of cholesterol. This compound undergoes sulfation at C-3, conjugation with N-
acetylglucosamine at C-7 (178), and amidation with glycine or taurine at C-24. In both circumstances,
the multiply-conjugated metabolites are eliminated in urine rather than bile, probably because they are not
substrates for the canalicular transporters. For UDCA, conjugation with N-acetylglucosamine is a very
minor pathway—less than 0.3% of the oral dose.
Glutathione conjugates of bile acids (presumably as thio-esters) have been identified in rat and human
infant bile by the group of Shigeo Ikegawa, but their concentration is several orders of magnitude less
than that of the major biliary bile acids (179).
To date all C27 bile acids have been found to be conjugated exclusively with taurine. Bile alcohols are
esterified with sulfate, and thus have a pKa similar to that of taurine conjugates.
E. Structure-function relationships.
In the steady state adult animal, cholesterol input – from de novo synthesis and dietary cholesterol –
must be balanced by excretion – cholesterol as such and bile acids. There is thus a mole fraction of total
cholesterol excretion composed of bile acids, fecal acidic steroids, divided by total (neutral plus acidic)
steroids. In man, based on balance studies, this figure is 0.46; in the mouse it is 0.45, whereas in the
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hamster it is 0.57, according to John Dietschy and Steve Turley (180). The very low cholesterol
proportions in the biliary lipids of many vertebrate species (181), suggests that in most vertebrates this
number should be well above 0.5. The route of bile acid excretion is solely via bile, as the amount of bile
acids in urine is negligible. In patients with complete biliary obstruction, fecal bile acid output is
extremely low, indicating that possible bile acid extruders in the small intestine epithelium are not
upregulated (182). In patients with biliary obstruction, bile acid elimination is by urinary excretion, albeit
with greatly increased levels in plasma and liver. In addition to cholesterol excretion in bile (as such and
after conversion to bile acids) there is a small flux of cholesterol directly from the small intestine (183).
For a bile acid to serve as the chemical form of cholesterol elimination there are only a few molecular
requirements. The molecule must be too polar to be absorbed passively and too large to pass through the
paracellular junctions between enterocytes. It also must be resistant to pancreatic enzymes.
The bile acid molecule is too large to pass via the tight junctions of the intestinal epithelium.
Amidation with glycine or taurine converts unconjugated bile acids from weak acids (pKa of 5) to a pKa of
4 for glycine amidates and a pKa of less than 2 for taurine conjugates (184). Thus, bile acids are present in
bile and intestinal content as organic anions and therefore will not cross membranes unless a transporter is
present. Bile acid glycine and taurine amidates are resistant to pancreatic carboxypeptidases, whereas bile
acids amidated with amino acids other than glycine or taurine (with the exception of aspartic acid and
cysteic acid) , are hydrolyzed rapidly by pancreatic carboxypeptidases (185).
In vertebrates with a cecum, deconjugation and dehydroxylation convert bile acids to membrane
permeable molecules and they are in part absorbed, as signaled by the presence of unconjugated bile acids
in the systemic circulation. Isao Makino, who had worked in the Sjövall laboratory in Stockholm and
returned to Japan, was among the first to report the presence in healthy individuals of unconjugated bile
acids in plasma using gas chromatography (186). Some decades later, this early finding was confirmed
by Kenneth Setchell et al, using gas chromatography-mass spectrometry (187). Nonetheless, absorption
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from the cecum is not complete, as unconjugated bile acids have poor solubility at the acidic pH (pH 5.5)
of the cecum and are bound to bacteria and unabsorbed dietary components.
Unconjugated (and conjugated) bile acids that are not absorbed are finally eliminated from the body by
the process of defecation. As there is negligible excretion of bile acids in urine, breath, and skin, and as
the bile acid nucleus is considered to be indestructible by bacterial enzymes, fecal excretion of bile acids
is equal to their synthesis from cholesterol. However, it must be stressed that there is little information on
the fecal bile acid composition across the vertebrate spectrum (188). There are some species that by our
analyses have very low fecal bile acids, suggesting that the bile acid molecule can in fact be degraded by
bacteria. The degradation of bile acids by individual bacterial strains with bile acids as the major carbon
source has been characterized in great detail by S. Hayakawa (189). The biological relevance of these
studies is unclear. To date, there are no studies in which bile acids tagged with 14C in the steroid nucleus
are administered and 14CO2 in breath is collected.
There is very little information on the fate of bile alcohols in the distal intestine. In the Asiatic carp we
found unchanged 5α-cyprinol sulfate in the distal intestine (190). Even if there were hydrolysis of the
ester sulfate, the liberated bile alcohol is likely to have negligible passive membrane permeability because
of the number of hydroxyl groups.
For solubilizing lipids in the form of mixed micelles, bile acids must be amphipathic, i.e. they must
have a polar side and a non-polar side. So far as is known, all major natural biliary bile acids and bile
alcohols are amphipathic, although the hydrophobic side (beta face) is somewhat reduced in UDCA.
Figure 5 shows a space-filling model of a bile acid molecule, emphasizing its amphipathic nature. To the
best of our knowledge, no one has synthetized a molecule unrelated to the bile acid molecule which has
the same physicochemical properties of bile acids as regards solubilizing membrane lipids.
III. Measurement
A. Isolation and chromatographic separation
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Elucidation of the metabolism of bile acids in health and disease requires accurate, reliable, and
sensitive methods for measurement, as well as techniques for extracting bile acids (or alcohols) from
complex biological matrices. The complexity of bile acid structure and the enormous range in polarity of
bile acids and their conjugates have always proved a challenge to the analytical chemist. A cogent review
by W. Griffiths and J. Sjövall on current analytical approaches was published in 2010 (9). These two
chemists have contributed greatly to the advances in bile acid analysis.
The modern era of bile acid analysis began with the development of improved chromatographic
methods for separation. Paper chromatography based on liquid-liquid partition was developed in the
1940’s by the great English chemists A.J. P. Martin and R.L. Synge who shared the Nobel Prize in 1952.
In the 1950’s it was first applied to bile acids by Jan Sjövall to separate individual unconjugated bile acids
(191). Sjövall and Haslewood then collaborated to apply the method to Haslewood’s vast collection of
bile samples from different animals (192).
Gas-liquid (partition) chromatography was developed some 10 years later by A.J.P Martin working
with A.T. James. The earliest application to bile acids came in the 1960’s (193,194), and Eneroth and
Sjövall tabulated retention times for different stationery phases in 1971 (195). A decade later capillary
gas chromatography was described, greatly increasing resolving power. Gas chromatography using flame
ionization detectors affords both separation and quantification. However, only when a peak is further
analyzed by mass spectrometry can one be one certain that the peak is truly a bile acid.
Thin-layer adsorption chromatography (TLC) became widely used when a commercial device for
coating the adsorbent (usually silicic acid or alumina) on glass plates became available and excellent
adsorbents were developed (196). TLC offered useful separations of bile acids by class (taurine
conjugates, glycine conjugates, unconjugates) but could not separate individual taurine dihydroxy-
conjugates from each other. For unconjugated bile acids, molecules separate according to the number and
orientation of hydroxyl groups, but a clear separation of DCA from CDCA with the commonly employed
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solvent systems does not occur. Moreover, quantification of individual spots was difficult. Nonetheless,
TLC is a highly useful quasi-quantitative method for assessing purity of bile acids and monitoring
chemical reactions.
The senior author was privileged to have access to the first TLC apparatus to enter Sweden,
purchased in 1960 by Borgström for the separation of lipids. Borgström had been impressed with the
remarkable separations of neutral lipids that were obtained using this technique by Malins and Mangold
(197), then working at the Hormel Institute in Minnesota. With TLC using silicic acid as the stationery
phase and phosphomolybdic acid (in ethanol) as the detection reagent most bile acids (as well as
unsaturated lipids) appear as deep blue spots on a brilliant yellow background (the colors of the Swedish
flag). A technique was described for coating silicic acid on microscope slides (198), and this in turn
permitted screening of multiple solvent systems for optimal bile acid separation. Systems were developed
(199) that our laboratory still uses today.
Reversed phase TLC has also been used for bile acids (200,201), but has not achieved popularity
because of the much lower capacity of the method, as well as difficulties in detecting bile acids after
separation. Eneroth and Sjövall tabulated RF values using TLC for a large spectrum of bile acids and a
number of solvent systems in their review in 1971 (195). Some of their values have been shown as actual
simulated chromatograms in a chapter by the senior author (202).
In our laboratory, we have found TLC to be of great value for quantifying hepatic biotransformation of
radioactive bile acids. Small (1 mm) bands of adsorbent can be directed into scintillation counter vials,
using the automatic device described by Snyder and Kimble (203), or manually, by transferring the
adsorbent to a vial using a brush.
HPLC was developed in the 1970’s and soon applied to bile acids. Today, the technique when used for
bile acid analysis is usually executed in the reversed phase mode, i.e. with C18 or C8 bonded silicic acid as
the lipophilic solid phase. Numerous laboratories have reported the power of this analytical method for
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separating and measuring conjugated bile acids in bile and intestinal content (9). Conjugated, i.e. N-acyl
amidated bile acids have an amide bond that has absorbance at 205 nm, permitting their measurement in
the effluent. Unconjugated bile acids, non-amidated bile acid sulfates, and bile alcohol sulfates are not
detected at 205 nm. HPLC with UV detection is too insensitive for analysis of serum bile acids unless
large volumes of serum are used (204). One solution to the non-detection of unconjugated bile acids and
bile alcohol sulfates is to use a light scattering detector that responds approximately equally to all bile
acids (205). A second approach, useful for unconjugated bile acids, is to couple the bile acid to a
chromophore such as the phenylacyl group (206).
In our laboratory, we developed an HPLC method for separating the 12 conjugated bile acids
predominant in human bile (165), and we have found this method to be essential in our analysis of
vertebrate bile samples (82). Nonetheless, when used alone for bile analysis, there are many unknown
peaks that may or may not be bile acids. Therefore peak identification requires confirmation by mass
spectrometry
B. Mass spectrometry
Mass spectrometry had been developed in the 1930’s and was used to analyze mixtures of
small molecules. Mass spectrometry of large organic molecules emerging from the gas chromatograph
was developed by R. Ryhage in Sweden, and the first commercial instrument for this purpose was
marketed by LKB in 1965. William Elliott wrote in 1988, “Two years later [1965], I returned to the
Karolinska to devote the summer to learning about the GC/MS which Ragner Ryhage had built, and was
then produced by LKB. Our LKB 9000 is still operating, beginning its 23rd year.” Since then, there have
been continuous improvements in techniques for sample volatilization and instrument sensitivity,
culminating in the MALDI analysis of proteins. In our opinion, anyone who wishes to do a complete bile
acid analysis must have HPLC/MS/MS available. It is also desirable to have GC/MS availability when
needed, as capillary gas chromatography has greater resolving power than HPLC.
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The wedding of HPLC and MS/MS is thus an essential step in the analysis of unconjugated and
conjugated bile acids. Currently, internal standards containing three or more deuterium atoms are not
available for many bile acids and are sorely needed. For a total analysis, class separation by ion exchange
chromatography developed by Setchell and Sjövall should be performed before HPLC/MS/MS (9). For
state of the art analysis, either capillary GC or capillary HPLC will be required to separate complex
mixtures of bile acids. For unknown bile acids, structure assignment requires NMR for elucidation of
structure, and ultimately synthesis for confirmation of assigned structure.
C. Enzymatic and competitive binding assays
Development of an enzymatic technique for determining total 3α-hydroxy bile acids based on
oxidation of the 3α-hydroxy group by a 3α-hydroxy steroid dehydrogenase was a major advance in bile
acid analysis (207), especially for workers in the field of bile acid physiology. This technique was based
on the pioneering work of Talalay (208), who developed enzymatic analysis of 3α- hydroxy hormonal
steroids using a steroid dehydrogenase isolated from Pseudomonas testosteroni. Details of the method
were clarified by Turley and Dietschy (209). The sensitivity of the method was increased by measuring
the reduced NAD by changes in fluorescence or by coupling the oxidation to reduction of a tetrazolium
dye (210). The enzyme technique using 3α-hydroxysteroid dehydrogenase does not measure 3β-hydroxy
bile acids. Thus, when used for determination of fecal bile acids, it will underestimate total bile acids
(119). An enzyme recycling system has recently been marketed by Diazyme™ and is quite sensitive.
In our laboratory, based on collaboration with Marlene DeLuca. Aldo Roda developed an enzymatic
bioluminescence assay for primary bile acids. The method was based on co-immobilizing on beads a 7α-
hydroxysteroid dehydrogenase, an oxido-reductase, and luciferase (211). The method was validated by
showing excellent agreement with GC/MS measurements (212). However, this method, despite its
simplicity and sensitivity, has not been widely adopted. Limitations in the enzymatic method are
summarized in the review by Griffiths and Sjövall (9).
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All of the chromatographic techniques discussed above are labor-intensive and time-consuming. For
measuring diurnal variations in plasma levels of total bile acids, two approaches have been taken to
develop a rapid, simple procedure for measuring total bile acids. The first was the enzyme method that
measures total 3α-hydroxy bile acids, as just discussed. The second approach was the development of
competitive binding techniques, such as radioimmunoassay. What is measured depends on the affinity of
the antibody, and thus one determines “immunoreactive” bile acids. At the Mayo Clinic, Wilfred
Simmonds et al developed a radioimmunoassay specific for cholyl conjugates (213) which was then used
by LaRusso et al to define the time course of the postprandial elevation of such cholyl conjugates in
health and in patients with bile acid malabsorption (214). Later, Schalm et al (215) developed a
radioimmunoassay for chenodeoxycholyl conjugates, and used it to show that the postprandial peak of
CDC conjugates occurs earlier than that of cholyl conjugates (216), as shown in Figure 6. Other
radioimmunoassays have been developed, as summarized in the review by Griffiths and Sjövall (9).
All of the above methods for measuring bile acids isolate the bile acids from the body tissue or fluid,
and then quantify the bile acids. We have long needed a bile acid-sensitive molecule whose signals would
provide information on the concentration of bile acids within the living cell. This need has now been
supplied by the development of an indicator molecule consisting of a protein possessing a bile acid
binding site (based on the binding site of FXR) and two reporter molecules whose fluorescence is
determined by the extent to which bile acids occupy the binding site -- the greater the binding, the less the
fluorescence. The technique, as described by van der Velden et al (217), uses the well-known Forster
Resonance Energy Transfer (FRET) principle, and has already been used to characterize bile acid
transport into isolated hepatocytes.
D. Diagnostic utility of plasma bile acid levels
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Despite the simplicity of competitive binding techniques for measuring conjugated bile acids, they
have not been widely adapted by the clinical chemists. The measurement of plasma bile acids is not part
of the usual panel of liver tests (aminotransferases, alkaline phosphatase, total bilirubin). The major
reason is that bile acid levels are probably inferior in sensitivity and specificity in detecting liver injury
when compared to the standard liver panel. This conclusion is based on clinical studies (218,219). In
addition, a recent animal study (220) in which liver injury was experimentally induced in rats by feeding a
variety of toxic molecules found that serum bile acid levels were not more sensitive than aminotransferase
levels. Thus, because of the lack of greater sensitivity of serum bile acid measurements, there is the belief
that the current liver tests are adequate. Moreover, the instruments that automate determination of a
serum “chemical panel” do not have a bile acid “channel”. For purely cholestatic diseases such as
primary cholestasis of pregnancy, the level of serum bile acids has value for predicting prognosis (221).
E. Determinants of bile acid profiles in body fluids
In assessing the significance of bile acid concentrations that in turn define the bile acid profile, it is
important to realize that plasma, hepatic, biliary, urinary and fecal bile acids all have a different
composition in health. Plasma bile acids, in health, represent the balance between instantaneous intestinal
input and net hepatic uptake, with first pass clearance ranging from 40 to 90%, depending on the bile
acid. Hepatic bile acids (as distinguished from hepatocyte bile acids) represent the balance between input
of unconjugated and conjugated bile acids, subsequent biotransformation of these molecules, as well as
bile acids that have been secreted by transporters into the canaliculus. Urinary bile acids represent
filtered bile acids plus any bile acids secreted by the tubules minus those absorbed by the tubules. Ductal
bile which is usually analyzed differs from canalicular bile by that fraction of conjugated and
unconjugated bile acids that are absorbed by the biliary ductules. Gallbladder bile is ductal bile minus bile
acids absorbed from the gallbladder. Fecal bile acids represent bile acids entering the colon with or
without modification by bacterial enzymes minus those that were absorbed.
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The power of current analytical techniques may result in an enormous variety of bile acids being
detected. When such results are tabulated, we suggest organizing bile acids by primary bile acids, and
then listing secondary bile acids derived from the primary bile acid underneath their precursor. Bile acids
that are present in trace proportions should be lumped together to simplify the presentation. The choice of
the analytical method should be determined by the biological question that is being asked. For
nomenclature in reporting bile acid analyses, the recommendations of an ad hoc nomenclature committee
(117) should be followed.
IV. Bile Acid Metabolism: transport and the enterohepatic circulation
A. Bile acid transport
1. Overview
Glycine and taurine conjugated bile acids are fully ionized at the pH conditions prevailing in plasma,
bile, and small intestinal content. Therefore, these molecules will not cross a cell membrane and enter a
cell unless a transporter is present. What is responsible for the enterohepatic circulation is the vectorial
transport of conjugated bile acids through the ileal enterocyte and the hepatocyte. The enterohepatic
circulation of bile acids results from the action of two chemical pumps and three fluid flows. The
chemical pumps are the vectorial transport systems of the ileal enterocyte and the hepatocyte – each of
which has basolateral and apical transporters. The fluid flows are the aboral movement of intestinal
content due to intestinal propulsive activity, portal venous blood flow that transports bile acids from the
intestine to the liver, and biliary flow that returns the bile acids to the intestine. The molecules that
constitute the circulating pool of bile acids must be substrates for the vectorial transport systems of both
the ileal enterocyte and the hepatocyte in order to circulate enterohepatically.
The initial input into the hepatic limb of the enterohepatic circulation consists of primary bile acids
formed and conjugated in the pericentral hepatocytes. The newly synthesized conjugated bile acids are
pumped across the canalicular membrane and flow in canalicular bile from the pericentral region to the
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periportal region, their direction of flow being opposite from that of sinusoidal blood. In the periportal
region, newly synthesized bile acids are joined by bile acids returning from the intestine and pumped into
bile.
The return pathway of the enterohepatic circulation features three inputs into the portal venous blood
from bile acids that have been absorbed by enterocytes. The dominant input into portal venous blood
consists of “undamaged” conjugated bile acids that have been absorbed by the ileal transport system. A
second input is that of unconjugated primary bile acids formed by bacterial deconjugation in the distal
small intestine and large intestine. A third input is that of “secondary (damaged) bile acids that are
formed by bacterial modification of the hydroxyl groups of primary bile acids. Only a fraction of the
secondary bile acids that are formed are absorbed, and those that are not absorbed are eventually
eliminated by defecation.
As discussed previously, those secondary bile acids that are absorbed from the distal intestine may
undergo uptake by the hepatocyte or in some cases, urinary excretion (or both). After hepatocyte uptake,
“damaged” bile acids undergo “repair” in the hepatocyte by “reconjugation” as well as re-epimerization
of hydroxy groups (from β to α) and reduction of oxo groups to hydroxy groups. They are then secreted
into bile to join the recycling primary bile acids. In man, the flux of bile acids returning from the intestine
is at least twentyfold greater than the input of newly synthesized bile acids. More details of bacterial
damage and hepatocyte repair are given in the introduction.
In man, the major secondary bile acids in colonic content are lithocholic acid, formed by 7-
dehydroxylation of CDCA, and DCA, formed similarly from cholic acid. In man, lithocholic acid is not
only N-acylamidated, but also sulfated in part. Such sulfo-lithocholyl amidates are secreted into bile, but
are poorly absorbed from the small intestine (222), so they do not have an enterohepatic circulation.
Those unsulfated amidates secreted in bile return to the liver, undergo sulfation in part, are re-secreted
into bile, and eliminated via the fecal route. The end result is that lithocholic acid has a very rapid
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turnover in man, and sulfated and unsulfated lithocholyl amidates constitute less than 5% of biliary bile
acids (165). In rodents, lithocholic acid is not sulfated but undergoes hydroxylation at C-6 or C-7 (15).
As noted previously, DCA may pass through the hepatocyte without further metabolism, or may be
rehydroxylated to varying degrees in a species dependent manner. The “repaired” and reconjugated bile
acids are secreted from the hepatocyte and join the cycling conjugated bile acids.
Reabsorption of bile acids from the small intestine gives a cycling “pool” for each bile acid. The size
of the bile acid pool and its turnover rate differ for each bile acid, although the turnover rate of CDCA and
DCA are about the same. Most of each individual bile acid pool is stored in the gallbladder during
overnight fasting, and biliary bile acid composition is determined by the relative proportions of each type
of bile acids, this in turn being determined by the sizes of the individual bile acid pools. .
2. Transport by the ileal enterocyte: the apical transporter
The first person to observe preferential absorption of conjugated bile acids by the ileum was
Hermann Tappeiner, who presented his findings to the Vienna Academy of Sciences in 1887 (223).
Tappeiner studied the absorption of taurocholate from different regions of the small intestine using
anesthetized dogs. He found that taurocholate was absorbed preferentially in the ileum. Rather than
concluding that the ileum had special properties, Tappeiner was puzzled by the failure of the proximal
intestine to absorb taurocholate. Verzar, working in Hungary, authored one of the first monographs on
intestinal absorption (224). He was skeptical of the results of Tappeiner and directed his colleague
Fröhlicher to repeat Tappeiner’s experiments. Fröhlicher obtained identical results (225), again showing
that ileal absorption of conjugated bile acids was greater than that of jejunal absorption. However, in
Verzar’s book on intestinal absorption the concept of specific transporters with differing substrate
affinities is not considered. At that time, the mechanism of absorption of small molecules was considered
to be passive, analogous to diffusion.
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Baker and Searle, working at the University of Iowa, rediscovered the work of Tappeiner and
Fröhlicher, and confirmed the selective ileal absorption of taurocholate in the rat ileum in 1960 (226).
A decade before, T.H. Wilson, an American working in the laboratory of G. Wiseman at the University
of Sheffield, developed the everted gut sack technique and used this method to show that glucose was
actively transported (227). (It is rumored that this technique was the result of a drunken party in Paris
some years before, where physiologists were bemoaning the difficulty of studying intestinal absorption in
the intact animal. Someone, never identified, said the only solution was to turn the animal inside out.
And someone else is supposed to have said, “Maybe that’s a good idea!” In 1960, Robert Crane, who had
studied glucose absorption since his postdoctoral studies with the Cori’s in St. Louis, published his idea
that glucose absorption was sodium dependent, and that the downhill transport of sodium served to
energize the uphill transport of glucose (228). (The late Robert Crane was a tall, handsome charmer who
always had an eye for attractive women. At a party, he tried to persuade a lovely young scientist to go
home with him; when she refused, he started attacking her data).
In 1961, Leon Lack and Ike Weiner, young faculty members at Johns Hopkins, were discussing how to
teach medical students about the enterohepatic circulation. They realized that nothing was known about
the mechanism of bile acid absorption. They used the everted sac technique to show that the last fourth of
the small intestine of rats and guinea pigs actively transport conjugated bile acids against a concentration
gradient (229). Their data are illustrated in Figure 7. As noted earlier, the senior author, working with
his mentor, Bengt Borgström and Göran Lundh (a surgeon and brother-in-law of Borgström) perfused the
small intestine of healthy volunteers and obtained strong evidence for ileal absorption of conjugated bile
acids in man (20). Peter Holt, working in the laboratory of Kurt Isselbacher at Harvard, showed that
conjugated bile acid absorption in the everted sac preparation was sodium-dependent (230). Thus, it
seemed that a Na+ dependent transporter for conjugated bile acids was present in ileal enterocytes. (The
term “enterocyte” was coined by Christopher Booth, a prominent English gastroenterologist and
bibliophile, who discovered that vitamin B12, like conjugated bile acids, was also absorbed
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predominantly in the ileum). In 1978, H. Lϋcke, G. Stange, R. Kinne and H. Murer, working at the Max-
Planck Institute in Frankfurt, prepared vesicles from ileal enterocyte brush borders, and used this
preparation to show that taurocholate uptake was carrier mediated and Na+ dependent (231). Attempts
were made to isolate the apical bile conjugated bile acid transporter by G. Kurz, working in Freiburg,
using the photoaffinity technique with 3H-tagged azido- bile acids (232). These studies were continued
by his student, Werner Kramer at the Hoechst Company in Frankfurt (233), but they failed to clearly
identify the ileal apical bile acid transporter.
During this time, ideas of cell polarity were developing (234). Thus, it seemed that at a minimum,
four transporters would be required for the enterohepatic circulation of bile acids. These would be an
apical and basolateral transporter in the ileal enterocyte, and an apical (canalicular) and basolateral
(sinusoidal) transporter in the hepatocyte.
The great breakthrough, in our judgment, came in 1987 with the use of expression cloning by Matthias
Hediger working in the laboratory of Ernie Wright at UCLA. Using the technique of injecting mRNA
into frog eggs (obtained from Xenopus laevis), they were able to report the cloning of the sodium coupled
transporter for glucose (235). Seven years later, Paul Dawson decided to attempt to clone the ileal bile
acid transporter. Dawson was well educated for the task, having worked with Joseph Goldstein, Michael
Brown, and David Russell at UT Southwestern in Dallas. Dawson, now relocated to Wake Forest
University, reported the successful cloning of the ileal apical sodium-dependent bile acid transporter
(protein, ASBT; gene, SLC10A2), using a somewhat different expression technique (236). This
achievement, more than a century after Tappeiner’s original study, permitted the testing of individuals for
defects in the gene encoding ASBT, as well as providing a molecular target for pharmaceutical companies
who might wish to develop an inhibitor.
In Dawson’s original paper, he found that ASBT was also present in the kidney, in agreement with
earlier studies by F. Wilson et al (237). Later, the laboratories of G. Alpini in Texas (238) and N. LaRusso
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(239) at the Mayo Clinic showed that ASBT was also present in cholangiocytes. ASBT is highly specific
for bile acids, although the integrity of the 7α-hydroxyl group seems more important than that of the 3α-
hydroxyl group, as some substituents can be added to the 3α- position without impairing transport (240).
As yet, the transporter has not been crystallized, although the structure of a bacterial homologue has
recently been published (241). That paper proposes that two sodium ions act allosterically to open a
binding pocket. An overview of the SLC10 transporter family is available (242).
3. Transport by the hepatocyte: bile acid uptake
Bile acid uptake by the liver was known to be highly efficient. For example, in an isolated perfused
dog preparation studied at Mayo Clinic by N. Hoffman et al, uptake of taurocholate was 100% and that of
the taurine conjugate of CDCA was 70% (243). In a study published that same year from the Erlinger
laboratory in Paris, first pass extraction of taurocholate in the dog was found to be 40-80% and saturable,
consistent with uptake being carrier mediated (244). Conjugated bile acid uptake was saturable, and
using either hepatocytes (245) or the isolated perfused rat liver (245) was sodium-dependent (246). Thus
it seemed highly likely that one or more sodium-coupled transporters was present in the sinusoidal
membrane and mediated the efficient uptake of bile acids.
A sodium-dependent basolateral hepatocyte protein, named NTCP (sodium taurocholate porter) was
cloned in 1991, again using expression cloning, by Bruno Hagenbuch, Bruno Stieger and colleagues
working in the laboratory of Peter Meier in Zurich (247). When the Na+ coupled ileal transporter ASBT
was cloned four years later, it was possible to compare the sequences of NTCP and ASBT and identify
some regions of homology (248). Thus, the ileal uptake protein and the hepatocyte removal protein were
related, and both used the sodium gradient to power the uptake. However, NTCP has a much broader
specificity than ASBT (249,250). NTCP can be considered an anion transporter that transports not only
bile acids but also other organic anions into the hepatocyte from plasma, whereas ASBT selectively
conserves solely conjugated bile acids, thereby generating their enterohepatic circulation.
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NTCP is not the only bile acid transporter in the basolateral (sinusoidal) membranes of the hepatocyte.
A family of Na+ independent transporters, organic anion transport polypeptide (OATP) was identified, and
several of these multi-specific transporters have the ability to transport bile acids, as tabulated in cogent
reviews by Hagenbuch and Stieger (249) and Klaassen and Alaskans (250). In man, these include
OATP1A2, OATP1B1, OATP1B3, and OATP1C1. The genes encoding OATP transporters are denoted by
the term solute carrier organic anion (SLCO).
To what extent OATP transporters contribute to the highly efficient uptake of conjugated bile acids
from sinusoidal plasma is not known, but it is believed that the majority of bile acid uptake is mediated by
hepatocyte NTCP. The efficient uptake of dihydroxy conjugated bile acids is truly remarkable, as these
are 99% bound to albumin (251), and presumably uptake is from the extremely low concentration (nM) of
unbound species. In our view, it is reasonable to think that more than one transporter may be involved in
the sinusoidal uptake of conjugated bile acids. Presumably the transporters involved in bile acid uptake
have a zonal distribution and are enriched in periportal hepatocytes.
During enterohepatic cycling, there is continuous bacterial deconjugation in the small intestine
followed by hepatic reconjugation, presumably occurring in the periportal hepatocytes. This was first
recognized in rat studies by Norman, working in Lund, Sweden in 1955 (252) and much later confirmed
for man in our own studies at the Mayo (253). The rate of “reconjugation” in the periportal cells is
several fold greater than the rate of conjugation of newly synthesized bile acids in the pericentral cells.
The uptake of unconjugated bile acids is by both passive and carrier-mediated mechanisms, and
depends on the number and orientation of hydroxyl substituents. Lithocholic acid, CDCA, DCA, UDCA,
and presumably, 3,6-dihydroxy bile acids are highly membrane permeable (221,245) and in our judgment
do not require a transporter for rapid uptake into the hepatocyte. Cholic acid, in contrast, passes across
cell membranes slowly and requires a transporter for rapid cellular entry. A systematic study of the
passive membrane permeability of the three muricholic acid epimers is lacking.
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At least two transporters appear to be involved in the uptake of unconjugated trihydroxy- bile acids.
The laboratory of Curt Klaassen in Kansas City showed that OATP1B2 (a sodium-independent
transporter), appears to be involved in the uptake of unconjugated bile acids that return from the intestine
(254). Knockout of the transporter in mice caused a marked elevation in the plasma level of unconjugated
bile acids. Hubbard et al (255) showed that fatty acid transport protein 5 (FATP5) knockout mice have a
major fraction of unconjugated bile acids in bile, and such unconjugated bile acids were enriched in
secondary bile acids. They suggested that FATP5 was involved in both the uptake and CoA formation of
unconjugated bile acids returning from the intestine. It is attractive to postulate that this transporter
mediates not only adenylate formation but also subsequent CoA thioester formation of the unconjugated
bile acids that enter the hepatocyte. Steinberg et al (256) characterized a homolog of a peroxisomal very
long chain CoA synthetase. This homolog was present in the smooth endoplasmic reticulum. They found
that it had CoA synthetase activity for cholic acid, and postulated a role for this enzyme in cholic acid
reconjugation.
In the isolated perfused liver, rapid uptake of DCA occurs, presumably followed immediately by CoA
formation. Formation of the adenylate and subsequent CoA thioester serves to prevent back diffusion
across the sinusoidal membrane and thereby traps the bile acid CoA in the hepatocyte. In contrast, 24-
norDCA, which does not form a CoA thioester, regurgitates back from the hepatocyte into the perfusate
(257).
The basolateral transporter multidrug resistant protein 4 (MRP4) has been shown to mediate the co-
efflux of conjugated bile acids and reduced glutathione, in work from the laboratory of Dieter Keppler
(258). For this work, we provided tritium-labeled conjugated bile acids of high specific activity (259).
Keppler has suggested that conjugated bile acids percolate in and out of the sinusoidal membrane, and
that such efflux from the hepatocyte into the space of Disse may serve to prevent toxic concentrations of
bile acids in the hepatocyte (260).
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4. Canalicular secretion of bile acids
The next challenge was to identify the canalicular transporter or transporters. This had to be a
powerful transporter, because the intracellular concentration of bile acids was thought to be in the low µM
range, whereas the concentration of monomeric bile acids in ductal bile, based on the dialysis studies of
Duane (261) was about 1000 µM. There was thus an uphill transport of three orders of magnitude.
To study this transport system, one needed vesicles consisting predominantly of canalicular
membranes. The first preparation was achieved by Inoue and Kinne, then working in the laboratory of
Irwin Arias in New York (262). The canalicular membranes were isolated by nitrogen cavitation and
calcium precipitation. At about the same time, Peter Meier working in the laboratory of James Boyer at
Yale developed a method for preparing canalicular vesicles using zonal flotation and high speed
centrifugation through discontinuous sucrose gradients (263). These vesicles were used by Meier and his
colleagues to show that taurocholate uptake by canalicular vesicles was carrier mediated and probably
electrogenic (264). A similar study was published by Inoue, Kinne, and the Arias group (265).
In 1986, Eamon O’Maille, who had studied bile acid secretion in the intact dog, was working in our
laboratory as a guest investigator. He carefully measured the Tmax of taurocholate transport in the rat and
concluded it was too great to be explained by membrane potential alone (266). Y. Adachi returned to
Japan from the Arias lab and in 1991 reported that taurocholate was actively transported into canalicular
vesicles if an ATP generating system was present (267) . That same year, the Arias lab (Nishida et al) also
reported that rat canalicular membrane vesicles contain an ATP-dependent bile acid transport system
(268).
A year or so later, the Zurich group began to work on the ATP-dependent process of conjugated bile
acid extrusion by the canaliculus. They studied a variety of cell systems to find one which did not display
ATP-stimulated bile acid transport and finally ended up with Sf9 (insect) cells. They noted a paper from
the laboratory of Victor Ling in Vancouver, Canada that showed a partial sequence of a transport protein
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named “sister of P-glycoprotein,” later named multidrug resistance 1 (MDR1) (269). Using this
sequence, an antibody to the putative canalicular transporter was generated. The Zurich group then used
phage libraries, their antibody, and Sf9 cells to monitor expression. They were ultimately successful in
cloning the bile salt export pump (BSEP) (protein, bile salt export pump; gene, Abc11A1) (270). Our
laboratory provided 3H-labeled bile acids with high specific activity (259) that were used to define its
substrate affinity.
It was not long until children with cholestatic liver disease were found to have mutations in Abcb11A1,
the gene encoding BSEP (271). This disease had already been named Progressive Familial Intrahepatic
cholestasis, type 2 (PFIC2), and as the defect is canalicular, markers of ductular injury such as γ-
glutamyl-transferase are not elevated in patients with this genetic defect. The name PFIC2 is widely
used, but it seems preferable to call the disease BSEP deficiency. There are other ABC-type canalicular
transporters such as MRP2, MDR1, and BCRP but these are not believed to be very important in the
canalicular transport of unsulfated bile acids in man, based on the severe cholestasis that occurs in
patients with BSEP deficiency as well as evidence from in vitro studies of MRP2 (272).
The laboratory of Victor Ling, in Vancouver, reported the creation of an Abcb11 knockout mouse,
which appeared to have only mild cholestasis. (273). The mildness of the mouse phenotype contrasted
strikingly with the severe cholestatic phenotype that occurs in infants with a genetic defect. This striking
difference between BSEP deficiency in humans and that in mice appears to have at least three
explanations. First, in mice, a major bile acid is β-muricholic acid, which is much less cytotoxic than
DCA and CDCA, these being major bile acids in man. Second, in mice, MDR1 is upregulated and is
likely to be an effective conjugated bile acid extruder (274). Third, BSEP deficient mice form tetra-
hydroxy and penta-hydroxy bile acids (275,276), some or all of which are likely to be transported by
MRP2 (277). Humans with cholestatic liver disease do not form tetra- or penta-hydroxy bile acids to any
extent. In mice, the defect in bile acid export from the hepatocyte is easily shown by feeding such mice
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cholic acid (278), which in this condition is highly toxic. Impaired β-fatty acid oxidation is also present
in in such cholic acid-fed animals (279).
5. The basolateral transporter of the ileal enterocyte
There was one last carrier to be identified, the basolateral transporter of the ileal enterocyte. Also, if
conjugated bile acids were absorbed by cholangiocytes using ASBT and returned via the periductular
capillary plexus to the hepatocyte, there had to be a basolateral transporter in cholangiocytes. The
hepatocyte did not require a basolateral efflux transporter, as it already had one in MRP4, and most bile
acid efflux from the hepatocyte was across the canalicular membrane into bile mediated by BSEP.
In 2001, Wei Wang working with James Boyer and Nazzareno Ballatori at the Mount Desert Island
identified two genes encoding a Na+ independent bile alcohol sulfate transporter in the skate (280). The
genes were isolated using the Xenopus laevis technique of expression cloning, and they mediated
transport only when they were expressed simultaneously. They were named organic solute transporters A
and B (OSTα/OSTβ). Subsequently, Ballatori collaborated with the laboratory of Paul Dawson and
identified this dimeric organic solute transporter as the long sought ileal enterocyte basolateral transporter
(281). The two proteins were present predominantly in ileum and kidney, and immunohistochemistry
showed that the dimeric transporter was present on the basolateral membrane of the ileal enterocyte and
renal proximal tubular cell. When OSTα/OSTβ was expressed in polarized monolayers at the basolateral
domain and ASBT was expressed at the apical domain, bile acid transport from the apical side to the
basolateral side was observed (281). Further study of OSTα/OSTβ showed that it was a facilitated
diffusion carrier and not entirely specific for bile acids, in contrast to ASBT, which is highly specific for
bile acids. In cholestasis, hepatocyte OSTα/OSTβ is upregulated on the basolateral (sinusoidal)
membranes, and mediates bile acid efflux from the hepatocyte into sinusoidal plasma (282).
To date, no diseases caused by defective OSTα/OSTβ have been identified. A mouse knockout has
been created, and it differs from the ASBT knockout in that with the OSTα/OSTβ knockout, there is no
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marked increase in bile acid biosynthesis despite bile acid malabsorption (283). In contrast, in the
Slc10A2 (ASBT) knockout, bile acid synthesis increases nearly twentyfold (284). This apparently
anomalous absence of a compensatory increase in bile acid synthesis is presumed to be the result of bile
acids accumulating in the ileal enterocyte and upregulating the nuclear receptor FXR, which in turn
stimulates the release of FGF15 into portal venous blood (285). Hepatocyte uptake of FGF15 acts to
downregulate bile acid synthesis, as noted in the Introduction. If there is no absorption of bile acids from
the ileum, yet synthesis does not increase, the net result should be a bile acid deficiency in the proximal
small intestine, as secretion will fall and become equal to synthesis. This in turn should cause
malabsorption of saturated fatty acids and fat soluble vitamins.
6. Imaging of bile acid transport
An 11C-labeled analogue of taurocholate (cholyltaurine) has been synthesized by the PET (positron
emission tomography) scan center of Aarhus University Hospital (286). The molecule, cholyl-N-11CH3 –
glycine (cholylsarcosine) permits the kinetics of conjugated bile acid hepatic uptake and excretion into the
biliary tract to be measured and visualized in real time. Because of the extremely short half-life of 11C (20
minutes), this compound can be used only in PET centers that have a reactor (needed for the production
of 11C) close to the patient. The clinical utility of this molecule in assessing cholestatic disease is under
active investigation.
While this review was being written, a book entitled, “Hepatobiliary Transport in Health and Disease”
was published (287). It should cover the topics discussed in our review in much more detail.
B. The enterohepatic circulation in health
1. History
The enterohepatic cycling of bile was clearly described by Borelli in 1681 (288). He wrote, “Bile is
collected in the portal vein [from the intestine] and separated from the blood again in the liver. It is then
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sent into the bile vessels. It leaves these and repeats its circuit thought the intestine and the mesenteric
veins again and again.” Liebig, a prominent German chemist who worked in the mid-19th century, was
fully aware of the enterohepatic circulation of some or all of the constituents of bile (289). Lehmann in
his text book of physiological chemistry, published in 1853, wrote,”the greater part of the bile is again
resorbed as it passes through the intestine. The chief biliary constituents which are resorbed are the
soluble salts and cholic acid” (290). Moritz Schiff, working in Florence, Italy, prepared anesthetized dogs
with an “amphibolic” catheter. When bile was diverted to the outside, bile flow stopped; when bile was
allowed to enter the intestine, bile flow continued. Schiff concluded that bile secretion requires the
intestinal absorption of bile acids (291). (Schiff was put on trial by the English in Florence for
unnecessary cruelty to animals, as he used anesthetized dogs in his experiments. He was found guilty,
and forced to leave Italy. Schiff established his laboratory in Geneva, Switzerland, and became a leading
physiologist in Europe in the 19th century). Additional information on the history of knowledge about the
enterohepatic circulation is given in the detailed review of the enterohepatic circulation by M. Carey and
W. Duane published in 1994 (292).
The enterohepatic circulation of bile acids denotes a mass of bile acids that is discharged by gallbladder
contraction into the small intestine during digestion, is absorbed from the distal intestine, returns to the
liver, and may either be stored in the gallbladder or resecreted into the intestine. When a meal is ingested,
the hormone cholecystokinin is released from the small intestine. Cholecystokinin induces gallbladder
contraction as well as relaxation of the valve (sphincter of Oddi) at the end of the common bile duct
where it empties into the small intestine. Bile then enters the duodenum. Some of the bile acids are
absorbed in the jejunum, but most are transported by intestinal peristalsis to the distal ileum where they
are efficiently absorbed. The bile acid molecules pass through ileal enterocytes, and enter portal venous
blood to return to the liver. One of the early illustrations of the enterohepatic circulation of bile acids with
values for man was presented by Sune Bergström in 1959 (8) and is shown in Figure 8. Whether ancient
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vertebrates, e.g. the coelacanth and the hagfish, have an enterohepatic circulation with their C27 bile
alcohol sulfates is not known.
2. Determinants, properties and measurement of the enterohepatic circulation
As noted previously, the enterohepatic circulation is the resultant of vectorial transport by the ileal
enterocyte and the hepatocyte, as well the flow in the portal venous system, the small intestine, and the
biliary tract. The only molecules that have an enterohepatic circulation are those that are transported by
the membrane transporters of the ileal enterocyte and the hepatocyte. Because only bile acids are
transported by specific transporters, only bile acids have an enterohepatic circulation. The possible
enterohepatic circulation of drugs is beyond the scope of this review.
The vast majority of the circulating bile acids are in the biliary tract and intestine, because plasma and
hepatocyte levels are very low. Plasma levels are low (< 10µM) because of efficient hepatic extraction as
well as dilution. Urinary levels are also low (< 10µM) because only a fraction of plasma bile acids enter
the glomerular filtrate, as bile acids are bound to plasma proteins, mostly to albumin (293). Moreover,
much of the fraction of bile acids that enters the glomerular filtrate is reabsorbed in the proximal tubules
via ASBT and OSTα/β. We found that cholyl conjugates were 70% bound, and dihydroxy conjugates
were >95% bound in human serum (251), so the glomerular filtrate should contain mostly conjugates of
cholic acid in man. The end result is that the concentration of bile acids in urine in healthy man is
extremely low, and urinary bile acids constitute a negligible fraction of bile acid excretion. .
The mass of cycling bile acids in man was first measured by Sven Lindstedt, a member of the
Bergström group, then in Lund (14). Lindstedt prepared cholic acid tagged with tritium and followed the
decay of bile acid specific activity (in bile) with time in human subjects. The specific activity of the
isolated cholic acid fell exponentially with time, indicating that loss of radioactivity followed first order
kinetics. The slope of the natural logarithm of the bile acid specific activity decay curve can be
extrapolated to zero time to permit calculation of the pool size. The slope of the specific activity decay
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curve times the pool size gives the synthesis rate. The technique of isotope dilution described by
Lindstedt has been used to measure the pool size and synthesis rate of the two primary bile acids – cholic
and CDCA repeatedly (294). Such studies have shown that the rate of cholic acid synthesis is about twice
that of CDCA.
The technique can also be performed with stable isotopes, measuring the atoms % excess in plasma
bile acids by mass spectrometry, approach developed by Franz Stellaard and his colleagues in Groningen
(295). When radioactive bile acids are used in the usual amounts, the specific activity of plasma bile acids
is too low to measure accurately.
The isotope dilution technique has also been used for DCA (296), but input now represents absorption
of newly formed DCA rather than synthesis. The input of DCA is some fraction of cholic acid synthesis,
and could therefore vary from 0 to nearly 1.0. This fraction – DCA input/cholic acid synthesis has been
termed the fdehydrox and ranges in healthy adults from 0.2 to 0.5 (297).
In the past, there has been concern because values for bile acid synthesis obtained by isotope dilution
were often somewhat higher than those obtained by analysis of fecal bile acids. The belief at present, is
that the discrepancy between these two methods, which should give identical results, is attributable to the
difficulty of analyzing all fecal bile acids. Thus some fecal bile acids are not measured, and a higher result
for bile acid synthesis is obtained by the isotope dilution technique. Also, if a considerable volume of bile
is removed during the daily duodenal bile sampling used in the isotope dilution technique, this will
increase bile acid synthesis.
No isotope dilution studies have been performed with C27 bile acids or bile alcohols.
3. Synthesis of labeled bile acids for metabolic studies
In the laboratory of the senior author at the Mayo Clinic, better radioactive tags for bile acids were
sought in order to characterize bile acid metabolism. The Bergström group was the first to synthesize 24-
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tagged bile acids that would also be stable during enterohepatic cycling, we used a method developed by
Peter Klein to exchange 3H into the C-2 and C-4 positions (299). N. LaRusso, as his first research
project, examined the stability of this label, and found it was good, but not perfect (300). For labeling
CDCA and lithocholic acid, we synthesized their Δ11 derivatives, and reduced them with 3H. Again, this
label was good, but not perfect (301). Finally, in San Diego, in work led by our chemist, Claudio
Schteingart, we prepared bile acids with a double bond between C-22 and C-23, and then reductively
tritiated these molecules (259). The 22-23 3H label was shown to be completely stable during
enterohepatic cycling in a collaborative study performed with W. Duane at the University of Minnesota
(302). Isotope dilution studies have not been performed with β-muricholic acid because of the non-
availability of labeled material. However, the Δ22 compound, the precursor for reductive tritiation, has
been synthesized by the Iida group (137).
4. The enterohepatic circulation in man
Because of bacterial deconjugation in the intestine followed by absorption from the intestine and
reconjugation in the liver, the turnover rate of the steroid moiety of C24 bile acids is considerably slower
than that of its glycine moiety. As noted previously, this deconjugation-reconjugation pathway was
identified in Sweden by A. Norman in rats (252), and also somewhat later by clinical studies in the
laboratory of the senior author at the Mayo Clinic (253). Deconjugation-reconjugation also occurs with
taurine conjugated bile acids, but the difference in turnover rates between the steroid and amino acid
moiety is less, because taurine conjugates are deconjugated more slowly than glycine conjugates (162).
These studies, performed at the Mayo Clinic in the laboratory of the senior author, have never been
repeated, so confirmation of their findings is desirable.
No information is available on the turnover rates of either the steroid moiety or the taurine moiety of
C27 bile acids. For most C27 bile alcohols, the process of deconjugation – reconjugation is unlikely, as the
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liberated bile alcohol, in contrast to that of unconjugated bile acids, is too polar to be passively absorbed
(190).
Early workers measured the bile acid pool by isotope dilution and guessed at the number of times that
the pool cycled, the product being bile acid secretion. In our view, this assumption is not valid, and is
analogous to trying to predict cardiac output from the measurement of blood volume. A flow can only be
estimated by indicator dilution techniques. Workers at the Mayo Clinic used such an indicator dilution
technique to measure bile acid secretion and found that it averaged about 12 g/day, rising during meal
digestion and falling during the interprandial interval, as well as during overnight fasting (304,305). Bile
acid secretion during digestion was about 0.3 µmol/kg-min, a value confirmed in a separate study of
gallbladder emptying by G. Van Berge Henegouwen and colleagues, also at the Mayo Clinic (306). Bile
acid secretion was also quantified in another intestinal perfusion study by T. Northfield and the senior
author, and similar results were obtained (307).
Portal venous blood delivers bile acids to the hepatic sinusoids, and bile acids are efficiently extracted,
first pass fractional extraction being 50-90%, depending on the bile acid. C. Einarsson, B. Angelin and
their colleagues performed experiments in which they measured bile acid concentrations in portal venous
blood and hepatic venous blood in patients at surgery and thus could measure directly hepatic extraction
(308). M. Korman and his colleagues found that hepatic extraction of glycocholate was identical during
both the fasting and postprandial state (309). This constancy of efficient hepatic extraction means that
hepatic extraction is blood-flow limited, and this view was confirmed in a careful study of hepatic bile
acid extraction in the dog from the laboratory of R. Hanson (310). First pass clearance for CDCA in
healthy volunteers was determined.by van Berge Henegouwen and colleagues. They compared the area
under the curve (AUC) after intravenous administration, with the AUC after oral administration. A value
of 62% was obtained (311). Unconjugated UDCA has a first pass extraction of about 50% (312). In
general, dihydroxy conjugates, i.e. the conjugates of DCA and CDCA have a lower first pass extraction
than cholyl conjugates, perhaps because the dihydroxy conjugates are bound much more tightly by
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albumin. For a given steroid moiety, conjugated bile acids are extracted more efficiently than the
unconjugated bile acid.
5. Absorption of bile acids in the biliary ductules; cholehepatic shunting
The discovery that cholangiocytes contain ASBT (238,239) is present in the ileal enterocyte,
meant that a fraction of conjugated bile acids secreted into canalicular bile is absorbed during passage
through the biliary ductules. The biliary ductules are nurtured by a periductular capillary plexus that
empties into the portal region of the hepatic sinusoids. If conjugation is not complete, unconjugated bile
acids will enter into canalicular bile. In addition, we found in our laboratory that 24-nor C23 dihydroxy
bile acids (norCDCA, norDCA, and norUDCA), which are inefficiently conjugated, are absorbed
passively from the biliary ductules (163,164). The absorbed molecules return to the hepatocyte and are
once again resecreted into canalicular bile, thus undergoing a cholehepatic circulation. The absorption of
the unconjugated bile acid removes a proton from the canalicular bile, generating a bicarbonate anion, the
result being no change in bile osmolarity. Lamri et al provided additional evidence for the validity of
cholehepatic shunting using immunohistochemical techniques (313). The existence of a “cholehepatic
shunt” for unconjugated mono- and dihydroxy bile acids has now been accepted by most hepatologists.
When the unconjugated bile acid returns to the sinusoid, it is taken up by the hepatocyte and
resecreted into bile, thereby inducing additional canalicular bile flow, as evidenced by increased mannitol
clearance. Mannitol clearance is considered a measure of canalicular secretion (314). The increase in
bile flow enriched in bicarbonate is termed “hypercholeresis”. Hypercholeresis was originally described
by the laboratory of Serge Erlinger (315). In their experiments in biliary fistula rats, UDCA was infused
at a rate exceeding the hepatocyte’s capacity to conjugate it. As a result, unconjugated UDCA was
secreted into bile, underwent cholehepatic shunting and induced a bicarbonate rich hypercholeresis. This
report from the Erlinger laboratory gave rise to the erroneous belief that UDCA had unique choleretic
properties. In cholestatic disease, most patients appear to conjugate their ingested UDCA completely
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based on analyses of biliary bile acids (316), but if conjugation were incomplete and unconjugated UDCA
were secreted into bile, it would most likely be absorbed in the biliary ductules, thereby undergoing
cholehepatic shunting. In further work from our laboratory, we showed that sulindac, but not other
NSAID’s, also underwent cholehepatic shunting (317).
6. Determinants of plasma bile acids
The plasma level of bile acids is determined by the instantaneous rates of intestinal absorption
and hepatic uptake. Dogs with a chronic biliary fistula have no detectable bile acids in their systemic
venous blood, thus confirming that intestinal absorption is the source of plasma bile acids, at least in
health (318). The profile of plasma bile acids is not identical to the profile of biliary bile acids. Plasma
bile acids are enriched in dihydroxy conjugates because of their lower hepatic extraction.
7. Regulation of the enterohepatic circulation
The enterohepatic circulation of bile acids must balance two conflicting physiological functions. On the
one hand, bile acids must serve as the end products of cholesterol metabolism and excretion is necessary
for cholesterol balance. For this function, enterohepatic cycling is unimportant. On the other hand, the
ileal conservation of bile acids is needed to provide ample bile acids for efficient lipid digestion, and bile
acid excretion is unimportant. Given these conflicting aims, it should be highly likely that the
enterohepatic circulation of bile acids would be tightly regulated.
The idea of regulation of the enterohepatic circulation is not new. Whipple in his 1922 review (91)
noted that there is little increase in total bile acids in dogs ingesting bile acids chronically, and suggested
that absorption must be regulated. Sten Eriksson, working in Lund, observed that bile acid synthesis
increased with time in the biliary fistula rat (17). Thus the working hypothesis was that bile acid
synthesis is determined by the hepatocyte concentration of bile acids. When this falls, bile acid synthesis
increases. When bile acids are fed, bile acid synthesis decreases. The senior investigator characterized
bile acid metabolism in patients with ileal resection. A marked increase – up to ten fold – was present in
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such patients, again attributed to bile acid malabsorption lowering the concentration of bile acids in the
hepatocyte (319). A similar increase in bile acid synthesis was induced by cholestyramine administration
(320). Studies in the rhesus monkey with an exteriorized enterohepatic circulation of bile acids, also
provided convincing evidence that bile acid synthesis was regulated in a negative feedback manner (321).
In these studies performed by R.H. Dowling, D. Small, and E. Mack (the surgeon who prepared the
monkeys), the return of bile acids to the liver could be systematically decreased, and an increase in
synthesis quantified.
However, two observations did not seem to support this useful hypothesis. The first was that bile acid
synthesis increased at least acutely when the bile duct is ligated (115). The second, made independently
by the group of Reno Vlahcevic in Richmond, VA, (322) and the group of Chiijiwa in Japan (323), was
that upregulated synthesis in the biliary fistula rat could be returned to normal levels by intestinal, but not
by intravenous infusion of bile acids. Both types of infusion should lead to an increase in the
intracellular concentration of bile acids. The Richmond group proposed that a blood-borne signal from
the intestine to the liver might down regulate bile acid synthesis.
The breakthrough came with the identification of FGF15 (in the rat) and FGF19 (in man) as a protein
released by the ileal enterocyte in response to FXR induction by bile acids. This observation by Inagaki
and Kliewer and colleagues at UT Southwestern (58) was a major advance in our understanding of the
regulation of the enterohepatic circulation.
The present view is that the enterohepatic circulation is regulated in two aspects. Input, i.e. bile acid
synthesis is regulated at the level of the hepatocyte. Conservation, i.e. ileal transport, is regulated at the
level of the ileal enterocyte. Both synthesis as well as ileal transport are downregulated by FGF19 (53).
FGF19 enters the hepatocyte via a receptor (FGFR4) and directly or indirectly downregulates bile acid
synthesis. Synthesis of FGF19 in the ileal enterocyte is under the control of FXR, the nuclear receptor
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activated by bile acids. Nonetheless, in some species, it remains possible that bile acid synthesis is
regulated by the bile acid concentration in the hepatocyte rather than by FGF19 (324).
The regulation of ileal conservation is poorly understood. J. Lillienau, working with the senior author,
found that bile salt feeding in the guinea pig downregulated ileal transport whereas administrations of a
bile acid sequestrant upregulated ileal transport (325). Upregulation of ileal bile acid transport was
associated with recruitment of more proximal enterocytes to transport bile acids. Nonetheless, with
complete cholestasis or with parenteral alimentation, bile acid transport by the ileal enterocyte decreases
(326,327). Thus there must be multiple mechanisms of control. It is likely that the down regulation of
the ileal bile acid transporter (ASBT) with bile acid feeding involves FXR agonism and FGF19 release.
C. Perturbations of the enterohepatic circulation in disease
Perturbations of the enterohepatic circulation result from defects in bile acid transport or flow,
assuming that bile acid synthesis and conjugation are normal. Bile flow begins at the canaliculus and
there can be ductular obstruction, as occurs in cholestatic liver disease such as primary biliary cirrhosis.
Physical obstruction can occur with choledocholithiasis or cholangiocarcinoma. Bile flow can be diverted
to the outside, as in patients with partial or complete biliary fistula. Bile acids can regurgitate into the
stomach and from there into the esophagus. In the small intestine, conjugated bile acids might leak across
the epithelium if increased paracellular permeability were present. (To date, such a defect has not been
identified). Bacterial overgrowth can be present, leading to deconjugation, and sometimes, to
dehydroxylation. Unconjugated bile acids are rapidly absorbed, leading to an intraluminal bile acid
deficiency, that in turn can cause lipid malabsorption.
Return of bile acids from the small intestine can be caused by defects in ileal transport, either at the
apical or basolateral domains of the ileal enterocyte, or by absence of the ileum. In addition, if rapid
intestinal transport propels bile acids past the terminal ileum at a rate exceeding its absorptive capacity,
spillover into the colon will occur, and such may induce diarrhea. Portal venous blood flow may be
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obstructed or portal-caval shunting may occur. Finally hepatocyte uptake, transport through the
hepatocyte, or canalicular secretion may be impaired. It is beyond the scope of this historical review to
deal with each of these perturbations on bile acid metabolism.
V. Physicochemical Aspects of Bile Acids
A. Overview
The physical chemistry of biliary lipids and digestive lipids are similar, in that both involve
solubilization of polar lipids in the form of mixed micelles. Mixed micelle formation – whether during
biliary lipid secretion or triglyceride digestion - have received a great deal of attention from physicians
working together with physical chemists, in an attempt to unravel the complexities of these processes.
In 1983, the Kroc Foundation sponsored a small conference (9 physicians, 15 chemists) entitled, “The
Physical Chemistry of Bile in Health and Disease”. Many of the topics discussed below are treated in
more detail in the published proceedings of that meeting (328). Sadly, it was to be the last Kroc
Foundation conference. Ray Kroc, patron of the foundation (and founder of McDonald’s, a global fast
food chain) died shortly thereafter, and his widow, Joan Kroc, had other priorities for her generous
philanthropy. The foundation was dissolved.
In 1966, the senior author collaborated with Donald Small in the writing of a brief review on the
physicochemical properties of bile acids (329). In 1985, Martin Carey authored a systematic review of
the physicochemical properties of C24 bile acids (330). His review included tabulation of physical
constants, crystal structure as determined by X-ray diffraction, surface chemistry of bile acids, pH-
solubility profiles, ionization behavior, and solubilization properties of bile acid solutions. Together, these
two reviews summarized existing knowledge of the physicochemical properties of bile acids, as
envisioned by medically trained physician-scientists. In the discussion below, only selected aspects of the
physicochemical properties are considered.
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B. Bile acids in aqueous solution
1. Solubility of the protonated acid
The first detailed studies on the solubility of protonated, unconjugated bile acids were performed by A.
Roda and A. Fini at the University of Bologna, Italy (331). They reported solubility values at pH 3 for ten
common bile acids. Values varied enormously. That of lithocholic acid was 0.05 µM whereas that of the
7β- epimer of cholic acid (so called “ursocholic” acid) was 1670 µM. Another observation of clinical
relevance was that the two bile acids used for gallstone dissolution had quite different aqueous
solubilities: that of CDCA was 27 µM whereas that of UDCA was 9 µM. The greater aqueous solubility
of CDCA contributes to its excellent gastrointestinal absorption (210); UDCA, with a lower monomeric
solubility is absorbed less efficiently and its crystal dissolution is rate limiting in its absorption (332).
The solubility of several protonated glycine conjugated bile acids have also been determined by A.
Roda and his colleagues. They are quite low (Aldo Roda, personal communication). Taurine conjugated
bile acids, in contrast, even in the acid form, are water soluble.
As yet, we have no solubility values for any of the unconjugated C27 bile acids. The solubility of 5α-
cyprinol was found to be 360 µM, thus being similar to that of cholic acid (190). No other solubility data
are available for C27 bile alcohols.
2. Ionization
One of the purposes of the Kroc Foundation meeting was to reach a consensus on the pKa values of
bile acids, as titration studies performed by D. Small (333) and H. Igimi and M.C. Carey (334) gave
conflicting results, suggesting that the pKa of an unconjugated bile acid was influenced by the number of
hydroxyl groups. B. Josephson, working in Stockholm in the 1930’s as a predoctoral student, determined
that taurine conjugated bile acids were strong acids, that glycine conjugates had a pKa of 4-5, and that
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unconjugated bile acids had a pKa of about 6 (335). A similar conclusion for unconjugated bile acids was
reached by the laboratory of Per Ekwall working in Turku, Finland (336).
At the Kroc meeting, Aldo Roda presented work performed by him and A. Fini which gave accurate
pKa values for unconjugated bile acids (331). Roda used a technique previously used to determine the
pKa of long chain fatty acids. One measures the apparent pKa in mixtures of increasing proportions of
water in methanol and extrapolates to 100% water. The values reported were about 5.1 for the common,
natural unconjugated bile acids with no substituents on the side chain. Later, Roda and Fini obtained
values of about 4 for glycine conjugates (337). The pKa of glycine conjugates is lower than that of
unconjugated bile acids, because the electron withdrawal effect of the amide bond enhances the loss of a
proton from the carboxyl group (338). The pKa of cholylsarcosine was determined by our laboratory to
be 3.7 (338), about 0.3 pKa units lower than that of cholylglycine.
For unconjugated bile acids, the pKa can be reduced by electronegative substituents on the side chain.
Roda et al found the pKa of 23R-hydroxy-CDCA to be 4.8, about 0.2 units lower than that of CDCA
(135). It should be possible to lower the pKa of unconjugated bile acids considerably by adding one or
two fluorine atoms to the C-23 carbon.
The physicochemical significance of conjugation is that it increases solubility markedly at intestinal
pH. Dihydroxy bile acids, which constitute 60% of human bile acids, are insoluble at the pH conditions
prevailing in the small intestine during digestion (pH 6-7). In vitro, the solubility of the bile acid ion
increases exponentially with increasing pH. When the concentration of bile acid reaches the critical
micellization concentration (CMC), solubility increases markedly, in that a crystalline excess becomes
transformed into micelles. There is thus a “critical micellization pH” (CMpH) which defines an
approximate concentration above which the bile acid is highly soluble and below which the bile acid is
insoluble (339). Lillienau et al, working in our laboratory, found the CMpH of glycocholate to be 4.8
(338). That of CDCA was found by van Berge Henegouwen to be about 6.8 (340) and that of UDCA has
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been calculated to be 7.9 (339). The converse of CMpH has been termed “the pH of precipitation” (333)
and denotes the pH at which bile acids begin to precipitate from a true solution of the bile acid, the value
being dependent on the bile acid concentration. The matter is discussed extensively in the review of
Donald Small which gives values for the pH of precipitation of the CDC, DCA, and cholic acid (333).
K.J. Mysels derived the following equation:
CMpH = log [CMC] + pKa + BApS
Where BApS is the negative logarithm of the aqueous solubility of the protonated monomer. The high
CMpH for UDCA is thus explained by its low aqueous solubility and a relatively high CMC.
Despite their excellent pH-solubility profile, glycine conjugated bile acids will precipitate in vivo if
the pH is sufficiently acidic. This occurs in patients with gastrin-secreting tumors in whom markedly
increased gastric acid secretion overwhelms pancreatic bicarbonate secretion and the small intestinal
content becomes acidic (341). In the porcupine, glycine conjugated bile acids reflux from the duodenum
into the stomach and precipitate, forming gastric bezoars (342).
3.Solubility of Calcium (Ca2+) Salts of Bile Acids
Conjugation also greatly increases the resistance to precipitation by divalent cations such as Ca2+.
Studies from our laboratory (343) in collaboration with K. Mysels (a legendary physical chemist) showed
that the calcium salts of taurine conjugates of common natural bile acids are water soluble. Glycine
conjugation has a very small effect on the ion product of Ca2+ ion x [BA-]2, and the solubility products of
Ca2+ salts of glycine conjugated bile acids are quite low. However, the supersaturated solutions of the
calcium salts of glycine conjugated bile acids are metastable, resisting formation of the insoluble Ca2+ salt
for many weeks. The solubilities of Ca2+ salts of unconjugated bile acids, like those of glycine conjugated
bile acids, are also quite low (344). However, metastability of supersaturated solutions is not observed
with unconjugated bile acids.
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In the enterohepatic axis, the most dangerous site for possible insoluble calcium salt formation is the
gallbladder, where ionized Ca2+ concentrations rise during the concentration of bile by the gallbladder
epithelium (345). Stone formation remains rare, as the concentration of ionized Ca2+ is kept rather low in
the biliary tract and small intestine. The low concentration of ionized Ca2+ is explained by the high Na+
concentration, which decreases the activity coefficient of calcium (339).
Formation of Ca2+ bile salt gallstones occurs when “calcium-sensitive” bile acids are present in
appreciable proportions in biliary bile acids. In general, this has occurred because of the feeding of
unusual bile acids or their precursors (346). Examples are the feeding of cholestanol (the saturated
derivative of cholesterol with an A/B trans structure) to the rabbit. The saturated sterol is converted to
allocholic acid, that in turn is converted to allodeoxycholic acid by intestinal bacteria. The
allodeoxycholic acid is conjugated with glycine and the insoluble glycine conjugate
(glycoallodeoxycholate) precipitates from solution forming concretions in the gallbladder (107). Other
instances of the formation of insoluble Ca2+ salts are exemplified by the feeding of lithocholic acid to the
taurine deficient rat (347,348), as well as the feeding of murideoxycholic acid to the prairie dog (349).
The biological significance of conjugation is that it enhances ionization at the pH present in jejunal
content during digestion, preventing passive absorption across the apical membrane of the enterocyte. The
bile acid molecule, whether unconjugated or conjugated is too large to traverse the paracellular junctions
of the healthy intestine. This “non-absorbability” of conjugated bile acids permits high concentrations of
conjugated bile acids to be maintained in the biliary tract and proximal small intestine where the pH
ranges from pH 6 to pH 7, and allows them to function as biological solubilizers. Conjugation is also
likely to prevent bile acids partitioning into the endoplasmic reticulum during their transit through the
hepatocyte.
4. Temperature effects on solubility
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Temperature effects are not very important in the biology of bile acids. Solutions of the common
primary bile acids are stable at 0o C. Friedrich Krafft, working in Heidelberg, Germany observed that
turbid dispersions of soaps became a clear solution over a very narrow temperature range, and his
observation has been immortalized in the term “Krafft point” which denotes this temperature of a phase
transition. One definition of the Krafft point, is that temperature when the concentration of the bile acid
monomer reaches the CMC. With a further increase in temperature, the excess solid phase disappears and
the suspension is transformed into a clear, micellar solution. A thorough discussion of the Krafft point
can be found in the monograph of Laughlin (350).
The term “critical micellization temperature” (CMT) was proposed, but has not been widely adapted.
Sodium cholanoate (no hydroxyl groups) and most mono-hydroxyl bile acids (including lithocholic acid)
have high CMT values, well above body temperature.
In contrast, primary dihydroxy- and trihydroxy-bile acid conjugates have very low CMT values. An
attempt was made by the senior author to define the CMT values of sodium taurodeoxycholate. This was
done by measuring the CMT of sodium taurodeoxycholate:sodium palmitate mixtures with a progressive
increase in the mole fraction of sodium taurodeoxycholate. One can then estimate the CMT value of pure
sodium taurodeoxycholate by extrapolation. The extrapolated values were well below 0oC (350). Thus,
because of their solubility at 0o C, the common natural polyhydroxy C24 bile acids may be considered
excellent “cold water detergents”.
The influence of the A/B ring juncture on the CMT of C24 bile acids has not been defined. There is also
no information on the CMT values of C27 bile acids as well as those of bile alcohol sulfates with the
single exception of 5α-cyprinol sulfate whose CMT was reported to be < 00 C (190).
5. Self-association in solution: Micelle formation
In 1936, G.S. Hartley, working in London, wrote a small lucid monograph entitled, “Aqueous
Solutions of Paraffin-Chain Salts: A study in micelle formation“(351). This work summarized the
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evidence, mostly based on conductivity measurements, for micelle formation by long chain fatty acid
anions as well as alkyl sulfates, and proposed a structure for the spherical micelle. A.S.C. Lawrence, a
colleague of Hartley’s once said to the senior author, “Hartley wrote down what everyone else was
thinking!” Hartley notes in the final chapter, “The bile salts are obviously amphipathic”. At the end of
the Second World War, Hartley returned to academic life and decided to take up the problem of micelle
formation in bile acid solutions. However, his postdoctoral student developed mental problems, and the
work stopped. Hartley himself left the academic world and became a scientist at Fisons, where he
invented novel sprayers for crops as well as developing new pesticides.
The concept of micelle formation had been first advanced by James McBain in 1913, who presented
this idea at a meeting of the Royal Society. The chairman listened carefully, and began the discussion
with, “Nonsense, McBain.” McBain, who spent the latter part of his scientific career at Stanford, spent
much of his subsequent years, characterizing soap solutions, and undertook limited studies with bile acid
solutions. McBain also noted in 1941 that detergent solutions can solubilize insoluble dyes. His widow
and E. Hutchinson published a book entitled, “Solubilization and Related Phenomena” in 1955, which
summarized many of the observations on this topic in the McBain laboratory (352). J. Bashour and L.
Bauman, working in New York, developed a method for synthesizing conjugated bile acids, and in 1937
used their synthetically prepared conjugates to show that bile acid solutions solubilize cholesterol in a
concentration-dependent manner (353).
In 1942, O. Mellander and E. Stenhagen, working in Sweden, performed conductivity studies with
sodium taurocholate and found evidence of aggregate formation (354). Per Ekwall, working in Turku,
Finland, summarized the evidence for micelle formation in sodium cholate solutions in 1951 (355).
Ekwall also characterized surface properties of bile acids and made many contributions to the physical
chemistry of bile acid solutions. (356).
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As noted above, a program on the biology and chemistry of bile acids was initiated by Sune Bergström
in Lund after the war. Arne Norman synthesized the glycine and taurine conjugates of most of the major
bile acids. He then collaborated with Per Ekwall to study the solubilization of 20-methylcholanthrene and
thus define the critical micellization concentration (CMC) for dilute solutions of pure, conjugated bile
acids (357). Shortly thereafter, and independently, the senior author, working in Lund, Sweden,
synthesized and measured the CMC of the major bile acids present in human bile using trans-azobenzene
as the solubilizate (358).
In contrast to typical ionic surfactants, the aggregation of bile acid anions is gradual, and there is no
well-defined CMC. Thus, the CMC for bile acid solutions is often taken as the midpoint of a small
concentration range over which micelles form. K.J. Mysels suggested the term “uncritical micellization
concentration”. P. Mukerjee has pointed out that bile acids are rigid molecules in contrast to typical ionic
detergents which have a flexible alkyl chain. Therefore, bile acid molecules, just as dye molecules, stack
rather than form spherical micelles. The aggregates (from tetramers up to 20 mers, depending on bile
acid structure and concentration) may be termed multimers rather than micelles. The size of the
aggregates was estimated by light scattering, especially by D. Small (329,333) and J. Krahtohvil (359).
However, as noted below, aggregates of bile acid molecules alone, i.e. without an accompanying lipid, do
not occur in nature, except when phospholipid extrusion into bile is defective.
The most recent measurement of CMC values for a large number of unconjugated and conjugated
bile acids was made by Aldo Roda in our laboratory, in collaboration with K. J. Mysels (360). This study
was successful because of two major advances. The first was the synthesis of a large number of bile acid
epimers by Takashi Iida and Frederick Chang (361), and in a few cases, by our laboratory. The second
was the availability of a maximum bubble pressure device designed, constructed, and characterized by
K.J. Mysels. A key aspect of this technique for measuring surface tension in relation to concentration, is
that a new bubble surface is constantly being formed whose composition consists of the dominant
constituent of the solution. In contrast, with surface tension methods using a tensiometer, a more surface
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active impurity may concentrate at the surface of the solution to be analyzed and lead to erroneous values
for the CMC. Roda validated his bubble pressure values by showing that identical values were obtained
with the technique of dye solubilization using Orange OT (1-O-tolyl-azo-2-naphthol) as the water-
insoluble dye. Values are reported in the absence and presence of 0.15 M Na+ concentration.
The senior author, when working in Sweden in the 1960’s observed that the aggregation of
monoglycerides and bile acids to form mixed micelles was cooperative, so that solubilization occurred at
a concentration well below the CMC of bile acids in the absence of such amphiphilic lipids (358). W.
Duane used dialysis to measure the intermicellar concentration of bile acids (monomers plus simple
micelles) in both model bile samples and human bile samples. He obtained values for the critical
micellization concentration as well as for the intermicellar concentration of bile acids which were quite
similar to the critical micellization concentration in bile acid-monoglyceride systems (261).
C. Solubilization of polar lipids by bile acid micelles.
1. Physical chemistry of biliary lipids
Bile acids are present at micellar concentrations only in bile and small intestinal content. In man, the
concentrations in plasma (2-10 µM) and in colonic content (400 µM) are well below the CMC. In health,
bile acids are always associated with a polar lipid. In bile, this is predominantly phosphatidylcholine
(PC). In small intestinal content, during digestion, the polar lipids are monoacyl glycerol and fatty acids
(partly ionized). The only circumstance in which bile acid micelles are present without an accompanying
polar lipid is in the Mdr2 knockout mouse and in patients with a defective Mdr3 protein. Such mice (and
patients) lack this ATP-activated PC extruder in the canaliculus of the hepatocyte and are believed to have
essentially no phospholipid in ductular bile. The micellar bile acids, if sufficiently hydrophobic, attack
the membranes of the cholangiocytes, leading to ductal inflammation in mice (362) and to a variety of
biliary problems in patients (363). Nonetheless, the bile of many vertebrate species has an extremely low
ratio of phospholipids to bile acids (181) without any sign of biliary ductule injury. In such species, it
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may be that phospholipid, after secretion into canalicular bile, is absorbed in the biliary ductules with the
result that it is not present in gallbladder bile in appreciable proportions, or that the ductules are highly
resistant to attack by bile acids or both.
The recognition that lecithin was present in bile occurred early in the 20th century. G. Whipple in his
1922 review (91) tabulated the reported analyses of biliary lecithin. In 1952, Polonovski and Bourillon,
working in Paris, confirmed that the dominant phospholipid in bile was lecithin, later to be named PC
(364). G. Phillips separated biliary phospholipids chromatographically and confirmed that the dominant
phospholipid in bile was PC (365). Another article in this series gives the history of phospholipids in
more detail.
Franz Ingelfinger, one of the dominant figures in American Gastroenterology in the mid 1900’s was
unhappy with the level of ignorance of the pathogenesis of the common disease of gallstones. He
arranged for Donald Small, one of his brightest fellows, to spend two years in Paris working under the
direction of a scholarly colloid chemist, Digren Dervichian. During those two years, the behavior of
model systems simulating bile was described clearly using triangular coordinates. The ternary systems –
water-lecithin-bile acid, water-cholesterol-bile acid, water-cholesterol-lecithin – were carefully defined
using sodium cholate, egg lecithin, and cholesterol. The behavior of the three lipids and water could then
be described by a tetrahedron. A slice across the tetrahedron at 10% total lipid concentration gave a
triangle with three coordinates – cholesterol, lecithin (PC) and bile acid. A line defined the limits of the
micellar zone. Accordingly, points lying above the line would be supersaturated in cholesterol either in
liquid crystalline or crystalline form. This classic work is summarized in reference 333 and illustrated in
Figure 9. The stage was set for applying this pure exercise in model systems to the pathogenesis of
cholesterol gallstone formation (see later).
That phospholipids play a key role in the solubilization of cholesterol in bile was perhaps first
recognized in the mid 1950’s by B. Isaksson, working in Sweden (366). Work on the solubilization of
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lecithin by bile acid solutions was carefully studied by S. Yoshimuta (367), a member of the Department
of Surgery at Kyushu University in Japan whose director, E. Miyake, had a longstanding interest in
gallstone formation. This work was published in the 1960’s about the same time that D. Small was
studying model systems simulating bile in Paris. Another physician with interest in this problem was
Kerrison Juniper who attributed cholesterol solubilization in bile to a macromolecular complex (368). A
review by D. Small and the senior author, published in 1967, advanced the idea that the physical
chemistry of micelle formation in bile and micelle formation in small intestinal content during triglyceride
digestion were similar physicochemical processes (329).
Thus by 1967, both bile and small intestinal content were considered micellar solutions, the only
difference being the nature of the accompanying lipids. Simple bile acid micelles are poor solubilizers for
cholesterol or non-polar dietary lipids. The mixed micelle provides a hydrocarbon interior in which
poorly soluble lipids can dissolve. In bile, this insoluble lipid is cholesterol; in small intestinal content,
insoluble lipids of physiological importance are the fat soluble vitamins. The remaining vexing problem
was the molecular arrangement of the mixed micelles present in bile and small intestinal content.
2. Molecular arrangement of mixed micelles
To solve the problem of the structural arrangement of the mixed micelles in bile, D. Small traveled to
England to the laboratory of Dennis Chapman (F.R.S.), a physical chemist with a deep interest in
membrane structure. They examined solutions by NMR and found that the angular carbons of the bile
salt molecule were in a hydrophobic environment. On the basis of the NMR spectra, they proposed the
“mixed disc” model in which a bilayer of PC molecules with a drum shaped perimeter was coated on its
surface with bile acid molecules (369). Later, dimers of bile acids interdigitating between the PC bilayer
were added (330). The model was widely accepted.
At the Kroc meeting in 1984, G. Lindblom of the University of Lund proposed that bile acids could
also interact with phospholipid bilayers by lying flat in the bilayer with the hydrophobic face of the bile
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acid molecule pushing the charged heads of PC apart (370). W. Nichols and J. Ozarowski proposed that
bile acids lie flat on the surface of worm-shaped micelles (371) in contrast to the mixed disc model. Rex
Hjelm, a biophysicist working at the Los Alamos National Laboratory, examined bile acid-PC-water
systems using small angle neutron scattering. Hjelm concluded that as the PC/bile salt ratio increased, the
change in scattering indicated that PC-bile acid micelles increase in size by the elongation of constant
diameter rods (372), providing additional support for the “radial shell model” structure advanced by
Nichols and Ozarowski. . Hjelm also concluded that the disk-shaped micelle originally proposed by
Small et al. could not be correct. Nonetheless, the conclusions of Hjelm are not widely known in the
medical community, because much of his work was published in chemical journals that are not included
in the PubMed data base. A depiction of the original model for the mixed micelle proposed by Small and
Carey is compared with that of the model inferred from the small angle neuron studies of Hjelm and his
colleagues in Figure 10.
In 2002, S. Marrink and A. Mark, working in the Netherlands used the technique of molecular
dynamic simulations, in which molecules aggregate in water to give the lowest free energy (373). They
concluded that, “phospholipids are packed radially with their headgroups at the surface and the
hydrophobic tales [sic] point…. toward the micellar center. The bile salts act as wedges between the
phospholipid headgroups”. They concluded that the structure of the micelles generated in their system
resemble the radial shell model proposed by Nichols and Hjelm. Additional work by Long et al (374)
using small angle X-ray scattering and quasi-elastic light scattering were also in agreement with the
model proposed by Nichols.
Another approach to study the interaction of bile acids and phospholipids was taken by Heuman et al
(375). They prepared single layer vesicles and characterized the binding of bile acids to them. They also
determined at what phospholipid/bile acid ratios, the vesicles were transformed into micelles.
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For teaching purposes, it would be nice to have a video with realistic molecular models showing the
transformation of lipid bilayers into mixed micelles by bile acid anions.
D. Physical chemistry of bile and cholesterol gallstone disease
1. Supersaturated bile in bile from gallstone patients
D. Small returned from France to Boston in 1965, and began to apply his studies on model systems
simulating bile to human biliary lipid compositions. Bile from radiolucent gallstone patients and healthy
control subjects was collected. The proportions of bile acid, PC, and cholesterol were plotted using
triangular coordinates in a study in which W. Admirand did much of the leg work. A line denoting
equilibrium cholesterol saturation had been obtained by D. Small based on his studies in Paris: samples
above the line were supersaturated with cholesterol whereas those below the line were unsaturated in
cholesterol. When the data were plotted, bile from patients with gallstones was supersaturated with or
without cholesterol microcrystals, whereas bile from most non-gallstone patients was unsaturated (376).
[The paper also re-introduced triangular (or ternary) coordinates to the medical audience. Triangular
coordinates can be used when there are three variables adding up to a constant sum, i.e. there are two
degrees of freedom, permitting the constructions of graphs in two dimensions. They were originally
proposed in 1873 by the great physical chemist J. Willard Gibbs, of Yale University and two decades later
popularized by a Dutch chemist, H.W.B. Roozeboom (377). Probably, their first usage in medical
journals was in a paper by E.H. Ahrens (378) published in 1957 to describe the proportions of fats,
carbohydrates, and protein in diets].
Cholesterol gallstone disease could now be considered a disease of defective bile secretion by the liver,
rather than being the result of a diseased gallbladder, as had often been proposed in the past. The paper
also indicated that biliary lipid composition should be expressed as a ratio of solute (cholesterol) to
solvent (bile acid-PC micelles). The story of the rise and fall of medical dissolution of cholesterol
gallstones is discussed later.
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2. Vesicle formation and gallstone disease
In the original phase diagram of D. Small, the physical state of cholesterol in supersaturated systems
depended on the ratio of PC to bile acid. At low ratios, cholesterol was crystalline. At higher ratios, the
excess was liquid crystalline, i.e. in lamellae of PC and cholesterol. Norman Mazer and Martin Carey
used quasi-elastic light scattering to characterize model systems simulating bile. At higher PC to bile acid
ratios, they found evidence for “microprecipitates” although they also suggested that these
microprecipitates were vesicles enriched in cholesterol compared to mixed micelles (379,380). About the
same time Giora Somjen and Tuvia Gilat, working in Israel, used gel permeation chromatography to
separate mixed micelles from vesicles (381). They equilibrated their columns with 10 mM cholate prior
to chromatography and thus the vesicular phase remained unchanged during the chromatographic
separation. They also noted that if the columns were preequilibrated with buffer alone, a bile sample
would become entirely vesicular, as the bile salt molecules moved into the aqueous spaces in the gel
permeation column, thereby increasing the phospholipid/bile acid ratio. Thomas Holzbach, working in
Cleveland, also found vesicles in human bile samples and showed that nucleation of cholesterol crystals
from the cholesterol-rich vesicles occurred rapidly (382). William Higuchi, working in Salt Lake City,
showed using dialysis techniques that model bile systems contained four constituents: monomers, simple
bile acid micelles, mixed micelles (containing bile acids, PC, and cholesterol) and vesicles also containing
the three biliary lipids. The vesicles were relatively enriched in cholesterol (383).
Because of the complexity of the physicochemical aspects of bile and because of the widespread
interest in gallstone pathogenesis and treatment, in 1989, the NIH sponsored a workshop entitled, “Biliary
Cholesterol Transport and Precipitation”. The proceedings (including much animated discussion) were
published as a supplement to Hepatology the following year (with financial support from the Ciba-Geigy
Corporation) (384). The meeting included a wide range of scientists from pure membrane biophysicists to
gastroenterologists and surgeons, and the discussion often indicated problems in communication. A
summary of the meeting was authored by Steven Strasberg, a scholarly surgeon, and his associate Robert
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Harvey. Their summary (385) presented a reasonably coherent view of bile supersaturation. Cholesterol
and phospholipids are secreted in vesicular form. Bile acids, initially present as simple bile acid micelles,
solubilize phospholipid and cholesterol. However, the transfer process, presumably occurring by
collision, solubilizes phospholipids preferentially, leaving behind vesicles containing an excess of
cholesterol (from which cholesterol crystallizes). Thus, there is a progressive change in the physical state
of bile as it moves from the canaliculus into the gallbladder.
At this meeting, J. Donovan, then working in the Carey laboratory, tabulated the existing clinical data
(386). The proportion of cholesterol in vesicles was higher in hepatic bile than in gallbladder bile in all
studies, indicating that the transformation of vesicles to mixed micelles continued during gallbladder
storage of bile. C.Schriever and D.Juengst showed, using bile samples from gallstone patients, that the
ratio of cholesterol to phospholipid in vesicles purified by gel filtration was 2.0 (387), a ratio that favors
formation of cholesterol crystals.
Since this meeting, especially because of the declining interest in medical dissolution of gallstones,
work on the physical chemistry of bile has waned. W. Higuchi continued his work on model systems,
emphasizing the “activity” of monomeric cholesterol (388). Cholesterol saturation continued to be
calculated from the original triangular figure of D. Small, even though the limit to the micellar zone was
moved downward, based on in vitro studies of model systems from the laboratories of Henrik Dam in
Copenhagen (389), and that of R.T. Holzbach in Cleveland (390). [Henrik Dam, a Dane, had a long
interest in cholesterol. He received the Nobel Prize in 1943 for his discovery of vitamin K. In the 1960’s,
he turned his enormous energy to the study of cholesterol gallstones and did both animal (391) and
clinical studies (392) of note. Sadly most of his publications are to be found in an obscure journal (Z. fur
Ernahrungswissenchaft) and are seldom cited]. Simple methods for calculating cholesterol saturation
based on the revised line of saturation were developed. M. Carey published “critical tables” that give a
value for cholesterol saturation for any molar ratio of phospholipids to cholesterol, as well as varying total
lipid concentration (393). The line of saturation is also influenced by bile acid type -- being lower when
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bile acids are enriched in conjugates of UDCA. M. Carey has also published critical tables for this special
circumstance (394).
E. Physical chemistry of fat digestion
1. Studies on model systems simulating triglyceride digestion
The physical chemistry of model systems simulating fat digestion has received much less attention.
The senior author, when working in Sweden, defined the behavior of each of the relevant constituents in
dilute bile acid solutions. Fatty acids (oleic) were readily available, but monoacyl glycerides
(monoglycerides) were not. Fortunately, they were generously supplied by Fred Mattson, a chemist
working at the Miami Valley laboratories of Proctor and Gamble in Ohio. [Mattson, a gifted biochemist,
discovered the positional specificity of pancreatic lipase (395) and then showed that triglyceride was
absorbed from the intestine in the chemical form of monoacylglycerol and fatty acid (396).] The solubility
of fatty acids in bile acid micelles was pH dependent, as protonated fatty acid has a rather low solubility
in bile acid micelles, whereas fully ionized fatty acid (soap) is infinitely soluble (350). Simple titration
experiments (397) indicated the pKa of fatty acids in a taurine conjugated bile acid micelle was about 6.9,
indicating that fatty acids at proximal small intestinal pH (where most fat absorption occurs) are about
half ionized. Mono-olein, whether the 1- or 2-isomer, was highly soluble, with the ratio of solubilized
monoolein to micellar bile acid approaching 1.8 (358). In contrast, di- and triacyl glycerides had little
solubility in bile acid micelles.
In 1988, M. Svärd working in the group of Björn Lindman in Lund, investigated the taurocholate-
monoolein-water system by a great variety of techniques, and constructed the complete phase diagram
(398). In her paper, she also noted minor, but consistent differences between this system and the bile
acid-PC-water system elucidated by the pioneering work of D. Small two decades before. What both
diagrams share in common is a large micellar zone, as shown for the taurocholate-monolein-water system
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illustrated in Figure 11. The paper of Svärd et al also proposes that bile acid molecules lie flat on the
surface of the bilayers.
2. Presence of a micellar phase in vivo during triglyceride digestion
The in vitro findings indicated that fatty acids and monoglycerides were highly soluble in bile acid
micelles, but that di- and triglycerides were not. These observations led to the prediction that during fat
digestion, glycerides and fatty acids would be in two phases. There would be an oil phase containing di-
and tri-acyl glycerides and a micellar phase consisting of bile acid micelles containing partly ionized fatty
acid and monoglyceride. The oil phase might also contain protonated fatty acids if these were generated
by pancreatic lipolysis at a rate exceeding that which could be solubilized in mixed micelles. Later,
healthy volunteers were intubated, fed a mixed meal, and intestinal content was collected. It was then
ultracentrifuged at body temperature, and both the oil phase and micellar phase analyzed. The results
confirmed the predictions derived from the in vitro experiments (399).
Some years later, a more detailed analysis of this system was performed by O. Hernell and J. Staggers
working in the M. Carey laboratory. They studied both model systems (400), as well as small intestinal
content obtained by intubation in healthy volunteers who had ingested a triglyceride rich meal (401).
They confirmed the older studies of the senior author, but noted in addition, that vesicles were present.
They proposed that vesicles form at the surface of the oil droplets, and that these are fairly rapidly
converted to mixed micelles when they encounter unsaturated bile acid micelles. In 1959, A.S.C.
Lawrence, a colloid chemist at Sheffield University, proposed that detergents adsorb to “dirt” and form
liquid crystalline myelin figures (multilayered vesicles). His analysis (402) is probably an apt description
of the formation of vesicles composed of fatty acid and monoacylglycerol at the surface of the triglyceride
droplet.
A closing chapter on this topic was authored by R. Hjelm, in which model mixtures simulating fat
digestion were prepared in our laboratory and examined by small angle neutron scattering. Hjelm
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concluded that mixed micelles containing a bile acid and either monoglyceride (monoacylglycerol) or
monoglycerides plus fatty acids had essentially the same structure as the mixed micelles containing bile
acids and PC that he had studied previously (403,404). In both, the bile acid molecules lie flat on the
surface of the spherical or worm shaped micelles.
3. Necessity of bile acids for efficient absorption of triglycerides
Numerous studies dating back to the 19th century had shown that triglyceride absorption efficiency
was impaired in animals with a biliary fistula and that feeding of bile acids would correct fat
malabsorption (405). Nonetheless, there were efforts to show that bile acids were unnecessary for fatty
acid absorption (406). The view of the senior author is that bile acids are essential for the efficient
absorption of those fatty acids that have very low aqueous solubilities, i.e. saturated fatty acids of C16
length or longer (407). Fatty acids with appreciable aqueous solubility (unsaturated fatty acids and
saturated acids having a chain length ‹ C16) do not require bile acids for efficient absorption. This view
that bile acids facilitate the absorption of saturated fatty acids (≥ C16 in length) is based on two
observations. First, fecal fatty acid analyses indicate that exogenous bile acids cause a decrease in the
proportion of long chain saturated fatty acids in short bowel syndrome patients (408). Second,
administration of cholestyramine to rats causes selective malabsorption of long chain saturated fatty acids
(409). It is well established that bile acids are required for the absorption of fat soluble vitamins.
The lack of requirement of bile acids for efficient absorption of fatty acids with appreciable aqueous
solubility provides the rationale for using medium chain triglycerides for nutritional support in patients
with a deficiency of bile acids in the small intestine. The liberated medium chain fatty acids are quite
water soluble and are rapidly absorbed in the complete absence of bile acids.
VI. Dissolution of cholesterol gallstones by oral bile acids and topical solvents
A. Gallstone dissolution by oral bile acids.
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As noted in the introduction, the famous triangle of D. Small (and W. Admirand) indicated how biliary
lipid composition could be plotted. (The term “biliary lipids” to include bile acids, PC (lecithin), and
cholesterol was probably introduced by D. Small in the 1960’s. It did not include bilirubin diglucuronide
(which gives bile its color), as this pigment derived from heme is a very minor constituent of bile. Biliary
lipids differ from plasma lipids in that biliary lipids include bile acids, and do not include glycerides and
fatty acids).
In the 1950’s, Charles Johnston, a surgeon at Wayne State University, was interested in determining
what he termed the “cholesterol holding capacity” of bile, that is, how much additional cholesterol could
be dissolved when added to bile samples. He observed that T-tube bile from gallstone patients was
“saturated”, whereas bile samples obtained from healthy subjects undergoing surgery was unsaturated.
With a young Japanese surgeon, Fumio Nakayama, he fed cholic acid to gallstone patients, but found that
it did not increase the “cholesterol holding capacity”. In the discussion of his paper, he noted that he
hoped to try CDCA, but of course, at that time, there was no CDCA to be had (410). [Fumio Nakayama
continued to study gallstone pathogenesis using chemical techniques and had a long and distinguished
career as a Professor of Surgery in Kyushu, Japan. After he retired he summarized his lifelong interest in
gallstone formation and treatment in a monograph (411)].
After J. Thistle and L. Schoenfield made the seminal discovery that CDCA but not cholic acid changed
bile from supersaturated to unsaturated, it was logical to perform a clinical trial of CDCA for gallstone
dissolution. The effort was spearheaded by J. Thistle, as L. Schoenfield took a new position in Los
Angeles in 1970. In 1971, the first observations were made that gallstones were decreasing in size,
probably the first witnesses being J. Thistle (and his radiological colleague) and the senior author. In
1972, successful gallstone dissolution was reported in May at the annual meeting of the American
Gastroenterological Association and later that year in Europe at the 2nd International Bile Acid Meeting
sponsored by the Falk Foundation of Freiburg, Germany. The early Mayo experience with gallstone
dissolution was published in the New England Journal of Medicine in 1972 and became a “Citation
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Classic” (37). Later that year, gallstone dissolution by CDCA was reported by the group of R.H.
Dowling, working at Guy’s Hospital in London (38). In 1973, Dr. Falk Pharma decided to market CDCA
for gallstone dissolution.
In February 1972, a small meeting was held at the NIH to discuss a national multicenter placebo-
controlled trial of CDCA. It was felt that the absence of patent protection would preclude development
by pharmaceutical companies. A “Request for Proposals” was issued in July 1972. L. Schoenfield
submitted a proposal which included committee structure for the proposed National Cooperative
Gallstone Study (NCGS). On the committees were most every American scientist or physician interested
in gallstone disease!
The study was delayed, because of the FDA concern that CDCA might cause liver damage. The
FDA demanded that liver biopsies be performed on the first 100 patients, including those on placebo.
Despite the ethical problems inherent in subjecting placebo group patients to liver biopsy, the study was
performed without any adverse events.
The concern of the FDA was based in part on two reports from the Huntington Laboratories in
England of severe toxicity induced by CDCA feeding in the rhesus monkey (412). As a result of these
reports, European studies were stopped in 1973. Report of the primate toxicity provided a justification for
liver biopsies in the patients when therapy was stopped (413). An international registry of biopsies was
set up, and a careful review of the biopsy histology showed no important histological changes. Trials in
Europe recommenced.
The answer to this remarkable species difference in CDCA toxicity came from work by the senior
investigator and his colleagues who showed that in man, lithocholic acid, the major bacterial metabolite
of CDCA, is sulfated and rapidly excreted from the body (414,415), whereas in the rhesus monkey,
lithocholic acid is extremely poorly sulfated, accumulates in the circulating bile acids, and induces liver
damage (416,417). Independent studies by the lab of E. Mosbach reported that CDCA also was toxic in
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the rabbit, again because of lithocholic acid accumulation (418). Similar findings were observed when
rhesus monkeys (419) and baboons (420) were given CDCA.
The NCGS began in 1976 and its results were published in 1981 (421). The study was a double blind
study with three groups -- placebo, 350 mg CDCA/day and 750 mg CDCA/day. Dissolution occurred in
the placebo group (11%), was greater (24%) in the low dose group and highest (41%) in the high dose
group. During the study it was recognized that the high dose was probably still too low a dosage (422),
but the study could not be changed. In the high dose group, 8% of patients had to discontinue the study
because of an increase in serum aminotransferase levels. Toxicity was shown to be attributable to CDCA
and not to lithocholic acid (423). While the NCGS was proceeding, controlled studies were being
performed in Europe which showed that CDCA had unequivocal efficacy and minimal toxicity (424,425).
An analysis of the kinetics of gallstone dissolution by William Higuchi indicated that the reduction in
diameter induced by the presence of unsaturated bile occurred independently of size (426). Thus, the
bigger the stone, the longer the time needed for complete dissolution. This prediction was later confirmed
by Senior et al in a thoughtful analysis of x-ray changes of gallstone size during medical dissolution
(427). .
Just as the NCGS was beginning, word came from Japan (a careful study led by Isao Makino and his
colleagues) that ingestion of ursodeoxycholic acid, the 7β- epimer of CDCA, would also induce gallstone
dissolution (40). Earlier, there were several anecdotal cases in which subjects had taken large doses of
UDCA and induced their own gallstone dissolution, and this was done without knowledge of the
American studies with CDCA. Thus, medical dissolution of gallstones must be considered an American
and Japanese discovery.
In the United States, the NCGS drafted a protocol that would permit a study similar to the original
NCGS, but would compare UDCA with CDCA; however, the study was not funded by NIH. CDCA was
approved for marketing in the United States by the FDA in 1983, and UDCA was approved a few years
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later. UDCA, like CDCA, was also toxic in the rhesus monkey (428) and rabbit (429) because of
lithocholic acid accumulation in the circulating bile acids.
Despite the safety and efficacy of CDCA and UDCA, there were some physicians who were
unimpressed by medical therapy as compared to surgical therapy. There were several problems with
medical therapy. First, it took a long time – months to years -- to obtain complete dissolution, especially
for larger stones. Second, studies by Wolpers suggested that with the first attack of biliary colic, the
resulting inflammation would cause a surface layer of non-cholesterol constituents on the gallstone,
slowing or precluding dissolution (430). Thus, there was no window of opportunity: when stones became
symptomatic, they had already become resistant to medical dissolution. Moreover, it took many months
of therapy before one could be sure that the stone was resistant. Finally, after stones dissolved, there was
recurrence (431). It was gallstone recurrence that led surgeons a half century before to switch from
cholecystotomy (removal of the stone but leaving the gallbladder in place) to cholecystectomy (432). A
thoughtful review by Gracie and Ransohoff concluded that it was better to “wait and cut” than to “screen
and treat” because of the benign natural history of most gallstones (433). An editorial by K. Isselbacher
was entitled “Disillusion with Dissolution” (434).
B. Acceleration of dissolution by extracorporeal shockwave lithotripsy
Attempts were made to speed up dissolution. One approach was to fragment the stones by using
extracorporeal shockwave lithotripsy, a technique that had been developed for the fragmentation of
kidney stones. For gallstones, the stone fragments would be smaller than the original stone(s), have
cholesterol surfaces exposed by the shattering of the stones, and thus, medical dissolution would occur
more rapidly. However, there was a major difference between kidney stones and gallbladder stones.
When kidney stones were fractured by this technique, the fragments passed spontaneously down the
urinary tract and were expelled during urination. With gallbladder stones, the fragments sunk to the
bottom of the gallbladder, so that dissolution by desaturating bile acids was needed. Another limitation of
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extracorpeal shock wave lithotripsy was that only certain types of stones (one or two in number) could be
treated. Thorough clinical studies of the efficacy and safety of this technique for gallstone dissolution
were conducted by the Munich group led by Gustav Paumgartner (435). Leslie Schoenfield led one of
several efforts in the United States (436).
C. Topical dissolution with organic solvents
Another approach to non-surgical treatment of cholesterol gallstones was contact (topical) dissolution
with organic solvents. The Mayo group, led by J. Thistle, introduced the idea of placing a percutaneous
transhepatic catheter into the gallbladder and then lavaging the stone with methyl tert-butyl ether (MTBE)
a solvent that had become available as a gasoline additive (437) and was a superb cholesterol solvent.
When the solvent was pumped into and then aspirated from the gallbladder, stones would dissolve in a
matter of a few days. A spark-free pump was designed and patented by the Mayo Clinic. European trials
commenced in a few centers (436,437).
In our laboratory at UCSD, we had not planned to become involved in this approach. A patient with
symptomatic gallstones who was a poor operative risk presented with symptomatic gallstones, and we
reluctantly carried out successful dissolution by manual instillation and aspiration of MTBE. A “smart”
pump was designed by S. Zakko so that solvent could be pumped into and out of the gallbladder at a high
rate. The pump was programmed to continuously sense intraluminal pressure and aspirate gallbladder
contents whenever the gallbladder started to contract, thus avoiding spillage of the solvent into the
common bile duct (438). A new solvent – ethyl propionate – was tested and found to dissolve gallstones
as nearly as rapidly as MTBE (439). Ethyl propionate did not induce nausea in contrast to MTBE
because of rapid removal from plasma (440). Patients were treated successfully (441).
However, there were multiple problems with contact dissolution. The solvents used were commercial
samples and they were not manufactured according to FDA standards. MTBE was flammable and
considered toxic (442). Preclinical studies had not been done. The procedure required some hours for
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complete dissolution, and as described, it required professional personnel to perform the infusion and
aspiration. In studies performed in pigs, the gallbladder epithelium was observed to be destroyed by
either solvent, although it recovered rapidly (443). There was a report from France of MTBE escaping
into the blood stream and causing severe renal damage (444). In the United States, two deaths occurred
unrelated to usage of the solvent, but nonetheless in association with solvent usage. A company was
formed to bring the pump and solvent into clinical practice. However, venture capital was never obtained,
and the approach slowly passed into oblivion. A review of topical dissolution by our group was published
(445).
D. Concurrent advances in gallstone management
Two other developments were occurring during this time that doomed widespread usage of medical
dissolution. The first was the development of laparoscopic cholecystectomy, a major surgical advance.
Laparoscopic surgery was an import from France, used first in gynecological procedures. Using this
technique, a gallbladder could be removed quickly, without a linear scar, and the patient could leave the
hospital in a few days. The gallbladder with its gallstones had been removed, and the patient was cured.
In experienced hands, the technique was as safe as the traditional open cholecystectomy which had been
performed widely for the past half century. The composition of the stone(s) was unimportant, whereas
with oral bile acid therapy only gallstones that had cholesterol as the major constituent could be
successfully treated...
The second advance was ultrasound imaging of the gallbladder and its contents. All types of stones
were detected, and there was no radiation exposure. In contrast, oral cholecystography required the
ingestion of iodine-rich contrast agents and subsequent X-ray to detect the presence of gallstones.
Thus in twenty years, medical dissolution had been discovered, prospered, and then slowly
disappeared. In the long history of biliary disease, medical dissolution was a “bubble”. Once again
symptomatic cholelithiasis became a surgical disease. Nonetheless, medical dissolution was “proof of
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principle”, and medical therapy with UDCA can be used to prevent cholesterol gallstone formation.
Gallstone prophylaxis might be useful in some clinical situations, for example, in patients with recurrent
gallstone pancreatitis who did not wish to have surgery.
The availability of UDCA led to the accidental discovery of its utility in the treatment of primary
biliary cirrhosis and cholestasis of pregnancy, as noted in the introduction.
VII. Epilogue: omissions and apologies
Space limitations means that historical aspects of many facets of biology and chemistry have been
omitted. We have not discussed the history of inborn errors of bile acid synthesis and conjugation. Our
understanding of these genetic defects is quite recent, and an excellent review (446) by Clayton is
available. In the United States, Ken Setchell, a talented mass spectroscopist, has described several new
inborn errors of bile acid biosynthesis, as well as a defect in bile acid conjugation. Identification of the
exact enzyme defect requires mass spectrometry to detect the presence of bile acid precursors and gene
sequencing to identify the defect (156). The rewards of discovery are great for the patient, as bile acid
replacement may be life-saving. Some of the early history is given in J. Sjövall’s retrospective review
(10).
A second area not discussed is the utility of bile acids and bile alcohols to disclose evolutionary
relationships, an area pioneered by G.A.D. Haslewood (21). Often bile acids agree with the perceived
evolutionary relationships, but sometimes they do not. It would be of interest to explore these
nonconcordancies in detail.
A third area not discussed is the role of bile acids in colonic secretion (447,448) and absorption (449),
as well as recent work suggesting that bile acid activation of TGR5 is essential for normal defecation, at
least in the mouse (70) . Details of the biochemical mechanism(s) for bile acid-induced intestinal
secretion are being elucidated by several laboratories, among these being that of Stephen Keely, in
Dublin, (448) and of Meena Rao in Chicago (450). Bile acid malabsorption causes diarrhea, and this can
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be treated by sequestrant administration (451,452). A new syndrome of defective FGF19 release causing
an inappropriate increase in synthesis of bile acids has been identified by Julian Walter and his colleagues
(453), and is considered to be present in many patients with irritable bowel syndrome of the diarrhea type
(454), as well as in patients with so-called primary bile acid malabsorption. Plasma levels of “C4” (7α-
hydroxy-cholest-4-ene-3-one), an intermediate in C24 bile acid biosynthesis, have been shown to correlate
highly with the rate of bile acid synthesis (455,456). The necessity of measuring C4 (as a marker of
increased bile acid synthesis) and FGF15/19 levels (to detect impaired FGF15/19 secretion from the ileal
enterocyte) in every patient with unexplained diarrhea is slowly being recognized by gastroenterologists
(457). The group of Michael Camilleri at Mayo is exploring the relationship of fecal bile acid profile to
constipation as well as diarrheal conditions (458). Our laboratory identified some children with
functional constipation whose fecal bile acids were predominantly the 3 sulfate of CDCA (459). The
absence of C-12 hydroxylation in these children could indicate a deficiency in cyp8B1, the enzyme that
hydroxylates C4 at the C-12 position.
A fourth area not discussed is the cellular toxicity of bile acids. Mechanisms of toxicity are multiple,
involving activation of the death receptor (460), as well as induction of reactive oxygen species (461).
UDCA is cytoprotective and mechanisms responsible for this effect are being studied (462). A new aspect
of bile acid toxicity is the proinflammatory action of retained bile acids (463).
One last area not discussed is the possible influence of bile acids on the microbiome. The presence of
secondary bile acids in bile has long been a sign that metabolic activity of the microbiome influences bile
acid metabolism. Indeed, characterization of bile acid metabolism in the germ free animal (464) by
Gustafsson and his colleagues in Lund aided in the initial distinction of primary and secondary bile acids
by the laboratory of Sune Bergström (8). There is still much is to be learned about the effect of individual
bile acids on the microbiome (465).
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The last two generations have had powerful tools to elucidate bile acid metabolism. One of these was
the discovery of 14C – a discovery that almost occurred by chance (466). The availability of 14C in
chemical form permitted 14C labeled bile acids to be prepared and their metabolism to be defined. A
second essential tool has been chromatography – adsorption, reversed phase (HPLC), and gas/liquid. A
final tool is mass spectrometry which when coupled with gas chromatography or HPLC provides both
qualitative and quantitative information on new molecules (9). For the assignment of structure to newly
discovered bile acids, proton and 13C nuclear magnetic resonance measurements are mandatory. Of
course, in today’s research, generation of specific knockouts, characterized by multisystem phenotyping,
combined with PCR, have enormous power in unraveling the recently discovered signaling properties of
bile acids.
At present (2014), at least two types of drugs based on natural bile acids are being tested for their
utility in the treatment of liver disease. The first, obeticholic acid, the 6α-ethyl derivative of CDCA, is a
potent FXR agonist. Its efficacy in the treatment of primary biliary cirrhosis (467) and nonalcoholic
steatohepatitis (468) is being tested in clinical trials. NorUDCA, the C23 homologue of UDCA, is also in
clinical trials for the treatment of cholestatic liver disease. In the bile duct ligated mouse, its feeding
causes less damage than UDCA feeding (469).
Inhibitors of ASBT, the sodium dependent bile acid transporter present in the apical domain of the ileal
enterocyte, are once again being developed. They were originally aimed at providing adjuvant therapy for
hypercholesterolemic patients who responded inadequately to statins (470). Now the target is cholestatic
liver disease or its symptom of pruritus as well as type 2 diabetes (471) and constipation (472).
Colesevelam is an improved bile acid sequestrant that should have a similar pharmacodynamic activity as
that of the ASBT inhibitors; however, it is only approved for type II diabetes, and its use to treat bile acid
diarrhea is off label. Bile acid derivatives that activate solely TGR5 or both TGR5 and FXR have been
synthesized (55) and may eventually enter clinical trials.
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In our opinion, the remarkable progress in our understanding of bile acid biology and chemistry is a
tribute to the hundreds of scientists who have worked with such diligence and talent throughout the world.
We have mentioned only a few in our brief history, and we have surely omitted some key advances.
There are many workers whose contributions have not been recognized because of space limitations. As
we noted in the introduction, this is a personal history, and other writers are likely to stress different
aspects of the historical record. We have probably overemphasized the contributions of our own
laboratory because we know them best. For those readers who would like more historical detail on some
of the topics discussed above, we refer them to a series of beautifully written historical reviews by Adrian
Reuben published in the journal Hepatology between 2002 and 2006.
Let us hope that future research in the chemistry and biology of bile acids will be as exciting as that of
the last 80 years!
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Acknowledgments: We are grateful to many of our colleagues who helped us in this review. In
particular, we thank Takashi Iida, Bruno Stieger, and David Mangelsdorf, as well as the anonymous
reviewers. We also acknowledge a grant-in-aid from Intercept Pharma, Inc. In the past, our laboratory
has been supported by the National Institutes of Health as well as a grant-in-aid from the Falk Foundation,
e.V., Freiburg, Germany.
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466. Kamen, M.D. 1963. The early history of carbon-14. J. Chem. Educ. 40:234-42.
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LEGENDS TO FIGURES
Figure 1. Participants in the first symposium in the United States dealing with bile acid metabolism. The
meeting was organized by Leon Schiff, a clinical hepatologist and took place in September, 1967. First
row, left to right. M. Shiner, L. Schiff (organizer), G. Haslewood, M. Siperstein, S. Tabaqchali, D. Small.
Second row, left to right. L. Lack, L. Schoenfield, P. Nair, J. Carey, A. Hofmann. Third row, left to right.
M. Stanley, S. Hsia, unidentified, N. Javitt, H. Danielsson, J. Dietschy, S. Emerman. Fourth row, left to
right. F. Kern, J. Sjövall, B. Drasar, R. Palmer, J. Senior, N. Myant, J. Wilson, E. Staple. Proceedings of
the meeting were published in 1969 (480).
Figure 2. Numbering system of bile acids and bile alcohols, and frontal view of CDCA. The numbering
system was developed in the 1930’s when the steroid structure was finally established.
Figure 3. Molecular structure of cholesterol (above) and CDCA (below) showing the changes in the
cholesterol molecule when a C24 bile acid is formed. The changes are (1) reduction of the double bond to
give a 5β (A/B cis) A/B ring juncture (2) a modified β- oxidation of the side chain to remove three carbon
atoms and convert the terminal carbon to a carboxyl group; and 3) epimerization of the 3β- hydroxy group
to a 3α- hydroxy group. For a discussion of the enzymes involved, see reference 155.
Figure 4. Depictions of the two epimers of decalin, a saturated C10 hydrocarbon that is used to illustrate
the two A/B ring juncture epimers of bile acids. When the two bridgehead hydrogen atoms are across
from each other, the juncture is called trans; when they are on the same side of the ring juncture, the
juncture is called cis. When bile acid structure is shown in frontal view (Figure 2), the only indication of
the stereochemistry of the A/B ring juncture is the orientation of the hydrogen atom, i.e. whether alpha or
beta. However, in bile acids, the substituent at the upper bridgehead carbon atom is the C-19 methyl
group. Often bile acid structure is shown without any indication as to the orientation of the hydrogen
atom at C-5. When this is done, it is understood that the molecule is A/B cis, as 5β- (A/B cis) bile acids
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are much more common in nature than 5α (A/B trans) bile acids. A/B trans bile acids are termed “allo”
bile acids.
Figure 5. Silhouette of a C24 conjugated bile acid molecule emphasizing its possession of a hydrophobic
side (beta face) and a hydrophilic side (alpha face). Common sites of hydroxylation in addition to the
default structure (3α, 7α) are at C-6, C-12, and C-16; all of these are on the hydrophilic side of the
molecule. Modified from Roda et al (360).
Figure 6. Time course in healthy subjects of levels in peripheral venous plasma of immunoreactive
conjugates of cholic acid (cholyl conjugates) and those of CDCA (here termed “chenyl” conjugates) in
response to meals; meals are indicated by the thick, vertical arrows. Levels of CDCA conjugates rise
sooner than those of cholyl conjugates indicating more proximal absorption. Levels of CDCA conjugates
are also higher than those of cholyl conjugates despite similar proportions in bile because of lower
fractional extraction by the liver. From reference 216.
Figure 7. Schematic depiction of the work of L. Lack and I. Weiner on conjugated bile acid (here,
taurocholate) absorption by everted sacs of rat intestine, showing preferential and active absorption by the
distal ileum. At time zero, bile acid concentrations are identical on both sides of the intestine. Modified
from reference 229.
Figure 8. Depiction of cholesterol and bile acid metabolism as well as the enterohepatic circulation by
Bergström, Danielsson, and Samuelsson in 1959 (8). In the accompanying text, they note that the mass of
cholesterol entering the small intestine in bile is greater than that of dietary cholesterol. The figure shows
neither the spillover of absorbed bile acids into the systemic circulation nor selective absorption of
conjugated bile acids by the terminal ileum. Values for biliary bile acid secretion and fecal bile acid
excretion are about two fold higher than those obtained in more recent studies.
Figure 9. Depiction of the use of triangular coordinates to plot biliary lipid composition and thereby to
define the saturation of cholesterol in bile. The triangle is derived from a plane parallel to the base
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indicating a constant water composition (10% solids, 90% water) in the tetrahedron consisting of the four
biliary components (water, lecithin, cholesterol, and bile acids). The micellar zone is shown in black.
Bile having a lipid composition falling within in the micellar zone is unsaturated. If the composition is
above the upper limit of the micellar zone, the sample is supersaturated in cholesterol and at risk for
cholesterol gallstone formation (376). If biliary lipids are known, the per cent saturation can also be
obtained from tables published by Carey (393,394).
Figure 10. Molecular arrangement of the drum shaped mixed micelle micelle originally proposed by
Small (369) and modified by Carey (330), as compared to the radial shell model proposed by Nichols and
Ozarowski (371), The radial shell model was confirmed by Hjelm et al using small angle neutron
scattering (372,403,404) and also by molecular dynamic studies of Marrink and Mark (373).
Figure 11. Phase diagram of the water-monoolein (monooleylglycerol)-taurocholate system, showing the
large micellar area (cross-hatched), thus depicting the excellent solubilizing potency of bile acids for
polar lipids. Liquid crystal states are indicated by vertical and horizontal cross hatching. Modified from
Svärd et al. (398).
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Table. Common and uncommon sites of additional nuclear hydroxylation (in addition to the default
structure of the 3α,7α-dihydroxy compound) in primary C24 bile acids, C27 bile acids, and C27 bile alcohols
+ Hydroxylation occurs at C-5 with 24-nor (C23) UDCA in hamsters (481).
*The bile acid described is a tetrahydroxy bile acid (3α,7α,12α,19-tetrahydroxy-)
Class Common Uncommon+
Site Natural occurrence References
C24 bile acids 6α,6β,12α,16α 1α Australian opossum 474 1β Pigeons, neonates 475, 476 2β Neonates 477 4β Chinese pheasant 473 9α Bear 478 11α Sunfish 472 15α Swan, goose 478,479 19* Neonates 480 C27 bile acids 12α 1α Tinamou 125 16α Ancient birds 473 C27 bile alcohols 12α 2α Arapaima 126 6α and 6β Elephants, hyrax, manatee 127 11α Sunfish 473 by guest, on F
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