thematic review
Thematic Review Series: Living History of Lipids
Key discoveries in bile acid chemistry and biology and
their clinical applications: history of the last
eight decades
Alan F. Hofmann1 and Lee R. Hagey1
Department of Medicine, University of California, San Diego, San Diego, CA
Abstract During the last 80 years there have been extraor-
dinary advances in our knowledge of the chemistry and biol-
ogy of bile acids. We present here a brief history of the
major achievements as we perceive them. Bernal, a physi-
cist, 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 conju-
gation are summarized. Bile acid measurement encom-
passes the techniques of GC, HPLC, and MS, as well as
enzymatic, bioluminescent, and competitive binding meth-
ods. 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 ac-
ids 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.—Hofmann, A. F., and L.
R. Hagey. Key discoveries in bile acid chemistry and biology
and their clinical applications: history of the last eight de-
cades. J. Lipid Res. 2014. 55: 1553–1595.
Supplementary key words bile acid transport • enterohepatic circula-
tion • bile acid physical chemistry • bile acid analysis • bile acid
metabolism
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 provoca-
tive judgments. Let us hope the omissions are minor, mis-
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, Ger-
many. The authors acknowledge a grant-in-aid from Intercept Pharma, Inc.
Manuscript received 24 March 2014 and in revised form 2 May 2014.
Published, JLR Papers in Press, May 17, 2014
DOI 10.1194/jlr.R049437
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
conceptions 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 ne-
cessity in many instances, consideration of only what we
perceive to be the highlights.
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 pla-
cebo-controlled studies showing efficacy. Indeed, bile ac-
ids 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 in-
duce bile flow in animals (2), and was occasionally used to
stimulate bile flow in patients; but again there were no con-
trolled studies showing efficacy in hepatobiliary disease.
Abbreviations: ASBT, apical sodium-codependent bile acid trans-
porter; 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; FDA, Food and Drug Administration; FGF,
fibroblast growth factor; FXR, farnesoid X receptor; IUPAC, Interna-
tional Union of Pure and Applied Chemistry; MDR, multidrug resis-
tance (protein); MRP, multidrug resistant protein; MTBE, methyl
tert-butyl ether; NCGS, National Cooperative Gallstone Study; NIH,
National Institutes of Health; NTCP, Na+ taurocholate cotransporting
polypeptide; OATP, organic anion transporting polypeptide; OST, or-
ganic solute transporter; PC, phosphatidylcholine; TGR5, transmem-
brane G protein-coupled receptor 5; UDCA, ursodeoxycholic acid.
1
To whom correspondence should be addressed.
e-mail: AHofmann@ucsd.edu (A.F.H.); LHagey@ucsd.edu
(L.R.H.)
Journal of Lipid Research Volume 55, 2014 1553
 Albert Boehringer founded a pharmaceutical company
in Ingelheim, Germany. His wife was a cousin of Heinrich
Wieland, then a professor in Munich. Wieland was success-
ful 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 doc-
toral studies. When they returned to Japan, they contin-
ued their studies on bile acid and bile alcohol structure.
Their work, as well as that occurring in Europe is summa-
rized in the book Ueber die Chemie and Physiologie der Gallen-
säuren authored by Shimizu in 1935 (3).
During the 1930s and 1940s, an enormous international
effort went into defining the structure of the major hor-
monal steroids. When it was recognized that cortisone had
a C-11 oxygen atom, it was logical to use deoxycholic acid
(DCA) (possessing a C-12 hydroxyl group) as a chemical
precursor for the synthesis of corticosteroids. DCA was eas-
ily 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 com-
mercial production on a small scale. Two 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 strik-
ing symptomatic improvement in patients with rheuma-
toid 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 enor-
mous 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 fun-
gal 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 me-
tabolism 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 liq-
uid 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 oth-
ers, who carried out fundamental studies of bile acid me-
tabolism in a variety of species including man (8). (It is
1554 Journal of Lipid Research Volume 55, 2014
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 GC and then later, after he had moved
to the Karolinska Institute in Stockholm, developed MS
for the measurement of bile acids (9). He used these tech-
niques and others to define important aspects of bile acid
metabolism over the next four decades (10). Norman de-
veloped a simple synthesis of conjugated bile acids, pre-
pared the conjugates of all the major bile acids known at
that time (11), and with Sjövall performed metabolic stud-
ies in animals which distinguished primary bile acids
(made in the liver from cholesterol) from secondary bile
acids (made from primary bile acids by intestinal bacte-
ria). 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 prostaglan-
dins. Their outstanding work in this field led to their re-
ceiving the Nobel Prize in 1982. However, at the Karolinska
Hospital, Swedish scientists continued to work on bile ac-
ids, 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. Borg-
ström directed the senior author, to perform studies show-
ing that in man, conjugated bile acids are absorbed mainly
in the ileum (20).
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 relation-
ships (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 identi-
fied the ␣-, ␤-, and ␻-muricholic acids (22). His student,
William Elliott synthesized numerous bile acids and de-
scribed 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 af-
ter 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 biosyn-
thesis from cholesterol (27).
In the United States, James Carey, working at the Uni-
versity of Minnesota, was directed by his mentor, Cecil
Watson, to pursue bile acid metabolism in man. He identi-
fied chenodeoxycholic acid (CDCA) as a major biliary bile
acid (28) and proposed that lithocholic acid, its bacte-
rial metabolite, caused liver injury in man (29). Edward
(“Pete”) Ahrens, working with Lyman Craig at the Rocke-
feller 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 chromato-
graphic 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 bal-
ance studies for much of his very productive career (33).
The first symposium devoted solely to bile acids was or-
ganized 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 Fig. 1.
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”.
Fig. 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).
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 measure-
ment of fecal bile acids and sterols, using the newly devel-
oped 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 g, and the synthesis from cholic acid was difficult.
However, in the 1960s, a small English pharmaceutical
company (Weddell Pharmaceuticals) began the manufac-
ture of CDCA for unknown reasons. A kilogram was pur-
chased 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 de-
creased biliary cholesterol saturation, whereas neither
cholic acid nor hyodeoxycholic acid had any effect (35).
In 1972, the first gallstone dissolution induced by the in-
gestion 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 arthri-
tis (38).]
The discovery that CDCA would induce gradual dissolu-
tion 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.
In Japan, UDCA had been brought to market in the
1950s by Tokyo Tanabe as a liver tonic, based on the leg-
endary 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 hepatotox-
icity. 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 1990s be-
cause of the development of laparoscopic cholecystectomy,
which promised a rapid cure for the problems of gallstone
disease, irrespective of gallstone type. Once again, bile
History of bile acid research 1555
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 hepatolo-
gist based in Frankfurt, Germany, noted that UDCA in-
gestion improved biochemical parameters in patients with
primary biliary cirrhosis (42). UDCA was subsequently
shown to slow disease progression in primary biliary cir-
rhosis, 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 stan-
dard of care. UDCA was also shown to be effective in cho-
lestasis of pregnancy (45). UDCA has been widely used
for other cholestatic diseases and to restore bile flow fol-
lowing liver transplantation, but proof of efficacy in these
conditions based on placebo-controlled trials has been
uncommon.
At present, it is estimated that about 1,000 metric tons,
or about 1,000,000 kg, of cholic acid are produced globally
from bovine bile, the majority of which is used for the pro-
duction of UDCA (S. Yorke, New Zealand Pharmaceuti-
cals, Ltd.; personal observation). Bile acids are also used as
precursors for the synthesis of nuclear receptor agonists
such as obeticholic acid (the 6␣-ethyl derivative of CDCA),
as building blocks for other molecules (46), and as key
constituents of some bacterial media. Bile acids have even
been used in the electronics industry as precursors of pho-
toresist molecules in integrated circuits (47). (This un-
likely technology transfer from a biological laboratory to
the electronic world resulted from one of the early chem-
ists 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 cho-
lesterol 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 hypercholesterol-
emia with a bile acid sequestrant had a rather modest ef-
fect 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 nu-
clear receptor farnesoid X receptor (FXR) (49–51), mean-
ing that bile acids regulate the activity of many genes
(52–54). Bile acid derivatives that are potent FXR agonists
have been shown to induce the synthesis and basolateral
release of the protein fibroblast growth factor (FGF)15/19
from the ileal enterocyte (55). FGF19 travels in portal ve-
nous blood to the liver and downregulates bile acid biosyn-
thesis (55, 56). FGF19 promotes hepatocyte growth (57),
and FXR agonism also has antidiabetic effects mediated by
multiple mechanisms (58), as well as anti-inflammatory
1556 Journal of Lipid Research Volume 55, 2014
activities (59). Several potent FXR agonists have been syn-
thesized (53, 60), and one of these, obeticholic acid (61),
is now being tested for its efficacy in primary biliary cirrho-
sis and nonalcoholic steatohepatitis (62).
Two Japanese groups discovered transmembrane G-
coupled protein receptor 5 (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 an-
tidiabetic effects (68). TGR5 also has effects on hepatic
blood flow (69), distal intestinal motility (70), and in addi-
tion, has anti-inflammatory effects (71) that are under ac-
tive investigation.
Discovery of the signaling properties of bile acids is cer-
tainly a new paradigm in the bile acid field. Bile acids have
become hormones. Now highly talented molecular biolo-
gists 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 Euro-
pean 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 proceed-
ings 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 mono-
graph summarizes current knowledge in 1966 (74) and
Haslewood later authored a monograph on the biologi-
cal importance of bile salts (75). Elsevier’s 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 Ste-
rols and Bile Acids is volume 12 of the series, New Compre-
hensive Biochemistry. This volume, published in 1985, has
six chapters dealing with varying aspects of bile acids
and bile alcohols (78). A multiauthor book describing
many aspects of bile acid research with particular em-
phasis 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 pub-
lished 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 se-
lected topics in more detail with an emphasis on struc-
ture-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.
BILE ACID STRUCTURE: HISTORY AND CURRENT
CONCEPTS
The carbon skeleton
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 fol-
lowing 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 labo-
rious attempts to define the structure of bile acids and pre-
sented his current vision of the structure of cholic acid
(83). Windaus presented his proposed structure of choles-
terol 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 struc-
ture 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 struc-
tures as “probable”.
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 stud-
ied 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 Rosen-
heim and King in London, and Wieland and Dane in Mu-
nich. Later, in that same year, Wieland and Dane followed
by Rosenheim and King published the application of Ber-
nal’s pioneering studies to the extensive literature on bile
acid structure (86). Each group proposed the cyclopen-
tanoperhydrophenanthrene structure for the steroid nu-
cleus. 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 1930s and has
been used ever since.
Bernal’s X-ray diffraction studies were the first bio-
logical 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
Fig. 2. Numbering system of bile acids and bile alcohols, and
frontal view of CDCA. The numbering system was developed in the
1930s when the steroid structure was finally established.
being the helical structure of DNA and RNA by Watson
and Crick. Bernal’s left wing political views and his Bohe-
mian life style probably kept him from being awarded a
Nobel Prize (89, 90).
The side chain. The structure of the C5 side chain of the
common natural C24 bile acids, isopentanoic acid or more
specifically ␥-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 ini-
tially that these molecules possessed the side chain of cho-
lesterol because it was quite uncertain whether these
compounds and even the common natural C24 bile acids
were derived from 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 Phys-
iologie 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 te-
traoxybufostan [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 Ru-
dolph Schoenheimer (93). Bloch, Berg, and Rittenberg
History of bile acid research 1557
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 struc-
ture, 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 synthe-
sis, his Nobel lecture does not even mention this key
experiment.)
The first bile acid with a longer side chain than the com-
mon natural C24 bile acids was isolated from the bile of the
toad Bufo vulgaris formosus by Shimizu and Oda (95) in
1934. The structure of this bile acid was established by
Takahiko Hoshita 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 verte-
brates. In particular, bile from the alligator, which was
readily available in large amounts (studies at that time be-
gan with up to 5 l 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 (98) synthesized a defined side chain starting
from the steroid nucleus. They then showed that their prod-
uct was identical to 3␣,7␣,12␣-trihydroxy-5␤-cholestan-27-
oic acid, the major bile acid isolated from the bile
of crocodiles and alligators, and they confirmed its struc-
ture by X-ray diffraction. Subsequently, the laboratory of
Fig. 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 dou-
ble bond to give a 5␤ (A/B cis) A/B ring juncture; 2) a modified
␤-oxidation of the side chain to remove three carbon atoms and
convert the terminal carbon to a carboxyl group; and 3) epimeriza-
tion of the 3␤-hydroxy group to a 3␣-hydroxy group. For a discus-
sion of the enzymes involved, see (155).
1558 Journal of Lipid Research Volume 55, 2014
Takashi Iida reported the synthesis of the 25R and 25S ste-
reoisomers of this compound (99).
The first C27 bile alcohol to be isolated was scymnol, ob-
tained from the bile of the Greenland shark (Scymnus bo-
realis) 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 combus-
tion 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 bull-
frog (Rana catesbeiana) by Kazuno, Masui, and Okuda
(101). Subsequently, Haslewood (102) found the same
bile alcohol in the European common frog (Rana tempo-
ria), 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 (␣,␧-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).
The A/B ring junction
In most natural bile acids, whether C24 or C27, the junc-
tion of the A and B rings is cis. This steric conformation is
indicated by depicting the bond connecting the C-5 hydro-
gen atom to the A/B ring 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 nonste-
roid 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 mark-
edly 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 Fig. 4. Every bile acid and bile alco-
hol has, in principle, a 5␣ and a 5␤ 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 Fie-
ser’s comprehensive text on steroids (7). Probably, it was
Windaus who introduced the prefix allo to denote 5␣ (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 5␣ bile acids from
5␤ 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 propor-
tions in the bile of many species (82). If cholestanol (the
saturated 5␣-derivative of cholesterol) is administered to
rabbits, it is converted to 5␣ bile acids (107). Allocholic
acid, the biological precursor of allodeoxycholic acid, has

Fig. 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 (Fig. 2), the only
indication of the stereochemistry of the A/B ring juncture is the
orientation of the hydrogen atom, i.e., whether ␣ or ␤. 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
5␤ (A/B cis) bile acids are much more common in nature than 5␣
(A/B trans) bile acids. A/B trans bile acids are termed “allo” bile
acids.
been prepared synthetically and some its biological prop-
erties explored (108). Allo bile acids have also been identi-
fied in fecal bile acids (109), presumably generated by
bacterial enzymes acting on the 3-oxo-⌬ intermediate
4,6
that is formed during the process of C-7 dehydroxylation
(110). Enantiomeric bile acids, in which the steric configu-
ration of all ring junctions is reversed, have recently been
synthesized (111).
Bile alcohols (C27) with a 5␣ configuration are not un-
common in nature. The simplest biotransformation of
cholesterol (⌬ ) into a bile alcohol results in a 5␣ nucleus,
5
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 5␣ and 5␤ epimers.
Within these three great classes, and for a given A/B ring
junction, the pattern of hydroxylation, on the steroid nu-
cleus and side chain or both, defines each individual bile
alcohol or bile acid.
Hydroxylation sites
The steroid nucleus. C24 PRIMARY BILE ACIDS. Bile acid no-
menclature, 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 cor-
rect 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, giv-
ing rise to the polysyllabic name of chenodeoxycholic
(acid). Other trihydroxy bile acids were named for their
animal source: muricholic acid (from rodents) and hyo-
cholic acid (from pigs).
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 7␣-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 7␤-hydroxy epimer of CDCA, is a pri-
mary bile acid. The route of formation of this 7␤-hydroxy
epimer of CDCA is unknown. One can speculate on the
existence of a cholesterol 7␤-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 ver-
tebrate species) of additional hydroxylation of CDCA is at
C-12 in cholic acid. The second most common site of hy-
droxylation is probably at C-16, because so many avian spe-
cies have the 3␣,7␣,16␣-trihydroxy bile acid as a major
biliary bile acid (82). Another common site of hydroxyl-
ation 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 (␣ or ␤), C-2 (␤), C-4 (␤), C-5
(␤), C-9 (␤), C-11 (␣), C-15 (␣), and C-19 (␤). Table 1
summarizes the species in which these rare biliary bile ac-
ids occur, and the appropriate citation.
C24 bile acids with four hydroxyl groups on the steroid
nucleus occur rarely. Some snakes have 3␣,7␣,12␣,16␣-
tetrahydroxy bile acids (82). Tetrahydroxy bile acids of
unknown structure are found in trace proportions in the
bile of many vertebrate species. After experimental liga-
tion of the common bile duct, tetrahydroxy and even pen-
tahydroxy 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 (BSEP) has been deleted
genetically (116).
History of bile acid research 1559
 TABLE 1. Common and uncommon sites of additional nuclear hydroxylation (in addition to the default
structure of the 3␣,7␣-dihydroxy compound) in primary C24 bile acids, C27 bile acids, and C27 bile alcohols
Class Common Site
C24 bile acids 6␣,6␤,12␣,16␣ 1␣
1␤
2␤
4␤
9␣
11␣
15␣
b
19
C27 bile acids 12␣ 1␣
16␣
C27 bile alcohols 12␣ 2␣
6␣ and 6␤
11␣
a
Hydroxylation occurs at C-5 with 24-nor (C23) UDCA in hamsters (481).
The bile acid described is a tetrahydroxy bile acid (3␣,7␣,12␣,19-tetrahydroxy).
b
An ad hoc committee of bile acid workers met in
Freiburg in 1980 under the auspices of the Falk Founda-
tion. The group proposed working rules for bile acid no-
menclature and bile acid abbreviations 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, ex-
cept for conversational purposes (117).
C24 SECONDARY BILE ACIDS. As noted before, bile acids are
modified by bacterial enzymes in the distal intestine. Mod-
ification 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 hy-
droxy-oxo bile acids, can be both primary and secondary.
A consideration of secondary bile acids is important be-
cause they can be major biliary bile acids. After their for-
mation in the distal intestine, they are absorbed in part.
They return to the liver in portal venous blood, and tra-
verse 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 enterohe-
patic circulation.
The major bacterial biotransformations of conjugated
bile acids are deconjugation, 7-dehydroxylation, oxido-
reduction, epimerization, and side chain desaturation [de-
tailed 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 medi-
ated by many bacterial species and does not require anaero-
bic 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-⌬4,6 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 ␻-
muricholic acid is termed hyodeoxycholic acid and that
of ␣- and ␤-muricholic acid is termed murideoxycholic
acid. To date, the C-7 deoxy derivative of “avicholic” (3␣,7␣,
1560 Journal of Lipid Research Volume 55, 2014
Uncommona
Natural occurrence References
Australian opossum 474
Pigeons, neonates 475, 476
Neonates 477
Chinese pheasant 473
Bear 478
Sunfish 472
Swan, goose 478, 479
Neonates 480
Tinamou 125
Ancient birds 473
Arapaima 126
Elephants, hyrax, manatee 127
Sunfish 473
16␣-trihydroxy) has not been identified, but bacterial
16-dehydroxylation has been reported for some steroids
(118), and is likely to occur in some birds. Such 16␣-
dehydroxylation would result in the formation of CDCA,
that in turn would likely undergo 7␣-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. 3␤-Hydroxy bile acids (“iso” bile acids) are major
fecal bile acids in man (119). 7␤-Hydroxy bile acids and
even 12␤-hydroxy bile acids are also formed, but to a far
lesser extent. Elimination of the 3-hydroxy group with for-
mation of a ⌬2 or ⌬3 bile acid may also occur (120).
Secondary bile acids may undergo additional hydroxyl-
ation 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, second-
ary bile acids may undergo hydroxylation at a new site,
generating a novel “tertiary” bile acid. One example is
16␣-hydroxylation of DCA to form “pythocholic” acid
(3␣,12␣,16␣-trihydroxy) that occurs in the python (75). In
the wombat, lithocholic acid undergoes hydroxylation at
C-15 (122). Iso (3␤-hydroxy) bile acids are epimerized to
3␣-hydroxy bile acids during transport through the hepa-
tocyte (123, 124). Biotransformation of the 3␤-epimers of
the muricholic and hyocholic acid families has not been re-
ported, but is likely to occur. In man, the 7␤-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 3␣,12␣,16␣-trihdroxy bile acid, which can be
formed by 16␣-hydroxylation of DCA, exceeds 50% (82).
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, hydroxyl-
ation at C-16 occurs (82). We have identified a 1␤,3␣,7␣-
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 2␤-hydroxy group has
been isolated from the bile of the large freshwater fish
Arapaima gigas (126). It seems likely that there are addi-
tional 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.
C27 BILE ALCOHOLS. In C27 bile alcohols, default nuclear
hydroxylation occurs at C-3 and C-7; additional hydroxyl-
ation occurs at C-12, and rarely at C-6 or C-16 (25, 26,
127). The C-3 hydroxyl group has a ␤-configuration in lat-
imerol 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 ␤-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. How-
ever, the presence of 7-deoxyscymnol in the megamouth
shark, as reported by Ishida et al. (128), suggests that bac-
terial dehydroxylation at C-7 may also occur for C27 bile
alcohols.
The side chain. C24 BILE ACIDS. With only four available
carbons in the side chain, C24 bile acids are likely to un-
dergo side chain hydroxylation only at C-23 or C-22, each
of which has potential R and S epimers. The most com-
mon 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 phocaecho-
lic 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 ad-
dition 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 3␣,7␣-dihydroxy bile acid with a hydroxy group at
C-22 was shown to occur in the fish Parapristipoma trilinea-
tum by the Hoshita laboratory (133) and given the awk-
ward 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 biologi-
cal 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 ⌬22 bile acids is a major
pathway in rats (136, 137) and cavimorphs (82). Such bile
acids could arise from further ␤-oxidation of the side
chain in the liver and/or by bacterial action in the distal
intestine.
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. Unfortu-
nately, 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.)
So far, all reported C27 bile acids have only a single hy-
droxyl 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 cho-
lestanoic acid epimers with a hydroxyl group at C-24 have
each been synthesized by Une et al. (138). The most com-
mon 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).
C27 BILE ALCOHOLS. As noted earlier, Hammarsten deter-
mined that he had isolated a C27 bile alcohol from the bile
of the Greenland shark (100). The challenge of determin-
ing the structure of this compound would occupy chemists
for many decades. Windaus, Bergmann, and König (140)
initially took up the problem of determining the structure
of Hammarsten’s compound (named scymnol) and pro-
posed 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 3␣, 7␣, and 12␣), as scymnol could
be converted into cholic acid by partial oxidation of the
side chain (141, 142). Because the side chain of choles-
terol has a C-27 methyl group, different paired combina-
tions 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 Ham-
marsten’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 struc-
ture of both scymnol and anhydroscymnol, the epoxide
formed during alkaline hydrolysis (145). He found that
the side chain hydroxyl group of scymnol was unexpect-
edly on C-26 (the only carbon with a single proton), con-
sistent 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
3␣,7␣,12␣,24,26,27-hexahydroxy-5␤-cholestane by com-
parison with a synthetic standard (143). C-24 is chiral, and
the configuration of the 24-hydroxyl group was deter-
mined to be 24R in the crystallographic study of Ishida
History of bile acid research 1561
et al. (146). This 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 re-
view (82). Additional information is available in the co-
gent review of Une and Hoshita (26).
Bile alcohols, just as bile acids, have been named for the spe-
cies in which they occur, and the name gives no clue what-
soever to the structure of the bile alcohol. It is convenient
to think of the trihydroxy bile alcohol with ␣-hydroxyl
groups at C-3 and 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).
Conjugation
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, am-
phibians, reptiles, and most avian species. It also occurs to
a variable extent in mammals (82). In some angelfish spe-
cies, 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 cysteino-
lic acid, a hydroxy-ethyl derivative of taurine that is of di-
etary origin (150). Glycine conjugation occurs in a few
avian species, some bovids, cavimorphs, and primates, in-
cluding humans (82). Sarcosine (N-methylglycine) conju-
gates have been synthesized and shown to be more
resistant to bacterial deconjugation during enterohepatic
cycling (151–154). Some workers have considered bile
acid amidation to be the final step in bile acid biosynthe-
sis (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 bio-
transformation 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 cholangio-
cytes. The conjugation process begins with the formation
of a bile acid adenylate (157) followed by formation of the
CoA thioester. 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). Conjuga-
tion 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 biologi-
cal properties of glycine- and taurine-conjugated bile ac-
ids, except that taurine conjugates are deconjugated by
bacteria more slowly than glycine conjugates (161, 162).
1562 Journal of Lipid Research Volume 55, 2014
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 mis-
leading name, in that “taurocholate” implies that the cho-
lic 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 ex-
tremely low proportion of unconjugated bile acids in biliary
bile acids has led some investigators to conclude that the
conjugation process is highly efficient. However, unconju-
gated 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.
Other types of bile acid conjugation. When the term “con-
jugated” was applied to bile acids, it denoted N-acyl amida-
tion with taurine or glycine. When bile acid sulfation and
glucuronidation were discovered to be important path-
ways under some circumstances, it was necessary to distin-
guish different modes of “conjugation” for bile acids. In
principle, there are six modes of bile acid conjugation: N-
acyl amidation (using glycine, taurine, or 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 sulfa-
tion (mostly at C-3) followed by urinary excretion becomes
a major pathway (167, 168). In the bile duct-ligated ham-
ster, 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 (3␣,6␣-
dihydroxy). Because hyodeoxycholic acid should be ab-
sorbed efficiently from the small intestine by passive
mechanisms, its absence in biliary bile acids was com-
pletely mystifying. In 1978, B. Almè and J. Sjövall reported
a patient with malabsorption who excreted 6-hydroxy bile
acids in urine that were present as O-linked (ethereal)
glucuronides (170). Five years later, Sacquet et al., work-
ing in the laboratory of Reca Infante in Paris, reported
that when hyodeoxycholic acid was administered to hu-
man subjects, it was largely excreted in urine as its glucuro-
nide (171), explaining its absence in biliary bile acids. This
group did not know the site of glucuronidation but pro-
posed 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 pre-
sumably results from defective N-acylamidation, and hyo-
deoxycholic 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 glucuroni-
dated (173).
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 effi-
cient for ␣-hydroxy bile acids (131, 132) and hydrophilic
epimers of cholic acid, for example 3␣,7␤,12␤-trihydroxy-
cholanoate (176).
In patients ingesting UDCA, UDCA also undergoes con-
jugation with N-acetylglucosamine at C-7, in addition to
amidation with glycine or taurine at C-24 (177). In Nie-
mann-Pick disease type C, a novel ⌬5 3␤,7␤-dihydroxy cho-
lestanoic acid is formed (178); this compound may be
considered an acidic 7␤-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 mul-
tiply-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
thioesters) have been identified in rat and human infant
bile by the group of Shigeo Ikegawa, but their concentra-
tion 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 conju-
gated exclusively with taurine. Bile alcohols are esterified
with sulfate, and thus have a pKa similar to that of taurine
conjugates.
Structure-function relationships
In the steady state adult animal, cholesterol input, from
de novo synthesis and dietary cholesterol, must be bal-
anced by fecal excretion as cholesterol and its bacterial
metabolites (neutral steroids) and bile acids (acidic ste-
roids). There is thus a mole fraction consisting of total bile
acids (numerator) divided by total cholesterol secretion
(acidic plus neutral steroids) (denominator). In man, based
on balance studies, this figure is 0.46; in the mouse it is
0.45, whereas in the 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), suggest 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 choles-
terol elimination, there are only a few molecular require-
ments. 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 gly-
cine 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 ami-
dates 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 carboxypepti-
dases (185).
In vertebrates with a cecum, deconjugation and dehy-
droxylation 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 circu-
lation. Isao Makino, who had worked in the Sjövall labora-
tory in Stockholm and returned to Japan, was among the
first to report the presence in healthy individuals of un-
conjugated bile acids in plasma using GC (186). Some de-
cades later, this early finding was confirmed by Kenneth
Setchell et al., using GC-MS (187). Nonetheless, absorp-
tion 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 pro-
cess of defecation. As there is negligible excretion of bile
acids in urine, breath, and skin, and as the bile acid nu-
cleus 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 bacte-
ria. 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
14
C in the steroid nucleus are administered and 14CO2 in
breath is collected.
History of bile acid research 1563
 There is very little information on the fate of bile alco-
hols in the distal intestine. In the Asiatic carp we found
unchanged 5␣-cyprinol sulfate in the distal intestine (190).
Even if there were hydrolysis of the ester sulfate, the liber-
ated bile alcohol is likely to have negligible passive mem-
brane 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 nonpolar side. So far as is known, all major nat-
ural biliary bile acids and bile alcohols are amphipathic,
although the hydrophobic side (␤ 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 mol-
ecule unrelated to the bile acid molecule which has the
same physicochemical properties of bile acids as regards
solubilizing membrane lipids.
MEASUREMENT
Isolation and chromatographic separation
Elucidation of the metabolism of bile acids in health
and disease requires accurate, reliable, and sensitive meth-
ods 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 co-
gent review by W. Griffiths and J. Sjövall on current analyti-
cal 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 de-
velopment of improved chromatographic methods for
separation. Paper chromatography based on liquid-liquid
partition was developed in the 1940s by the great English
chemists A. J. P. Martin and R. L. Synge, who shared the
Nobel Prize in 1952. In the 1950s, it was first applied to
Fig. 5. Silhouette of a C24 conjugated bile acid molecule empha-
sizing its possession of a hydrophobic side (␤ face) and a hydro-
philic side (␣ face). Common sites of hydroxylation in addition to
the default structure (3␣, 7␣) 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).
1564 Journal of Lipid Research Volume 55, 2014
bile acids by Jan Sjövall to separate individual unconju-
gated bile acids (191). Sjövall and Haslewood then collab-
orated 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
1960s (193, 194), and Eneroth and Sjövall tabulated reten-
tion times for different stationery phases in 1971 (195). A
decade later, capillary GC was described, greatly increas-
ing resolving power. GC using flame ionization detectors
affords both separation and quantification. However, only
when a peak is further analyzed by MS can one be certain
that the peak is truly a bile acid.
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 solvent
systems does not occur. Moreover, quantification of indi-
vidual spots was difficult. Nonetheless, TLC is a highly use-
ful quasi-quantitative method for assessing the 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 neu-
tral 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 unsatu-
rated 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.
Reverse-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 re-
view in 1971 (195). Some of their values have been shown
as actual simulated chromatograms in a chapter by the se-
nior author (202).
In our laboratory, we have found TLC to be of great
value for quantifying hepatic biotransformation of radio-
active bile acids. Small (1 mm) bands of adsorbent can be
directed into scintillation counter vials, using the auto-
matic device described by Snyder and Kimble (203), or
manually, by transferring the adsorbent to a vial using a
brush.
 HPLC was developed in the 1970s and was soon applied
to bile acids. Today, the technique, when used for bile acid
analysis, is usually executed in the reverse-phase mode,
i.e., with C18 or C8 alkyl-bonded silicic acid as the lipophilic
solid phase. Numerous laboratories have reported the
power of this analytical method for separating and mea-
suring 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,
nonamidated 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 vol-
umes of serum are used (204). One solution to the nonde-
tection of unconjugated bile acids and bile alcohol sulfates
is to use a light scattering detector that responds approxi-
mately 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 MS.
MS
MS had been developed in the 1930s and was used to
analyze mixtures of small molecules. MS of large organic
molecules emerging from the gas chromatograph was de-
veloped 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 learn-
ing about the GC-MS which Ragner Ryhage had built, and
was then produced by LKB. Our LKB 9000 is still operat-
ing, beginning its 23rd year.” Since then, there have been
continuous improvements in techniques for sample vola-
tilization 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 GC has greater
resolving power than HPLC.
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 sepa-
ration 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 com-
plex mixtures of bile acids. For unknown bile acids,
structure assignment requires NMR for elucidation of
structure, and ultimately synthesis for confirmation of as-
signed structure.
Enzymatic and competitive binding assays
Development of an enzymatic technique for determin-
ing total 3␣-hydroxy bile acids based on oxidation of the
3␣-hydroxy group by a 3␣-hydroxy steroid dehydrogenase
was a major advance in bile acid analysis (207), especially
for workers in the field of bile acid physiology. This tech-
nique was based on the pioneering work of Talalay (208),
who developed enzymatic analysis of 3␣-hydroxy hor-
monal 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 tech-
nique using 3␣-hydroxysteroid dehydrogenase does not
measure 3␤-hydroxy bile acids. Thus, when used for deter-
mination 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 biolumines-
cence assay for primary bile acids. The method was based
on coimmobilizing on beads a 7␣-hydroxysteroid dehydro-
genase, 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 sum-
marized in the review by Griffiths and Sjövall (9).
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 were taken to develop a rapid simple proce-
dure for measuring total bile acids. The first was the en-
zyme method that measures total 3␣-hydroxy bile acids, as
just discussed. The second approach was the development
of competitive binding techniques, such as radioimmuno-
assay. What is measured depends on the affinity of the an-
tibody, and thus one determines “immunoreactive” bile
acids. At the Mayo Clinic, Wilfred Simmonds et al. (213)
developed a radioimmunoassay specific for cholyl conju-
gates, which was then used by LaRusso et al. (214) to de-
fine the time course of the postprandial elevation of such
cholyl conjugates in health and in patients with bile acid
malabsorption. Later, Schalm et al. (215) developed a ra-
dioimmunoassay for chenodeoxycholyl conjugates, and
used it to show that the postprandial peak of CDC conju-
gates occurs earlier than that of cholyl conjugates (216), as
shown in Fig. 6. Other radioimmunoassays have been de-
veloped, as summarized in the review by Griffiths and
Sjövall (9).
All of the above methods for measuring bile acids iso-
late 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 informa-
tion 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
History of bile acid research 1565

a bile acid binding site (based on the binding site of FXR)
and two reporter molecules whose fluorescence is deter-
mined by the extent to which bile acids occupy the bind-
ing 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 character-
ize bile acid transport into isolated hepatocytes.
Diagnostic utility of plasma bile acid levels
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 bili-
rubin). The major reason is that bile acid levels are prob-
ably inferior in sensitivity and specificity in detecting liver
injury when compared with the standard liver panel. This
conclusion is based on clinical studies (218, 219). In addi-
tion, 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 measure-
ments, there is the belief that the current liver tests are
adequate. Moreover, the instruments that automate deter-
mination of a serum “chemical panel” do not have a bile
acid “channel”. For purely cholestatic diseases, such as pri-
mary cholestasis of pregnancy, the level of serum bile acids
has value for predicting prognosis (221).
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 instan-
taneous 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 hepato-
cyte bile acids) represent the balance between input of
unconjugated and conjugated bile acids, subsequent bio-
transformation of these molecules, as well as bile acids that
have been secreted by transporters into the canaliculus.
1566 Journal of Lipid Research Volume 55, 2014
Fig. 6. Time course in healthy subjects of levels in
peripheral venous plasma of immunoreactive conju-
gates of cholic acid (cholyl conjugates) and those of
CDCA (here termed “chenyl” conjugates) in re-
sponse 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 conju-
gates are also higher than those of cholyl conjugates
despite similar proportions in bile because of lower
fractional extraction by the liver. From (216).
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 uncon-
jugated 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 bacte-
rial enzymes minus those that were absorbed.
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 pri-
mary 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 ana-
lytical method should be determined by the biological ques-
tion that is being asked. For nomenclature in reporting bile
acid analyses, the recommendations of an ad hoc nomencla-
ture committee (117) should be followed.
BILE ACID METABOLISM: TRANSPORT AND THE
ENTEROHEPATIC CIRCULATION
Bile acid transport
Overview. Glycine and taurine conjugated bile acids
are fully ionized at the pH conditions prevailing in plasma,
bile, and small intestinal content. Therefore, these mole-
cules will not cross a cell membrane and enter a cell unless
a transporter is present. What is responsible for the en-
terohepatic circulation is the vectorial transport of conju-
gated 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 trans-
port 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 in-
testine. 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 enterohe-
patic circulation consists of primary bile acids formed and
conjugated in the pericentral hepatocytes. The newly synthe-
sized conjugated bile acids are pumped across the canalicular
membrane and flow in canalicular bile from the pericentral
region to the periportal region, their direction of flow being
opposite from that of sinusoidal blood. In the periportal re-
gion, newly synthesized bile acids are joined by bile acids re-
turning 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 “un-
damaged” conjugated bile acids that have been absorbed
by the ileal transport system. A second input is that of un-
conjugated primary bile acids formed by bacterial decon-
jugation 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 second-
ary 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 up-
take 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 reepimerization of hydroxy groups (from ␤ to ␣)
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 in-
testine is at least 20-fold 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 con-
tent 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 se-
creted into bile, but are poorly absorbed from the small
intestine (222), so they do not have an enterohepatic cir-
culation. Those unsulfated amidates secreted in bile re-
turn to the liver, undergo sulfation in part, are resecreted
into bile, and eliminated via the fecal route. The end re-
sult is that lithocholic acid has a very rapid turnover in
man, and sulfated and unsulfated lithocholyl amidates
constitute less than 5% of biliary bile acids (165). In ro-
dents, lithocholic acid is not sulfated but undergoes hy-
droxylation at C-6 or C-7 (15).
As noted previously, DCA may pass through the hepato-
cyte without further metabolism, or may be rehydroxyl-
ated 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, al-
though 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 propor-
tions of each type of bile acid, this in turn being deter-
mined by the sizes of the individual bile acid pools.
Transport by the ileal enterocyte: the apical transporter. The
first person to observe preferential absorption of conju-
gated bile acids by the ileum was Hermann Tappeiner,
who presented his findings to the Vienna Academy of Sci-
ences 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 conclud-
ing 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 transport-
ers 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.
Baker and Searle, working at the University of Iowa, re-
discovered 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 Shef-
field, 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 physi-
ologists were bemoaning the difficulty of studying intesti-
nal 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 attrac-
tive 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 mem-
bers at Johns Hopkins, were discussing how to teach medi-
cal students about the enterohepatic circulation. They
realized that nothing was known about the mechanism of
History of bile acid research 1567
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 transports conjugated bile acids
against a concentration gradient (229). Their data are il-
lustrated in Fig. 7. As noted earlier, the senior author,
working with his mentor Bengt Borgström and Göran
Lundh (a surgeon and brother-in-law of Borgström), per-
fused the small intestine of healthy volunteers and ob-
tained strong evidence for ileal absorption of conjugated
bile acids in man (20). Peter Holt, working in the labora-
tory of Kurt Isselbacher at Harvard, showed that conju-
gated 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 pres-
ent in ileal enterocytes. (The term “enterocyte” was coined
by Christopher Booth, a prominent English gastroenter-
ologist and bibliophile, who discovered that vitamin B12,
like conjugated bile acids, was also absorbed predomi-
nantly in the ileum.) In 1978, H. Lücke, 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 taurocho-
late uptake was carrier mediated and Na+ dependent
(231). Attempts were made to isolate the apical conju-
gated 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 stu-
dent, Werner Kramer at the Hoechst Company in Frank-
furt (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 baso-
lateral (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 University of
California, Los Angeles. 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
Fig. 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 (229).
1568 Journal of Lipid Research Volume 55, 2014
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
University of Texas Southwestern in Dallas. Dawson, now
relocated to Wake Forest University, reported the success-
ful cloning of the ileal apical sodium-dependent bile acid
transporter [protein, apical sodium-codependent bile acid
transporter (ASBT); gene, SLC10A2], using a somewhat
different expression technique (236). This achievement,
more than a century after Tappeiner’s original study, per-
mitted 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 stud-
ies by F. Wilson et al. (237). Later, the laboratories of G.
Alpini in Texas (238) and N. LaRusso (239) at the Mayo
Clinic showed that ASBT was also present in cholangio-
cytes. ASBT is highly specific for bile acids, although the
integrity of the 7␣-hydroxyl group seems more important
than that of the 3␣-hydroxyl group, as some substituents
can be added to the 3␣- position without impairing trans-
port (240). As yet, the transporter has not been crystal-
lized, although the structure of a bacterial homolog 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).
Transport by the hepatocyte: bile acid uptake. Bile acid up-
take by the liver was known to be highly efficient. For ex-
ample, in an isolated perfused dog preparation studied at
the Mayo Clinic by N. Hoffman et al., uptake of taurocho-
late 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 tau-
rocholate in the dog was found to be 40–80% and satura-
ble, consistent with uptake being carrier mediated (244).
Conjugated bile acid uptake was saturable, and using ei-
ther 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 were
present in the sinusoidal membrane and mediated the ef-
ficient uptake of bile acids.
A sodium-dependent basolateral hepatocyte protein,
named Na+ taurocholate cotransporting polypeptide
(NTCP) (sodium taurocholate porter), was cloned in
1991, again using expression cloning, by Bruno Hagen-
buch, Bruno Stieger, and colleagues working in the labo-
ratory of Peter Meier in Zurich (247). When the Na+
coupled ileal transporter ASBT was cloned 4 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 specific-
ity than ASBT (249, 250). NTCP can be considered an an-
ion 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.
NTCP is not the only bile acid transporter in the baso-
lateral (sinusoidal) membranes of the hepatocyte. A family
of Na+-independent transporters, organic anion trans-
porting 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 or-
ganic anion (SLCO).
To what extent OATP transporters contribute to the
highly efficient uptake of conjugated bile acids from sinu-
soidal plasma is not known, but it is believed that the ma-
jority 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 (nanomolar) of unbound species. In our
view, it is reasonable to think that more than one trans-
porter may be involved in the sinusoidal uptake of conju-
gated 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 peri-
portal hepatocytes. This was first recognized in rat stud-
ies by Norman, working in Lund, Sweden in 1955 (252),
and much later confirmed for man in our own studies at
the Mayo Clinic (253). The rate of “reconjugation” in the
periportal cells is several fold greater than the rate of con-
jugation 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. Litho-
cholic acid, CDCA, DCA, UDCA, and presumably 3,6-dihy-
droxy 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 con-
trast, passes across cell membranes slowly and requires a
transporter for rapid cellular entry. A systematic study of
the passive membrane permeability of the three muricho-
lic acid epimers is lacking.
At least two transporters appear to be involved in the
uptake of unconjugated trihydroxy bile acids. The labora-
tory of Curt Klaassen in Kansas City showed that OATP1B2
(a sodium-independent transporter) appears to be in-
volved in the uptake of unconjugated bile acids that re-
turn 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 pos-
tulate 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 per-
oxisomal very long chain CoA synthetase. This homo-
log 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 oc-
curs, presumably followed immediately by CoA formation.
Formation of the adenylate and subsequent CoA thioester
serves to prevent back diffusion across the sinusoidal mem-
brane and thereby traps the bile acid CoA in the hepato-
cyte. 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
(MRP)4, has been shown to mediate the coefflux of conju-
gated bile acids and reduced glutathione, in work from
the laboratory of Dieter Keppler and colleagues (258). For
this work, we provided tritium-labeled conjugated bile ac-
ids of high specific activity (259). Keppler (260) has sug-
gested 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.
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 intra-
cellular concentration of bile acids was thought to be in
the low micromolar range, whereas the concentration of
monomeric bile acids in ductal bile, based on the dialysis
studies of Duane (261), was about 1,000 ␮M. There was
thus an uphill transport of three orders of magnitude.
To study this transport system, one needed vesicles con-
sisting predominantly of canalicular membranes. The first
preparation was achieved by Inoue and Kinne, then work-
ing in the laboratory of Irwin Arias in New York (262). The
canalicular membranes were isolated by nitrogen cavita-
tion 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 vesi-
cles 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 me-
diated and probably electrogenic (264). A similar study
was published by Inoue et al. (265).
In 1986, Eamon O’Maille, who had studied bile acid se-
cretion in the intact dog, was working in our laboratory as
a guest investigator. He carefully measured the Tmax of tau-
rocholate 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
History of bile acid research 1569
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. (268)] also reported that rat canalicular membrane
vesicles contain an ATP-dependent bile acid transport
system.
A year or so later, the Zurich group began to work on
the ATP-dependent process of conjugated bile acid extru-
sion by the canaliculus. They studied a variety of cell sys-
tems 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 named “sister of P-glycoprotein,” later
named multidrug resistance (MDR)1 (269). Using this
sequence, an antibody to the putative canalicular trans-
porter was generated. The Zurich group then used phage
libraries, their antibody, and Sf9 cells to monitor expres-
sion. They were ultimately successful in cloning the BSEP
(protein, BSEP; gene, Abcb11) (270). Our laboratory pro-
vided 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 dis-
ease were found to have mutations in Abcb11, the gene en-
coding 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 ␥-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 oc-
curs 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 ap-
peared to have only mild cholestasis (273). The mildness
of the mouse phenotype contrasted strikingly with the se-
vere cholestatic phenotype that occurs in infants with a
genetic defect. This striking difference between BSEP de-
ficiency in humans and that in mice appears to have at
least three explanations. First, in mice, a major bile acid is
␤-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 effec-
tive conjugated bile acid extruder (274). Third, BSEP-defi-
cient mice form tetra-hydroxy and penta-hydroxy bile
acids (275, 276), some or all of which are likely to be trans-
ported by MRP2 (277). Humans with cholestatic liver dis-
ease do not form tetra- or penta-hydroxy bile acids to any
extent. In mice, the defect in bile acid export from the he-
patocyte is easily shown by feeding such mice cholic acid
(278), which in this condition is highly toxic. Impaired
␤-fatty acid oxidation is also present in in such cholic acid-
fed animals (279).
1570 Journal of Lipid Research Volume 55, 2014
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 laboratory,
+
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 expres-
sion cloning, and they mediated transport only when they
were expressed simultaneously. They were named organic
solute transporters A and B (OST␣/OST␤). 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 di-
meric transporter was present on the basolateral membrane
of the ileal enterocyte and renal proximal tubular cell.
When OST␣/OST␤ 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
OST␣/OST␤ showed that it was a facilitated diffusion car-
rier and not entirely specific for bile acids, in contrast to
ASBT, which is highly specific for bile acids. In cholestasis,
hepatocyte OST␣/OST␤ 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 OST␣/OST␤
have been identified. A mouse knockout has been created,
and it differs from the ASBT knockout in that with the
OST␣/OST␤ knockout, there is no marked increase in
bile acid biosynthesis despite bile acid malabsorption
(283). In contrast, in the Slc10A2 (ASBT) knockout, bile
acid synthesis increases nearly 20-fold (284). This appar-
ently 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 up-
take 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 satu-
rated fatty acids and fat soluble vitamins.
Imaging of bile acid transport. An 11C-labeled analog of
taurocholate (cholyltaurine) has been synthesized by the
positron emission tomography scan center of Aarhus Uni-
11
versity Hospital (286). The molecule, cholyl-N- CH3-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 min), this compound
can be used only in positron emission tomography 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 Hep-
atobiliary Transport in Health and Disease was published
(287). It should cover the topics discussed in our review in
much more detail.
The enterohepatic circulation in health
History. The enterohepatic cycling of bile was clearly
described by Borelli in 1681 (288). He wrote: “Bile is col-
lected in the portal vein [from the intestine] and sepa-
rated from the blood again in the liver. It is then sent into
the bile vessels. It leaves these and repeats its circuit
though 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 enterohe-
patic circulation of some or all of the constituents of bile
(289). Lehmann, in his text book of physiological chemis-
try 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, work-
ing in Florence, Italy, prepared anesthetized dogs with an
“amphibolic” catheter. When bile was diverted to the out-
side, 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 anesthe-
tized 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 entero-
hepatic 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 contrac-
tion into the small intestine during digestion, is absorbed
from the distal intestine, returns to the liver, and may ei-
ther be stored in the gallbladder or resecreted into the
intestine. When a meal is ingested, the hormone cholecys-
tokinin is released from the small intestine. Cholecystoki-
nin 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 ab-
sorbed in the jejunum, but most are transported by intesti-
nal peristalsis to the distal ileum where they are efficiently
absorbed. The bile acid molecules pass through ileal en-
terocytes 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 Fig. 8.
Whether ancient vertebrates, e.g., the coelacanth and the
hagfish, have an enterohepatic circulation with their C27
bile alcohol sulfates is not known.
Determinants, properties, and measurement of the enterohepatic
circulation. As noted previously, the enterohepatic circu-
lation is the resultant of vectorial transport by the ileal en-
terocyte 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 transport-
ers of the ileal enterocyte and the hepatocyte. Because
only bile acids are transported by specific transporters,
only bile acids have an enterohepatic circulation. The pos-
sible 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 (<10 ␮M) because
of efficient hepatic extraction as well as dilution. Urinary
levels are also low (<10 ␮M) 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 tu-
bules via ASBT and OST␣/␤. We found that cholyl conju-
gates were 70% bound, and dihydroxy conjugates were
>95% bound in human serum (251), so the glomerular
Fig. 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 spill-
over of absorbed bile acids into the systemic circulation nor se-
lective absorption of conjugated bile acids by the terminal ileum.
Values for biliary bile acid secretion and fecal bile acid excre-
tion are about 2-fold higher than those obtained in more recent
studies.
History of bile acid research 1571
filtrate should contain mostly conjugates of cholic acid in
humans. The end result is that the concentration of bile
acids in urine in healthy humans is extremely low, and uri-
nary bile acids constitute a negligible fraction of bile acid
excretion.
The mass of cycling bile acids in humans was first mea-
sured 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 expo-
nentially with time, indicating that loss of radioactivity
followed first order kinetics. The slope of the natural loga-
rithm 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 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 ac-
ids, cholic acid 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 iso-
topes, measuring the atoms percent excess in plasma bile
acids by MS, an approach developed by Franz Stellaard
and his colleagues in Groningen (295). When radioactive
bile acids are used in the usual amounts, the specific activ-
ity 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 vol-
ume of bile is removed during the daily duodenal bile sam-
pling 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.
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 char-
acterize bile acid metabolism. The Bergström group was
the first to synthesize 24-14C-tagged bile acids (298), and
this label was stable during enterohepatic cycling. In order
3
to prepare H-tagged bile acids that would also be stable
during enterohepatic cycling, we used a method devel-
oped by Peter Klein to exchange 3H into the C-2 and C-4
1572 Journal of Lipid Research Volume 55, 2014
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 ⌬ derivatives, and reduced
11
3
them with H. 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 triti-
ated 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 Univer-
sity of Minnesota (302). Isotope dilution studies have not
been performed with ␤-muricholic acid because of the non-
availability of labeled material. However, the ⌬ compound,
22
the precursor for reductive tritiation, has been synthesized
by the Iida group (137).
The enterohepatic circulation in humans. Because of bac-
terial deconjugation in the intestine followed by absorp-
tion from the intestine and reconjugation in the liver, the
turnover rate of the steroid moiety of C24 bile acids is con-
siderably slower than that of its glycine moiety. As noted
previously, this deconjugation-reconjugation pathway was
identified in Sweden by A. Norman in rats (252), by Yamsaki
in Japan (303), and also somewhat later by clinical studies
in the laboratory of the senior author at the Mayo Clinic
(253). Deconjugation-reconjugation also occurs with tau-
rine-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 find-
ings is desirable.
No information is available on the turnover rates of ei-
ther the steroid moiety or the taurine moiety of C27 bile
acids. For most C27 bile alcohols, the process of deconjuga-
tion-reconjugation is unlikely, as the liberated bile alco-
hol, 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 dilu-
tion and guessed at the number of times that the pool cy-
cled, 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 di-
gestion was about 0.3 ␮mol/kg/min, a value confirmed in a
separate study of gallbladder emptying by G. Van Berge He-
negouwen 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 si-
nusoids, 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 directly mea-
sure hepatic extraction (308). M. Korman and his col-
leagues 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 bloodflow limited, and this view
was confirmed in a careful study of hepatic bile acid ex-
traction 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
(311). They compared the area under the curve (AUC)
after intravenous administration with the AUC after oral
administration. A value of 62% was obtained. Unconju-
gated 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 conju-
gates are bound much more tightly by albumin. For a
given steroid moiety, conjugated bile acids are extracted
more efficiently than the unconjugated bile acid.
Absorption of bile acids in the biliary ductules, cholehepatic
shunting. The discovery that cholangiocytes contain
ASBT (238, 239) meant that a fraction of conjugated bile
acids secreted into canalicular bile is absorbed during pas-
sage 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 conjuga-
tion is not complete, unconjugated bile acids will enter
into canalicular bile. In addition, we found in our labora-
tory that 24-nor C23 dihydroxy bile acids (norCDCA, nor-
DCA, 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 undergo-
ing a cholehepatic circulation. The absorption of the un-
conjugated bile acid removes a proton from the canalicular
bile, generating a bicarbonate anion, the result being no
change in bile osmolarity. Lamri et al. (313) provided
additional evidence for the validity of cholehepatic shunt-
ing using immunohistochemical techniques. 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 sinu-
soid, 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 bicar-
bonate-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 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 ab-
sorbed in the biliary ductules, thereby undergoing chole-
hepatic shunting. In further work from our laboratory, we
showed that sulindac, but not other NSAIDs, also under-
went cholehepatic shunting (317).
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 ve-
nous 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 pro-
file of biliary bile acids. Plasma bile acids are enriched
in dihydroxy conjugates because of their lower hepatic
extraction.
Regulation of the enterohepatic circulation. The enterohe-
patic 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 con-
flicting aims, it should be highly likely that the enterohe-
patic 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 concen-
tration of bile acids. When this falls, bile acid synthesis
increases. When bile acids are fed, bile acid synthesis de-
creases. The senior investigator characterized bile acid
metabolism in patients with ileal resection. A marked in-
crease, up to 10-fold, was present in such patients, again
attributed to bile acid malabsorption lowering the concen-
tration of bile acids in the hepatocyte (319). A similar in-
crease 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 re-
turn of bile acids to the liver could be systematically de-
creased, and an increase in synthesis quantified.
History of bile acid research 1573
 However, two observations did not seem to support this
useful hypothesis. The first was that bile acid synthesis in-
creased, at least acutely, when the bile duct was 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 intes-
tine to the liver might downregulate bile acid synthesis.
The breakthrough came with the identification of FGF15
(in the rat) and FGF19 (in humans) as a protein released by
the ileal enterocyte in response to FXR induction by bile
acids. This observation by Inagaki and Kliewer and col-
leagues at University of Texas Southwestern (58) was a ma-
jor 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; and conservation,
i.e., ileal transport is regulated at the level of the ileal en-
terocyte. Both synthesis as well as ileal transport are down-
regulated by FGF19 (53). FGF19 enters the hepatocyte via
a receptor [fibroblast growth factor receptor 4 (FGFR4)]
and directly or indirectly downregulates bile acid synthe-
sis. Synthesis of FGF19 in the ileal enterocyte is under the
control of FXR, the nuclear receptor activated by bile ac-
ids. Nonetheless, in some species, it remains possible that
bile acid synthesis is regulated by the bile acid concentra-
tion in the hepatocyte rather than by FGF19 (324).
The regulation of ileal conservation is poorly under-
stood. J. Lillienau, working with the senior author, found
that bile salt feeding in the guinea pig downregulated ileal
transport, whereas administration of a bile acid seques-
trant upregulated ileal transport (325). Upregulation of
ileal bile acid transport was associated with recruitment of
more proximal enterocytes to transport bile acids. None-
theless, with complete cholestasis or with parenteral ali-
mentation, bile acid transport by the ileal enterocyte
decreases (326, 327). Thus, there must be multiple mecha-
nisms of control. It is likely that the downregulation of the
ileal bile acid transporter (ASBT) with bile acid feeding
involves FXR agonism and FGF19 release.
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 ob-
struction, as occurs in cholestatic liver disease such as pri-
mary 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
1574 Journal of Lipid Research Volume 55, 2014
present. (To date, such a defect has not been identified.)
Bacterial overgrowth can be present, leading to deconju-
gation, and sometimes, to dehydroxylation. Unconjugated
bile acids are rapidly absorbed, leading to an intralumi-
nal bile acid deficiency, which 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 pro-
pels 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 bloodflow
may be obstructed or portal-caval shunting may occur. Fi-
nally 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.
PHYSICOCHEMICAL ASPECTS OF BILE ACIDS
Overview
The physical chemistry of biliary lipids and digestive lip-
ids are similar, in that both involve solubilization of polar
lipids in the form of mixed micelles. Mixed micelle forma-
tion, whether during biliary lipid secretion or triglyceride
digestion, has received a great deal of attention from phy-
sicians working together with physical chemists in an at-
tempt to unravel the complexities of these processes.
In 1983, the Kroc Foundation sponsored a small confer-
ence (9 physicians, 15 chemists) entitled, “The Physical
Chemistry of Bile in Health and Disease”. Many of the top-
ics discussed below are treated in more detail in the pub-
lished 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 philan-
thropy. The foundation was dissolved.
In 1966, the senior author collaborated with Donald
Small in the writing of a brief review on the physicochemi-
cal properties of bile acids (329). In 1985, Martin Carey
authored a systematic review of the physicochemical prop-
erties of C24 bile acids (330). His review included tabula-
tion 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 re-
views summarized existing knowledge of the physicochem-
ical properties of bile acids, as envisioned by medically
trained physician-scientists. In the discussion below, only
selected aspects of the physicochemical properties are
considered.
Bile acids in aqueous solution
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 in Italy (331). They reported solubility values at
pH 3 for 10 common bile acids. Values varied enormously.
That of lithocholic acid was 0.05 ␮M whereas that of the
7␤ epimer of cholic acid (so called “ursocholic” acid) was
1,670 ␮M. 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 ␮M, whereas that of UDCA was 9 ␮M. The greater
aqueous solubility of CDCA contributes to its excellent
gastrointestinal absorption (210); UDCA, with a lower mo-
nomeric solubility, is absorbed less efficiently and its crys-
tal dissolution is rate limiting in its absorption (332).
The solubility of several protonated glycine-conjugated
bile acids has also been determined by A. Roda and his
colleagues. They are quite low (A. Roda, personal observa-
tion). 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 uncon-
jugated C27 bile acids. The solubility of 5␣-cyprinol was
found to be 360 ␮M, thus being similar to that of cholic
acid (190). No other solubility data are available for C27
bile alcohols.
Ionization. One of the purposes of the Kroc Founda-
tion 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 re-
sults, suggesting that the pKa of an unconjugated bile acid was
influenced by the number of hydroxyl groups. B. Josephson,
working in Stockholm in the 1930s as a predoctoral stu-
dent, determined that taurine-conjugated bile acids were
strong acids, that glycine conjugates had a pKa of 4–5, and
that 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 per-
formed 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 in-
creasing proportions of water in methanol and extrapo-
lates 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 ob-
tained values of about 4 for glycine conjugates (337). The
pKa of glycine conjugates is lower than that of unconju-
gated 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 de-
termined 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.
(135) found the pKa of 23R-hydroxy-CDCA to be 4.8,
about 0.2 units lower than that of CDCA. It should be pos-
sible 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 in-
testine 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 criti-
cal 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, Schteingart, and
Hofmann (338), working in our laboratory, found the
CMpH of glycocholate to be 4.8. That of CDCA was found
by van Berge Henegouwen to be about 6.8 (340) and that
of UDCA has been calculated to be 7.9 (339). The con-
verse of CMpH has been termed “the pH of precipitation”
(333) and denotes the pH at which bile acids begin to pre-
cipitate from a true solution of the bile acid, the value be-
ing 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 loga-
rithm of the aqueous solubility of the protonated mono-
mer. 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-se-
creting 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).
Solubility of calcium (Ca2+) salts of bile acids. Conjugation
also greatly increases the resistance to precipitation by di-
2+
valent cations such as Ca . Studies from our laboratory
(343) in collaboration with K. Mysels (a legendary physical
chemist) showed that the calcium salts of taurine conju-
gates of common natural bile acids are water soluble. Gly-
cine conjugation has a very small effect on the ion product
2+ 2 2+
of Ca ion × [BA-] , and the solubility products of Ca
salts of glycine-conjugated bile acids are quite low. How-
ever, the supersaturated solutions of the calcium salts of
glycine-conjugated bile acids are metastable, resisting for-
mation of the insoluble Ca2+ salt for many weeks. The solu-
2+
bilities of Ca 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.
In the enterohepatic axis, the most dangerous site for
possible insoluble calcium salt formation is the gallblad-
der, where ionized Ca2+ concentrations rise during the
concentration of bile by the gallbladder epithelium (345).
History of bile acid research 1575
Stone formation remains rare, as the concentration of ion-
ized Ca2+ is kept rather low in the biliary tract and small
2+
intestine. The low concentration of ionized Ca is ex-
+
plained by the high Na concentration, which decreases
the activity coefficient of calcium (339).
Formation of Ca2+ bile salt gallstones occurs when “cal-
cium-sensitive” bile acids are present in appreciable pro-
portions in biliary bile acids. In general, this has occurred
because of the feeding of unusual bile acids or their pre-
cursors (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, which in turn is converted to allodeoxy-
cholic acid by intestinal bacteria. The allodeoxycholic acid
is conjugated with glycine and the insoluble glycine conju-
gate (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 en-
hances ionization at the pH present in jejunal content
during digestion, preventing passive absorption across the
apical membrane of the enterocyte. The bile acid mole-
cule, whether unconjugated or conjugated, is too large to
traverse the paracellular junctions of the healthy intes-
tine. This “nonabsorbability” of conjugated bile acids per-
mits high concentrations of conjugated bile acids to be
maintained in the biliary tract and proximal small intes-
tine 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 from partitioning into the
endoplasmic reticulum during their transit through the
hepatocyte.
Temperature effects on solubility. Temperature effects are
not very important in the biology of bile acids. Solutions of
the common primary bile acids are stable at 0°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 de-
notes this temperature of a phase transition. One defini-
tion of the Krafft point is the temperature at which 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 val-
ues, 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
1576 Journal of Lipid Research Volume 55, 2014
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 extrapo-
lation. The extrapolated values were well below 0°C (350).
Thus, because of their solubility at 0°C, the common natu-
ral 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 in-
formation on the CMT values of C27 bile acids, as well as
those of bile alcohol sulfates, with the single exception of
5␣-cyprinol sulfate, whose CMT was reported to be <0°C
(190).
Self-association in solution: micelle formation. In 1936, G.
S. Hartley, working in London, wrote a small lucid mono-
graph entitled Aqueous Solutions of Paraffin-Chain Salts: A
Study in Micelle Formation (351). This work summarized the
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 every-
one 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. Hart-
ley 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 ad-
vanced 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 stud-
ies 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 Solu-
bilization and Related Phenomena in 1955, which summarized
many of the observations on this topic in the McBain labo-
ratory (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 cho-
lesterol in a concentration-dependent manner (353).
In 1942, O. Mellander and E. Stenhagen, working in
Sweden, performed conductivity studies with sodium tau-
rocholate and found evidence of aggregate formation
(354). Per Ekwall, working in Turku, Finland, summarized
the evidence for micelle formation in sodium cholate solu-
tions in 1951 (355). Ekwall also characterized surface
properties of bile acids and made many contributions to
the physical chemistry of bile acid solutions (356).
 As noted above, a program on the biology and chemis-
try of bile acids was initiated by Sune Bergström in Lund,
Sweden after the war. Arne Norman synthesized the gly-
cine and taurine conjugates of most of the major bile ac-
ids. He then collaborated with Per Ekwall to study the
solubilization of 20-methylcholanthrene and thus define
the CMC for dilute solutions of pure conjugated bile acids
(357). Shortly thereafter, and independently, the senior
author, working in Lund, Sweden, synthesized and mea-
sured 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. There-
fore, 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 sur-
face tension in relation to concentration is that a new bub-
ble surface is constantly being formed whose composition
consists of the dominant constituent of the solution. In
contrast, with surface tension methods using a tensiome-
ter, a more surface-active impurity may concentrate at the
surface of the solution to be analyzed and lead to errone-
ous values for the CMC. Roda validated his bubble pres-
sure 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. Val-
ues are reported in the absence and presence of 0.15 M
Na+ concentration.
The senior author, when working in Sweden in the
1960s 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 inter-
micellar concentration of bile acids (monomers plus sim-
ple micelles) in both model bile samples and human bile
samples. He obtained values for the CMC as well as for the
intermicellar concentration of bile acids, which were quite
similar to the CMC in bile acid-monoglyceride systems
(261).
Solubilization of polar lipids by bile acid micelles
Physical chemistry of biliary lipids. Bile acids are present
at micellar concentrations only in bile and small intestinal
content. In humans, the concentrations in plasma (2–10
␮M) and in colonic content (400 ␮M) are well below the
CMC. In health, bile acids are always associated with a po-
lar lipid. In bile, this is predominantly phosphatidylcho-
line (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 de-
fective Mdr3 protein. Such mice (and patients) lack this
ATP-activated PC extruder in the canaliculus of the hepa-
tocyte and are believed to have essentially no phospho-
lipid 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 may be that
phospholipid, after secretion into canalicular bile, is ab-
sorbed 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 oc-
curred early in the 20th century. G. Whipple in his 1922
review (91) tabulated the reported analyses of biliary leci-
thin. 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). An-
other article in this series will give the history of phospho-
lipids in more detail.
Franz Ingelfinger, one of the dominant figures in Amer-
ican Gastroenterology in the mid 1900s, 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 triangu-
lar coordinates. The ternary systems, water-lecithin-bile
acid, water-cholesterol-bile acid, and 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 concentra-
tion 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
History of bile acid research 1577
would be supersaturated in cholesterol, either in liquid
crystalline or crystalline form. This classic work is sum-
marized in (333) and illustrated in Fig. 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 1950s by B. Isaksson, working in Sweden (366). Work
on the solubilization of 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 1960s, about the
same time that D. Small was studying model systems simulat-
ing 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 mi-
celle formation in bile and micelle formation in small in-
testinal 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 be-
ing 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 hydro-
carbon interior in which poorly soluble lipids can dissolve.
In bile, this insoluble lipid is cholesterol; in small intesti-
nal content, insoluble lipids of physiological importance
are the fat soluble vitamins. The remaining vexing prob-
lem was the molecular arrangement of the mixed micelles
present in bile and small intestinal content.
Molecular arrangement of mixed micelles. To solve the
problem of the structural arrangement of the mixed mi-
celles in bile, D. Small traveled to England to the labora-
tory 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
1578 Journal of Lipid Research Volume 55, 2014
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 Uni-
versity 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 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 con-
cluded 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 pro-
posed by Small et al. (369) could not be correct (372).
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 in-
cluded in the PubMed database. A depiction of the origi-
nal model for the mixed micelle proposed by Small and
Carey (330, 369) is compared with that of the model in-
ferred from the small angle neuron studies of Hjelm and
colleagues in Fig. 10.
In 2002, S. Marrink and A. Mark, working in the Nether-
lands, used the technique of molecular dynamic simula-
tions, in which molecules aggregate in water to give the
lowest free energy (373). They concluded that: “… phos-
pholipids 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 re-
semble the radial shell model proposed by Nichols and
Hjelm. Additional work by Long, Kaler, and Lee (374) us-
ing small angle X-ray scattering and quasi-elastic light scat-
tering were also in agreement with the model proposed by
Nichols.
Fig. 9. Depiction of the use of triangular coordi-
nates 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
indicating a constant water composition (10% sol-
ids, 90% water) in the tetrahedron consisting of the
four biliary components (water, lecithin, choles-
terol, 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 composi-
tion is above the upper limit of the micellar zone,
the sample is supersaturated in cholesterol and at
risk for cholesterol gallstone formation (376). If bili-
ary lipids are known, the percent saturation can also
be obtained from tables published by Carey and col-
league (393, 394).

Fig. 10. Molecular arrangement of the drum-shaped mixed mi-
celle micelle originally proposed by Small, Penkett, and Chapman
(369) and modified by Carey (330), as compared with the radial
shell model proposed by Nichols and Ozarowski (371). The radial
shell model was confirmed by Hjelm and colleagues using small
angle neutron scattering (372, 403, 404) and also by molecular dy-
namic studies of Marrink and Mark (373).
Another approach to study the interaction of bile acids
and phospholipids was taken by Heuman, Bajaj, and Lin
(375). They prepared single layer vesicles and character-
ized the binding of bile acids to them. They also deter-
mined at what phospholipid/bile acid ratios the vesicles
were transformed into micelles.
For teaching purposes, it would be nice to have a video
with realistic molecular models showing the transfor-
mation of lipid bilayers into mixed micelles by bile acid
anions.
Physical chemistry of bile and cholesterol
gallstone disease
Supersaturated bile in bile from gallstone patients. D. Small
returned from France to Boston in 1965, and began to ap-
ply 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. Admi-
rand did much of the leg work. A line denoting equilib-
rium 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 supersatu-
rated with or without cholesterol microcrystals, whereas
bile from most nongallstone patients was unsaturated
(376). [The paper also reintroduced triangular (or ter-
nary) coordinates to the medical audience. Triangular co-
ordinates 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 di-
mensions. 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) pub-
lished in 1957 to describe the proportions of fats, carbohy-
drates, 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 choles-
terol gallstones is discussed later.
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 la-
mellae 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,” al-
though they also suggested that these microprecipitates
were vesicles enriched in cholesterol compared with
mixed micelles (379, 380). About the same time, Giora
Somjen and Tuvia Gilat, working in Israel, used gel perme-
ation chromatography to separate mixed micelles from
vesicles (381). They equilibrated their columns with
10 mM cholate prior to chromatography and thus the ve-
sicular phase remained unchanged during the chromato-
graphic 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 col-
umn, thereby increasing the phospholipid/bile acid ratio.
Thomas Holzbach, working in Cleveland, also found vesi-
cles in human bile samples and showed that nucleation of
cholesterol crystals from the cholesterol-rich vesicles oc-
curred rapidly (382). William Higuchi, working in Salt
Lake City, showed, using dialysis techniques, that model
bile systems contained four constituents: monomers, sim-
ple bile acid micelles, mixed micelles (containing bile ac-
ids, 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 as-
pects of bile and because of the widespread interest in gall-
stone pathogenesis and treatment, in 1989, the National
Institutes of Health (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 prob-
lems in communication. A summary of the meeting was
authored by Steven Strasberg, a scholarly surgeon, and his
History of bile acid research 1579
associate Robert Harvey. Their summary (385) presented
a reasonably coherent view of bile supersaturation. Cho-
lesterol and phospholipids are secreted in vesicular form.
Bile acids, initially present as simple bile acid micelles,
solubilize phospholipids and cholesterol. However, the
transfer process, presumably occurring by collision, solubi-
lizes phospholipids preferentially, leaving behind vesicles
containing an excess of cholesterol (from which choles-
terol 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 pu-
rified by gel filtration was 2.0 (387), a ratio that favors for-
mation 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 contin-
ued 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 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 1960s, 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 (Zeitschrift
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 choles-
terol, as well as varying total lipid concentration (393).
The line of saturation is also influenced by bile acid type,
being lower when bile acids are enriched in conjugates of
UDCA. M. Carey has also published critical tables for this
special circumstance (394).
Physical chemistry of fat digestion
Studies on model systems simulating triglyceride digestion. The
physical chemistry of model systems simulating fat diges-
tion 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 glycer-
ides (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
1580 Journal of Lipid Research Volume 55, 2014
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 titra-
tion 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. Monoolein, 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 triacylglycerides had little solubility in bile acid
micelles.
In 1988, M. Svärd, working in the group of Björn Lind-
man in Lund, investigated the taurocholate-monoolein-wa-
ter 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 sys-
tem and the bile acid-PC-water system elucidated by the pio-
neering 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 illus-
trated in Fig. 11. The paper of Svärd et al. (398) also pro-
poses that bile acid molecules lie flat on the surface of the
bilayers.
Presence of a micellar phase in vivo during triglyceride diges-
tion. 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
Fig. 11. Phase diagram of the water-monoolein (monooleylgl-
ycerol)-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).
oil phase containing di- and triacylglycerides and a micel-
lar 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 tem-
perature, and both the oil phase and micellar phase ana-
lyzed. 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 intu-
bation in healthy volunteers who had ingested a triglycer-
ide-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 (multilay-
ered vesicles). His analysis (402) is probably an apt de-
scription of the formation of vesicles composed of fatty
acid and monoacylglycerol at the surface of the triglycer-
ide 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 concluded that mixed micelles
containing a bile acid and either monoglyceride (monoac-
ylglycerol) or monoglycerides plus fatty acids had essen-
tially 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 sur-
face of the spherical or worm shaped micelles.
Necessity of bile acids for efficient absorption of triglycerides. Nu-
merous studies dating back to the 19th century had shown
that triglyceride absorption efficiency was impaired in ani-
mals 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 solubili-
ties, i.e., saturated fatty acids of C16 length or longer (407).
Fatty acids with appreciable aqueous solubility (unsatu-
rated 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 satu-
rated fatty acids (⭓C16 in length) is based on two observa-
tions. 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 ab-
sorption of fatty acids with appreciable aqueous solubility
provides the rationale for using medium chain triglycer-
ides 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.
DISSOLUTION OF CHOLESTEROL GALLSTONES
BY ORAL BILE ACIDS AND TOPICAL SOLVENTS
Gallstone dissolution by oral bile acids
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 prob-
ably introduced by D. Small in the 1960s. It did not in-
clude bilirubin diglucuronide (which gives bile its color),
as this pigment, derived from heme, is a very minor con-
stituent 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 1950s, 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 Na-
kayama, 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 gall-
stone 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 life-
long interest in gallstone formation and treatment in a
monograph (411).]
After J. Thistle and L. Schoenfield made the seminal dis-
covery 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 observa-
tions were made that gallstones were decreasing in size,
probably the first witnesses being J. Thistle (and his radio-
logical colleague) and the senior author. In 1972, success-
ful gallstone dissolution was reported in May at the annual
meeting of the American Gastroenterological Associa-
tion and later that year in Europe at the 2nd Interna-
tional Bile Acid Meeting sponsored by the Falk Foundation
of Freiburg, Germany. The early Mayo Clinic experi-
ence with gallstone dissolution was published in the New
History of bile acid research 1581
England Journal of Medicine in 1972 and became a “Cita-
tion 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 compa-
nies. A “Request for Proposals” was issued in July 1972. L.
Schoenfield submitted a proposal which included commit-
tee structure for the proposed National Cooperative Gall-
stone Study (NCGS). On the committees were most every
American scientist or physician interested in gallstone
disease!
The study was delayed, because of the Food and Drug
Administration (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 subject-
ing 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 re-
ports from the Huntington Laboratories in England of se-
vere toxicity induced by CDCA feeding in the rhesus
monkey (412). As a result of these reports, European stud-
ies 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. Tri-
als 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 humans, lithocho-
lic acid, the major bacterial metabolite of CDCA, is sul-
fated and rapidly excreted from the body (414, 415);
whereas in the rhesus monkey, lithocholic acid is ex-
tremely poorly sulfated, accumulates in the circulating
bile acids, and induces liver damage (416, 417). Indepen-
dent studies by the laboratory of E. Mosbach reported that
CDCA was also toxic in the rabbit, again because of litho-
cholic acid accumulation (418). Similar findings were ob-
served 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 aminotransfer-
ase 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
1582 Journal of Lipid Research Volume 55, 2014
Europe which showed that CDCA had unequivocal effi-
cacy 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 in-
dependently 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. (427) in a
thoughtful analysis of X-ray changes of gallstone size dur-
ing medical dissolution.
Just as the NCGS was beginning, word came from Japan
(a careful study led by Isao Makino and his colleagues)
that ingestion of UDCA, the 7␤ 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 dissolu-
tion, and this was done without knowledge of the Ameri-
can 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 the NIH. CDCA was approved for market-
ing in the United States by the FDA in 1983, and UDCA
was approved a few years later. UDCA, like CDCA, was also
toxic in the rhesus monkey (428) and rabbit (153, 429)
because of lithocholic acid accumulation in the circulat-
ing bile acids.
Despite the safety and efficacy of CDCA and UDCA,
there were some physicians who were unimpressed by
medical therapy as compared with 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 and Hofmann (430) suggested that with the
first attack of biliary colic, the resulting inflammation
would cause a surface layer of noncholesterol constitu-
ents on the gallstone, slowing or precluding dissolution.
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 recur-
rence (431). It was gallstone recurrence that led surgeons
a half century before to switch from cholecystotomy (re-
moval 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).
Acceleration of dissolution by extracorporeal shockwave
lithotripsy
Attempts were made to speed up dissolution. One ap-
proach was to fragment the stones by using extracor-
poreal 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 dif-
ference 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. An-
other limitation of 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).
Topical dissolution with organic solvents
Another approach to nonsurgical treatment of choles-
terol gallstones was contact (topical) dissolution with or-
ganic solvents. The Mayo Clinic group, led by J. Thistle,
introduced the idea of placing a percutaneous transhe-
patic 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. Eu-
ropean trials commenced in a few centers (438).
In our laboratory at University of California, San Diego,
we had not planned to become involved in this approach.
A patient with symptomatic gallstones who was a poor op-
erative 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 gallblad-
der started to contract, thus avoiding spillage of the sol-
vent into the common bile duct (439). A new solvent, ethyl
propionate, was tested and found to dissolve gallstones
nearly as rapidly as MTBE. Ethyl propionate did not in-
duce nausea in contrast to MTBE because of rapid re-
moval 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 stan-
dards. MTBE was flammable and considered toxic (442).
Preclinical studies had not been done. The procedure re-
quired some hours for complete dissolution, and as de-
scribed, 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 us-
age. 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).
Concurrent advances in gallstone management
Two other developments were occurring during this
time that doomed widespread usage of medical dissolu-
tion. The first was the development of laparoscopic chole-
cystectomy, a major surgical advance. Laparoscopic
surgery was an import from France, used first in gyneco-
logical 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 per-
formed 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 gall-
bladder and its contents. All types of stones were detected,
and there was no radiation exposure. In contrast, oral cho-
lecystography required the ingestion of iodine-rich con-
trast agents and subsequent X-ray to detect the presence
of gallstones.
Thus in twenty years, medical dissolution had been dis-
covered, 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 principle”, and medical therapy with UDCA can
be used to prevent cholesterol gallstone formation. Gall-
stone prophylaxis might be useful in some clinical situa-
tions, for example, in patients with recurrent gallstone
pancreatitis who did not wish to have surgery.
The availability of UDCA led to the accidental discov-
ery of its utility in the treatment of primary biliary cir-
rhosis and cholestasis of pregnancy, as noted in the
introduction.
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
History of bile acid research 1583
exact enzyme defect requires MS 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 retrospec-
tive 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 ex-
plore these nonconcordancies in detail.
A third area not discussed is the role of bile acids in co-
lonic 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-in-
duced intestinal secretion are being elucidated by several
laboratories, among these being that of Stephen Keely in
Dublin (448), and that of Mrinalini Rao in Chicago (450).
Bile acid malabsorption causes diarrhea, and this can be
treated by sequestrant administration (451, 452). A new
syndrome of defective FGF19 release causing an inappro-
priate increase in synthesis of bile acids has been identi-
fied by Julian Walters et al. (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”
(7␣-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 neces-
sity 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 pa-
tient with unexplained diarrhea is slowly being recognized
by gastroenterologists (457). The group of Michael Camil-
leri at the Mayo Clinic is exploring the relationship of fe-
cal 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 (119). 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 (459).
A fourth area not discussed is the cellular toxicity of bile
acids. Mechanisms of toxicity are multiple, involving acti-
vation 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 proinflam-
matory 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 activ-
ity of the microbiome influences bile acid metabolism. In-
deed, 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 sec-
ondary bile acids by the laboratory of Sune Bergström (8).
1584 Journal of Lipid Research Volume 55, 2014
There is still much is to be learned about the effect of in-
dividual bile acids on the microbiome (465).
The last two generations have had powerful tools to elu-
cidate bile acid metabolism. One of these was the discov-
ery of 14C, a discovery that almost occurred by chance
(466). The availability of 14C in chemical form permitted
14
C-labeled bile acids to be prepared and their metabolism
to be defined. A second essential tool has been chroma-
tography, adsorption, reverse-phase (HPLC), and gas/liq-
uid. A final tool is MS, which when coupled with GC or
HPLC provides both qualitative and quantitative informa-
tion on new molecules (9). For the assignment of struc-
ture to newly discovered bile acids, proton and 13C NMR
measurements are mandatory. Of course, in today’s re-
search, generation of specific knockouts, characterized by
multisystem phenotyping, combined with PCR, have enor-
mous power in unraveling the recently discovered signal-
ing 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
6␣-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 homolog of UDCA, is also
in clinical trials for the treatment of primary sclerosing
cholangitis. 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 hyper-
cholesterolemic 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 pharma-
codynamic activity as that of the ASBT inhibitors; how-
ever, 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.
In our opinion, the remarkable progress in our under-
standing of bile acid biology and chemistry is a tribute to
the hundreds of scientists who have worked with such dili-
gence and talent throughout the world. We have men-
tioned 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 dif-
ferent 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 his-
torical reviews by Adrian Reuben published in the journal,
Hepatology, between 2002 and 2006 (c.f. 482).
 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!
The authors 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.
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