Identification of a novel bile acid in swans, tree ducks, and geese : 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid

By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by 1H and 13C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3α,7α,15α-Trihydroxy-5β-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15α-hydroxy-chenodeoxycholic acid.

Abstract By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by 1 H and 13 C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3a,7a,15a-trihydroxy-5b-cholan-24oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes.
Supplementary key words Anseriformes . bile acid evolution . bile acid conjugation Bile acids are the amphipathic end products of cholesterol metabolism. In the small intestine, luminal bile acids promote lipid absorption by forming mixed micelles with dietary lipids (1) and also have antimicrobial effects (2). In addition, plasma bile acids appear to promote thermogenesis by interacting with a G protein-coupled receptor present on the surface of adipocytes in brown adipose tissue (3).
C 24 bile acids are found in most mammals and are present in bile as N-acyl amidates (conjugates) of taurine or glycine (4). Individual C 24 bile acids are distinguished by their pattern of hydroxylation on the C 19 nucleus or the C 5 side chain. The default hydroxylation pattern of bile acids is at carbon 3 (C-3) (because bile acids are formed from cholesterol) and at C-7 [because hydroxylation at C-7 (catalyzed by cholesterol 7a-hydroxylase or sterol 7-hydroxylase) is believed to be an essential step in the biosynthesis of all bile acids] (5). Thus, chenodeoxycholic acid (3a,7adihydroxy) may be considered the root C 24 bile acid (6).
In most mammals, the majority of biliary bile acids are trihydroxy bile acids (4,7). Hydroxylation at a third nuclear site, presumably mediated by cytochrome P450 hydroxylases, varies considerably between species. Hydroxylation at C-12 (cholic acid) and at C-6 (hyocholic acid and the muricholic acid epimers) has long been known (7). The third most common site of hydroxylation is probably at C-16, a hydroxylation site discovered many years ago in snakes by Haslewood (7) but now known to occur frequently in avian species (8). Hydroxylation at C-1 also has been reported. In the Australian opossum, hydroxylation at C-1 is in the a-configuration (9), whereas in certain fruit doves and pigeons, C-1 hydroxylation is in the b-configuration (10).
Identification of novel bile acids is based on nuclear magnetic resonance spectroscopy and mass spectrometry, but chemical synthesis is highly desirable for confirmation of the assigned structure. We recently reported the synthesis of 3a,7a,16a-trihydroxy-5b-cholan-24-oic acid (11). In the process of that work, we were able to synthesize 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid and 3a,7a, 15b-trihydroxy-5b-cholan-24-oic acid. It seemed likely to us that bile acids hydroxylated at C-15 should occur in nature, because hydroxylation at C-15 of a bile acid analog sulfonate during enterohepatic cycling in the hamster had already been observed (12). We report here the isolation of a new natural bile acid, 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid, from the bile of the black-necked swan, where it occurs as its taurine conjugate. We also show that this novel bile acid is present in the biliary bile acids of swans, tree ducks, and geese.

Biological material
Bile from the black-necked swan, Cygnus melanocoryphus, and from most other species mentioned in this study was obtained by aspiration from the gallbladder of deceased birds during autopsy by the Pathology Department of the Zoological Society of San Diego. Bile from the barnacle goose was provided by Sea World (San Diego CA), and bile from two other goose species was provided by Dr. Valentine Lance of San Diego State University. Bile samples were dispersed in 4 volumes of reagent-grade isopropanol and kept at 48C until analysis.
Biliary bile acid analysis for screening of individual species was performed by HPLC as described (4).

HPLC analysis of gallbladder bile of the black-necked swan
The apparatus used in this work consisted of a Jasco Gulliver Series HPLC system (two PU-2080 intelligent high-pressure pumps, an HG-980-31 solvent mixing module, and an HG-980-50 degasser; Tokyo, Japan) equipped with a Shimadzu C-R8A dataprocessing system (Kyoto, Japan). An Alltech 2000ES evaporative light-scattering detection (ELSD) system (Deerfield, IL) was used under the following conditions: flow rate of purified compressed air as a nebulizing gas, 1.9 l/min; temperature of the heated drift tube, 758C; gain, 16. A Capcell Pak type MG II column (5 mm, 250 mm 3 3.0 mm inner diameter; Shiseido, Tokyo, Japan) was used as an analytical C 18 reverse-phase column. The column temperature was kept at 378C using a Sugai u-620 type 30 V column heater (Wakayama, Japan). A mixture (43:68, v/v) of 15 mM ammonium acetate buffer (pH 5.4)-methanol was used as the mobile phase. The flow rate was kept constant at 0.4 ml/min.
The gallbladder bile of the black-necked swan in isopropanol (100 ml) was centrifuged for 10 min at 10,000 rpm. The supernatant liquid was applied to a preconditioned Sep-PakR C 18 cartridge (360 mg; Waters, Milford, MA). After the cartridge was washed with water (1 ml) and 20% methanol (1 ml), the bile acid fraction was eluted with 90% methanol (2 ml). The solvent was evaporated under reduced pressure, and the residue was dissolved in methanol (100 ml). An aliquot (1 ml) of the sample solution was injected into the HPLC-ELSD system.

Isolation of compound A by HPLC-ELSD
A Capcell Pak type MG II column (5 mm, 250 mm 3 10 mm inner diameter) was used as a preparative C 18 reverse-phase column. The ELSD system was set to the following conditions: drift tube temperature, 708C; gas (air) flow rate, 1.5 l/min; gain, 1; splitting ratio, 30:1. Aqueous methanol (70%) containing 0.01% trifluoroacetic acid (pH 2.7) was used as the mobile phase at a flow rate of 2.0 ml/min.

Measurement of mass spectra
Low-resolution mass spectra (LR-MS) were recorded on a JEOL JMS-303 mass spectrometer with electron ionization (EI) at 70 eV using the positive ion mode (PIM). Liquid chromatography-mass spectrometry (LC-MS) was performed and high-resolution mass spectra (HR-MS) were obtained on a JEOL JMS-LCmate doublefocusing magnetic mass spectrometer equipped with electrospray ionization (ESI) using the negative ion mode (NIM). Chromatographic separation was carried out on a YMC Pack ProC 18 column (3 mm, 100 3 2.0 mm inner diameter; YMC, Kyoto, Japan) using a 20 mM ammonium acetate (pH 7)-methanol mixture (35:65, v/v) as the mobile phase at a flow rate of 0.16 ml/min. The mass detector was set to the following conditions: needle voltage, 22.5 kV; orifice-1 temperature, 1508C; desolvating plate temperature, 2508C; ring lens voltages, 30 V/100 V or 200 V/250 V.

H and 13 C NMR
NMR spectra were recorded at 238C in CD 3 OD in a 5 mm tube on a JEOL EX-270 instrument (270 and 68.8 MHz for 1 H and 13 C, respectively) or a JEOL ECA-600 instrument (600 and 149.4 MHz for 1 H and 13 C, respectively); 1 H and 13 C chemical shifts were expressed in ppm. Complete 1 H and 13 C resonance assignments were made using a combination of two-dimensional homonuclear ( 1 H-1 H) and heteronuclear ( 1 H-13 C) shift-correlated techniques, which include 1 H-1 H homonuclear correlation spectroscopy (COSY), long-range 1 H-1 H COSY, 1 H-1 H nuclear Overhauser effect spectroscopy (NOESY), 1 H-13 C heteronuclear correlation spectroscopy (HETCOR), 1 H detected heteronuclear multiple quantum correlation (HMQC), and 1 H detected heteronuclear multiple bond correlation (HMBC) experiments. These two-dimensional NMR spectra were recorded using standard pulse sequences and parameters recommended by the manufacturer. One-dimensional 13 C distortionless enhancement by polarization transfer (DEPT; 1358, 908, and 458) spectra were also measured to determine the exact 1 H signal multiplicity and to differentiate between CH 3 , CH 2 , CH, and C based on their proton environments.

RESULTS
Isolation of 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid from swan bile and proof of structure As shown in Fig. 1, HPLC-ELSD analysis of the bile acid fraction obtained from the gallbladder bile of the black-necked swan showed two major peaks, which were designated compounds A (74%) and C (25%), accompanied by a trace amount of compound B (1%).
Major peaks A and C were subjected to LC-ESI-MS analysis with selected ion monitoring (SIM) under the NIM. Figure 2 shows SIM chromatograms for peaks A and C and authentic taurocholate and taurochenodeoxycholate. The deprotonated molecules, [M-H] 2 , obtained were as follows: a) m/z 514.3, a trihydroxy C 24 bile acid; b) m/z 496.3, a disubstituted (hydroxyl, oxo) C 24 bile acid (not shown in Fig. 2); and c) m/z 498.3, a dihydroxy C 24 bile acid. All of these structures were assumed to be conjugated in N-acyl linkage with taurine, based on the m/z number of the deprotonated molecules and the known dominance of taurine conjugation in vertebrate bile acids (4).
Peak C was readily identifiable as the common 3a,7adihydroxy primary bile acid conjugate, taurochenodeoxycholate. Identification was made by direct comparison of the HPLC-ELSD retention time, the m/z value (498.4) of the deprotonated molecule, and the retention time and fragmentation pattern during the LC-ESI-MS measurement, using an authentic standard.
The largest peak, A, had a retention time less than that of authentic taurocholate. Figure 3 shows the negative ion LC-ESI-MS fragmentation pattern of the isolated compound A, which gave a deprotonated m/z of 514.5. A fragment ion with strong intensity was observed at m/z 79.9, corresponding to SO 3 2 , supporting the presence of an N-acylamido linkage with taurine in the side chain. However, no other significant ion serving to characterize the structure of compound A, particularly the position of three hydroxyl groups, appeared in the spectrum.
To identify the location and orientation of the hydroxyl groups, compound A was isolated by preparative HPLC-ELSD and analyzed by 1 H and 13 C NMR. The chemical shifts, spectral pattern, and signal multiplicity of the 1 H  15a-hydroxy-CDCA in swans, tree ducks, and geese and 13 C signals suggested that carbons 3, 7, and 15 were the sites of hydroxylation and that all of the hydroxyl groups were in the a-configuration. Table 1 shows 1 H and 13 C NMR spectral data for compound A, together with those of authentic 3a,7a,15atrihydroxy-5b-cholan-24-oyl acid. Complete 1 H and 13 C NMR spectral assignments of the synthetic taurine conjugate of 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid were also made on the basis of several two-dimensional NMR techniques: COSY, long-range COSY, NOESY, HETCOR, HMQC, and HMBC ( Table 1). Differentiation of CH 3 , CH 2 , CH, and C was made with the 13 C DEPT spectra. The  correlation of the 1 H and 13 C signals by the HETCOR spectrum is illustrated in Fig. 4. As expected, the spectral pattern, chemical shifts, and signal multiplicity of the major 1 H and 13 C signals of compound A were in good agreement with those of synthetic 3a,7a,15a-trihydroxy-5bcholan-24-oyl taurine. A particularly noteworthy feature is that in compound A, the CH signals at the 3, 7, and 15 positions resonate at 72.72, 68.95, and 72.86 ppm, respectively, whereas the neighboring protons appear at 3.41 (3a-H, broad multiplet), 4.00 (7a-H, multiplet), and 3.97 (15a-H, broad multiplet) ppm. These 1 H and 13 C resonances for the 5b-steroid moiety were in good agreement with those reported for sodium 3a,7a,15a-trihydroxy-5, 26-bishomo-5b-cholestane-26-sulfonate (12) but not for methyl 3a,7a,16a-trihydroxy-5b-cholane-24-oate triacetate (8). (Further discussion of the NMR spectra of C-15 hydroxy bile acids is available in references 8 and 12). In addition, 13 C signals were present at 36.62 and 51.48 ppm; these were assigned to CH 2 N and CH 2 S, respectively, and the adjacent protons appeared at 3.57 (25-H 2 , t) and 2.95 (26-H 2 , t). These 1 H and 13 C data for compound A also agreed very well with those reported for 3a-sulfo-7b-(2acetamido-2-deoxy-a-D-glucopyranosyl)-5-cholen-24-oyl taurine (14), again providing evidence for the presence of the N-acylamido linkage with taurine in the side chain.
This new bile acid may be named 15a-hydroxy-chenodeoxycholic acid (CDCA) (15). For conversational purposes only, we suggest the trivial name "cygnocholic acid," in keeping with the longstanding tradition of assigning bile acid trivial names according to their biological source (15).
Peak B showed a deprotonated molecule at m/z 496.4 by LC-ESI-MS, suggesting that this compound is the taurine conjugate of 3a-hydroxy-15-oxo-cholanoate, or less likely, 3-oxo-15a-hydroxy-cholanoate (or both). Such a com-  15a-hydroxy-CDCA in swans, tree ducks, and geese pound could be formed by bacterial enzymes during the enterohepatic cycling of bile acids, as intestinal bacteria are well known to affect deconjugation, dehydroxylation at C-7, and dehydrogenation at any of the nuclear hydroxy groups (16).

Occurrence of 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid in birds
The black-necked swan is a member of the subfamily Anserinae of the family Anatidae. The family Anatidae in turn is a member of the order Anseriformes. [For a review of the phylogeny of Anserinae, see Livezey (17) and Donne-Goussé, Laudet, and Hänni (18).] In the order Anseriformes, there are two additional families, Anhimidae (three species of screamers) and Anserindae (only one species, the magpie goose). The 15a-hydroxy bile acid was not present in the biliary bile acids of the Southern screamer (Chauna torquata) or in the magpie goose (unpublished observation).
The family Anatidae can be divided into 10 subfamilies. The 15a-hydroxy compound was present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese). HPLC analyses are presented in Tables 2 and 3. The 15ahydroxy compound was present in the greatest proportion in the whooper swan, a common swan of Europe. The proportion of the 15a-hydroxy compound was highly variable in those species in which multiple birds were examined, suggesting marked polymorphism in the cytochrome P450 enzyme responsible for hydroxylation at C-15 (19). No sample from the closely related subfamily Thalassorninae (white-backed duck) was available to us.
The 15a-hydroxy compound was not present in the remaining seven subfamilies in the family Anatidae. These subfamilies comprise Stictonettinae (freckled ducks), Plectropterinae (spur-wing geese), Tadorninae (transition species between geese and ducks), Anatinae (dabbling and diving ducks), Merginae (eiders, scooters, and mergansers), and Oxyurinae (stiff-tail ducks). In these species, the third site of hydroxylation was at C-23(R), the a-carbon of the side chain.

DISCUSSION
In this work, we isolated an unknown conjugated bile acid from the bile of the black-necked swan and show that it is the taurine conjugate of a new natural bile acid, 3a,7a,15atrihydroxy-5b-cholan-24-oic acid, a bile acid previously synthesized by us. We also found that this new trihydroxy bile acid is present in the biliary bile acids of tree ducks, swans, and geese, in which it occurs as its taurine conjugate. All bile acids were conjugated with taurine. a Only one bird in each species was analyzed, with the exception of the white-faced whistling tree duck (n 5 5), with values shown as means 6 SD, and the black-bellied tree duck (n 5 3), for which values of individual birds are given. Phocacholic acid (3a,7a,23R-trihydroxy-5b-cholan-24-oic acid) was not present in any species. All bile acids were conjugated with taurine. a Only one bird was available for each species, with the exception of the black-necked swan (n 5 9), the red-breasted goose (n 5 2), the barheaded goose (n 5 2), and the Canada goose (n 5 2). Individual values for the latter three species are given.
b For the black-necked swan, in seven of nine birds, the proportion of 15a-hydroxy-chenodeoxycholic acid ranged from 0 to 4%; the eighth bird contained 58.7%; and the ninth bird, which was used for isolation of the 15-hydroxy compound, contained 74%. The mean value for all nine birds are given.
In 1991, Pyrek (20) (now deceased) reported in a review article on mass spectrometry of natural products that 3a,15a-dihydroxy-5b-cholan-24-oic acid was present in the biliary bile acids of the wombat. This compound, which has not been synthesized to date, would be formed by bacterial 7-dehydroxylation of the primary, trihydroxy bile acid reported in this paper. We analyzed one sample of wombat bile by HPLC and found compounds with retention times corresponding to that of the taurine conjugate of 3a,7a,15a-trihydroxy-5b-cholan-24-oic acid (5% of total biliary bile acids) and consistent with that predicted for the taurine conjugate of 3a,15a-dihydroxy-5bcholan-24-oic acid (56% of total biliary bile acids). Proof of structure of the compound tentatively identified as the taurine conjugate of 3a,15a-dihydroxy-5b-cholan-24-oic acid is beyond the scope of this paper.
Hydroxylation of a bile acid analog sulfonate at C-15 in the hamster has been reported previously, as noted above (12). In addition, Lund et al. (21) reported the formation from cholesterol of C 21 bile acids hydroxylated at C-15 in either the 15a-or 15b-configuration by the isolated perfused female rat liver. Mouse cytochrome P450 2A6 when mutated (phenylalanine-209 to asparagine) acquires the ability to 15a-hydroxylate corticosterone (22). Xenobiotics may also undergo 15a-hydroxylation. For example, desogestrel, an orally active progestogen, is converted to a 15ahydroxy metabolite in rats (23).
The biological forces responsible for the evolution of C-15 hydroxy bile acids are not known. Perhaps specific cytochrome P450 hydroxylases for the detoxification of plant toxins evolved and were adapted for bile acid biosynthesis. We have speculated elsewhere that the formation of trihydroxy bile acids by the hydroxylation of CDCA or a precursor of CDCA was desirable, at least in vertebrates possessing a cecum, because of the marked hepatotoxicity of lithocholic acid, the bacterial metabolite of CDCA (24).
In Fig. 6, we depict changes in biliary bile acid composition in relation to a reasonable evolutionary scheme for the order Anseriformes. The scheme for evolution is based on current views of the phylogenetic relationships derived from morphological studies (17) or analysis of mitochondrial DNA (18). The oldest family, Anhimidae, has allochenodeoxycholic acid as its major biliary bile acid, suggesting evolution from reptiles in which such bile acids occur (7). This was followed by the development of enzymes for the formation of 5b bile acids (A/B ring juncture cis), with the result that allochenodeoxycholic acid was replaced by chenodeoxycholic acid, the root bile acid in most vertebrates. Hydroxylation at 15a occurs transiently in tree ducks, swans, and geese and is eventually replaced by hydroxylation at C-23. The reason for the changes in the position of hydroxylation with evolution are not known, although C-23 conjugated bile acids have been shown to be more resistant to bile acid Fig. 6. Biliary bile acid composition denoted by nuclear substituents in relation to an evolutionary scheme for the order Anseriformes based on morphological studies (17) and mitochondrial DNA analysis (18). 15a-Hydroxylation arose in the subfamilies of Anatidae but was replaced by 23(R)-hydroxylation in later evolving families. Cereopsis is an exception, but its evolutionary position is controversial. deconjugation than bile acids with an unsubstituted side chain (25).
With this report, the number of additional hydroxylation sites on the cholanoic acid nucleus in primary bile acids is now seven (6a, 6b, 12a, 1a, 1b, 15a, and 16a). Additional sites of hydroxylation, such as C-2 or C-4, occur during experimental cholestasis (26); hydroxylation at C-4 also occurs during fetal development in human (27). Hydroxylation at C-5 of C 23 (C 24 nor) bile acids has also been reported to occur in the hamster (28). Thus, it seems likely that natural trihydroxy bile acids hydroxylated at C-2, at C-4, or at C-5 in addition to C-3 and C-7 will be identified as primary bile acids in vertebrates in the future.