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Originally published In Press as doi:10.1194/jlr.M700340-JLR200 on September 4, 2007

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Journal of Lipid Research, Vol. 48, 2682-2692, December 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15{alpha}-hydroxylithocholic acid

Genta Kakiyama*, Hideyuki Tamegai*, Takashi Iida1,*, Kuniko Mitamura{dagger}, Shigeo Ikegawa{dagger}, Takaaki Goto§, Nariyasu Mano§, Junichi Goto§, Peter Holz**, Lee R. Hagey{dagger}{dagger} and Alan F. Hofmann1,{dagger}{dagger}

* Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajosui, Setagaya, Tokyo 156-8550, Japan
{dagger} Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi-Osaka 577-8502, Japan
§ Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 980-8578, Japan
** Healesville Sanctuary, Healesville, Victoria 3777, Australia
{dagger}{dagger} Division of Gastroenterology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0063

Published, JLR Papers in Press, September 4, 2007.

1 To whom correspondence should be addressed. e-mail: takaiida{at}chs.nihon-u.ac.jp (T.I.); ahofmann{at}ucsd.edu (A.F.H.)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15{alpha}-hydroxylithocholic acid (3{alpha},15{alpha}-dihydroxy-5ß-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15{alpha}-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a {Delta}14 intermediate. The synthesis of its C-15 epimer, 15ß-hydroxylithocholic acid (3{alpha},15ß-dihydroxy-5ß-cholan-24-oic acid), is also reported. The taurine conjugate of 15{alpha}-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15{alpha}-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15{alpha}-hydroxylation of lithocholic acid that was formed by bacterial 7{alpha}-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15{alpha}-hydroxylation as a novel detoxification mechanism for lithocholic acid.

Supplementary key words bile acid synthesis • detoxification of bile acids • bile acid metabolism in marsupials • 15-hydroxylation • lithocholic acid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Bile acids are amphipathic end products of cholesterol metabolism that mediate numerous physiological functions in the liver, biliary tract, and intestine. Bile acids are of two types: C24 bile acids [with a C5 (isopentanoic acid) side chain] and C27 bile acids [with a C8 (isooctanoic acid) side chain]. The C24 and C27 bile acids, together with C27 bile alcohols, are the predominant chemical metabolites of cholesterol and are the major chemical form in which cholesterol is eliminated in most vertebrates (1). All primary C24 bile acids have a hydroxyl group at position C-3 (from cholesterol) and at C-7, as cholesterol 7{alpha}-hydroxylation is the rate-limiting step in bile acid biosynthesis. Thus, chenodeoxycholic acid (CDCA; 3{alpha},7{alpha}-dihydroxy-5ß-cholan-24-oic acid) is the root C24 bile acid.

In the majority of vertebrates, hydroxylation of CDCA (or an intermediate in the synthesis of CDCA) occurs at an additional site on the steroid nucleus. The sites of such hydroxylation have been identified at C-1, C-4, C-5, C-6, C-12, C-15, and C-16 (for original references, see Ref. 2). We have suggested that the ability to form such trihydroxy bile acids by the hepatocyte may have evolved as a response to the acquisition of a bacterial flora in the cecum mediating dehydroxylation at C-7. Bacterial dehydroxylation of CDCA results in the formation of lithocholic acid, a 3{alpha}-monohydroxy secondary bile acid (3). Lithocholic acid is toxic when it accumulates in the enterohepatic circulation of bile acids (4–12), causing segmental bile duct obstruction and destructive cholangitis in the mouse (12) and bile duct hyperplasia and choledocholithiasis in the rat (6, 7). On the other hand, 7-dehydroxylation of a trihydroxy bile acid results in the formation of a dihydroxy bile acid; the dihydroxy bile acids formed by bacterial 7-dehydroxylation of the common natural bile acids are known to be less toxic than lithocholic acid when they accumulate in the enterohepatic circulation.

We report here the structure of a novel dihydroxy bile acid, 15{alpha}-hydroxylithocholic acid, which is a major biliary bile acid in the wombat (Vombatus ursinus), a common Australian marsupial. We have confirmed its structure by direct synthesis of this bile acid from CDCA. The 15{alpha}-hydroxylithocholic acid occurs in bile as its taurine conjugate, which was also synthesized and shown to have identical chromatographic and spectroscopic properties as the compound isolated from bile.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Biological material
Wombat bile was obtained by aspiration of the gallbladder at autopsy from two wombats that died as a result of being struck by automobiles. The wombat cadavers had been transported to the Healesville Sanctuary (Victoria, Australia), where the autopsy disclosed no tissue abnormalities other than those caused by trauma. Bile samples were diluted in three volumes of isopropanol and mailed to the laboratory of T.I. for analysis.

Chemistry: standard analytical methods
Melting point (mp) values were determined on a micro hot-stage apparatus and are uncorrected. Infrared (IR) spectra were obtained in KBr discs on a Jasco FT-IR 4100 spectrometer (Tokyo, Japan). 1H- and 13C-NMR spectra were obtained on a JEOL JNM-EX 270 FT instrument at 270 and 68.8 MHz, respectively; in this report, only values for critical functionalities are given. EI low-resolution (LR) mass spectra were determined on a JEOL JMS-303 mass spectrometer at 70 eV. High-resolution (HR) mass spectra were measured using a JEOL LCmate double-focusing magnetic mass spectrometer equipped with an EI or fast-atom bombardment (FAB) probe under the positive ion mode. HR-MS spectra were also obtained on a JEOL JMS-700 mass spectrometer with an EI or FAB probe under the positive ion mode. Normal-phase TLC was performed on precoated silica gel plates (0.25 mm layer thickness; E. Merck, Darmstadt, Germany) using hexane-ethyl acetate-acetic acid mixtures (40:60:1–60:40:1, v/v/v) as the developing solvent. Reverse-phase TLC was carried out on precoated RP-18F254S plates using methanol-water-acetic acid mixtures (90:10:1, v/v/v) as the developing solvent. Sep-Pak Vac tC18 cartridges (adsorbent weight, 5 g) for solid-phase extraction were purchased from Waters Associates (Milford, MA); they were washed successively with 50 ml of methanol and 50 ml of distilled water before use.

Bile acid standards
CDCA was kindly supplied by Mitsubishi Pharma (Osaka, Japan). Cholyl taurine, chenodeoxycholyl taurine, ursodeoxycholyl taurine, deoxycholyl taurine, and lithocholyl taurine were from our laboratory collection.

HPLC with an evaporative light-scattering detector
The apparatus used was a Jasco HPLC system (two PU-2085 high-pressure pumps, an MX-2080-32 solvent mixing module, and an HG-980-50 degasser; Tokyo, Japan) equipped with a Shimadzu C-R8A data-processing system (Kyoto, Japan). A Capcell Pak-type MGII column [250 mm x 3.0 mm inner diameter (ID); particle size, 5 µm; Shiseido, Tokyo, Japan] was used for analytical purposes. The column temperature was kept at 37°C using a Sugai u-620-type 30V column heater (Wakayama, Japan). An Alltech 2000ES evaporative light-scattering detector (Deerfield, IL) was used; the flow rate of purified compressed air as a nebulizing gas was 1.6 l/min, and the temperature of the heated drift tube was 82°C.

For simultaneous separation of unconjugated, glycine-amidated, and taurine-amidated bile acids, an isocratic elution mode was used according to the procedure of Roda et al. (13). The mobile phase used was 65% (v/v) aqueous methanol containing 15 mM ammonium acetate adjusted to pH 5.4 with acetic acid, and the flow rate was kept at 400 µl/min. Figure 1 shows the HPLC result that was obtained; the identities of individual peaks are discussed in Results.


Figure 1
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  		Fig. 1. HPLC-evaporative light-scattering detection profile of the bile acids of the common wombat. For each peak (A–G), the compound, its retention time, and its percentage of total biliary bile acids are given. Peak A, unknown (9.2 min), 2.3%; peak B, unknown (13.1 min), 2.3%; peak C, 3{alpha},15{alpha}-dihydroxy-5ß-chol-24-oyl taurine (20.1 min), 11.2%, peak D, chenodeoxycholyl taurine (24.0 min), 77.9%; peak E, unknown (26.8 min), 2.1%; peak F, unknown (44.6 min), 1.7%; peak G, lithocholyl taurine (53.3 min), 2.5%.

 
Isolation of a major, unidentified bile acid from the bile of wombats
The bile of the wombats was diluted with isopropanol (~10 ml) and filtered, and the filtrate was evaporated under a nitrogen stream at 35°C. The residue was dissolved in methanol-water (1:9) (5 ml) and then applied to a preconditioned Sep-Pak tC18 cartridge (400 mg; Waters). After the cartridge was washed successively with water (2 ml) and 20% methanol (2 ml), the bile acid fraction was eluted with methanol. The methanol eluate was evaporated under a nitrogen stream at 35°C; the residue was then dissolved in 200 µl of methanol, and its individual bile acids were isolated by preparative HPLC. The apparatus consisted of a Jasco Gulliver series HPLC system (two PU-980 high-pressure pumps, an HG-980-31 solvent mixing module, and an HG-980-50 degasser). Reverse-phase chromatographic separation was carried out by stepwise gradient elution on a Capcell Pak C18-type MGII column (5 µm, 250 mm x 10 mm ID) using methanol-water mixtures as the mobile phase. The methanol composition was gradually increased at a flow rate of 2 ml/min as follows: 48% (0–15 min) -> 50% (15.1–30 min) -> 52% (30.1–45 min) -> 54% (45.1–60 min). The 52% methanol fraction, which contained compound C (the unknown bile acid), was evaporated under a nitrogen stream at 35°C.

LC-ESI-MS of wombat bile components
LC-MS analyses of wombat bile components were performed on a JEOL JMS-LCmate double focusing magnetic mass spectrometer equipped with an ESI probe using the negative ion mode. Chromatographic separation was carried out on a YMC Pack Pro C18 column (3 µm, 100 x 2.0 mm ID; 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 200 µl/min. The mass detector was set to the following conditions: needle voltage, –2.5 kV; orifice 1 temperature, 150°C; desolvating plate temperature, 250°C; ring lens voltages, 30 V/100 V.

LC-ESI-MS/MS of compound C
Negative ion LC-ESI-MS/MS spectra of compounds C and 1d (see below) were obtained on a Finnigan LTQ (Thermo Fisher Scientific, Inc., Waltham, MA) equipped with a Paradigm MS4 HPLC system (AMR, Inc., Tokyo, Japan). Chromatographic separation was carried out on a TSKgel ODS-100V (5 µm, 150 x 2.0 mm ID) using 5 mM ammonium acetate (pH 6)-acetonitrile mixtures as the mobile phase. A linear gradient was used: 30% CH3CN (0 min) -> 80% CH3CN (30 min); the flow rate was kept constant at 200 µl/min. The mass detector was set as follows: capillary temperature, 270°C; sheath gas flow rate, 50 arbitrary units; auxiliary gas flow rate, 5 arbitrary units; source voltage, ±4 kV; capillary voltage, ±30 V; tube lens offset voltage, ±100 V.

Synthesis of methyl 3{alpha}-cathyloxy-5ß-chol-7-en-24-oate (4)
The synthetic scheme used to prepare 15{alpha}- and 15ß-hydroxylithocholic acid (and their methyl esters) is shown in Fig. 2 (compounds 1–8).


Figure 2
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  		Fig. 2. Synthetic route to epimeric 3{alpha},15{xi}-dihydroxy-5ß-cholan-24-oic acids (1 and 2) from chenodeoxycholic acid 3-cathylate methyl ester (3).

 
To a magnetically stirred solution of methyl chenodeoxycholate 3-cathylate (3; 4.4 g) in pyridine (50 ml), phosphoryl chloride (15 ml) was slowly added with ice bath cooling. After being allowed to stand overnight at room temperature, the mixture was poured into ice water (0°C) and extracted with CH2Cl2. The combined CH2Cl2 extract was washed with 10% HCl and then with water to neutrality, dried with Drierite, and evaporated to an oily residue, which, when treated with CH2Cl2-methanol, crystallized as colorless crystals: yield, 3.6 g (86%); mp 119–123°C [literature value (14) mp 120–121°C]. IR (KBr) {nu}maxcm–1; 1,738 (C=O). 1H-NMR (CDCl3) {delta}; 0.54 (3H, s, 18-CH3), 0.87 (3H, s, 19-CH3), 0.93 (3H, d, J = 6.2 Hz, 21-CH3), 1.29 (3H, t, J = 7.0 Hz, COOCH2CH3), 3.67 (3H, s, -COOCH3), 4.16 (2H, m, COOCH2CH3), 4.58 (1H, brm, 3ß-H), 5.09 (1H, m, 7-H). 13C-NMR (CDCl3) {delta}; 11.8 (C-18), 14.2 (COOCH2CH3), 18.4 (C-21), 24.3 (C-19), 51.4 (COOCH3), 63.5 (COOCH2CH3), 77.3 (C-3), 115.2 (C-7), 137.2 (C-8), 154.6 (COOCH2CH3), 174.7 (C-24). LR-MS (EI) m/z: 370 (M-CtOH, 100%), 355 (M-CtOH-CH3, 49%), 255 [M-CtOH-CH3-side chain (S.C.)-ring D, 32%], 228 (M-CtOH-S.C.-part of ring D, 11%), 213 (M-CtOH-S.C.-ring D, 21%).

Synthesis of 3{alpha}-hydroxy-5ß-chol-7-en-24-oic acid (5a) and its methyl ester (5b)
The 3{alpha}-cathyloxy-{Delta}7 ester 4 (2.65 g) was hydrolyzed using 5% methanolic KOH (35 ml), followed by acidification with H2SO4 and recrystallization from methanol. The {Delta}7-unsaturated acid 5a was isolated as colorless needles: yield, 1.98 g (92%); mp 183–185°C [literature value (14) mp 183–184°C]. IR (KBr) {nu}maxcm–1; 1,708 (C=O), 3,352 (OH). 1H-NMR (CDCl3) {delta}; 0.55 (3H, s, 18-CH3), 0.86 (3H, s, 19-CH3), 0.95 (3H, d, J = 6.2 Hz, 21-CH3), 3.63 (1H, brm, 3ß-H), 5.10 (1H, m, 7-H). 13C-NMR (CDCl3) {delta}; 11.9 (C-18), 18.4 (C-21), 24.5 (C-19), 71.4 (C-3), 115.4 (C-7), 137.2 (C-8), 179.3 (C-24). LR-MS (EI) m/z: 374 (M, 25%), 356 (M-H2O, 100%), 341 (M-H2O-CH3, 69%), 302 (16%), 255 [M-H2O-S.C., 32%], 228 (M-H2O-part of ring D, 14%), 213 (M-H2O-S.C.-ring D, 30%). HR-MS (EI), calculated for 374.2821 [M]; found m/z, 374.2799.

The corresponding {Delta}7 ester 5b, prepared from 5a by the usual methanol and p-toluenesulfonic acid method, was recrystallized from methanol as colorless thin plates: yield, 60%; mp 88–92°C [literature value (14) mp 104–105°C]. IR (KBr) {nu}maxcm–1; 1,614 (C=C), 1,734 (C=O), 3,017 (=C-H), 3,337 (OH). 1H-NMR (CDCl3) {delta}; 0.54 (3H, s, 18-CH3), 0.85 (3H, s, 19-CH3), 0.94 (3H, d, J = 5.9 Hz, 21-CH3), 3.62 (1H, brm, 3ß-H), 3.67 (3H, s, -COOCH3), 5.10 (1H, m, 7-H). 13C-NMR (CDCl3) {delta}; 11.9 (C-18), 18.4 (C-21), 24.4 (C-19), 51.4 (COOCH3), 71.3 (C-3), 115.4 (C-7), 137.2 (C-8), 174.7 (C-24). LR-MS (EI) m/z: 388 (M, 27%), 370 (M-H2O, 100%), 316 (M-H2O-ring A, 14%), 355 (M-H2O-CH3, 75%), 255 (M-H2O-S.C., 42%), 228 (M-H2O-S.C.-part of ring D, 18%), 213 (M-H2O-S.C.-ring D, 41%). HR-MS (EI), calculated for C25H40O3: 388.2977; found m/z, 388.2974.

Synthesis of methyl 3{alpha}-hydroxy-5ß-chol-14-en-24-oate (6)
A solution of the {Delta}7 ester 5b (300 mg) in dry CHCl3 (10 ml) was prepared, and dry HCl gas was bubbled through it for 2 h in an ice bath. The CHCl3 solution was washed successively with water, with 5% NaHCO3 solution, and then again with water to neutrality. It was then dried with Drierite and evaporated to an oily residue, which, according to capillary GC analysis, consisted of a mixture of three components in an approximate ratio of 17:44:39 (%). The mixture was chromatographed on a column of 25% AgNO3-impregnated silica gel (30 g) (15). Elution with hexane-EtOAc (4:1 -> 7:3, v/v) provided three well-separated fractions. The less polar fraction was identified as methyl 3{alpha}-hydroxy-5ß-chol-8(9)-en-24-oate (9; Fig. 3 ), which resisted crystallization attempts: yield, 36 mg (12%); viscous oil [literature value (16)] mp 116–117°C). IR (KBr) {nu}maxcm–1; 1,737 (C=O), 3,420 (OH). 1H-NMR (CDCl3) {delta}; 0.87 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 0.99 (3H, s, 19-CH3), 3.59 (1H, brm, 3ß-H), 3.66 (3H, s, -COOCH3). 13C-NMR (CDCl3) {delta}; 19.3 (CH3), 23.6 (CH3), 28.6 (CH3), 51.3 (COOCH3), 70.9 (C-3), 130.1 and 131.7 (C-8 and C-9, respectively, or vice versa), 174.6 (C-24). LR-MS (EI) m/z: 388 (M, 27%), 370 (M-H2O, 100%), 355 (M-H2O-CH3, 59%), 316 (79%), 273 (M-S.C., 18%), 255 (M-H2O-S.C., 83%), 229 (M-H2O-S.C.-part of ring D, 17%), 213 (M-H2O-S.C.-ring D, 26%). HR-MS (EI), calculated for C25H40O3, 388.2977; found m/z, 388.2981.


Figure 3
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  		Fig. 3. Left: Chemical structures of epimeric 3{alpha},15{xi}-dihydroxy-5ß-cholan-24-oic acids (1a and 2a) and the glycine (1c) and taurine (1d) N-acylamidates (conjugates) of 1a. Right: Chemical structures of methyl 3{alpha}-hydroxy-5ß-chol-8(9)-en-24-oate (9) (above) and 3{alpha}-hydroxy-5ß-chol-8(14)-en-24-oate (10) (below), side products formed by the attempted acid-catalyzed dehydration procedure of Yamasaki and colleagues (14, 22) on compound 5b.

 
Continued elution gave a homogeneous viscous oil, which was characterized as methyl 3{alpha}-hydroxy-5ß-chol-8(14)-en-24-oate (10; Fig. 3): yield, 115 mg (38%). IR (KBr) {nu}maxcm–1; 1,739 (C=O), 3,393 (OH). 1H-NMR (CDCl3) {delta}; 0.80 (3H, s, 18-CH3), 0.83 (3H, s, 19-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 3.64 (1H, brm, 3ß-H), 3.66 (3H, s, -COOCH3). 13C-NMR (CDCl3) {delta}; 18.0 (C-18), 18.6 (C-21), 23.6 (C-19), 51.4 (COOCH3), 127.0 (C-8), 141.5 (C-14), 174.7 (C-24). LR-MS (EI) m/z: 388 (M, 35%), 370 (M-H2O, 100%), 355 (M-H2O-CH3, 78%), 316 (13%), 255 (M-H2O-S.C., 40%), 229 (M-H2O-S.C.-part of ring D, 23%), 215 (46%), 213 (M-H2O-S.C.-ring D, 39%). HR-MS (EI), calculated for C25H40O3, 388.2977; found m/z, 388.2993.

The most polar fraction was crystallized from aqueous ethanol to give the desired methyl 3{alpha}-hydroxy-5ß-chol-14-en-24-oate (6) as colorless amorphous solids; yield, 92 mg (31%); mp 140–141°C [literature value (14) mp 140–141°C]. IR (KBr) {nu}maxcm–1; 1,740 (C=O), 3,048 (=C-H), 3,255 (OH). 1H-NMR (CDCl3) {delta}; 0.89 (3H, s, 18-CH3), 0.92 (3H, d, J = 5.9 Hz, 21-CH3), 0.93 (3H, s, 19-CH3), 3.62 (1H, brm, 3ß-H), 3.67 (3H, s, -COOCH3), 5.15 (1H, brs, 15-H). 13C-NMR (CDCl3) {delta}; 16.8 (C-18), 18.5 (C-21), 23.1 (C-19), 51.5 (COOCH3), 71.9 (C-3), 116.9 (C-15), 155.4 (C-14), 174.8 (C-24). LR-MS (EI) m/z: 388 (M, 9%), 370 (M-H2O, 21%), 355 (M-H2O-CH3, 11%), 273 (M-S.C., 57%), 255 (M-H2O-S.C., 100%), 208 (part of ring C+ring D+CH3+S.C., 13%). HR-MS (EI), calculated for C25H40O3, 388.2977; found m/z, 388.2961.

Synthesis of 3{alpha},15{alpha},24-trihydroxy-5ß-cholane (7)
To a stirred solution of the {Delta}14 ester (6) (30 mg) in dry tetrahydrofuran (THF) (900 µl), a solution of BH3/THF (1.0 mol/l, 390 µl) was added gradually, and the mixture was stirred at room temperature under a stream of N2. After 5 h, 3 N NaOH (300 µl) and then 30% H2O2 (300 µl) were added with ice-bath cooling, and the mixture was stirred overnight at room temperature. The resulting solution was acidified with 10% HCl, and the reaction product was extracted with EtOAc. The combined EtOAc layers were washed with saturated brine and evaporated to dryness. The oily residue was chromatographed on a reverse-phase C18-bonded silica gel (10 g; particle size, 50 µm). Elution with methanol-water (7:3, v/v) afforded the title compound 7, which was recrystallized from EtOAc-hexane as colorless amorphous solids: yield, 21 mg (75%); mp 102–105°C. IR (KBr) {nu}maxcm–1; 3,340 (OH). 1H-NMR (CDCl3) {delta}; 0.68 (3H, s, 18-CH3), 0.92 (3H, d, J = 7.0 Hz, 21-CH3), 0.94 (3H, s, 19-CH3), 3.61 (2H, t, J = 6.6 Hz, 24-H2), 3.63 (1H, brm, 3ß-H), 3.94 (1H, brm, 15ß-H). 13C-NMR (CDCl3) {delta}; 13.3 (C-18), 18.4 (C-21), 23.4 (C-19), 63.5 (C-24), 71.8 (C-3), 73.8 (C-15). LR-MS (EI) m/z: 360 (45%, M-H2O), 342 (96%, M-2H2O), 327 (M-2H2O-CH3, 44%), 288 (M-H2O-ring A, 30%), 273 (M-H2O-S.C., 86%), 255 (M-2H2O-S.C., 100%) 217 (ring A+B+C, 62%). HR-MS (FAB+), calculated for C24H42O3Na [M+Na], 401.3032; found m/z, 401.3045.

Synthesis of 3,15-dioxo-5ß-cholan-24-oic acid (8a) and its methyl ester (8b)
Jones reagent (500 µl) was added gradually to a solution of the 3{alpha},15{alpha},24-triol 7 (30 mg) in acetone (4 ml) under 10°C, and the mixture was stirred for 30 min at room temperature. Methanol (500 µl) was added, and the oxidation product was extracted with EtOAc. The combined EtOAc extracts were washed with saturated brine, dried over Drierite, and evaporated to dryness. The oily residue was chromatographed using a column of C18-bonded silica gel (10 g; particle size, 50 µm). Elution with methanol-water (7:3, v/v) afforded the title dioxo acid 8a, which was recrystallized from EtOAc-hexane as colorless amorphous solids: yield, 22 mg (72%); mp 165–166°C. IR (KBr) {nu}maxcm–1; 1,705 (C=O, ketone), 1,736 (C=O, carboxyl group). 1H-NMR (CDCl3) {delta}; 0.78 (3H, s, 18-CH3), 1.02 (3H, d, J = 5.9 Hz, 21-CH3), 1.03 (3H, s, 19-CH3). 13C-NMR (CDCl3) {delta}; 13.0 (C-18), 18.5 (C-21), 22.5 (C-19), 177.9 (C-24), 212.9 (C-3), 214.9 (C-15). LR-MS (EI) m/z: 388 (M, 61%), 370 (19%), 318 (M-ring A, 10%), 287 (M-S.C., 44%), 259 (M-S.C.-part of ring D, 48%), 232 (M-S.C.-ring D, 15%), 215 (16%), 197 (S.C.+ring D+CH3, 100%). HR-MS (EI), calculated for C24H36O4 [M], 388.2614; found m/z, 388.2587.

The free acid 8a, reesterified with methanol and p-toluenesulfonic acid and processed by the usual work-up, yielded the corresponding methyl ester. Recrystallization from EtOAc-hexane gave the dioxo ester 8b as colorless amorphous solids: yield, 91%; mp 110–112°C. IR (KBr) {nu}maxcm–1; 1,713 (C=O, ketone), 1,735 (C=O, ester). 1H-NMR (CDCl3) {delta}; 0.78 (3H, s, 18-CH3), 1.00 (3H, d, J = 6.2 Hz, 21-CH3), 1.03 (3H, s, 19-CH3), 3.67 (3H, s, COOCH3). 13C-NMR (CDCl3) {delta}; 12.9 (C-18), 18.5 (C-21), 22.5 (C-19), 51.6 (COOCH3), 174.1 (C-24), 212.8 (C-3), 214.9 (C-15). LR-MS (EI) m/z: 402 (M, 45%), 387 (M-CH3, 34%), 371 (20%) 329 (16%), 287 (M-S.C., 100%), 259 (M-S.C.-part of ring D, 54%), 211 (S.C.+CH3+ring D, 33%). HR-MS (EI), calculated for C25H38O4 [M+], 402.2770; found m/z, 402.2762.

Synthesis of methyl 3{alpha},15{alpha}-dihydroxy-5ß-cholan-24-oate (1b) and its 15ß-epimer (2b)
tert-Butylamineborane complex (30 mg) was added to a stirred solution of the 3,15-dioxo ester 8b (30 mg) in CH2Cl2 (3 ml), and the mixture was refluxed overnight. After cooling the mixture, 10% HCl (900 µl) was added, and the solution was stirred for 30 min. The CH2Cl2 layer was washed with 5% NaHCO3 and water, dried over Drierite, and evaporated to dryness. The oily residue, which consisted essentially of two components on normal-phase TLC, was chromatographed on a column of silica gel (10 g). Elution with EtOAc-hexane (1:1–3:2, v/v) provided two well-separated fractions. The less polar fraction was identified as methyl 3{alpha},15ß-dihydroxy-5ß-cholanoate (2b), which recrystallized from aqueous methanol as colorless amorphous solids: yield, 17 mg (56%); mp 64–67°C. IR (KBr) {nu}maxcm–1; 1,739 (C=O), 3,409 (OH). 1H-NMR (CDCl3) {delta}; 0.92 (3H, d, J = 5.7 Hz, 21-CH3), 0.93 (3H, s, 19-CH3), 0.96 (3H, s, 18-CH3), 3.64 (1H, brm, 3ß-H) 3.67 (3H, m, -COOCH3), 4.19 (1H, t, J = 5.7 Hz, 15{alpha}-H). 13C-NMR (CDCl3) {delta}; 14.6 (C-18), 18.2 (C-21), 23.3 (C-19), 51.5 (COOCH3), 70.2 (C-15), 71.7 (C-3), 174.6 (C-24). LR-MS (EI) m/z: 388 (M-H2O, 59%), 370 (M-2H2O, 42%), 355 (M-2H2O-CH3, 22%), 273 (M-H2O-S.C., 34%), 255 (M-2H2O-S.C., 33%), 217 (ring A+B+C, 43%), 208 (100%). HR-MS (EI), calculated for C25H40O3, 388.2977 [M]; found m/z, 388.2976.

The more polar fraction was characterized as the desired 3{alpha},15{alpha}-dihydroxy ester 1b, which resisted crystallization attempts: yield, 10 mg (33%). IR (KBr) {nu}maxcm–1; 1,737 (C=O), 3,350 (OH). 1H-NMR (CDCl3) {delta}; 0.68 (3H, s, 18-CH3), 0.90 (3H, d, J = 5.7 Hz, 21-CH3), 0.93 (3H, s, 19-CH3), 3.62 (1H, brm, 3ß-H), 3.66 (3H, m, -COOCH3), 3.94 (1H, brm, 15ß-H). 13C-NMR (CDCl3) {delta}; 13.3 (C-18), 18.1 (C-21), 23.4 (C-19), 51.5 (COOCH3), 71.7 (C-3), 73.6 (C-15), 174.6 (C-24). LR-MS (EI) m/z: 388 (M-H2O, 72%), 370 (M-2H2O, 42%), 355 (M-2H2O-CH3, 50%), 273 (M-H2O-S.C., 98%), 255 (M-2H2O-S.C., 88%), 217 (ring A+B+C, 41%), 208 (100%). HR-MS (EI), calculated for C25H40O3, 388.2977 [M]; found m/z, 388.2969.

Synthesis of 3{alpha},15{alpha}-dihydroxy-5ß-cholan-24-oic acid (1a) and its 15ß-epimer (2a)
The 3{alpha},15{alpha}-dihydroxy ester 1b (50 mg) was refluxed in 5% methanolic KOH (1 ml) for 1 h. Solvent was removed by evaporation, and the residue was dissolved in water and acidified with 10% H2SO4 with stirring and ice-bath cooling. The precipitate was collected by filtration, washed with water, and recrystallized from EtOAc as colorless amorphous solids of 3{alpha},15{alpha}-dihydroxy acid 1a (analytically pure): yield, 46 mg (95%); mp 206–208°C. IR (KBr) {nu}maxcm–1; 1,703 (C=O), 3,245 (OH). 1H NMR (CD3OD) {delta}; 0.71 (3H, s, 18-CH3), 0.94 (3H, d, J = 5.9 Hz, 21-CH3), 0.96 (3H, s, 19-CH3), 3.54 (1H, brm, 3ß-H), 3.84 (1H, brm, 15ß-H). 13C NMR (CD3OD) {delta}; 13.7 (C-18), 18.6 (C-21), 21.8 (C-11), 24.0 (C-19), 27.6 (C-6), 28.3 (C-7), 31.2 (C-2), 31.9 (C-22), 32.2 (C-23), 35.7 (C-10), 36.1 (C-20), 36.6 (C-1), 36.9 (C-8), 37.2 (C-4), 41.6 (C-16), 41.7 (C-12), 42.0 (C-5), 43.5 (C-9) 44.9 (C-13), 54.8 (C-17), 64.0 (C-14), 72.4 (C-3), 74.2 (C-5) 178.0 (C-24). LR-MS (EI) m/z: 374 (M-H2O, 36%), 356 (M-2H2O, 100%), 341 (M-2H2O-CH3, 43%), 302 (M-H2O-ring A, 32%), 273 (M-H2O-S.C, 48%), 255 (M-2H2O-S.C., 60%), 217 (ring A+B+C, 51%). HR-MS (FAB+), calculated for C24H40O4Na, 415.2824 [M+Na]; found m/z, 415.2788.

The 3{alpha},15ß-dihydroxy ester 2b (50 mg) was hydrolyzed with 5% methanolic KOH (1 ml) and processed as described for the preparation of 1a to yield the crude acid. Recrystallization from EtOAc gave the 3{alpha},15ß-dihydroxy acid 2a as colorless amorphous solids: yield, 44 mg (91%); mp 119–121°C [literature value (17) mp 185.5–186.5°C]. IR (KBr) {nu}maxcm–1; 1,710 (C=O), 3,321 (OH). 1H NMR (CD3OD) {delta}; 0.94 (3H, s, 18-CH3), 0.95 (3H, d, J = 7.6 Hz, 21-CH3), 0.98 (3H, s, 19-CH3), 3.53 (1H, brm, 3ß-H), 4.16 (1H, brm, 15{alpha}-H). 13C NMR (CD3OD) {delta} ; 15.2 (C-18), 18.8 (C-21), 21.8 (C-11), 24.0 (C-19), 26.8 (C-7), 28.3 (C-6), 31.2 (C-16), 32.0 (C-23), 32.3 (C-2), 33.0 (C-20), 35.9 (C-10), 36.4 (C-8), 36.6 (C-22), 37.3 (C-1), 42.1 (C-4), 42.2 (C-9), 42.9 (C-12), 43.6 (C-5 and C-13), 57.6 (C-17), 62.4 (C-14), 70.6 (C-15), 72.5 (C-3), 178.2 (C-24). LR-MS (FAB) m/z: 374 (M-H2O, 39%), 356 (M-2H2O, 54%), 341 (M-2H2O-CH3, 27%), 302 (M-H2O-ring A, 11%), 273 (M-H2O-S.C., 34%), 263 (38%), 255 (M-2H2O-S.C., 54%), 217 (M-H2O-S.C.-ring D, 100%). HR-MS (FAB+), calculated for C24H40O4Na, 415.2824 [M+Na]; found m/z, 415.2802.

Synthesis of 3{alpha},15{alpha}-dihydroxy-5ß-cholan-24-oyl glycine (1c; sodium salt)
To a magnetically stirred solution of 3{alpha},15{alpha}-dihydroxy acid 1a (30 mg) in dry dimethylformamide (3 ml), glycine methyl ester hydrochloride (25 mg), diethylphosphorocyanide (30 µl), and triethylamine (125 µl) were added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was extracted with EtOAc, and the combined extracts were washed with saturated brine and evaporated to dryness. The residue was then refluxed in 5% methanolic NaOH (4 ml) for 1 h, and the solution was adjusted to pH 9 by 10% HCl. Most of the solvent was evaporated under reduced pressure, and the residue was dissolved in water (5 ml). The aqueous solution was loaded onto a Sep-Pak Vac tC18 cartridge, which was washed successively with water (20 ml), 30% methanol (20 ml), and 40% methanol (20 ml). Elution with 50% methanol (60 ml) gave the glycine conjugate of the title compound (1c), after the solvent was evaporated under reduced pressure. The residue was recrystallized from methanol-Et2O in the form of colorless amorphous solids: yield, 22 mg (61%); mp 170–173°C. IR (KBr) {nu}maxcm–1; 1,593 (C=O), 3,394 (OH). 1H-NMR (CD3OD) {delta}; 0.71 (3H, s, 18-CH3), 0.96 (3H, d, J = 5.4 Hz, 21-CH3), 0.96 (3H, s, 19-CH3), 3.53 (1H, brm, 3ß-H), 3.74 (2H, s, -NHCH2-) 3.84 (1H, brm, 15ß-H). 13C-NMR (CD3OD) {delta}; 13.7 (C-18), 18.8 (C-21), 21.8 (C-11), 24.0 (C-19), 27.7 (C-6), 28.3 (C-7), 31.2 (C-2), 33.0 (C-22), 34.1 (C-23), 35.7 (C-10), 36.4 (C-20), 36.6 (C-1), 37.0 (C-8), 37.2 (C-4), 41.6 (C-16), 41.7 (C-12), 42.0 (C-5), 43.6 (C-9), 44.5 (-CH2N-), 44.9 (C-13), 54.9 (C-17), 64.1 (C-14), 72.5 (C-3), 74.2 (C-15), 176.2, 176.3 (C-24, -COONa). LR-MS (FAB+) m/z: 494 (M+Na, 100%). HR-MS (FAB+), calculated for C26H42O5NNa2, 494.2858 [M+Na]; found m/z, 494.2832.

Synthesis of 3{alpha},15{alpha}-dihydroxy-5ß-cholan-24-oyl taurine (1d; sodium salt)
A suspension of 1a (50 mg), powdered taurine (40 mg), diethylphosphorocyanide (40 µl), and triethylamine (75 µl) in dry dimethylformamide (5 ml) was stirred at room temperature. After 1 h, the mixture was adjusted to pH 12–14 with 1 M NaOH and then to pH 8–9 with 10% HCl. The resulting solution was diluted with water (45 ml), passed through a Sep-Pak Vac tC18 cartridge (50 ml), and washed successively with water (20 ml) and 20% methanol (20 ml). Elution with 50% methanol (60 ml) gave the desired taurine conjugate 1d, after evaporation of the solvent under reduced pressure. The residue was recrystallized from methanol-Et2O as colorless amorphous solids of 1d: yield, 40 mg (59%); mp 157–160°C. IR (KBr) {nu}maxcm–1; 1,636 (C=O), 3,421 (OH). LR-MS (FAB+) m/z: 544 (M+Na, 100%). HR-MS (FAB+), calculated for C26H44O6NSNa2, 544.2685 [M+Na]; found m/z, 544.2665.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Synthesis of 15{alpha}-hydroxylithocholic acid
Figure 1 shows a representative HPLC-evaporative light-scattering detection chromatogram of the bile acid composition in the gallbladder bile of the wombat. Identification of the major peaks was made by a direct comparison with authentic reference compounds available in our laboratory. The largest peak, designated D (78% of total bile acids), was proven to be taurine-conjugated CDCA. The structure of the second most abundant component, peak C (11%), was tentatively identified as 15{xi}-hydroxylithocholyl taurine (3{alpha},15{xi}-dihydroxy-5ß-cholan-24-oyl taurine), based on the HPLC and LC-MS data (see below). Wombat bile also contained minor components labeled A, B, E, F, and G (total amounts, less than ~11%). Peak G (2.5%) was identified as taurine-conjugated lithocholic acid. LC-ESI-MS under negative ion mode exhibited a signal arising from a deprotonated molecule [M-H]– at m/z 498 for peak A, m/z 496 for peak B, m/z 498 for peak E, and m/z 480 for peak F. However, definitive identification of the minor conjugated bile acids present in wombat bile is beyond the scope of the present paper.

Identification of 15{alpha}-hydroxylithocholic acid (3{alpha},15{alpha}-dihydroxy-5ß-cholan-24-oic acid) in the biliary bile acids of the wombat was mentioned in a review by J. S. Pyrek (18) many years ago, but details were never published because of his untimely death. To verify the structure, in particular the position and the stereochemical configuration of hydroxyl groups as well as the nature of the N-acylamidated moiety in the side chain at C-24, we decided to undertake the synthesis of the two stereoisomeric 3{alpha},15{xi}-dihydroxy-5ß-cholan-24-oic acids (1a and 2a) and the glycine (1c) and taurine (1d) conjugates of 1a as authentic reference specimens (Fig. 3).

The formation of 3{alpha},15ß-dihydroxy-5ß-cholanoic acid (2a) from lithocholic acid by fungal enzymes was reported previously by two groups (17, 19–21). However, the preparation of the corresponding 15{alpha}-hydroxy epimer (1a) has hitherto been unreported. On attempting to repeat the preparation of 2a from lithocholic acid with Penicillium species ATCC 12556 (21), we encountered difficulties in isolating sufficient 2a to be adequate for chemical epimerization to 1a.

Our aim, therefore, was to develop an alternative synthesis of 1a and 2a, as summarized in Fig. 2. A key intermediate in our work was methyl 3{alpha}-hydroxy-{Delta}14-5ß-cholenoate (6). To prepare this compound, the 3{alpha}-cathyloxy-7{alpha}-hydroxy ester 3, prepared from CDCA (14), was converted to the 3{alpha}-cathyloxy-{Delta}7 ester 4 by an elimination reaction with phosphoryl chloride. Subsequent alkaline hydrolysis of 4, followed by the usual esterification of the resulting 3{alpha}-hydroxy-{Delta}7 acid 5a, gave the corresponding 3{alpha}-hydroxy-{Delta}7 ester 5b.

The most promising method reported in the literature for the preparation of the unsaturated {Delta}14 ester 6 from 5b on a substantial scale appeared to be the procedure of Yamasaki and colleagues (14, 22), in which acid-catalyzed isomerization of a double bond in 5b with dry HCl gas was reported to give 6, together with the {Delta}8(14) isomer 9. On repeating this work using modern instrumentation (NMR, MS, HPLC, and capillary GC) that was unavailable at the time of their efforts, we encountered difficulties that led us to investigate the reaction in detail. We found that the reaction product consisted of a mixture of three unsaturated compounds, {Delta}8 (9), {Delta}8(14) (10), and {Delta}14 (6) isomers, in an approximate ratio of 20:40:40 (%) as estimated by capillary GC. (The structure of compound 6 is shown in Fig. 2, and those of compounds 9 and 10 are shown in Fig. 3.) The three positional isomers were not resolved by conventional silica gel column chromatography but were efficiently separated by chromatography on 25% AgNO3-impregnated silica gels (15); compounds were eluted in the order {Delta}8 before {Delta}8(14) before {Delta}14 isomer. The individual isomers were distinguished by their 1H- and 13C-NMR spectra. The 13C-NMR spectra revealed that compounds 9 and 10 had a tetrasubstituted double bond, whereas compound 6 was trisubstituted. The chemical shifts of 13C signals in 6 and 10 were in good agreement with those reported in the literature (23). The remaining tetrasubstituted alkene 9, therefore, was deduced to have a double bond between C-8 and C-9 in the 5ß-steroid nucleus. The 13C chemical shifts of the olefinic carbons at C-8 (130.1 ppm) and C-9 (131.7 ppm) in 9 were similar to those reported (128.3 and 134.9 ppm, respectively) for 3{alpha}-hydroxy-5{alpha}-cholest-8-ene (24).

Hydroboration of the 3{alpha}-hydroxy-{Delta}14 ester 6 with B2H6/THF and subsequent treatment of the resulting alkylborane with H2O2/NaOH resulted in simultaneous hydroxylation at C-15 and C-24 to give 5ß-cholane 3{alpha},15{alpha},24-triol (7). Attempted selective oxidation of the hydroxyl group at the C-24 position in 7 with pyridinium chlorochromate, pyridinium dichromate, or Swern oxidation was unsuccessful, yielding complicated mixtures, even under milder conditions than are conventionally used. Hence, 7 was completely oxidized with Jones reagent to give the 3,15-dioxo acid 8a.

After esterification of 8a in the usual manner, reduction of the resulting 3,15-diketone 8b with tert-butylamineborane complex yielded a mixture of 3{alpha},15{alpha}-dihydroxy (1b) and 3{alpha},15ß-dihydroxy (2b) esters. The reduction was stereoselective, as the 3ß-hydroxy isomers (i.e., 3ß,15{alpha}- and 3ß,15ß-dihydroxyls) were not formed, in agreement with our previous work on this topic (25). The two stereoisomeric pairs were cleanly separated by column chromatography on silica gel to give 1b and 2b in isolated yields of 56% and 33%, respectively. The stereochemical nature of the 15-hydroxyl group in 1b and 2b was confirmed by 1H- and 13C-NMR. In the 1H-NMR spectra, the 18-methyl proton signal (0.96 ppm) in 2b is deshielded to a large extent by ~0.3 ppm, compared with that (0.64 ppm) of the parent compound methyl lithocholate, probably owing to quasi-1,3-diaxial interaction between the 18-methyl and 15ß-hydroxyl groups, whereas the corresponding 18-methyl signal (0.68 ppm) in 1b is barely shifted. The 3ß-H in both 1b and 2b showed essentially identical chemical shifts (3.62 and 3.64 ppm, respectively) and signal multiplicity (a broad multiplet). The chemical shifts and the signal multiplicity for the 15-proton signal in both isomers differed considerably from each other; the 15ß-H in 1b appeared at 3.94 ppm as a broad multiplet, whereas the 15{alpha}-H in 2b resonated at 4.19 ppm as a triplet (J, 5.4 Hz). The 1H-NMR data are consistent with those reported for epimeric 3{alpha},7{alpha},15{xi}-trihydroxy bile acids (25) and a 3{alpha},12{alpha},15ß-trihydroxy analog (21). On the other hand, the 13C-NMR chemical shifts of the C-24 free acid (2a) (see below) of 3{alpha},15ß-dihydroxy epimer 2b agreed completely with the previously described product obtained by microbiological transformation of lithocholic acid (17, 19, 20).

Alkaline hydrolysis of 1b followed by acidification afforded the desired 3{alpha},15{alpha}-dihydroxy-5ß-cholanoic acid (1a) in an overall yield of 2.3% starting from 3. The free 3{alpha},15ß-hydroxy acid 2a was also similarly obtained from the ester 2b. The mp (119–121°C) of the synthetic 2a differed markedly from that (186–187°C) reported by Kulprecha et al. (17). Although the reason for this discrepancy is unclear, the 13C-NMR spectral data are completely consistent with each other. Polymorphism in crystal habit is well known for C24 bile acids (26).

Synthesis of the taurine and glycine conjugates of 15{alpha}-hydroxylithocholic acid
N-Acylamidation of the C-24 carboxyl group in 1a with glycine methyl ester hydrochloride or freshly powdered taurine was effectively attained using diethyl phosphocyanide as a condensing reagent and triethylamine as a catalyst (27). After the condensation reaction, the resulting solutions were adjusted to appropriate pH with the treatment of acid and/or base (see Methods), and the desired glycine (1c) and taurine (1d) conjugates (as the sodium salts) were recovered efficiently by applying a reverse-phase prepacked cartridge (Sep-Pak Vac tC18) for solid-phase extraction.

Proof of structure of peak C in the HPLC result of wombat bile
As shown in Fig. 4 , LC-ESI-MS/MS of peak C under negative ion mode gave only one peak, corresponding to that of the deprotonated molecule [M-H]– at m/z 498. When the collision-induced dissociation spectrum of the signal as a precursor ion was measured, an intense peak appeared at m/z 480, corresponding to the elimination of one molecule of water from the [M-H]–, accompanied by minor fragment ions at m/z 432, 416, 386, 355, and 327. An essentially identical collision-induced dissociation spectrum was obtained for synthetic 1d.


Figure 4
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  		Fig. 4. Negative ion LC-ESI-MS/MS fragmentation pattern of synthetic 1d (left) and compound C (right). CID, collision-induced dissociation.

 
Compound C was isolated from a mixture of bile acids in wombat bile by preparative HPLC. Figure 5 shows the 1H-NMR spectra of isolated compound C, and Table 1 compiles the 1H and 13C signal assignments of compound C and 1d. The 1H- and 13C-NMR spectral patterns of both compounds were essentially identical. Particularly noteworthy were signals occurring at 2.97 ppm (t, J = 6.4 Hz, -CH2S-) and 3.60 ppm (t, J = 6.4 Hz, -CH2N-) by 1H-NMR, indicating the presence of a taurine-conjugate moiety (2, 27). As mentioned above, the 1H signals occurring at 3.53 and 3.83 ppm, owing to the 3ß-H and 15ß-H, respectively, also provide the confirmatory evidence for the structure of compound C as 15{alpha}-hydroxylithocholyl taurine.


Figure 5
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  		Fig. 5. 1H-NMR spectrum of compound C isolated from wombat bile. TMS, tetramethylsilane.

 

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  		TABLE 1. 1H- and 13C-NMR data for synthetic 1d and compound C

 
Biological aspects
Because of its geological history, the continent of Australia has a unique fauna. It is generally considered that Australia separated from what became Antarctica some 60 million years ago. Marsupials, which had originated in the land mass becoming South America, migrated via a land bridge connecting South America to the land mass eventually becoming Antarctica and, in turn, via a land bridge to the land mass that became Australia (28). Among these marsupials, the Australian opossum Trichosurus vulpecula was shown by Lee, Lester, and Pyrek (29) to have a novel bile acid, 1{alpha},3{alpha},7{alpha}-trihydroxy-5ß-cholan-24-oic acid.

The wombat, another Australian marsupial, is an herbivore and has a voluminous large intestine (30). Based on studies of fecal bile acid composition in numerous mammals, it is highly likely that in the distal intestine of the wombat, the taurine conjugate of CDCA, the major bile acid of the animal, undergoes bacterial deconjugation and 7-dehydroxylation to form lithocholic acid (31). We hypothesize that lithocholic acid is absorbed and returned to the liver, where it undergoes 15{alpha}-hydroxylation to form 15{alpha}-hydroxylithocholic acid. If this hypothesis is correct, the wombat resembles other rodents [mouse (9–12, 32), rat (33–38), and guinea pig (39, 40)], species in which lithocholic acid is efficiently hydroxylated at C-6, C-7, or both during hepatocyte transport. Sulfation (36) and glucuronidation (35, 37) of lithocholic acid also occur in these species, but to a much smaller extent. N-Acylamidation (mostly with taurine) of the dihydroxy and trihydroxy metabolites of lithocholic acid occurs, but their sulfates and glucuronides have not been reported. In contrast, in human (40, 41) and nonhuman primates [chimpanzee (42), baboon (43, 44), and rhesus monkey (45)], lithocholic acid is not hydroxylated but is excreted into bile as such and in part in sulfated form. The extent of sulfation is much less in the rhesus monkey and baboon than in human, explaining the toxicity of lithocholic acid when its precursor, CDCA, is administered chronically (46–49).

The proportion of 15{alpha}-hydroxylithocholic acid in the biliary bile acids of the wombat was more than four times greater than that of lithocholic acid, its probable precursor. The proportion of a given bile acid within the biliary bile acids depends on the size of its individual pool divided by the sum of the total bile acid pool. The size of the bile acid pool of any individual bile acid depends, in turn, on the magnitude of its input and of its intestinal conservation; intestinal conservation, which is responsible for the development of a recirculating bile acid pool, may be considered to amplify the input (3). For primary bile acids, input is from biosynthesis (from cholesterol); for secondary bile acids, input is from the absorption of newly formed molecules from the distal intestine. Intestinal conservation occurs by active and passive absorption. In the steady state, input is balanced by loss attributable to fecal excretion or biotransformation or both. For primary bile acids, biotransformation involves mostly bacterial 7-dehydroxylation; for secondary bile acids, biotransformation involves hepatocyte hydroxylation, as reported here. It is likely that the far larger pool of 15{alpha}-hydroxylithocholic acid compared with that of its precursor is the result of greater intestinal conservation of the dihydroxy bile acid, as well as its lack of conversion to any other bile acid. In the rabbit, the pool of deoxycholic acid is >20 times larger than that of cholic acid, its precursor (50).

For trihydroxy bile acids, bacterial 7{alpha}-dehydroxylation generates a 3{alpha},X-dihydroxy bile acid, where X denotes the additional hydroxylation site on CDCA. Thus, 7-deoxydihydroxy bile acids may arise either by hepatic hydroxylation of lithocholic acid or by bacterial 7-dehydroxylation of a trihydroxy bile acid (or both). The 7{alpha}-deoxy derivatives of 1ß,3{alpha},7{alpha}-trihydroxy acid (1ß,3{alpha}-dihydroxy) have been reported (51–53), as has that of 3{alpha},4ß,7{alpha}-trihydroxy acid (3{alpha},4ß-dihydroxy) (54, 55), but they are not known to be present in biliary bile acids in appreciable proportions. The 7-deoxy derivatives of hyocholic acid (3{alpha},6{alpha}-dihydroxy, hyodeoxycholic acid) and muricholic acid (3{alpha},6ß, murideoxycholic acid) are well known biliary bile acids, as is deoxycholic acid. As yet, the 7-deoxy derivatives of bile acids hydroxylated at C-5 or C-16 have not been reported.

We previously reported that 15{alpha}-hydroxylation occurs in swans, tree ducks, and geese, in whom 3{alpha},7{alpha},15{alpha}-trihydroxy-5ß-cholan-24-oic acid is a primary bile acid (2). We think it highly unlikely that 15{alpha}-hydroxylithocholic acid arose by bacterial 7{alpha}-dehydroxylation of this primary, trihydroxy bile acid, as it was not present in the biliary bile acids of the wombat. Additional supporting evidence for 15{alpha}-hydroxylithocholic acid originating from the hydroxylation of lithocholic acid is the concurrent occurrence of lithocholic acid (in taurine-conjugated form) itself in the biliary bile acids of the wombat.

Because metabolic studies were not performed, we cannot exclude the highly unlikely possibility that 15{alpha}-hydroxylithocholic acid is a primary bile acid (i.e., that it is formed from cholesterol in the hepatocyte). However, all primary bile acids (and bile alcohols) reported to date in healthy vertebrates have a hydroxy or oxo group at C-7.

Hydroxylation at C-15 of a bile acid sulfonate analog occurs in the hamster (56), and hydroxylation at C-15 of pregnanoic acids (C21 steroids that may also be considered bile acid homologs with a two carbon side chain) occurs in the rat (57). Thus, the enzyme(s) mediating 15-hydroxylation appear(s) to have evolved in parallel in multiple vertebrate species. The CYP isoforms involved in the hydroxylation of estradiol at C-15 have been identified as CYP 1A1, 1B1, and 3A4 (58). Whether these CYP isoforms and/or others are involved in the formation of 15{alpha}-hydroxylithocholic acid in the wombat remains to be determined.


   ACKNOWLEDGMENTS
 
This work was supported in part by the Nihon University Multidisciplinary Research Grant for 2007 and a Grant-in-Aid for Scientific Research from the Ministry of Education, Sciences, Sports, and Culture of Japan (Grant 19510223) for 2007–2008. Work at Kinki University was supported by a Sunbor Grant for the Suntory Institute for Bioorganic Research (2007) and a Grant-in-Aid for Scientific Research (C) (Grant 19590165) from the Japan Society for the Promotion of Science for 2007–2008. Work at the University of California, San Diego, was supported by National Institutes of Health Grant DK-64891.

Manuscript received July 27, 2007 and in revised form August 28, 2007 and in re-revised form September 4, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
  1. Moschetta, A., F. Xu, L. R. Hagey, G. P. van Berge-Henegouwen, K. J. van Erpecum, J. F. Brouwers, J. C. Cohen, M. Bierman, H. H. Hobbs, J. H. Steinbach, et al. 2005. A phylogenetic survey of biliary lipids in vertebrates. J. Lipid Res. 46: 2221–2232.[Abstract/Free Full Text]

  2. Kakiyama, G., T. Iida, T. Goto, N. Mano, J. Goto, T. Nambara, L. R. Hagey, C. D. Schteingart, and A. F. Hofmann. 2006. Identification of a novel bile acid in swans, tree ducks, and geese: 3{alpha},7{alpha},15{alpha}-trihydroxy-5ß-cholan-24-oic acid. J. Lipid Res. 47: 1551–1558.[Abstract/Free Full Text]

  3. Hofmann, A. F. 1994. Bile acids. In The Liver: Biology and Pathobiology. 3rd edition. I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. A. Schacter, and D. A. Shafritz, editors. Raven Press, New York. 677–718.

  4. Holsti, P. 1960. Cirrhosis of the liver induced in rabbits by gastric instillation of 3-monohydroxycholanic acid. Nature. 186: 250.[Medline]

  5. Fischer, C. D., N. S. Cooper, M. A. Rothschild, and E. H. Mosbach. 1974. Effect of dietary chenodeoxycholic acid and lithocholic acid in the rabbit. Am. J. Dig. Dis. 19: 877–886.[CrossRef][Medline]

  6. Palmer, R. H., and Z. Hruban. 1966. Production of bile duct hyperplasia and gallstones by lithocholic acid. J. Clin. Invest. 45: 1255–1267.[Medline]

  7. Zaki, F. G., J. B. Carey, F. W. Hoffbauer, and C. Nwokolo. 1967. Biliary reaction and choledocholithiasis induced in the rat by lithocholic acid. J. Lab. Clin. Med. 69: 737–748.[Medline]

  8. Palmer, R. H. 1976. Toxic effects of lithocholate on the liver and biliary tree. In The Hepatobiliary System. Fundamental and Pathological Mechanisms. W. Taylor, editor. Plenum Press, New York. 227–240.

  9. Zhang, J., W. Huang, M. Qatanani, R. M. Evans, and D. D. Moore. 2004. The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid- induced toxicity. J. Biol. Chem. 279: 49517–49522.[Abstract/Free Full Text]

  10. Kitada, H., M. Miyata, T. Nakamura, A. Tozawa, W. Honma, M. Shimada, K. Nagata, C. J. Sinal, G. I. Guo, F. J. Gonzalez, et al. 2003. Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity. J. Biol. Chem. 278: 17838–17844.[Abstract/Free Full Text]

  11. Stedman, C. A., G. R. Robertson, S. A. Coulter, and C. Liddle. 2004. Feed-forward regulation of bile acid detoxification by CYP3A4: studies in humanized transgenic mice. J. Biol. Chem. 279: 11336–11433.[Abstract/Free Full Text]

  12. Fickert, P., A. Fuchsbichler, H-U. Marschall, M. Wagner, G. Zollner, R. Krause, K. Zatloukal, H. Jaeschke, H. Denk, and M. Trauner. 2006. Lithocholic acid feeding induces segmental bile duct obstruction and destructive cholangitis in mice. Am. J. Pathol. 168: 410–422.[Abstract/Free Full Text]

  13. Roda, A., C. Cerrè, P. Simoni, C. Polimeni, C. Vaccari, and A. Pistillo. 1992. Determination of free and amidated bile acids by high-performance liquid chromatography with evaporative light-scattering mass detection. J. Lipid Res. 33: 1393–1402.[Abstract]

  14. Osawa, R., and K. Yamasaki. 1959. Dehydration of bile acids and their derivatives. VII. Isomerization of methyl 3{alpha}-hydroxy-{Delta}7-cholenate to methyl 3{alpha}-hydroxy-{Delta}14-cholenate. Bull. Chem. Soc. Jpn. 32: 1302–1306.[CrossRef]

  15. Vroman, H. E., and C. Cohen. 1967. Separation of sterol acetates by column and thin-layer chromatography. J. Lipid Res. 8: 150–152.[Abstract]

  16. Nakada, F. 1963. Dehydration of bile acids and their derivatives. XIII. Isomerization of chola-7,11-dienic acid derivatives to 7,9-diene compounds and dehydration of 3{alpha},12{alpha}-dihydroxy-chol-8(14)-enic acid. Steroids. 2: 403–416.[CrossRef]

  17. Kulprecha, S., T. Nihira, C. Shimomura, K. Yamada, N. Nilubol, T. Yoshida, and H. Taguchi. 1984. 15ß-Hydroxylation of lithocholic acid by Cunninghamella sp. Tetrahedron. 40: 2843–2846.[CrossRef]

  18. Pyrek, J. S. 1991. Mass spectrometry in the chemistry of natural products. In Modern Phytochemical Methods. N. H. Fischer, M. B. Isman, and H. A. Stafford, editors. Plenum Press, New York. 176–249.

  19. Jodoi, Y., T. Nihira, H. Naoki, Y. Yamada, and H. Taguchi. 1987. New trihydroxy bile acids produced by Cunninghamella blakesleeana ST-22 from lithocholic acid. Tetrahedron. 43: 487–492.

  20. Kulprecha, S., T. Nihira, K. Yamada, T. Yoshida, N. Nilubol, and H. Taguchi. 1985. Transformation of lithocholic acid to a new bile acid, 3{alpha},15ß-dihydroxy-5ß-cholanic acid by Cunninghamella blakesleeana St-22. Appl. Microbiol. Biotechnol. 22: 211–216.

  21. Carlström, K., D. N. Kirk, and J. Sjövall. 1981. Microbial synthesis of 1ß- and 15ß-hydroxylated bile acids. J. Lipid Res. 22: 1225–1234.[Abstract]

  22. Nakada, F., R. Osawa, and K. Yamasaki. 1961. Dehydration of bile acid and their derivatives. VIII. Synthesis of 3{alpha}-hydroxy-{Delta}8(14)-cholenic and 3{alpha}-hydroxy-{Delta}14-cholenic acids. A note on "ß"-cholenic acid. Bull. Chem. Soc. Jpn. 34: 538–541.[CrossRef]

  23. Iida, T., J. Goto, and T. Nambara. 1993. Carbon-13 NMR spectra of bile acid derivatives. III. Unsaturated 5ß-cholenoic acids. Magn. Reson. Chem. 31: 421–424.[CrossRef]

  24. Wilson, W. K., R. M. Sumpter, J. J. Warren, P. S. Rogers, B. Ruan, and G. J. Schroepfer, Jr. 1996. Analysis of unsaturated C27 sterols by nuclear magnetic resonance spectroscopy. J. Lipid Res. 37: 1529–1555.[Abstract]

  25. Iida, T., M. Hikosaka, G. Kakiyama, K. Shiraishi, C. D. Schteingart, L. R. Hagey, H-T. Ton-Nu, A. F. Hofmann, N. Mano, J. Goto, et al. 2002. Potential bile acid metabolites. XXV. Synthesis and chemical properties of stereoisomeric 3{alpha},7{alpha},16- and 3{alpha},7{alpha},15-trihydroxy-5ß-cholan-24-oic acids. Chem. Pharm. Bull. (Tokyo). 50: 1327–1334.[Medline]

  26. Giiuseppetti, G., and M. Paciotti. 1978. Polymorphism of chenodeoxycholic acid. Farmaco [Sci]. 33: 64–72.[Medline]

  27. Momose, T., T. Tsubaki, T. Iida, and T. Nambara. 1997. An improved synthesis of taurine- and glycine-conjugated bile acids. Lipids. 32: 775–778.[Medline]

  28. Macdougall, J. D. 1966. A Short History of Planet Earth. John Wiley & Sons, New York. 183–185.

  29. Lee, S. P., R. Lester, and J. S. Pyrek. 1987. Vulpecholic acid (1{alpha},3{alpha},7{alpha}-trihydroxy-5ß-cholan-24-oic acid): a novel bile acid from a marsupial, Trichosurus vulpecula. J. Lipid Res. 28: 19–31.[Medline]

  30. Stevens, C. E. 1988. Comparative Physiology of the Vertebrate Digestive System. Cambridge University Press, New York.

  31. Ridlon, J. M., D. J. Kang, and P. B. Hylemon. 2006. Bile salt biotransformations by human intestinal bacteria. J. Lipid Res. 47: 241–259.[Abstract/Free Full Text]

  32. Miyata, M., H. Watase, W. Hori, M. Shimada, K. Nagata, F. J. Gonzalez, and Y. Yamazoe. 2006. Role for enhanced faecal excretion of bile acid in hydroxysteroid sulfotransferase-mediated protection against lithocholic acid-induced liver toxicity. Xenobiotica. 36: 631–644.[CrossRef][Medline]

  33. Thomas, P. J., S. L. Hsia, J. T. Matschiner, E. A. Doisy, Jr., W. H. Elliott, S. A. Thayer, and E. A. Doisy. 1964. Bile acids. XIX. Metabolism of lithocholic acid -24-14C in the rat. J. Biol. Chem. 239: 102–105.[Free Full Text]

  34. Einarsson, K. 1966. On the formation of hyodeoxycholic acid in the rat. J. Biol. Chem. 241: 534–539.[Abstract/Free Full Text]

  35. Little, J. M., P. Zimniak, K. E. Shattuck, R. Lester, and A. Radominska. 1990. Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-O-glucuronide in vivo. J. Lipid Res. 31: 615–622.[Abstract]

  36. Palmer, R. H. 1971. Bile acid sulfates. II. Formation, metabolism, and excretion of lithocholic acid sulfates in the rat. J. Lipid Res. 12: 680–687.[Abstract]

  37. Dionne, S., B. Tuchweber, G. L. Plaa, and I. M. Yousef. 1994. Phase I and phase II metabolism of lithocholic acid in hepatic acinar zone 3 necrosis. Evaluation in rats by combined radiochromatography and gas-liquid chromatography-mass spectrometry. Biochem. Pharmacol. 48: 1187–1197.[CrossRef][Medline]

  38. Okuda, K., and T. Kazuno. 1961. Stero-bile acids and bile sterols. XXXIX. Metabolism of lithocholic acid. J. Biochem. (Jpn.) 50: 20–23.

  39. Crombie, D. L., L. R. Hagey, J. Lillienau, K. Miyai, and A. F. Hofmann. 1992. Toxicity of orally administered cholyltaurine in the guinea pig: discovery of a rodent that cannot detoxify deoxycholic acid (Abstract). Gastroenterology. 102: A922.

  40. Cowen, A.E., M.G. Korman, A.F. Hofmann, and O. W. Cass. 1975. Metabolism of lithocholate in healthy man. I. Biotransformation and biliary excretion of intravenously administered lithocholate, lithocholylglycine, and their sulfates. Gastroenterology. 69: 59–66.[Medline]

  41. Fisher, R. L., A. F. Hofmann, J. L. Converse, S. S. Rossi, and S-P. Lan. 1991. The lack of relationship between hepatotoxicity and lithocholic acid sulfation in biliary bile acids during chenodiol therapy in the National Cooperative Gallstone Study. Hepatology. 14: 454–463.

  42. Schwenk, M., A. F. Hofmann, G. L. Carlson, J. A. Carter, F. Coulston, and H. Greim. 1978. Bile acid conjugation in the chimpanzee: effective sulfation of lithocholic acid. Arch. Toxicol. 40: 109–118.[CrossRef][Medline]

  43. Palmer, R. H., and C. K. McSherry. 1982. Lithocholate metabolism in baboons fed chenodeoxycholate. J. Lab. Clin. Med. 99: 533–538.[Medline]

  44. Dew, M. J., K. James, D. Gatehouse, N. J. Dorricott, and R. N. Allan. 1982. Lithocholate sulphation in the baboon. J. Med. Primatol. 11: 59–64.[Medline]

  45. Gadacz, T. R., R. N. Allan, E. Mack, and A. F. Hofmann. 1976. Impaired lithocholate sulfation in the rhesus monkey: a possible mechanism for chenodeoxycholate toxicity. Gastroenterology. 70: 1125–1129.[Medline]

  46. Webster, K. H., M. C. Lancaster, A. F. Hofmann, D. F. Wease, and A. H. Baggenstoss. 1975. Influence of primary bile acid feeding on cholesterol metabolism and hepatic function in the rhesus monkey. Mayo Clin. Proc. 50: 134–138.[Medline]

  47. Dyrszka, H., G. Salen, F. G. Zaki, T. Chen, and E. H. Mosbach. 1976. Hepatic toxicity in the rhesus monkey treated with chenodeoxycholic acid for 6 months: biochemical and ultrastructural studies. Gastroenterology. 70: 93–104.[Medline]

  48. Federowski, T., G. Salen, F. G. Zaki, S. Shefer, and E. H. Mosbach. 1978. Comparative effects of ursodeoxycholic acid and chenodeoxycholic acid in the rhesus monkey. Gastroenterology. 74: 75–81.[Medline]

  49. Morrissey, K. P., C. K. McSherry, R. W. Swarm, W. H. Nieman, and J. E. Deitrick. 1975. Toxicity of chenodeoxycholic acid in the nonhuman primate. Surgery. 77: 851–860.[Medline]

  50. Hagey, L. R., C. D. Schteingart, S. S. Rossi, H-T. Ton-Nu, and A. F. Hofmann. 1998. An N-acyl glycyltaurine conjugate of deoxycholic acid in the biliary bile acids of the rabbit. J. Lipid Res. 39: 2119–2124.[Abstract/Free Full Text]

  51. Gustafsson, J., S. Anderson, and J. Sjövall. 1987. Bile acid metabolism during development: metabolism of lithocholic acid in human fetal liver. Pediatr. Res. 21: 99–103.[CrossRef][Medline]

  52. Miyara, T., N. Shindo, M. Tohma, and K. Murayama. 1990. Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1ß-hydroxylated and unsaturated bile acids in serum and urine. Biomed. Chromatogr. 4: 56–60.[CrossRef][Medline]

  53. Obinata, K., H. Nittono, K. Yabuta, R. Mahara, and M. Tohma. 1992. 1ß-Hydroxylated bile acids in the urine of healthy neonates. J. Pediatr. Gastroenterol. Nutr. 15: 1–5.[Medline]

  54. Nakagawa, M., and K. D. R. Setchell. 1990. Bile acid metabolism in early life: studies of amniotic fluid. J. Lipid Res. 31: 1089–1098.[Abstract]

  55. Meng, L. J., H. Reyes, J. Palma, I. Hernadez, J. Ribalta, and J. Sjövall. 1997. Effects of ursodeoxycholic acid on conjugated bile acids and progesterone metabolites in serum and urine of patients with intrahepatic cholestasis of pregnancy. J. Hepatol. 27: 1029–1040.[CrossRef][Medline]

  56. Mikami, T., A. Ohshima, E. H. Mosbach, B. I. Cohen, N. Ayyad, M. Yoshii, K. Ohtani, K. Kihira, C. D. Schteingart, and T. Hoshita. 1996. 15{alpha}-Hydroxylation of a bile acid analogue, sodium 3{alpha},7{alpha}-dihydroxy-25,26-bishomo-5ß-cholane-26-sulfonate in the hamster. J. Lipid Res. 37: 1189–1197.[Abstract]

  57. Lund, E., K. M. Boberg, S. Byström, J. Ölund, K. Carlström, and I. Björkhem. 1991. Formation of novel C21-bile acids from cholesterol in the rat. J. Biol. Chem. 266: 4929–4937.[Abstract/Free Full Text]

  58. Lee, A. J., M. X. Cai, P. E. Thomas, A. H. Conney, and B. T. Zhu. 2003. Characterization of the oxidative metabolites of 17ß-estradiol and estrone formed by 15 selectively expressed human cytochrome P450 isoforms. Endocrinology. 144: 3382–3398.[Abstract/Free Full Text]


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