Ursodeoxycholic acid in the Ursidae: biliary bile acids of bears, pandas, and related carnivores Lee R. Hagey: Diane L. Crombie, 1. * Edgard Espinosa, r Martin C. Carey, § Hirotsune 19imi,~ and Alan F. Hofmann3• * Division of Gastroenterology, Department of Medicine,* University of California, San Diego, La Jolla, CA 92093-0813; National Fish and Wildlife Forensics Laboratory, t Ashland OR; and Division of Gastroenterology, Brigham and Women's Hospital,S Harvard Medical School, Boston, MA Abstract The biliary bile acid composition of gallbladder bile bladders of the Polar bear (Thalarctos maritimus) and obtained from six species of bears (U rsidae), the Giant panda, several other arctic mammals and delivered these to Olof the Red panda, and 11 related carnivores were determined by Hammarsten, Professor of Biochemistry, in Uppsala, reversed phase liquid chromatography and gas chromatogra- Sweden: Hammarsten (2, 3) isolated an unknown bile phy-mass spectrometry. Bile acids were conjugated solely with taurine (in N-acyllinkage) in all species. Ursodeoxycholic acid acid from the bile of the Polar bear and named it "ur- (3a,7/3-dihydroxy-5/3-cholan-24-oic acid) was present in all Ur- socholeinsiiure", indicating. that it was a new bile acid sidae, averaging 1-39% of biliary bile acids depending on the from the bear. Twenty-five years later, Shod a (4) crystal- species; it was not detected or present as a trace constituent lized this bile acid from a commercial preparation of bile « 0.5%) in all other species, including the Giant panda. Ur- of the Black bear and Shoda renamed' sodeoxycholic acid was present in 73 of 75 American Black (Ursus amen'canus), bears, and its proportion averaged 34% (range 0-62%). Ur- it ursodeoxycholic acid (UDCA), as it was an isomer of sodeoxycholic acid averaged 17% of biliary bile acids in the Po- deoxycholic acid. Oxidation of UDCA was shown by lar bear (n - 4) and 18% in the Brown bear (n - 6). Lower Iwasaki (5) to yield the same 3,7-dioxo derivative as'ob- proportions (1-8%) were present in the Sun bear (n 2), Cey- = tained by oxidation of chenodeoxycholic acid, and he can- lon Sloth bear (n - 1), and the Spectacled bear (n - 1). Bile of all species contained taurine-conjugated chenodeoxycholic acid eluded that UDCA is the 7j3-epimer of chenodeoxycholic and cholic acid. In some related carnivores, deoxycholic acid, acid. Later, Kanazawa et al. (6) succeeded in synthesizing the 7-dehydroxylation product of cholic acid, was also present. UDCA from cholic acid. In 1959, UDCA was identified To determine whether the 7/3 hydroxy group ofursodeoxycholic as a minor constituent in human bile by Sjovall (7). Ex- acid was formed by hepatic or bacterial enzymes, bile acids were tensive studies by Tammar (8), Haslewood (9), and Hagey determined in hepatic bile obtained from bears with chronic biliary fistulae. Fistula bile samples contained ursodeoxycholic (10) on the biliary bile acid composition of vertebrates acid, chenodeoxycholic acid, and a trace amount of cholic acid, have shown that although UDCA is present in the biliary all as taurine conjugates, indicating that ursodeoxycholic acid is bile acids of a number of vertebrates, it generally consti- a primary bile acid formed in the liver in Ursidae. Additional tutes < 1-5%. At present, the only species besides the evidence for ursodeoxycholic acid being a primary bile acid was bear known to have an appreciable portion of UDCA in provided by fecal bile acid analyses in six bear species. These showed the presence of ursodeoxycholic acid in five of six species biliary bile acids is the nutria (Myocastor cqypus), in which and absence of its 3a-hydroxy-7-oxo precursor in all. IIlI It is UDCA is a dominant primary biliary bile acid (11, 12). concluded that ursodeoxycholic acid occurs in all Ursidae, but In this paper, we report a systematic examination of the in few other species, and is formed in the liver.-Hagey, L. R., biliary bile acid composition of the family Ursidae. Ana- D. L. Crombie, E. Espinosa, M. C. Carey, H. Igimi, and lyses of the biliary bile acid composition of the Giant A. F. Hofmann. U rsodeoxycholic acid in the U rsidae: biliary bile acids of bears, pandas, and related carnivores. J Lipid Res. 1993. 34: 1911-1917. Supplementary key words bile salts • bile acid metabolism • tau- Abbreviations: UDCA, ursodeoxycholic acid; GLC, gas-liquid chro- rine • 7,3-hydroxylation matography; HPLC, high performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry. lCurrent address: Ligand Pharmaceuticals, LaJolla, CA 92037. 2Associate Director, Shianogi Research Laboratory, Osaka, Japan. In 1900-1901, two expeditions set out to explore Green- 'To whom reprint requests should be addressed at: Department of land (1). The Swedish party was led by Kolthoff and the Medicine, 0813, University of California, San Diego, 9500 Gilman Danish party by Amdrup. The two groups obtained gall- Drive, La Jolla, CA 92093-0813. panda (Ailuropoda melanoleuca) and Red panda (Ailurusful- Analyses gens) are also included, as these have been considered to Bile acid conjugates were analyzed by HPLC of gall- be closely related to the Ursidae. Data on biliary and fecal bladder bile essentially as described by Rossi, Converse, bile acid composition from a number of additional carni- and Hofmann (15). The method uses an octadecylsilane vores have also been included for comparison with those column (RP C-18) with elution at 0.75 ml/min with an of Ursidae. Biliary bile' acids in chronic biliary fistula isocratic 50 mM KH2PO+-K2HPO+ buffer, apparent pH bears were examined in order to ascertain whether 5.4, in methanol-water 68.2:31.8 (v/v). The efHuent was UDCA is formed by hepatic enzymes and is a primary monitored at 205 nm (amide bond of conjugated bile bile acid, or is formed by bacterial enzymes in which case acids) to quantify bile acids. Peaks were tentatively as- it would be a secondary bile acid (13). signed by comparison of the relative retention times with those of known standards. Biliary bile acids were also ana- lyzed by GC-MS using a Hewlett-Packard 5890 Gas Chromatograph-5970 MSD, controlled by an HP/UX Chern Station program. The 30-meter capillary column Samples was a Supelco 0.25-mm ID SPB-35 (35% phenyl methyl Gallbladder bile from bears and related carnivores was silicone) operated at 275°C (isothermal). A splitless injec- obtained from deceased animals at the San Diego Zoo un- tion was used with an injection temperature of 295°C and der an approved protocol of the Zoological Society of San interface temperature of 290°C. Helium served as the Diego. Bile samples from 11 North American Black bears carrier gas with a column head pressure of 6 pounds per that were killed by accident or by hunting in Maine and square inch. To prepare for GC-MS, bile acids were Minnesota were analyzed by GLC at the Brigham and deconjugated chemically (1.0 N NaOH, 130°C, 4 h). Women's Hospital, Boston, MA. An additional 57 HPLC Deconjugated bile acids were esterified with methanol (di- analyses of American Black bears, 3 Polar bears, and 4 azomethane) and acetylated by the perchloric acid- North American Brown bears (Ursus arctos; also known as catalyzed method of Roovers, Evrard, and Vanderhaeghe grizzly bear) were provided by the National Fish and (16); bile acids were incubated for 1.5 h in 2 ml of the Wildlife Forensics Laboratory, Ashland, OR. This acetylation fluid (14 ml acetic acid, 10 ml acetic an- laboratory also provided HPLC analyses of bile obtained hydride, 0.1 ml perch10ricacid), and then extracted with ethyl from 23 bears with chronic biliary fistulae maintained in acetate; the ethyl acetate phase was then reduced to dryness. the Peoples Republic of China. Details on the main- The unconjugated bile acids, which constitute> 90% tenance of these animals has been published in the popu- of all fecal bile acids, were analyzed in six bear species. lar press (14). Capsules (n = 20) of "bear bile" were pur- Samples were refluxed for 1 h in methanolic sodium chased in traditional Chinese pharmacies. Bile from the hydroxide solution (85 ml methanol, 10 ml of 10 N sloth bear (Melursus ursinus) was provided by the Philadel- NaOH), and after the addition of water, neutral sterols phia Zoo (Penrose research laboratory). Bile samples were were extracted with hexanes. The remaining aqueous dispersed in several volumes of reagent grade isopropanol fraction was acidified (pH < 2) with dilute hydrochloric immediately after collection to prevent bacterial degrada- acid and the unconjugated bile acids were extracted into tion. Fecal samples were frozen immediately after collec- ethyl acetate. Fecal bile acids were then derivatized and tion and kept at - 20°C until analysis. examined by GC-MS as described above. TABLE 1. HPLC analysis of the biliary bile acids present in gallbladder bile of adult bears (Ursidae) Bile Acid Composition, 'lI> Common Name Latin Name N 3a7a' 3a7{3 3a7a12a 3a12a' Black bear Ursus ammcanus 7 29.6 ± 5.2 38.8 ± 7.4 31.0 ± 5.2 0.6 ± 0.37 Brown bear Ursus arctos 6 5.7 ± 1.5 18.6 ± 7.3 75.7 ± 14.3 0.0 Polar bear Tha[q,rctos 71UIritimus 4 19.6 ± 7.8 17.4 ± 6.9 62.8 ± 14.4 0.2 ± 0.2 Sun bear Hewctos 71UI[q,yanus 2 63.6 ± 8.7 8.6 ± 3.1 27.8 ± 5.6 0.0 Sloth bear Melursus ursinus 1 31.3 1.4 67.3 0.0 Spectacled bear TrC71Ulrctosornatus 1 23.6 6.3 70.1 0.0 Bile acids were present in bile as the taurine (N-acyl) conjugates.