Beta-Phocacholic Acid in Bile; Biochemical Evidence That The

The Condor92593-591 0 TheCooper Ornithological Society 1990

,&PHOCACHOLIC ACID IN BILE; BIOCHEMICAL EVIDENCE THAT THE FLAMINGO IS RELATED TO AN ANCIENT GOOSE ’

L. R. HAGEY, C. D. SCHTEINGART, H-T. TON-Nu, S. S. Ross, D. ODELL,~ AND A. F. HOFMANN Physiology-Pharmacology Program and the Division of Gastroenterology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093

Abstract. The biliary bile acid composition of 10 anseriform and three flamingo species was determined usinga variety of chromatographictechniques. Some bile samplescontained an unusual 23-hydroxylated derivative of chenodeoxycholic acid, (23R)-3a,7a,23_trihy- droxy-5P-cholan-24-oic acid (P-phocacholicacid). The proportion of this unusualbile acid correlated highly with the order of evolutionary appearance.&Phocacholic acid was found to constitute a major proportion of biliary bile acids in three flamingo species.The bile acid spectrapresent in these flamingoswere similar to those of several ducksfrom the subfamily Anatinae; those of the Greater Flamingo (Phoenicopterus ruber) were virtually identical to those ofthe Nene or Hawaiian Goose (Branta sandvicensis). It is suggestedthat the common presenceof a new biochemical character,@-phocacholic acid, in both ducks (Anatinae) and flamingos (Phoenicopteridae)provides evidence for a close evolutionary link between these bird families. Key words: Bile acids; fl-phocacholic; flamingo; phylogeny.

INTRODUCTION MATERIALS AND METHODS Traditional morphological considerations (Wet- Anseriform and flamingo gall bladder bile sam- more 1960, Cracraft 1981) and a recent DNA ples obtained from dead animals were provided hybridization study (Sibley et al. 1988) have been by the pathology laboratories of the San Diego used as evidence for assigning flamingos together Zoo and by Sea World Florida. Samples were with storks, herons, and ibises to the order Ci- placed in several volumes of isopropanol im- coniiformes. Nonetheless, others have proposed mediately after collection to prevent bacterial that flamingos are more closely related to the degradation. Charadriiformes (Olson and Feduccia 1980) or Thin layer chromatography (TLC) of the whole Anseriformes (Delacour 1961). Although it is bile was performed on silica gel G (E. Merck, hazardous to use a single biochemical character Darmstadt, Federal Republic of Germany) using as a criterion for evolutionary relationships, the two solvent systems: (1) isoamylacetate: pro- available bile acid analyses of vertebrate species pionic acid : 1-propanol : water, 4:3:2: 1, v/v have been shown to agree well with assignments (Hofmann 1962), for free and conjugated bile based on morphology and palaeontology (Hasle- acids; (2) a double development system for the wood 1978a). We reasoned that bile acid struc- taurine conjugates, in which plates were devel- ture could provide useful information for defin- oped first in chloroform : acetone : methanol : ing the phylogenetic position of the flamingo. TO propionic acid : water, 10:4:2:2: 1, v/v and after test this hypothesis, conventional and spectro- drying overnight, in 1-butanol: propionic acid : scopic techniques developed in the process of water 10: 1: 1 v/v (Elferink et al. 1989). (The latter identifying the unusual bile alcohols of the man- system separates glucuronide conjugates from atee (Kuroki et al. 1988) were used to analyze taurine conjugates.) Bile acids were isolated by the bile salts of a variety of birds and flamingos. scraping selected bands off a TLC plate and elut- ing with CHCl, : MeOH, 2: 1, v/v or collecting selected peaks in the high pressure liquid chromatography (HPLC) eluent. Bile acids were vi- Received 5 October 1989. Final acceptance26 Feb- ’ ’ sualized by spray reagents for hydroxyl groups ruary 1990. 2Sea World Florida, 7007 Sea World Drive, Orlan- (phosphomolybdic acid), 0x0 groups (2,4 dini- do, FL 32821. trophenyl-hydrazine), glucuronides (naphthore-

15931 594 L. R. HAGEY ET AL.

ONHCH,CH,SO; esterof 23R-phocacholic acid (RRT 1.45 relative to cholic methyl ester acetate). This derivative was found by IH-NMR to be methyl (23R) 3a,7o1,23-triacetyloxy-5P-cholan-24-oate:6 0.663 (s, 3H, Me-18) 0.933 (s, 3H, Me-19) 0.967 (d, 3H, J = 6.1 Hz, Me-21) 2.030 (s, 3H, CH,COO-), 2.051 (s, 3H, CH,COO-), 2.145 (s, 3H, 23- CH,COO-), 3.732 (s, 3H, -COOCH,), 4.587 (m, H lH, H-3), 4.880 (m, lH, H-7), 5.05 (dd, lH, J FIGURE 1. Structuralformula of @-phocacholicacid = 11.0 and 1.6 Hz, H-23). This spectrum was present in flamingo bile as its taurine (N-acyl) conju- identical to that reported by Klinot et al. (1986). gate. Especially diagnostic is the signal of H-23 which appeared as a double doublet (J = 11.O and 1.6 sorcinol), or vicinal hydroxyl groups (lead tet- Hz) at 5.05 ppm, indicating a configuration of raacetate) (Dawson et al. 1969). Conjugated bile 23R for the acetate and the original hydroxyl acids were analyzed by HPLC of whole bile using group. In contrast, in 23s derivatives H-23 ap- a modification of the technique of Rossi et al. pears as a triplet with J = 11 Hz. (1987) which uses a reversed phase octadecyl- Analyses of the (biliary) bile acids from the silane column (RP C- 18) and elution at 0.75 ml/ Greater Flamingo (Phoenicopterusruber), Lesser min with an isocratic buffer, pH 5.4, composed Flamingo (Phoeniconaias minor), and Chilean of a mixture of methanol (67.4%) and 0.01 M Flamingo (Phoenicopteruschilensis) indicated the KH,PO,; the effluent was monitored at 205 nm presence of a substantial proportion of /3-pho- (amide bond). Bile acids were deconjugated catholic acid (Fig. 1) in each of the three species. chemically (1.0 NaOH, 130°C 4 hr), and the This compound was present, as were the other resulting unconjugated bile acids were analyzed identified bile acids, in the form of its N-acyl by gas liquid chromatography (GLC) as their taut-me conjugate. methyl ester acetate derivatives using a Hewlett- Unlike the great majority of vertebrate species Packard 5840A instrument fitted with a 0.63 cm in which dihydroxy bile acids are further hy- x 2 m SP-2250 (methyl : phenyl) column packed droxylated at position 12 (on the steroid nucleus) on Supelcoport 100-120 at 270°C (isothermal). to form cholic acid, /3-phocacholic acid is hy- IH-NMR was recorded in Cl,CD on a 360 MHz droxylated in position 23 (on the side chain), a instrument equippedwith a modified Varian MR- trait shared in common with the Anatinae 220 console, Oxford magnet and Nicolet 1180-E subfamily (ducks) of Anseriformes (Klinot et al. computer system. Chemical shifts are in ppm 1986). relative to tetramethylsilane. Table 1 lists the biliary bile acid composition of these three flamingo species,as well as other RESULTS AND DISCUSSION selectedwading birds with which flamingos have The chemical identity of P-phocacholyltaurine been traditionally associated.From this table, it (Fig. 1) was determined from the material ob- is quite clear that the presenceof P-phocacholic tained by collecting the appropriate peak from acid, as well as the low proportion or absenceof an HPLC chromatogram of flamingo bile. By low cholic acid, groups the flamingos with the an- resolution secondary ion mass spectroscopy seriforms and differentiates them from other ci- (SIMS) the compound’s quasimolecular ion at coniiforms and the charadriforms. Additional M/2 5 14 indicated that it was the taurine con- support in favor of this grouping is the sharing jugate of a trihydroxy C,, bile acid (Whitney and of common anatomical and physiological fea- Burlingame 1985). It had a TLC (Rf 0.12 System tures between ducks and flamingos, including la- 1, Rf 0.37 System 2) and HPLC (RRT 0.34 com- mellate bill, feather structure, voice patterns, a pared to chenodeoxycholyl-glycine) identical to large cecum, preen wax composition, and mal- that of an authentic sample of P-phocacholyl- lophagan parasites (Sibley et al. 1969). taurine. Following deconjugation and formation In Table 2, 10 anseriforms are listed in order of the methyl ester acetate derivative, material of evolutionary appearance, as assembled by from peak (A) (Fig. 2) had a GLC RRT identical Livezey (1986), based on his phylogenetic anal- to that of the triacetyl derivative of the methyl ysis using morphological characters.A high cor- BIOCHEMICAL EVIDENCE FOR PHYLOGENY 595

TABLE 1. Biliary bile acid compositionGb of flamingos and other selectedwading birds by HPLC.

Biliarybile acid composition,% Species COlll- Order Commonname Latin name n 3a7& 3a7u12& 3a7o123Rcpound Ed

Chilean Flamingo Pheonicopterus chilensis 6 25.2 7.1 62.3 0 Lesser Flamingo Phoeniconaias minor 2 60.8 7.3 29.5 2.4 Greater Flamingo Phoenicopterus ruber 7 82.8 1.3 13.6 2.3 Anseriformes Nene Branta sandvicensis 2 84.7 0 10.5 4.8 Anseriformes Ringed Teal Callonetta leucophrys 1 64.3 2.9 30.1 2.7 Charadriformes American Avocet Recurvirostra americana 2 30.8 69.2 0 0 Ciconiiformes Marabou Stork Leptoptilos crumeniferus 1 35.3 64.7 0 0 Ciconiiformes Scarlet Ibis Eudocimus ruber 1 21.5 78.5 0 0 ’ ’ Bile acidswere presentin bile in all birds as the taurine#V-acyl) conjugates. bBiliiry bile acid compositionhas been normalized to 10 %. ’ ’ The posit@ of the hydroxylsubstituents is indicated.3a7a, chenodeoxycholicacid; 3a7a12a, cholicacid, 3u7a23R, fl-phocacholicacid. ’ A prebmmarydetermination of the structureof this compoundindicates that it is allo-chenodeoxycholicacid (3a,7a-dibydroxy-5ol-cholan-24- oic acid). relation between the relative percentage of the flamingo-duck families. This is true even if P-phocacholic acid and the ranking of the birds one assumes that the genetic course of 23-hy- is readily apparent (Spearman’s rank correlation droxylase development proceeded differently in test, P < 0.001) (Brown and Hollander 1977). flamingos and geeseonce these two families di- Including the flamingo in this statistical test (rank verged. value of 0 assigned)resulted in a P-value c 0.0 15, Using rank correlation based on the percentage still statistically significant. In this correlation, of phocacholic acid, it is possible to assign the the relative amount of @-phocacholicacid in- origin of the Phoenicopteridae within the phy- creasesin bile over time in a manner which is logeny of extant anseriforms. Figure 2 contains independent of diet and morphology; evidently an abbreviated phylogenetic tree of anseriform the genes encoding enzymes responsible for 23- family names assembled by Livezey (1986) into hydroxylation have been evolutionarily success- which the Phoenicopteridae has been inserted ful. next to the Branta. In addition to the presence If it is true that the flamingos and anseriforms of P-phocacholic acid, a high degree of concor- are directly related (as evidenced by the common dance is also present in the overall bile acid pro- presenceof a bile acid 23-hydroxylase), the rel- files of the Greater Flamingo and the Nene, two atively low level of P-phocacholic acid expres- birds with low but proportionately equal amounts sion in the Greater Flamingo suggeststhat among of P-phocacholicacid. As shown by Figure 3, this the flamingos it is closestto the branch point of similarity included not only the common pres-

TABLE 2. Biliary bile acid compositiona,bof selectedanseriformsc and the Greater Flamingo by HPLC.

Species Biliary bile acid composition,%d Commonname Rank Latin name 3u7a 3a7a23R ComooundE

Southern Screamer 1 Chauna torquata 0 63 Pied Goose 2 Anseranus semipalmata 0 7 Java Tree Duck 3 Dendrocygna javanica 0 2 Black-Neck Swan 5 Cygnus melanocoryphus 4 Bar-Head Goose 5 Anser indicus i 4 Red-Breasted Goose 5 Branta rufcollis 5 5 Nene 5 Branta sandvicensis 10 4 Greater Flamingo Phoenicopterus ruber 13 3 Mandarin Duck 9 Aix galericulata 27 5 Crested Duck 12 Anas specularidides 34 4 Tufted Duck 13 Aythya &l&da 69 4 1Bile acidswere presentin bile in all birds as the taurinef$N-acyl) conjugates. bBiliary bile acid compositionhas been normalized to 1 %. EAnserifonns are listedin order of evolutionaryappearance. d For abbreviations.see Table I 596 L. R. HAGEY ET AL.

ANCESTOR (0)

PA?“* Goose q--~E~;~;l:, IA)

CYGNUS

CC)

ANSER* (E) BRANTA* z PHOENICOPTERIDAE* 0 10 20 30 40 Time, Ini” 1.,!1!: FIGURE 2. Addition of the flamingo family Phoeni- copteridae into the phylogenetictree of recent anseri- FIGURE 3. Similar high pressureliquid chromatog- form genera adopted from Livezey (1986). The pres- raphy (HPLC) bile acid profiles of the Greater Fla- ence of p-phocacholic acid in bile is indicated by an mingo and Nene. Peak identification: (A) phocacholyl- asterisk. taurine, (B) phocadeoxycholyl-taurine,(C) unknown, (D) chenodeoxycholyl-taurine (chemical structure is given in Table l), and (E) allo-chenodeoxycholyl-taurine. ence of a dominant amount of chenodeoxycholic acid (Peak D) and the unusual bile acid fi-pho- catholic acid (Peak A), but in addition included of compound E (tentatively identified to be 3cu,7a- most of the unidentified peaks seen in the HPLC dihydroxy-5cY-cholan-24-oicacid), so prominent chromatograms. The unexpected resemblance in the relatively “ancient” screamer, and shared between these two biliary profiles offers further by all the anseriforms (including the Greater and support for both a close relationship and a sim- Lesser flamingos), is also observed in certain ilar time of divergence from a common ancestor. “older” members of the Galliformes. The occurrenceof a similar bile acid spectrum The hepatic biosynthesis of bile acids from (presumably inherited from a common ancestor) cholesterol and the formation of urea from am- in two birds which exhibit dissimilar morphol- monia (Mommsen and Walsh 1989) are exam- ogy and diets implies that morphological and ples of stable end products of metabolism which dietary changesare evolving at a faster rate than appear useful to deducetaxonomic relationships. are changesin bile acids. This stability in the bile Bile acids are the metabolic end products of cho- acid profile is present even though steady state lesterol found in all vertebrates, and their for- biliary bile acid composition is the result of not mation is essential to life (Haslewood 1978b). only hepatic biosynthesis rate, but also canalic- Moreover, there are a variety of vertebrate bile ular secretion rate, as well as intestinal conser- acids, and all of these function adequately as vation and subsequentbacterial and hepatic bio- digestive surfactants (Hofmann and Mysels transformation pathways (Hofmann 1989). In 1988). The biliary bile acid pattern observed in vitro, microorganisms can be shown to hydrox- each speciesis determined predominantly by he- ylate bile acids at a variety of positions on the patic synthesis, in which specific hepatic hy- steroid ring (Hayakawa 1982), but such com- droxylasesoperate on a finite number of sites on pounds are never observed in significant pro- the steroid molecule. The relationship between portions in bile. Side-chain hydroxylation of bile biosynthetic intermediates and functioning bile acids by microorganisms has not been observed acids is biochemically very close and appears to (MacDonald et al. 1983). conform to a biochemical analogy of Haeckel’s The presenceof 23-hydroxylated bile acids in law in which the bile acids present in adults of only a few genera that belong to unrelated groups older vertebrates have become intermediates in (ducks,pinnipeds, snakes)is further evidence that the synthesis of the more complex bile acids of phocacholic acid is derived, rather than primi- more recently evolved vertebrates. Whether the tive. Although it is possible that p-phocacholic precise molecular differences seen in the stable acid could have arisen independently in flamin- bile acid spectrum can ultimately be directly re- gos and anseriforms, the rarity of 23-hydroxyl- lated to the genes of each species can only be ation in vertebrate bile would appear to justify decided by further investigation. our proposal of the existence of one common While evolutionary biologists have long dis- ancestor that experienced the event de novo. trusted conclusions based on a single character, It is also of interest to note that the presence the bile acid profiles reported here are objective BIOCHEMICAL EVIDENCE FOR PHYLOGENY 597 and quantifiable data that link the origins of the HOFMANN,A. F. 1989. Enterohepatic circulation of flamingo to the Anseriformes, albeit a currently bile acids, p. 567-596. In S. G. Schultz [ed.], Handbook of physiology.American Physiological unfashionable view. Society, Bethesda,MD. Ho-, A. F., AND K. J. MYSEIS. 1988. Bile salts ACKNOWLEDGMENTS as biological surfactants.Colloids and Surfaces30: 145-173. Bile samplesfor these studieswere obtained from the KLINOT, J., M. JIRSA,E. KLINOTOVA,K. UBIK, AND J. Pathology Department, San Diego Zoological Society. PROTIVA. 1986. Isolation of (23R) 301,70(, 23- We would like to thank M. P. Anderson and K. G. trihydroxy-5@-cholan-24-oic(P-phocaecholic) acid Osbom for collectingthese samples. S. Branch(Curator from duck bile. ‘H-NMR spectraof its derivatives. of Birds) and M. T. Walsh (Staff Veterinarian) at Sea Collection Czechoslovak Chem. Commun. 5 1: World of Florida provided additional bile samples. 1722-l 730. This is Sea World of Florida Technical Contribution KUROKI, S., C. D. SCHTEINGART,L. R. HAGEY, B. I. No. 8904-F. We would also like to acknowledge D. COHEN, E. H. MOSBACH,S. S. ROSSI, A. F. Maltby and the Bio-Organic, Biomedical Mass Spec- Ho-, N. MATOBA,M. UNE, T. HOSHITA,AND trometry Resource(A. L. Burlingame, Director), sup- D. K. 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