Bile salts of vertebrates: structural variation and possible evolutionary significance

Abstract

Biliary bile salt composition of 677 vertebrate species (103 fish, 130 reptiles, 271 birds, 173 mammals) was determined. Bile salts were of three types: C(27) bile alcohols, C(27) bile acids, or C(24) bile acids, with default hydroxylation at C-3 and C-7. C(27) bile alcohols dominated in early evolving fish and amphibians; C(27) bile acids, in reptiles and early evolving birds. C(24) bile acids were present in all vertebrate classes, often with C(27) alcohols or with C(27) acids, indicating two evolutionary pathways from C(27) bile alcohols to C(24) bile acids: a) a 'direct' pathway and b) an 'indirect' pathway with C(27) bile acids as intermediates. Hydroxylation at C-12 occurred in all orders and at C-16 in snakes and birds. Minor hydroxylation sites were C-1, C-2, C-5, C-6, and C-15. Side chain hydroxylation in C(27) bile salts occurred at C-22, C-24, C-25, and C-26, and in C(24) bile acids, at C-23 (snakes, birds, and pinnipeds). Unexpected was the presence of C(27) bile alcohols in four early evolving mammals. Bile salt composition showed significant variation between orders but not between families, genera, or species. Bile salt composition is a biochemical trait providing clues to evolutionary relationships, complementing anatomical and genetic analyses.

Fig. 1.

Chemical structure of cholesterol (A) and the three main classes of bile salts: C₂₇ bile alcohols (B), C₂₇ bile acids (C), and C₂₄ bile acids (D). Bile salts are shown as their natural conjugates: C₂₇ bile alcohols are shown esterified with sulfate; C₂₇ bile acids are shown as their N-acyl amidate conjugates with taurine. Glycine conjugation is not shown. Also not shown is modification of the 7α-hydroxy group; in some caviomorphs, it is oxidized to a 7-oxo group, and in some rodents and bears, it is epimerized to a 7β-hydroxy group. C₂₇ bile alcohols are shown as either 5α (trans A/B ring juncture) or 5β (cis A/B ring juncture). C₂₇ and C₂₄ bile acids are shown only in the 5β configuration, although 5α-C₂₇ bile acids are likely to occur, and 5α-C₂₄ bile acids (allo bile acids) are common in nature. The large arrows indicate sites of hydroxylation on the nucleus or side chain that occur in many species. The small arrows indicate sites of hydroxylation that occur in only a few species. For C₂₄ bile acids, some sites of hydroxylation that occur in only a few species and constitute <10% of biliary bile acids are not shown, for example, hydroxylation at C-4. Additional sites of hydroxylation are likely to be discovered in the future.

Fig. 2.

Variation of bile salt structures across vertebrate species. The phylogeny is a general one for vertebrates (69) with debated evolutionary relationships (e.g., frogs, salamanders, caecilians) depicted as polyotomies. Each bar chart shows the proportion of animals in each vertebrate group (using data from animal species analyzed so far) that are classified into one of six bile salt profiles based on the one or two bile salt classes (C₂₇ bile alcohols, C₂₇ bile acids, C₂₄ bile acids) that account for 10% or more of the total biliary bile salt pool (see key). There are a small number of fish and amphibian species that have all three major classes of bile salts present in bile, each at 10% or more of the total bile salt pool. For simplicity, these species are still classified based on the two bile salt types that account for the greatest percentage of the bile salt pool. The inferred bile salt profiles for the last common ancestor to all living vertebrates and the last common ancestor to bony fish and land vertebrates (basal gnathostome) are indicated.

Fig. 3.

Variation of bile salt structures across reptile groups in comparison with birds and mammals. The major bile salts of each group are indicated with hydroxylation patterns in addition to the default hydroxylation for each bile salt class noted. The evolutionary relationships of living reptile groups are still debated, especially with regard to the placement of Testudines (turtles and tortoises) (69, 78). The traditional hypothesis, based on morphological and paleontological data, placed Testudines as the most basal group (A). More recent molecular phylogenies challenge this hypothesis and place Testudines as a sister-group to crocodiles (B) (69). In either arrangement, one may hypothesize that the common ancestor to all living reptile groups possessed a bile salt profile consisting mainly of C₂₇ bile acids (possibly with C₂₇ bile alcohols as well). In this case, four main “innovations” (indicated by, *, **, ***, and ****) with regard to evolution of bile salt structures in reptiles are postulated: *, ability to synthesize C₂₄ bile acids; **, formation of 5α (allo) C₂₄ bile acids in some lizard lineages; ***, novel additional modifications to nucleus and side-chain (e.g., C-16 and C-23 hydroxylation; introduction of double bond between C-22 and C-23) to C₂₄ bile acids in snakes; ****, 15- and 22-hydroxylation in the C₂₇ bile salts of turtles and tortoises.

Fig. 4.

Variation of bile salt structures across birds. The evolutionary relationships of living bird groups are controversial, especially within the large number of passeriform birds (69, 79, 80). Nevertheless, recent large-scale morphological/paleontological (80) and molecular phylogenies (79) share agreement in placing ratite birds (cassowaries, rheas, emus, kiwis, ostriches) and tinamous (collectively called paleognathic birds) at the base of the bird evolutionary tree. Paleognathic birds share with crocodiles (hypothesized to be the closest living reptile relatives to modern birds) the phenotype of having bile salt profiles consisting largely of C₂₇ bile acids. Each pie chart shows the proportion of birds in each group (using data from species analyzed so far) that are classified into one of six bile salt profiles based on the one or two bile salt classes (C₂₇ bile alcohols, C₂₇ bile acids, C₂₄ bile acids) that account for 10% or more of the total biliary bile salt pool (see key). No bird species analyzed so far have a type I (C₂₇ bile alcohols only) profile, so this type is not included in the plots.

Fig. 5.

Variation of bile salt structures across mammals. The evolutionary relationships of living mammals are still actively researched (69, 98). The phylogeny depicted is based on variation of 20 nuclear DNA sequences (100). The phylogeny is annotated with the major bile salts of each mammalian group. CA, CDCA, and DCA refer to cholic acid, chenodeoxycholic acid, and deoxycholic acid, respectively. C₂₇ acids refers to the presence of C₂₇ bile acids at more than 10% of the total bile salt pool. Modifications to the stem C₂₄ bile acid including additional hydroxylation (1α-OH, 6α-OH, 6β-OH, 7β-OH, 15α-OH) and conversion of the 7α-hydroxyl group to a 7-oxo group are indicated.