**2. Bile acids**

Bile acids are the main components of bile and are among the first products isolated in a pure form, generally from gallbladder bile where it is present in high concentrations. They are acidic steroids produced during cholesterol metabolism in the liver and are secreted in the gallbladder or in the intestine.

Bile acids are produced by all vertebrates and show a great structural diversity among different species [9, 10]. Indeed, no other class of small molecules shows such striking variety across vertebrates. The diversity in bile salt chemical structures originates from differences in the two basic structural components of bile salt molecules: the 19-carbon (C19) steroid nucleus and a side-chain. In all bile salts characterized to date, the four-ring cyclopentanophenanthrene ('steroid') nucleus (rings labelled A, B, C and D) is fully saturated. The A/B ring juncture is *cis* in most bile salts but *trans* in some species, a shift that greatly influences the overall shape of the steroid nucleus. A/B *trans* (5α) bile salts have an extended, planar orientation of the steroid rings, while A/B cis 5β) bile salts have a 'bent' orientation of the A ring relative to the other three rings [10].

The structural variation in the C19 of the steroid nucleus would be stereochemistry of the A/B ring juncture, sites of the oxo or hydroxy groups and orientation of hydroxyl groups (*α* or *β*). The structural variation in the side chain includes the length, the presence and orientation of OH groups, the presence of unsaturation, the stereochemistry of the C25 carbon atom and the site of carboxyl group in bile acids and OH group in bile alcohols [9, 10]. Other than length, further structural variation in the side-chain includes the presence and orientation of hydroxyl groups, the presence of unsaturation in the side-chain, and above all, the substituent on the terminal carbon atom, which are a hydroxyl group in bile alcohols and a carboxyl group in bile acids. Side-chain length and the state of oxidation at C27 is used to assign bile salts to three broad classes: C27 bile alcohols, C27 bile acids and C24 bile acids [10]. The structure of the side chain determines the class of compound; bile acids have a carboxyl group at the end of the chain and bile alcohols have a primary alcohol group [11] (Fig. 1).

Unlike the majority of biological small molecules, whose structures have remained constant since the formation of prokaryotic and eukaryotic cells, the molecular structure of bile acids show a distinctive evolution which parallels that of the vertebrate species which formed them (from C27 bile alcohols, C27 bile acids to C24 bile acids). The progressive nature of bile acid evolution is detectable between different genera, between members of different families and members of different orders [10].

#### Use of Chromatography in Animal Ecology 37

36 Chromatography – The Most Versatile Method of Chemical Analysis

orientation of the A ring relative to the other three rings [10].

(Mammalia).

**2. Bile acids** 

group [11] (Fig. 1).

and members of different orders [10].

the gallbladder or in the intestine.

In this chapter we will discuss the general chromatographic methods commonly used for the analysis of bile acids in biological samples. Moreover, we will emphasize the TLC and HPLC methodologies we used at our laboratory and the most relevant results we obtained, applied to the identification of mammal species, particularly of the Magnaorden Xenarthra

Bile acids are the main components of bile and are among the first products isolated in a pure form, generally from gallbladder bile where it is present in high concentrations. They are acidic steroids produced during cholesterol metabolism in the liver and are secreted in

Bile acids are produced by all vertebrates and show a great structural diversity among different species [9, 10]. Indeed, no other class of small molecules shows such striking variety across vertebrates. The diversity in bile salt chemical structures originates from differences in the two basic structural components of bile salt molecules: the 19-carbon (C19) steroid nucleus and a side-chain. In all bile salts characterized to date, the four-ring cyclopentanophenanthrene ('steroid') nucleus (rings labelled A, B, C and D) is fully saturated. The A/B ring juncture is *cis* in most bile salts but *trans* in some species, a shift that greatly influences the overall shape of the steroid nucleus. A/B *trans* (5α) bile salts have an extended, planar orientation of the steroid rings, while A/B cis 5β) bile salts have a 'bent'

The structural variation in the C19 of the steroid nucleus would be stereochemistry of the A/B ring juncture, sites of the oxo or hydroxy groups and orientation of hydroxyl groups (*α* or *β*). The structural variation in the side chain includes the length, the presence and orientation of OH groups, the presence of unsaturation, the stereochemistry of the C25 carbon atom and the site of carboxyl group in bile acids and OH group in bile alcohols [9, 10]. Other than length, further structural variation in the side-chain includes the presence and orientation of hydroxyl groups, the presence of unsaturation in the side-chain, and above all, the substituent on the terminal carbon atom, which are a hydroxyl group in bile alcohols and a carboxyl group in bile acids. Side-chain length and the state of oxidation at C27 is used to assign bile salts to three broad classes: C27 bile alcohols, C27 bile acids and C24 bile acids [10]. The structure of the side chain determines the class of compound; bile acids have a carboxyl group at the end of the chain and bile alcohols have a primary alcohol

Unlike the majority of biological small molecules, whose structures have remained constant since the formation of prokaryotic and eukaryotic cells, the molecular structure of bile acids show a distinctive evolution which parallels that of the vertebrate species which formed them (from C27 bile alcohols, C27 bile acids to C24 bile acids). The progressive nature of bile acid evolution is detectable between different genera, between members of different families

**Figure 1.** Sites of hydroxylation of the three main classes of bile salts. A: cholesterol; B: C27 bile alcohols (sulfates); C: C27 bile acids (as tauroamidated) and D: C24 bile acids (also as tauroamidated) (from [11]).

Conjugation of bile acids with the amino acids glycine and taurine occurs in the liver, before storage in the gallbladder and subsequent secretion into the duodenum via the bile duct. Within the intestinal lumen, bile acids interact with lipases and assist the lipolysis and absorption of fats, including fat-soluble vitamins, by the formation of mixed micelles. During enterohepatic circulation, the primary bile acids, CA and CDCA which are both synthesized in the liver may be modified by intestinal bacteria to form secondary bile acids, mainly DCA, LCA and UDCA (Fig. 2). In the colon of animals with a cecum, anaerobic bacteria remove the hydroxyl group at C7 to form 7-deoxy bile acids. In the side chain, bile acids suffer deconjugation [11, 12-14].

**Figure 2.** Structure of the main primary and secondary bile acids in mammals (from [15]).

Fecal bile acid patterns are complex due to bacterial metabolism during intestinal transit, which gives mono-, di- and/or –trioxo compounds, and also iso-(3β-hydroxy), urso-(7βhydroxy) and lago-(12β-hydroxy) bile acids [16-18]. The colon bacteria deconjugate bile acids. The majority of the unconjugated bile acids are 7α-dehydroxylated, being LCA and DCA the predominant secondary fecal bile acids. They are reabsorbed from the colon, modified by hepatic enzymes and circulate in the enterohepatic circulation. Bile acids in the jejunum remain conjugated because of the absence of bacteria in the small intestine [19]. Moreover, in feces, certain bile acids are firmly bound to bacteria [12].
