**1. Introduction**

34 Chromatography – The Most Versatile Method of Chemical Analysis

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There are several applications of chromatographic science in the field of animal ecology, biology and physiology. One of the most spread uses is identifying the bile acid profiles either by Thin Layer Chromatography, Gas chromatography or High Performance Liquid Chromatography in wild collected feces.

The analysis of wild collected feces is a fundamental ecological tool, applied in studies about population size, diet analysis and to identify the presence of a species in a certain area. Moreover, it is useful when it is necessary to monitor those species which are elusive, difficult to observe, threatened, nocturnal or sympatric, or exist in low densities. The identification can be done by external physical characteristics such as size, shape, odor and color, or through specific signals associated with the deposition of feces, for example tracks and scrapes [1,2]. However, this technique is sometimes useless because of the difficulties that exist in the correct identification of feces. Often, this sort of evidence is not present mainly because many of these external characteristics are sensitive to environmental conditions such as heat, desiccation or fast decomposition in humid and rainy regions, and can be affected by another type of factors: health, diet, size and age of the individual [3, 4].

Because of these reasons is that other techniques become necessary. The use of chromatographic techniques to identify or confirm the identity of wild collected feces is of great importance for biologists, because invasive procedures such as capture and manipulation are avoided. During the last years, the chromatographic determination of fecal bile acids has become a more precise method to identify unknown feces from the wild. The comparison of the whole pattern of fecal bile acids between field-collected scats and scats with known origin allows identifying the species. It has been demonstrated in several studies that fecal bile acids and their relative concentration follow patterns that are species-specific, particularly for mammals [5-7], including our recent studies in Xenarthra species [8].

© 2012 Casanave et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Casanave et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 (Mammalia).

Use of Chromatography in Animal Ecology 37

**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

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

Moreover, in feces, certain bile acids are firmly bound to bacteria [12].

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].

acids suffer deconjugation [11, 12-14].
