Hagey et al. BMC Evolutionary Biology 2010, 10:133 http://www.biomedcentral.com/1471-2148/10/133 Open Access RESEARCH ARTICLE BioMed Central © 2010 Hagey et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Evolutionary diversity of bile salts in reptiles and mammals, including analysis of ancient human and extinct giant ground sloth coprolites Lee R Hagey* 1 , Nicolas Vidal 2 , Alan F Hofmann 1 and Matthew D Krasowski* 3 Abstract Background: Bile salts are the major end-metabolites of cholesterol and are also important in lipid and protein digestion and in influencing the intestinal microflora. We greatly extend prior surveys of bile salt diversity in both reptiles and mammals, including analysis of 8,000 year old human coprolites and coprolites from the extinct Shasta ground sloth (Nothrotherium shastense). Results: While there is significant variation of bile salts across species, bile salt profiles are generally stable within families and often within orders of reptiles and mammals, and do not directly correlate with differences in diet. The variation of bile salts generally accords with current molecular phylogenies of reptiles and mammals, including more recent groupings of squamate reptiles. For mammals, the most unusual finding was that the Paenungulates (elephants, manatees, and the rock hyrax) have a very different bile salt profile from the Rufous sengi and South American aardvark, two other mammals classified with Paenungulates in the cohort Afrotheria in molecular phylogenies. Analyses of the approximately 8,000 year old human coprolites yielded a bile salt profile very similar to that found in modern human feces. Analysis of the Shasta ground sloth coprolites (approximately 12,000 years old) showed the predominant presence of glycine-conjugated bile acids, similar to analyses of bile and feces of living sloths, in addition to a complex mixture of plant sterols and stanols expected from an herbivorous diet. Conclusions: The bile salt synthetic pathway has become longer and more complex throughout vertebrate evolution, with some bile salt modifications only found within single groups such as marsupials. Analysis of the evolution of bile salt structures in different species provides a potentially rich model system for the evolution of a complex biochemical pathway in vertebrates. Our results also demonstrate the stability of bile salts in coprolites preserved in arid climates, suggesting that bile salt analysis may have utility in selected paleontological research. Background Bile salts are amphipathic, water-soluble end-metabolites of cholesterol that facilitate intestinal absorption of lipids, enhance proteolytic cleavage of dietary proteins, and exert potent antimicrobial activity in the small intestine [1]. The synthesis of bile salts is the major route for elimi- nation of cholesterol (a water-insoluble molecule) from the body [2]. Bile salts are produced by every class of ver- tebrate animals and show substantial structural diversity across species [1-5]. The two basic structural components of bile salts are the 19-carbon (C 19 ) steroid nucleus and a side-chain (Fig- ure 1). In all bile salts characterized to date, the four-ring cyclopentanophenanthrene ('steroid') nucleus (with rings labelled A, B, C, and D as in cholesterol in Figure 1B) is fully saturated (i.e., the cholesterol double bond at C5-C6 has been reduced). The A/B ring juncture is variable, being cis in most bile salts but trans in some species (e.g., jawless fish, lobe-finned fish, agamid lizards), a shift that greatly influences the overall shape of the steroid nucleus. A/B trans (5α) bile salts have an extended, planar orienta- tion 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. Virtually all bile salts have a hydroxyl group at C-3 (like cholesterol) and at C-7, because cholesterol 7α- * Correspondence: lhagey@ucsd.edu, mkrasows@healthcare.uiowa.edu 1 Department of Medicine, University of California - San Diego, La Jolla, CA, USA 3 Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, IA, USA Full list of author information is available at the end of the article