Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca*+ ions

The principles governing the in vitro solubility of the common natural conjugated and unconjugated bile acids and salts in relation to pH, micelle formation, and Ca2' concentration arc considered from a theoretical standpoint and then correlated first with experimental observations on model systems and second with the formation of precipitates containing bile acids in health and disease. In vitro, taurine-conjugated bile acids are soluble at strongly acidic pH; glycine-conjugated bile acids are poorly soluble at moderately acidic pH; and many of the common, natural unconjugated bile acids are insoluble at neutral pH. For both glycine-conjugated and unconjugated bile acids, solubility rises exponentially, with increasing pH, until the concentration of the anion reaches the critical micellization concentration (CMC) when micelle formation occurs and solubility becomes practically unlimited. In vivo, in health, conjugated bile acids are present in micellar form in the biliary and intestinal tract. Unconjugated bile acids formed in the large intestine remain at low monomeric concentrations because of the acidic pH of the proximal colon, binding to bacteria, and absorption across the intestinal mucosa. In diseases in which proximal small intestinal content is abnormally acidic, precipitation of glycine-conjugated bile acids (in protonated form) occurs. Increased bacterial formation of unconjugated bile acids occurs with stasis in the biliary tract and small intestine; in the intestine, unconjugated bile acids precipitate in the protonated form. If the precipitates aggregate, an enterolith may be formed. In vitro, the calcium salts of taurine conjugates are highly water soluble, whereas the calcium salts of glycine conjugates and unconjugated bile acids possess limited aqueous solubility that is strongly influenced by bile acid structure. Precipitation occurs extremely slowly from supersaturated solutions of glycineconjugated bile acids because of metastability, whereas supersaturated solutions of unconjugated bile acids rapidly form precipitates of the calcium salt. In systems containing CaZ+ ions and unconjugated bile acids, pH is important, since it is the key determinant of the anion concentration. For bile acids with relatively soluble calcium salts (or with a low CMC), the concentration of the anion will reach the CMC and micelles will form, thus precluding formation of the insoluble calcium salt. For bile acids, with relatively insoluble calcium salts (or with a high CMC), the effect of increasing pH is to cause the anion to reach the solubility product of the calcium salt before reaching the CMC so that precipitation of the calcium salt occurs instead of micelle formation. In vivo, in health, precipitation of the calcium salts of the common, natural conjugated bile acids does not occur. In experimental animals, concretions composed chiefly of insoluble calcium bile salts (unconjugated or conjugated) can be induced to form in the biliary tract by chronic oral administration of uncommon secondary bile acids (or their precursors) whose calcium salts are extremely insoluble (lithocholate, murideoxycholate, allodeoxycholate). It is concluded that the pattern of bile acid hydroxylation and conjugation (with taurine or glycine) of the common natural primary bile acids has resulted in formation of recycling anionic surfactants that are quite resistant to formation of insoluble calcium salts under the usual physiological conditions where they function as effective solubilizers of biliary and digestive lipids. -Hofmann, A. F., and K. J. Mysels. Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2' ions. J Lipid Res. 1992. 33: 617-626. Supplementary key words cholelithiasis enteroliths gallstones Bile acids are not only the water-soluble end products of cholesterol metabolism, but are also amphipathic molecules with multiple physiological functions. Bile acids are secreted into bile in the form of their N-acyl conjugates with glycine or taurine. In bile, they solubilize cholesterol as mixed micelles, enhancing its elimination; in small intestinal content, bile acids solubilize dietary lipids and their digestion products in mixed micelles, enhancing their absorption (1). After promoting the absorption of lipids from the small intestine, bile acids are actively absorbed from the terminal ileum and resecreted into bile; because of their efficient intestinal conservation, a large pool of bile acids accumulates and undergoes a number of enterohepatic cycles daily (2). Calcium ions are ubiquitous and present at relatively high concentrations (mM) in extracellular fluids. Calcium ions form insoluble salts with many inorganic and organic anions including bile acid anions. Precipitation of insoluble calcium salts of bile acids or other anions in the biliary tract is of medical interest primarily because of the mechAbbreviations: C, cholic; LC, lithocholic; CDC, chenodeoxycholic; DC, deoxycholic; CMC, critical micellization concentration; CMpH, critical micellization pH; CMT, critical micellization temperature. IAddress correspondence on biological aspects to author at: Department of Medicine, 0813, University of California, San Diego, La Jolla, CA 92093. *Address correspondence on physiochemical aspects to author at: 8327 La Jolla Scenic Drive, La Jolla, CA 92037. Journal of Lipid Research Volume 33, 1992 617 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom anical effects caused by the presence of fine precipitates (sludge) or stones (gallstones). In addition, formation of precipitates of calcium bile salts could remove bile acids from solution, thus lowering their concentration and decreasing their solubilizing activity. Lastly, precipitation of bile acids in the large intestine has been thought to decrease the intrinsic cytotoxicity of bile acids and thus influence the turnover of colonic epithelial cells (3). This could occur because of formation of the insoluble calcium salt or by a less direct mechanism proposed by van der Meer et al. (4), in which bile acid anions adsorb to calcium phosphate particles present in colonic content. Rile acids, as many weak organic acids whose anions are amphipathic, can exist in several states in biological systems: an insoluble, protonated acid; a simply dissolved protonated acid and/or its anion; a simple micelle; a constituent of mixed micelles or vesicles; or an insoluble calcium salt. A paper published elsewhere in this issue (5) and a previous publication (6) from this laboratory reported the solubility of the calcium salts of a number of unconjugated bile acids and their corresponding N-acyl glycine and taurine conjugates. Those studies also described, by means of phase maps, the behavior of dilute aqueous solutions when bile acid concentration, sodium concentration, and calcium activity were varied simultaneously. Those studies showed that the solubilities of the calcium salts of bile acids varied widely and were influenced by the number, position, and orientation of hydroxylic substituents on the steroid moiety as well as by the side chain structure (unconjugated, glycine-con.jugated, or taurine-conjugated). This paper is a review of the general problem of bile acid solubility and precipitation in vitro in model systems and in vivo in health and disease. The emphasis is mainly on bile acid concentration and type, but also considers the role of pH, micelle formation, and Ca2' concentration, all of which are potentially key determinants of bile acid solubility. The review is based on our own work as well as the work from other laboratories on the thermodynamic and kinetic aspects of the solubility of bile acids and their calcium salts. As will be shown, precipitation of bile acids in the form of the insoluble, protonated acid is mainly controlled by pH, whereas precipitation of the insoluble calcium salt is controlled by the activity of Ca2' ions and by the concentration and structure of the monomeric bile acid anion (BA-). In turn, the activity of Ca2' ions in the biliary and gastrointestinal tract is controlled by many factors including sodium ion concentration, Donnan equilibrium effects (because of the presence of micelles, vesicles, and proteins), as well as the presence of anions such as phosphate, carbonate, and fatty acid anions that may complex with bile acids in solution or form insoluble calcium salts. Accordingly, Ca2+ activity shall be considered an independent variable, and the behavior of various bile acids in its presence will be discussed. The emphasis is largely on events in the biliary and intestinal tract in mammals, with particular emphasis on humans. The chemical structures of the bile acids (and salts) under consideration are shown in Fig. 1 of the accompanying