Free bile acids

Table 1.1 lists some of the characteristics of the more common bile acids, which are divided into 3 main classes free bile acids, glycine and taurine conjugates. 

Bile acids are recycled via the enterohepatic circulation, with less than 5% of the total bile acid pool entering the colon.Bile adds are reabsorbed by ileum columnar epithelium cells and are transported back to the liver by the portal vein where they are extracted by hepatocytes. Approximately 6-12 enterohepatic circulations occur daily. Free bile acids, like DCA, are partly absorbed into the colon and enter the enterohepatic circulation, where they are... 

Acid suppression is probably responsible for deconjugation of bile acids in the upper GI tract and hence oesophageal exposure to free bile acids. 

Free bile acids Glycine conjugates Taurine conjugates... 

Scalia and Games developed a packed column SFC method for the analysis of free bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) [32]. The baseline separation of all five bile acids was achieved on a packed phenyl column with a methanol-modified carbon dioxide in less than 4 min. The elution order showed a normal-phase mechanism because the solutes eluted in the order of increasing polarity following the number of hydroxyl groups on the steroid nucleus. The method was also applied to the assay of UDCA and CDCA in capsule and tablet formulations. The method was found to be linear in the range 1.5-7.5 ng/ml (r > 0.99, n = 6). The average recoveries (n= 10) for UDCA and CDCA were 100.2% with a RSD of 1.7% and 101.5% with a RSD of 2.2%, respectively. The reproducibility of the method was less than 1.5% (n = 10) for both UDCA and CDCA. 

S. Scalia and D.E. Games, Determination of free bile acids in pharmaceutical preparation by packed column supercritical fluid chromatography, J. Pharm. Sci., 82 44 (1993). 

Fig. 6.15. Normal-phase CEC negative ion ES1-MS separation of a mixture of free bile acids, glycine, and taurine conjugates using monolithic poly (acrylamide-co-methylene bisacrylamide-co-3-aminopropane vinyl ether-co-[2-(acryloyloxy)-ethyl]-trimethyl...

The secondary bile acids result from the activity of anaerobic intestinal microorganisms in the ileum, caecum and colon, (s. fig. 3.3) Deconjugation, with the subsequent release of free bile acids, is a prerequisite for these reactions. This is followed by 7a-dehydroxylation of cholic acid and chenodeoxycholic acid to yield deoxy-cholic acid and lithocholic acid, respectively. 7a-de-hydrogenation and oxidation of chenodeoxycholic acid also yield ketolithocholic acid ... 

Unlike enzymatic or radioimmunoassay methods, GLC requires lengthy sample preparation before bile acid concentrations can be determined. In the case of serum, bile acids must be extracted (see Section 6.1) and hydrolysis carried out to remove glycine and taurine, and also sul te groups, if they are likely to be present (see Section 6.2). The free bile acids are then converted to volatile derivatives. 

Because HPLC ofiers the possibility of rapid separation, quantification, and recovery of both free bile acids and bile acid conjugates, much interest is currently centered on this technique. A significant advant e of HPLC compared to CLC is that potentially destructive hydrolysis steps can be avoided and bile acid conjugates can be analyzed as they occur in bile or serum. The method is relatively fast as the major bile acids in human bile have been separated in less than 1 hour by reverse-phase HPLC (B25, M8). 

M36, Musial, B. C., and Williams, C. N., Quantitative assay of conjugated and free bile acids as heptafluorobutyrate derivates by gas-liquid chromatography. /. Lipid Res. 20, 78-85... 

The intestinal microflora of man and animals can biotransform bile acids into a number of different metabolites. Normal human feces may contain more than 20 different bile acids which have been formed from the primary bile acids, cholic acid and chenodeoxycholic acid [1-5], Known microbial biotransformations of these bile acids include the hydrolysis of bile acid conjugates yielding free bile acids, oxidation of hydroxyl groups at C-3, C-6, C-7 and C-12 and reduction of oxo groups to give epimeric hydroxy bile acids. In addition, certain members of the intestinal microflora la- and 7j8-dehydroxylate primary bile acids yielding deoxycholic acid and lithocholic acid (Fig. 1). Moreover, 3-sulfated bile acids are converted into a variety of different metabolites by the intestinal microflora [6,7]. 

The hydrolysis of bile acid conjugates is probably the initial reaction catalyzed by intestinal bacteria. Therefore, primarily free bile acids are isolated from the feces of man and animals [1-5]. The bulk of the free bile acids in feces of man is deoxycholic acid and lithocholic acid which are generated by the 7 -dehydroxylation of cholic acid and chenodeoxycholic acid, respectively. A portion of fecal acids is absorbed from the intestinal tract, returned to the liver where they are conjugated and again secreted via biliary bile. Therefore, the final composition of biliary bile acids is the result of a complex interaction between liver enzymes and enzymes in intestinal bacteria. 

Fig. 11. Effects of temperature on water solubility (in negative logarithmic units, moles/l) of undissociated free bile acids at pH 3. (Modified from ref. 90.) Numbers and Greek letters denote position and orientation, respectively, of hydroxyl groups in each 5/3-cholanoic acid. Mean m.p.s [34-41, and private communications from A. Roda and A.F. Hofmann] are indicated on each curve.

It is quite probable that enterolith formation in man is a consequence of stasis and bacterial proliferation in the intestine. Microbial enzymes deconjugate bile salts, which raise their pK to a value of about 6, so that much of the free bile acid is un-ionized and precipitates at the / H of intestinal content. When microbial enzymes also convert cholic acid to deoxycholic acid, the latter compound forms choleic acids, which have an even higher pKa and thus precipitate more readily. 

Deconjugation with rapid absorption of free bile acids... 

The efficient intestinal absorption of bile acids involves both active and passive absorption, but little information on the relative sites and mechanisms of absorption and on their contribution to the entire enterohepatic cycle of bile acids exists. Although the contribution of passive and active absorption of bile acids in the rat small intestine has been measured (20), no data are available for other species. The major site of absorption in all vertebrates appears to be the ileum, where an active transport site exists (14,15). Free bile acids are absorbed passively in the jejunum by nonionic diffusion, dihydroxy acids being absorbed more rapidly than trihydroxy acids (21,22). Perfusion studies in the human jejunum have suggested that glycine dihydroxy bile acids may be absorbed to some extent, and additional evidence for jejunal absorption of bile acids has been obtained in patients and animals with ileal resection (97,98). No information exists on the importance of jejunal bile acid absorption in health in man. Taurine-conjugated bile acids do not appear to be absorbed in the human jejunum (24). 

Taurine conjugates are not absorbed in the upper intestine of human subjects (31,32), the major transport taking place in the lower ileum by both an active mechanism and passive ionic diffusion. Glycine conjugates, particularly those of dihydroxy bile acids, on the other hand, are absorbed also in the jejunum by passive ionic diffusion (33). Negligible amounts of free bile acids are normally found in the upper small intestine (23), while deconjugation is known to occur in the lumen of the terminal ileum. Absorption of free bile acids appears to take place by both ionic and nonionic diffusion, the transport for dihydroxy bile acids being particularly rapid even in the upper intestine (33). 

Reabsorption of bile acids is very effective, so that only a small percentage escapes into the cecum. The rest returns via the portal circulation as conjugates [or as unconjugated bile salts if free bile acids were present in the gut lumen (34)] into the liver, thus completing the enterohepatic circulation. Before resecretion, free bile salts are conjugated in the liver with taurine and glycine. 

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