Hydrolysis of Conjugated Bile Acids and Subsequent Purification Steps

Hydrolysis of Conjugated Bile Acids and Subsequent Purification Steps 

In many studies it is not necessary to isolate conjugated bile acid. It is simpler to work with free bile acids and a hydrolysis can be made at different stages of the work-up procedure. One should realize, however, that the nature of conjugated bile acids is not known in all details ornithine-conjugated bile acids have been described (57), esterified bile acids occur in feces (19), and sulfates were recently found in bile (47).Information about these compounds is lost upon hydrolysis. 

Enzymatic or alkaline hydrolysis can be made (acid hydrolysis to obtain the amino acid). The enzymatic method is described in detail by Nair in another chapter. It is a mild method, and should be preferred to the alkaline hydrolysis in many analytical procedures. All details on the structural requirements are not yet known and some bile acid types are not hydrolyzed. For this reason both enzymatic and alkaline hydrolysis should be used in parallel in identification work. 

The alkaline hydrolysis is vigorous, and some loss of bile acid occurs. Ketonic bile acids (particularly 3-ketones) are most likely to give low recoveries. In our experience the losses are larger with biological extracts than when pure bile acids are subjected to these conditions. The possibility of artifact formation should always be kept in mind. Two methods are used more commonly than others 1.25 m NaOH, 3 hr, 15 psi, and 15% NaOH in 50% aqueous ethanol 10 hr, 110°C (I, 27, 58). Teflon or nickel vessels should be used instead of glass. Some authors have hydrolyzed bile acids with 20% KOH in ethylene glycol at reflux temperature. This method was carefully tested by Evrard and Janssen who found 80-100% recovery after 15-20 min of refluxing (23). 

The rate of hydrolysis may vary with the bile acid structure. The hindered nature of the peptide bond in the taurine-conjugated 3 ,7a, l2a-trihydroxy-5 3-cholestanoic acid may explain why this conjugate is more resistant to saponification than C24 acids (59). 

---