Quantitative determination of the major 3-hydroxy bile acids in biological material after thin-layer chromatographic separation

Abstract

A micromethod is described for the quantitative determination of the major conjugated and free 3-hydroxy bile acids in biological material after thin-layer chromatographic separation, using a NAD-linked 3-hydroxysteroid dehydrogenase with spectrophotometric determination of the resultant NADH. As neither the taurine nor the glycine conjugates of the dihydroxycholanic acids can be separated by thin-layer chromatography but are found as common spots of the taurine and the glycine conjugates respectively, a special procedure is necessary for the separate determination of these conjugates. The sum of the dihydroxycholanic acid conjugates (taurine and glycine respectively) is measured enzymatically, while the deoxycholic acid conjugate is determined colorimetrically after reaction with salicylaldehyde. The amount of the chenodeoxycholic acid conjugate is calculated by subtraction of the value for the deoxycholic acid conjugate from the sum.