Bile acid conjugates, quantitation separation

In quantitative studies, Stiehl et al. (91) extracted bile acids from duodenal bile with methanol-acetone and the conjugates were hydrolyzed according to the method of Nair et al. (118). The free bile acids were then separated with thin-layer chromatography and determined spectrophoto-metrically after reaction with a sulfuric acid reagent. 

Some further examples of the application of TLC may be cited a procedure for two dimensional separation [179] analysis of bile from various species [76] fractionation of bile lipids into groups [128] metabolism [73] and analysis of human faecal bile acids [153 a] quantitative determination of free [203] and bile acid conjugates [202] in serum. 

Gas-liquid chromatography (GLC). The use of capillary columns has markedly increased sensitivity and specificity of GLC in the determination of the bile acids (see Fig. 1). Owing to high resolution power great many individual bile acids can be separated and quantitated in each run yet the complex mixtures of fecal and urinary bile acids still show some overlapping and require preliminary group separation. Also, free and conjugated bile acids... 

There is still no perfect method for quantitative determination of the bile acids. Ganshirt, Koss and Mobianz [62] have used two systems together, one for separation of the free acids and one for the conjugates the zones were localised by spraying with water, eluted with 65 % sulphuric acid and determined spectrophotometricaUy. Forth et al. [201] have extended this method. Hara et al. [77] and Semenuk and Beher [226] have tried densitometric evaluation Spritz [153a], titrimetric and Iwata and Yamasaki [212], enzymatic. Frosch and Wagener [59] have made use of the differences in absorption of partially separated mixtures to estimate the components. 

The quantitative determination of both conjugated and free bile acids must be preceded by elimination of other lipids and the separation of individual bile acids. This can be achieved with the use of chromatographic procedures as already described. 

Since free and conjugated dihydroxy cholanic acids cannot be separated completely by paper and thin-layer chromatography they must be differentiated by specific reactions. The following procedures are suitable for quantitative determination of free bile acids reaction with concentrated sulfuric acid for determination of cholic acid (Hammarsten 1925), reaction with salicylic aldehyde and sulfuric acid for determination of deoxycholic acid (Szalkowski and Mader 1952), reaction with ethyl acetate, concentrated sulfuric acid and acetic acid anhydride for chenodeoxycholic acid (Isaksson 1954). 

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