Bile acid conjugates, quantitation thin-layer chromatography

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. 

After deproteinization of bile, Turnberg and Anthony-Mote (94) were able to quantitate bile acids directly with a NAD-dependent steroid oxido-reductase from a Pseudomonas strain. The suitable concentration range was 10-80 mg of bile acids per 100 ml. The amounts of individual bile acids could be determined after preparative thin-layer chromatography, either before or after hydrolysis of bile acid conjugates. 

Ali et al. (128 and 129) suspended freeze-dried human feces in alkaline-50 % ethanol and extracted with petroleum ether-diethyl ether, 1 1, to remove neutral lipids. The bile acids in the aqueous phase were next subjected to conditions for hydrolysis of conjugates. After acidification, the free bile acids were extracted with diethyl ether, methylated, and purified by preparative thin-layer chromatography. Mono-, di-, and trisubstituted methyl cholanoates are eluted together from the thin-layer plate and then quantitated on QF-1 columns before and after conversion into trifluoroacetates. 

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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