DIFFERENTIAL MICRODETERMINATION OF BILE ACIDS IN BILE BY PAPER CHROMATOGRAPHY
1959 Volume 46 Issue 6 Pages 681-693
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1959 Volume 46 Issue 6 Pages 681-693
1. A method for micro differential determination of bile acids by use of p. p. c. and ultraviolet absorption was devised. With this method, free, glyeine-conjugated and taurine-conjugated bile acids were determined quantitatively without any interference by lipids.
2. To remove the lipids in bile which interfere with the determination, two-step-p. p, c. with 50 per cent methanol (pH 12) or an extraction procedure with 50 per cent ethanol and petroleum ether was performed and after this treatment, p. p. c. was allowed to proceed.
3. Taurine-conjugates were separated by p. p. c. with a new solvent containing ethyl butyrate (or isoamyl formate)/heptane/70 per cent acetic acid.
4. After heating at 60° for 60 minutes, conjugated chenodeoxycholic acid and conjugated deoxycholic acid which could not be separated by p. p. c. were determined differentially by utilizing a quality of absorption spectra.
5. The optimum heating conditions for free and conjugated cholic acids were investigated and it was found that the heating condition of 28° for 60 minutes gave more precise results than that of 60° for 15 minutes. 6. The maxima for the spectra of free and conjugated cholic acid, were found at 320mμ and 386mμ, not at 320mμ and 389mμ as in previously published reports.
7. As determinations of free and conjugated cholic acid by p. p. c. were much influenced by ferric-ion in the filter paper, Toyo filter paper No. 53 which contained less ashes and ferric-ion was preferable.
8. Recoveries of pure bile acids by this method were as follows: Deoxycholic acid (98.6 per cent), cholic acid (93.2 per cent), glycodeoxycholic acid (98.4 per cent), and Glycocholic acid (93.4 per cent).
6. Recoveries of bile acid added to human bile were as follows: Deoxycholic acid (97.1 per cent), and cholic acid (90.7 per cent).
10. The reports published before said that in Jananese bile there were no taurine-conjugates, but as a result of this experiment, taurine-conjugates were found in all examples.
11. In bladder bile of some cases of cholecystitis with gall stone, small amounts of free bile acids were also detected.
The author wished to thank Prof. H. Miyake, Faculty of Medicine, Kyushu University, for his interests in this work and many valuable instructions. Thanks are also due to Prof. Y. Oshim a and Assistant Prof. M. Funatsu, Faculty of Agriculture, Kyushu University, for their valuable discussions.
