Determination of Cholic Acid in Bile and in Duodenal Drainage

The Gregory-Pascoe 1 reaction was found to be specific for the determination of cholic acid. The Reinhold and Wilson 2 modification of this method was combined with the procedure of Harwood 3 in such a fashion as to obviate both the use of the color filter and the necessity of carrying out the reaction in the presence of alcohol. In addition it was found that the large amount of deoxycholic acid present in human bile, although not taking part in the reaction, formed a precipitate which interfered markedly with the colorimetric procedure. This difficulty was overcome by the addition of alcohol after the Gregory-Pascoe reaction had been carried out to completion.

Method. 1-2 cc. of human or canine gall bladder bile, 3-5 cc. of canine fistula bile, 10-30 cc. of human fistula bile or 20-35 cc. of duodenal drainage material are the usual quantities measured out into a 50 cc. centrifuge tube. Three cc. of 2N KOH is then added and thoroughly mixed with the bile. When small quantities of concentrated bile are used, 15-20 cc. of water is added, the presence of alkali preventing the precipitation of protein. Following this, 3 cc. of 40% zinc sulphate is added drop by drop with constant stirring. When the contents are thoroughly mixed, the supernatant fluid is separated by centrifuging and the precipitate washed 3 times with 15 cc. quantities of hot water. The 4 water washings are poured into a 100 cc. volumetric flask. The precipitate is then washed 3 times with 20 cc. portions of 95% alcohol. For the first washing cold alcohol is used, and for the subsequent washings hot alcohol. The alcohol washings are all transferred to a 100 cc. beaker and dried on a water bath. The residue, taken up with 5 cc. 2N K₂CO₃, is combined with the water washings in the 100 cc. flask, which is made up to volume.