The Quantitative Determination of Bile Acids by Means of a New Color Reaction and Monochromatic Light

A survey of the literature reveals the fact that numerous methods, direct and indirect, have been employed by various workers for the determination of bile acids. Due to the many advantages offered by a calorimetric method for the determination of small quantities of a substance, the Pettenkofer reaction (1) as modified by Mylius (2) seemed most worthy of consideration. A critical study of the quantitative possibilities of this reaction, however, soon revealed several undesirable features: (1) The red or reddish violet color varied greatly as to color shade, making comparison in the calorimeter very difficult; (2) the quantitative recovery of pure sodium glycocholate was very irregular with errors of from 1 to 12 per cent; (3) the color reaction was not specific for bile acids, Mylius finding 150 other substances which gave the Pettenkofer reaction. It was obvious that the Pettenkofer reaction, as commonly described, was wholly unsuitable for quantitative purposes even on pure solutions of bile acids and unreliable for even qualitative work on body fluids. During the course of a spectrophotometric study of the reaction between glycocholic and sulfuric acids and furfural, a blue-colored compound was discovered. When to a dilute bile acid solution were added 34 volume per cent of HzS04 and 0.05 volume per cent of furfural and the mixture heated for 30 minutes at 65”, a pure blue color results. This color is different from any that has been reported in the literature. Pettenkofer (1) described the color he obtained as violet-red, Vogel and Dragendorff (3) obtained a brown color, Mylius (2) describes the end-product as “blood red,” Jolles (4) and Hirschfelder (5) reported a red to brown color, Gilbert,