Studies on the Determination of Bile Pigments. Vi. Urobilinogen in Urine as Urobilinogen-aldehyde.

THE photometric determination of urobilinogen was proposed by Terwen (i) and employs the reaction of native urobilinogen, plus that derived from ferrous hydroxide reduction of urobilin, with Ehrlich’s reagent, p-dimethylaminobenzaldehyde. This basic approach culminated in the “quantitative” and “semiquantitative” methods of Watson (2,3) with minor modifications by various authors (4). Efforts in this laboratory directed at proper methods of standardization have provided absorptivities of the best available urobilin preparations and their respective urobilinogen-aldehydes (5). Phenol red (PSP) was shown to be superior to the Pontacyl dye mixture or to phenolphthalein as a secondary standard for the urobilinogen-aldehyde color. Also, increased stability of urobilinogen was achieved in the urobilinogen-aldehyde reaction by the addition of ascorbic acid to the reaction mixture. It was then our purpose to complete the standardization of the “quantitative” procedure with recovery experiments for urobilin added to urine and to redetermine normal values using the proposed method of standardization (5). The results obtained revealed the apparent inaccuracy of these technics. The problems encountered incidental to the purpose at hand will be described in this paper.