Bilirubin Interference in the Diphenylamine Reaction

Abstract

IN the course of analysis of human bile for the presence of mucosaccharide and deoxyribose-reacting material by the diphenylamine reaction^1,2, an unusual peak at 650 mµ was found. It was thought possible that this peak might be due to the yellow-green colour of bile, resulting from the presence of high concentrations of bilirubin and other pyrrolic breakdown products. This possibility was checked using pure bilirubin, deoxyribonucleic acid (DNA), and a mixture of bilirubin and DNA (Fig. 1). It can be seen that a solution of bilirubin (after heating at 90° C with 5 per cent trichloroacetic acid (TCA)) produces a peak at 650 mµ on boiling for 20 min with the diphenylamine reagent. DNA gives a peak at 600 mµ under the same conditions, whereas a mixture of bilirubin and DNA gives a peak at 620 mµ The extinctions of DNA and bilirubin appear to be additive. Bilirubin also interferes in the Burton³ procedure for deoxyribose, producing a strong green colour.