The identification of direct-reacting bilirubin as bilirubin glucuronide.

It has long been recognized that, with p-diazobenzenesulfonic acid (van den Bergh reagent), bilirubin in serum of patients with jaundice may exhibit two types of reaction (1, 2). If coupling with the diazo reagent occurs promptly in aqueous solution, the reaction is said to be “direct;” if, however, coupling t.akes place only after prior addition of alcohol, the reaction is said to be “indirect.” The factors responsible for this difference in behavior have been a matter of much controversy (3)) but, in recent years, evidence has rapidly accumulated to suggest that the two types of reaction observed reflect the presence of two different forms of the pigment in the serum (4-6). Thus, bilirubin giving an “indirect” diazo reaction appeared to be similar to crystalline bilirubin which is soluble in chloroform, but insoluble in water (7)) whereas “direct-reacting” bilirubin is more water-soluble (6). The two pigment fractions could be separated by paper chroma.tography (5), by column chromatography (8)) or by reverse phase column chromatography (6)) but the more water-soluble of the two compounds was found to be so unstable that satisfactory purification and isolation could not be achieved (6). A possible way of overcoming this difficulty was indicated by the observation of Ekuni (9) and Kawai (lo), later confirmed by Billing (11)) that the two forms of bilirubin gave rise to two more stable ‘Lazobilirubins,” which could be separated by chromatography. In the present investigation, the bilirubin present in normal bile and in serum and urine of jaundiced patients was converted to the respective hydroxypyrromethene azo derivatives (12)) chromatographed on paper, purified, and studied. In preliminary communications, it has been reported that the azo derivative of water-soluble “directreacting” bilirubin contains a mole of glucuronic acid, whereas the derivative of water-insoluble “indirect-reacting” bilirubin lacks this component (13, 14).