Determination of Bilirubin with Precipitation of the Plasma Proteins

Methods for the estimation of bilirubin are of two general types: those in which the bilirubin is caused to react with diazotized sulphanilic acid through the agency of added ethanol, which causes precipitation of the plasma proteins, and those in which some other agent is used to bring the bilirubin into reaction, without pre- cipitating the proteins. The former methods (for example, those of van den Bergh, 1918, quoted by Thannhauser and Andersen, 1921; Jendrassik and Czike, 1928; Haslewood and King, 1937) all suffer from the disadvantage that some bilirubin may be lost in th-protein precipitate. Van den Bergh recognized the difficulty, and Hasle- wood and King measured the loss to be about 10% in the case of highly icteric sera, but pointed out the impossibility of estimating what it might be with sera containing only small amounts of the pigment. Methods not entailing precipitation of the plasma proteins are most prominently represented by the procedures of Malloy and Evelyn (1937), Gray and Whidborne found an average difference of 40% between a "precipitation" method on the one hand and two "non- precipitation" methods on the other, when these were applied to sera and plasma from cases of jaundice. Because we have been dissatisfied with the performance of the " non-precipitation " methods thus far described, for example, on account of the very pale colours produced in the Malloy and Evelyn method, which employs a very high dilution (1 in 25), and the frequently " impure " colours produced by the Rappaport and Eichhorn method, we have sought another procedure.