A Method for Determination of Total Pigment in Bile Which Is Applicable to “Biliverdin Biles.”

Methods for quantitation of bile pigments, with the exception of the methods of Hooper and Whipple, 1 Beccari, 2 Kerppola and Leikola, 3 and Peterman and Cooley 3 have been confined to modifications of the van den Bergh method 4 for the determination of bilirubin. That the pigment in the bile of carnivora is present chiefly as bilirubin and that of herbivora as biliverdin 5 is a conception based partly upon physical appearances of the respective bile and partly upon specific tests for these particular constituents. Modifications of the van den Bergh method are satisfactory for clinical identification or quantitation of bilirubin; however, such methods are without value when used for the quantitation of pigment in bile in which the pigment is present in the form of oxidized bilirubin derivatives. Furthermore, Müller and Engel 6 have shown spectrophotometrically that the diazo reaction of the van den Bergh test detects only 30 to 60% of the bilirubin present in a solution, while Beccari 7 has reported that a number of compounds other than bilirubin, and present both in bile and serum, gave color reactions which were indistinguishable from that given by bilirubin.

In attempting to use the method of Hooper and Whipple 1 for the determination of total pigment, it was found that the method was uncertain and has objectionable features which have been pointed out by Peterman and Cooley. 8 These investigators have shown that the blue-green color taken as an end-point in the Hooper-Whipple and their own methods is a mixture of unoxidized bilirubin and bilicyanin, the combination of which constitutes the green pigment of bile heretofore considered as an individual pigment, “biliverdin.”