THE SYNTHESIS OF BILIRUBIN GLUCURONIDE BY TISSUE HOMOGENATES

Abstract

For many years the van den Bergh react'ion has been utilized for the differentiation and quantitative measurement of t,he two forms of bilirubin found in the blood.One form, "direct" bilirubin, reacts instantaneously with diazotized sulfanilic acid.The second pigment, bilirubin or so called "indirect" bilirubin, reacts with the diazo reagent after the addition of alcohol (l), caffeine urea (2), or caffeine sodium benzoate (3).Despite the wide clinical use of the test for the differential diagnosis of jaundice, the chemical difference between these two pigments has not yet been established.Direct bilirubin has been postulated to be a protein complex (I), a heavy metal complex (4, 5), or an ester (6, 7) of bilirubin.Some authors have believed that only one pigment exists, the differences in reaction in the van den Bergh test being caused by substances in the serum (8-10).An excellent critical review of these interpretations has been made by With (11).Recently, Billing and.coworkers (12, 13) isolated and separated the two pigments on a kieselguhr column.The two compounds retained their specific diazo reactions despite the complete absence of all proteins.Similarly, Najjar and Childs ( 14) crystallized the two pigments and found that the protein-free crystals still possessed their original diazo reactions.Within the last few mont)hs Billing and Lathe (15) and Schmid ( 16) have isolated glucuronic: acid after t,he hydrolysis of "direct"' hilirubin azo pigment with alkali or p-glucuronidase.These aut)horx concluded that "direct" bilirubin is t,he product of the conjugation of bilirubin with glucuronic acid and is presumably formed in the liver.Dutton and Storey (17,18) have studied the synthesis in vitro of glucuronides of various phenols in liver homogenat,es.We have investigated the conversion of bilirubin to "direct" bilirubin in a similar system in order to demonstrate that glucuronide conjugation is the primary mechanism involved in the synthesis of "direct" bilirubin by the liver.EXPERIMESTAL Preparation oj Bilirubin Solution-A stock bilirubin solution was prepared by dissolving 10 mg. of bilirubin (Eastman 2101) in 100 ml. of chloro-