The crystallization and properties of serum bilirubin.

It is generally accepted that in serum two types of bilirubin may exist, the so called direct and indirect. The differentiation is based upon the van den Bergh (1) reaction which is essentially a coupling reaction between the pigments and freshly diazotized sulfanilic acid. The indirect bilirubin is the normally occurring pigment and does not couple readily with the diazo reagent except upon the addition of alcohol to the reactants. The direct, on the other hand, couples with the reagent rapidly and completely within 30 minutes and produces the same color without the addition of alcohol. This latter appears in serum only when the bile duct system is blocked and the pigment is thereby reintroduced into the blood stream. The importance of these two types of pigments lies in the fact that they indicate different types of pathology and therefore assume diagnostic significance in clinical medicine. The results of the chemical and clinical studies on these pigments are still controversial and have been adequately reviewed recently by Lemberg and Legge (2). The difficulty was largely due to the fact that it was not possible to obtain native pure bilirubin from serum for comparative study with that available from bile. We have recently succeeded in obtaining indirect serum bilirubin in crystalline form (3) with relative ease and in yields as high as 85 per cent of that present originally in serum. The purpose of this report is to describe in detail the method of crystallization and the properties of the direct and indirect pigments. A brief report on this work was presented earlier (3).