The separation of serum pigments giving the direct and indirect van den Bergh reaction.

Forty years ago van den Bergh developed a method for the estimation of bilirubin in serum by combining it in alcoholic solution with freshly diazotized sulphanilic acid. Shortly afterwards van den Bergh and Muller (1916) noted that by the omission of alcohol they could distinguish two types of reaction. Pigments from bile and serum from cases of obstructive jaundice developed a colour in the absence of alcohol. This was called a " direct " reaction. Normal serum, and that of patients with haemolytic jaundice, required alcohol for the development of colour, and they were said to give an " indirect " reaction. Many attempts have been made to explain the difference between the " direct " and the " indirect " reactions, but the explanations fall mainly into two groups. The first attributes the difference to the existence of two separate pigments. On the basis of clinical and physiological evidence it is considered that bilirubin is formed through the breakdown of haemoglobin. Bilirubin reacts indirectly in the van den Bergh reaction, but on passage through the parenchymal cells of the liver it is transformed into an unknown, direct-reacting pigment which appears in the bile, or in the serum when the flow of bile is obstructed. The second group considers that the difference between the two types of reaction is due to the presence of either inhibiting substances or of catalysts. Particular attention has been paid to the possible role of proteins, and it is widely held that in the indirect reaction alcohol is necessary to release bilirubin from plasma proteins to which it is bound. This fundamental uncertainty as to the nature of the van den Bergh reaction has lasted for a quarter of a century. Recently our attention was directed to a study of the bile pigments present in kernicterus, and methods have been devised for fractionating bile pigments by "reverse phase" chromatography.