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The more important methods used and the problems involved in the estimation of bilirubin in serum have been adequately reviewed in recent times by Lemberg & Legge (1949) and by King & Coxon (1950). The methods are of two types: those based essentially on the original van den Bergh technique for the so-called 'indirect reaction' (Thannhauser & Andersen, 1921; Haslewood & King, 1937), and those which promote coupling between van den Bergh reagent and bilirubin without precipitation of proteins. In the latter type, special catalysts or accelerators are employed, e.g. sodium benzoate and caffeine (Jendrassik & Cleghorn, 1936), urea and caffeine (Rappaport & Eichhorn, 1943), thereby avoiding the use of alcohol or other protein precipitants. Alternatively, alcohol is used in relatively low concentration in the presence of diluted serum in such a way that no precipitation occurs (Malloy & Evelyn, 1937). The main defect of the first type of method lies in the fact that, in the course of protein precipitation and its subsequent removal, considerable loss of either bilirubin or azobilirubin is incurred. On the other hand, methods of the second class suffer from the fact that the clarity ofthe final coloured solutions leaves much to be desired. In some cases, indirect bilirubin fails to react completely in a reasonable space of time (Gray & Whidborne, 1946). King & Coxon (1950), dissatisfied with the performance of the non-precipitation methods, have reverted to the original type ofprocedure with a modification which employs a higher dilution of serum and reagents in the presence of ethanol than is customary, thereby minimizing the loss of azobilirubin. We have investigated the van den Bergh reaction to see whetherwe could arrive at conditions whereby real clarity could be achieved in the final solution prepared for colorimetric analysis without recourse to any step involving separation of proteins, and at the same time utilize a reaction which would proceed rapidly to completion in all types of case, no matter what the form of bilirubin present. Rapid coupling of indirect bilirubin with the diazo reagent is achieved by use of urea-ethanol solutions in conditions which introduce only slight opacity, representing a very small ddgree of protein precipitation. This cloudiness completely disappears when concentrated phenol solution is added. Employment of phenol in connexion with the development of a diazo colour requires considerable care. Regard must be paid to the necessity of inactivating the excess of diazo reagent before the phenol is added, and also to avoidance ofthe formation ofany pink oxidation product from the phenol used. Following the observation of Lees (1950) that azide would react with the diazonium component of the Griess-Ilosva reagent to diminish the colour intensity produced by subsequent coupling with 1naphthylamine, azide was tried as a simple means of inactivating excess of the van den Bergh reagent and was found highly effective. The addition, therefore, ofa trace of azide, and the removal of excess of nitrous acid by the usual sulphamate addition, overcome the objections to the introduction of phenol into the van den Bergh reactions, and allows the completion of a method which in practice has proved satisfactory in its application to all clinical cases so far investigated.

The estimation of serum bilirubin