Estimation of Bilirubin in Serum

Most methods of estimating the concentration of bilirubin in serum are based on the van den Bergh reaction in which bilirubin couples with diazotized sulphanilic acid to give red " azobilirubin," the reaction being facilitated by the addition of an activator or catalyst such as ethanol. The many modifications of this reaction can be classified into two groups: (a) those in which ethanol is used as the catalyst, with consequent precipitation of the serum proteins, and (b) those in which some sub- stance, other than ethanol, acts as the catalyst and protein precipitation is avoided. Precipitation methods, such as those described by McNee and Keefer (1925), Haslewood and King (1937), and King and Coxon (1950), suffer from the disadvan- tage that some " azobilirubin " is lost on the protein precipitate; and further, the volume of coloured supernatant fluid or filtrate is unknown and varies from test to test. Consequently such methods can- not give really accurate results.