Determination of serum bilirubin based on the measurement of azobilirubin produced from the cleavage of bilirubin

The diazo coupling reaction of bilirubin is a consecutive reaction involving the first and second reaction, and in each reaction one molecule of azobilirubin is produced. All the diazo coupling method previously reported are based on the measurement of two molecules of azobilirubin produced at the completion of the second reaction. We studied the determination of serum bilirubin based on the measurement of azobilirubin formed in the first reaction which is a cleavage reaction of its molecule. As a result, it was assumed that if the ratio (k1/k2) of the rate constants of the first and second reaction is more than 500, the azobilirubin produced from the first reaction is measured separately without the influence of the azobilirubin formed in the second reaction. In the measurement usingthe diazonium salt of 4-anisidine whose ratio of the rate constants was 941 at 25°C, the measurement values obtained correlated well with the Michaëlsson method and the enzymatic method. From these results, it was found that serum bilirubin can be determined on the basis of colorimetric measurement of one molecule of azobilirubin produced from the first reaction, the cleavage reaction of bilirubin.