Sulfanilic acid reacts with sodium nitrite to form diazotized Sulfanilic acid. In the presence of accelerator (cetrimide), conjugated and unconjugated bilirubin reacts with diazotized Sulfanilic acid to form azobilirubin (Bilirubin Total). In the absence of accelerator only conjugated bilirubin reacts (Bilirubin Direct). The increase of absorbance at 546 nm is proportional to bilirubin concentration.