Materials and Method

An automated procedure is presented, based on the method of Bruchner [Amer. J. Gun. Pathol. 32, 513 (1959)), for determination of both total and conjugated bilirubin. The method is standardized by use of bilirubin in human serum, which is stabilized by a mixture of formamide and cyanide. Hemoglobin does not interfere at concentrations less than 500 mg/100 ml, and the effect of lipemia is minimized. Additional Keyphrases formamide-cyanide stabilizer #{149}AutoAnalyzer spectrophotometry of azo derivative #{149}hemoglobin and lipemia, interference by Bruchner (1) described a method for the deter-mination of serum bilirubin based on the work of Jendrassik and Grof (2). Serum total bilirubin was reacted with diazotized sulfanilic acid in the pres-ence of a buffered urea-antipyrine mixture. Phos-phoric acid was added to form the blue azo deriva-tive, which was measured spectrophotometrically. To determine conjugated bilirubin, he added phos-phoric acid before the diazo reagent, to suppress reaction with the unconjugated form. Bruchner claimed that in his procedure there was minimal interference by hemoglobin and lipemia, but no experimental evidence was pre-sented to support this. In this paper, Bruchner’s method has been adapted for use on the AutoAnalyzer and the effects of hemoglobin and lipemia have been in-vestigated. The suggestion of Dybkaer and Hertz (3) to stabilize bilirubin with formamide and cyanide has been used to standardize the method