Studies on the Determination of Bile Pigments

The absorbance at 450 mµ of serum diluted with acidified ethylene glycol is used as a measure of the serum's total bilirubin content in a single-tube, single-extraction method for rapid determination of free and total bilirubin in 0.1 ml. of serum. With the addition of chloroform, free bilirubin is extracted into the chloroform but conjugated bilirubin remains in the ethylene glycol. Free bilirubin is determined from the absorbance at 450 mµ in the chloroform layer. Conjugated bilirubin is calculated from the difference. Absorbance readings at 520 mµ compensate for the absorbance at 450 mµ, due to hemoglobin in the serum. Comparison with the Malloy-Evelyn procedure showed equivalent levels of total but higher levels of conjugated bilirubin.