Determination of Plasma Bilirubin;a Modified van den Bergh Procedure

Abstract

The diazotization of bilirubin in the van den Bergh estimation is strictly proportioned to the intensity of the color produced. 1 However, the use of the artificial color standards and the stratification of the plasma-ammonium sulphate and alcohol in the determination afford a valid criticism of the procedures ordinarily employed. Some three years ago we adapted the Thannhauser-Anderson 2 method to avoid these difficulties. A strongly acidified solution of azobilirubin, which remains unchanged for 3 to 6 months if kept in a cold refrigerator, is employed as a standard. A proportional concentration of acid is added to the unknown. There is no stratification of the mixture, and the red reaction changes to an intense blue. 3 Publication of our procedure has been delayed until a ready source of bilirubin for the standard could be located. Failing in this, we have prepared our own bilirubin from bile rather simply. Foweather 4 recently has given a short procedure for obtaining bilirubin from gall stones. Our method of preparing bilirubin and the technique for the quantitative test follow:

Preparation of bilirubin. 500 cc. of bile from surgical drainage patients or 50 cc. of post mortem gall bladder bile (diluted 2–3 times) is allowed to stand a few hours in the refrigerator and the supernatant fluid is decanted. This is diluted several times with water. Barium chloride solution is added with stirring. If the precipitate of barium bilirubinate does not flocculate immediately, addition of a few drops of alkali usually suffices. When the precipitate settles the supernatant fluid is siphoned off. The precipitate is poured on a filter and is washed with water on the paper; it is air dried and pulverized in a mortar. The powder is extracted with warm alcohol and with ether or chloroform and again air dried.