METHODS FOR DETERMINATION OF CONJUGATED BILIRUBIN IN RAT FECES

Conjugated bilirubin was prepared from the feces of germ-free (GF) rats by 3 different preparative methods. The bilirubin conjugate preparations were coupled with diazotized ethyl anthranilate and the formed ethyl anthranilate azopigments were quantified spectrophotometrically and separated by TLC. The most polar azopigment was purified by TLC and subjected to ammonolysis followed by TLC of the released saccharide. As a result of this procedure, only glucuronic acid was detected as the conjugating saccharide, indicating that the most polar azopigment prepared from GF rat feces was the .delta. ethyl anthranilate azopigment. Reference azopigments were prepared from GF rat small intestinal contents and subjected to separation by TLC. The azopigment pattern was very similar to the pattern obtained with the fecal azopigment preparations and a maximum of 10 separated azopigment spots were detected. In addition to bilirubin glucuronides, other bilirubin conjugates with unknown structure apparently are excreted with the feces of GF rats. One of the preparative methods used for the preparation of conjugated bilirubin from GF rat feces was tested on feces from conventional (CONV) rats. From these preparations, no ethyl anthranilate azopigments were formed, indicating that feces from CONV rats is devoid of conjugated bilirubin.