Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography

Abstract

Conjugated bile pigments, separated in 2 fractions by semi-quantitative TLC performed on silicic acid with phenol/water as the developing solvent, were treated with diazotized ethyl anthranilate. Resulting dipyrrylazo derivatives were analyzed by quantitative TLC. The tentative structure elucidation of tetrapyrrolic bilirubin conjugates and semi-quantitative evaluation of rat bile, post-obstructive human bile and dog bile composition is presented. Homogeneous and mixed hexuronic acid diesters of bilirubin containing glucuronic acid constitute 51% of the total conjugates in normal rat bile, 45% of those in human post-obstructive bile and 38% of those in obstructed rat biles. Monoconjugated bilirubin amounts to 33% of total conjugated bile pigments in normal rat bile and 17 and 14% in post-obstructive hepatic human bile and dog gall-bladder bile, respectively. After loading with unconjugated bilirubin a greater amount of monoconjugates (56%) occur in the rat bile; bilirubin diglucuronide excretion is decreased (34%). In normal dog gall-bladder bile 40% of glucose-containing diconjugates, 32% of homogeneous and/or mixed hexuronic acid (mainly glucuronic acid) diesters of bilirubin and 14% of xylose-containing diconjugates are estimated. Increased amounts of bilirubin conjugates, including some with unidentified uronic acid groups, were observed in cholestatic rat biles and quantities of conjugates with glucuronic acid were decreased.