Inaccuracies in measurement of conjugated and unconjugated bilirubin in bile with ethyl anthranilate diazo and solvent-partition methods.

A criticial evaluation was made of the ethyl anthranilate diazo and two solvent-partition methods for the determination of conjugated and unconjugated bilirubin in human and rat bile. The ethyl anthranilate diazo reagent, which reacts only with conjugated bilirubin in serum, also diazotized a variable proportion of unconjugated bilirubin in bile and thus overestimated the concentration of monoconjugates. With the Weber-Schalm and modified Folsch solvent-partition methods applied to human or rat bile, 4--9% of added 14C-labelled unconjugated bilirubin partitioned with the conjugated bilirubin in the upper phase, and 4--9% of added 14C-labelled conjugated bilirubin partitioned into the lower phase. With dog bile, the spill-over of 14C-labelled bilirubin into the lower phase was 9--11%. Analysis of azopigments from the Weber-Schalm partition confirmed that over two-thirds of the bilirubin in the lower phase represents monoconjugates, principally the less-polar monoxylosides and monoglucosides. These solvent-partition methods thus overestimate the concentration of unconjugated bilirubin in bile.

Measurement of conjugated and unconjugated bilirubin in bile. II. A new thin-layer chromatographic method.

A new thin-layer chromatographic (TLC) method is described for measurement of conjugated (CB) and unconjugated (UCB) bilirubin in bile. Bile is streaked on a silica-gel plate and developed with chloroform. Bilirubin in the mobile UCB band and stationary CB band is quantitated by diazotization from the scraped gel with p-iodoaniline reagent. Both CB and UCB in bile yielded identical azopigment absorbance. There was no contamination of the UBC band with either CB or oxidative derivatives of bilirubin, and over 98 per cent added 14C-UBC migrated with the mobile band. Recoveries from TLC averaged 98.2 +/- 4.3 per cent of the applied pigment, duplicated agreed within 10 per cent, and the method accurately detected the enzymatic and alkaline hydrolysis of CB to UCB. Concentrations of UCB in nine samples of normal human gallbladder bile were found to be less than 1.9 mg./dl. and never exceeded 1.2 per cent of the total bilirubin.