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

Abstract

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.

Uridine Diphosphate-Glucuronic Acid-independent Conversion of Bilirubin Monoglucuronides to Diglucuronide in Presence of Plasma Membranes from Rat Liver is Nonenzymic

“…It is essential to buffer the sample first to pH 2.7 before adding the diazo-reagent, because otherwise the reaction mixture may temporarily be at pH above 3, causing diazocleavage of unconjugated bilirubin (24). Also, for bile, appropriate dilution (20-fold or more) is required because otherwise added or endogenous bilirubin may react to a considerable extent (26), probably owing to the reactionpromoting effect of bile salts. By subsequent TLC of the azopigment extracts (25), unconjugated azodipyrrole was separated from azodipyrrole glucuronide and the ratio: r = unconjugated azodipyrrole/azodipyrrole glucuronide (Eq.…”

Bilirubin kinetics in intact rats and isolated perfused liver. Evidence for hepatic deconjugation of bilirubin glucuronides.

“…In the present study a new and more sensitive procedure for quantitation of plasma bilirubin has been used, obviating the use of the Weber-Schalm solventpartition method (24) that has been widely adopted (8,22,25) despite its documented inaccuracies (8,(26)(27)(28). Using this procedure we have examined the hepatic transport kinetics of radiobilirubin in 30 intact rats and developed a compartmental model that described all experimental data obtained from plasma, liver, and bile.…”

A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in bile

“…Unfortunately, it has been shown that both methods are inadequate. The solvent partition method does not separate conjugated and unconjugated bilirubin adequately and does not distinguish between the various conjugated forms of bilirubin (7,8). Although the formation of the diazonium salts has been applied successfully to both clinical and research problems for a number of years, it has become apparent that the diazo procedures directed at separating bilirubin and its conjugates have also been hampered by the participation of unconjugated bilirubin, to a small extent, in the diazo reaction under conditions initidly considered to be conducive of reaction only with the conjugated bilirubins (9).…”