Measurement of bilirubin and its monoconjugates and diconjugates in human serum by alkaline methanolysis and high-performance liquid chromatography.

No accurate methods are available for specific determination of unconjugated and conjugated bilirubin. Using a novel approach, we now have developed such an assay which permits direct individual measurement of bilirubin and its isomeric monoconjugates and diconjugates in serum. The monosugar and disugar conjugates are quantitatively converted to the corresponding methyl esters, which are readily extractable into chloroform. These monomethyl and dimethyl esters and unconjugated bilirubin are then separated by HPLC and detected spectrophotometrically in the effluent. Use of an internal standard and calibration of the method with crystalline reference bilirubin and bilirubin methyl esters permit direct measurement of the individual pigment fractions in the sample. The acuracy of this procedure was verified by a radioisotope dilution method. In sera of 22 healthy adults and six patients with Gilbert syndrome, only unconjugated bilirubin was detected. In 42 serum samples of jaundiced patients with hepatobiliary disease, unconjugated and all conjugated bilirubin fractions were increased, with the monoconjugates generally predominating. The total concentration of bilirubin and its carbohydrate conjugates, as determined by the new method, was considerably lower than the TB obtained with conventional diazo procedures. Contrariwise, both the new method and the diazo procedures gave comparable resuts when normal serum enriched with purified bilirubin glucuronides was assayed. Our findings thus indicate that unidentified, diazo-positive compounds distinct from bilirubin and its ester conjugates are present in pathlogical serum samples. The reported asssay is expectd to serve as a reference method for measurement of bilirubin and its carbohydrate conjugates in serum and to find general application in the study of bilirubin metabolism.