MEASUREMENT OF BILIRUBIN AND ITS MONOCONJUGATES AND DICONJUGATES IN HUMAN-SERUM BY ALKALINE METHANOLYSIS AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

  • 1 January 1980
    • journal article
    • research article
    • Vol. 96  (2) , 198-212
Abstract
No accurate methods are available for specific determination of unconjugated and conjugated bilirubin. Using a novel approach, an assay was developed which permits direct individual measurement of bilirubin and its isomeric monoconjugates and disconjugates 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 high-performance liquid chromatography 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 accuracy of this procedure was verified by a radioisotope dilution method. In sera of 22 healthy adults and 6 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 [total bilirubins] obtained with conventional diazo procedures. The new method and the diazo procedures gave comparable results when normal serum enriched with purified bilirubin glucuronides was assayed. Unidentified, diazo-positive compounds distinct from bilirubin and its ester conjugates are present in pathological serum samples. The reported assay is expected 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.