Biliprotein in adult icteric serum--demonstrated by extension of the alkaline methanolysis procedure.

We confirmed that the alkaline methanolysis procedure of Blanckaert (Biochem. J. 185: 115-128, 1980) converts the sugar conjugates of bilirubin (Bc) into their corresponding methyl and dimethyl esters, which can be extracted into chloroform along with underivatized unconjugated bilirubin (Bu). By this procedure, we accounted for Bu nearly quantitatively, but only 76-83% of total Bc. By pretreating samples containing Bu and (or) Bc with a caffeine/benzoate reagent, we improved the analytical recovery of Bc to 85-93% without affecting the Bu. When the method (+ caffeine/benzoate) was applied to adult icteric serum, a variable fraction (20-75%) of the original total bilirubin (based on diazo reactivity) remained with the protein pellet, which is routinely discarded in the original methanolysis procedure. In this pellet we demonstrated the occurrence of a strongly protein-bonded bilirubin fraction (biliprotein) similar to the recently described "delta" fraction (Clin. Chem. 28: 629-637, 1982). The analytical and clinical implications of our findings are discussed.