Chemical nature of a synthetic bilirubin conjugate and its reactivities in the total and direct reactions by the Jendrassik-Gróf method.

Using liquid chromatography, nuclear magnetic resonance spectroscopy, and elemental analyses, we verified that a commercially available synthetic bilirubin conjugate is predominantly a ditaurobilirubin (DTB) disodium salt. The Jendrassik-Gróf total bilirubin (TBIL) method quantitatively measures unconjugated bilirubin (Bu) and the Bu-equivalent content in DTB, from which we infer that the azopigments of Bu and DTB have identical molar absorptivities, which do not change in the presence of either serum or serum albumin of human or bovine origin. However, based on the ratio of direct bilirubin (DBIL) to TBIL, the DBIL reaction in dilute HCI is incomplete (even up to 20 min), with lower yields in a matrix of bovine serum albumin than in human serum. By contrast, the DBIL reaction in acetate buffer (pH 4.75) is quantitative for DTB in human serum (or its albumin), but less so in bovine serum (or its albumin). DTB is water soluble, is relatively stable when lyophilized with human serum, and is determined with such high precision in the above-mentioned endpoint assays that it may be a suitable surrogate calibrator for conjugated bilirubin.