Caffeine-splitting of bilirubin/albumin complex: its relevance to the spectrophotometry of bilirubin in serum

By means of gel-filtration of bilirubin/albumin mixtures, it is shown that unconjugated bilirubin remains completely linked to albumin (both human and bovine) in tetraborate buffer (pH 9.3), protein-free bilirubin appearing only when the bilirubin/albumin molar ratio exceeds two. On the other hand, bilirubin is completely set free from its protein link in the caffeine reagent. Additional chromatographic and spectrophotometric evidence is reported indicating the formation of a low-affinity complex between bilirubin and caffeine. These data explain why the spectrophotometric properties of bilirubin/albumin mixtures are matrix-dependent if measured in the tetraborate buffer but are no longer so when measured in the caffeine reagent. The relevance of these findings to the spectrophotometric "direct" assay of bilirubin in serum is discussed with reference to the occurrence of "delta-bilirubin" in pathological sera: this tightly protein-bound bilirubin fraction does not split in the presence of caffeine.