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EXAMPLE 16. We have previously shown that unbound bilirubin (also referred to as non-albumin bound or free bilirubin) is a more sensitive and specific predictor than total serum bilirubin or the Bilirubin:Albumin (B:A) molar ratio.
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.