Before unconjugated bilirubin can react with Ehrlich diazo reagent, which must be added?

• A. Acetone
• B. Ether
• C. Distilled water
• D. Caffeine

Answer: (D)

Unconjugated bilirubin is hydrophobic and binds tightly to albumin, so it won’t react with Ehrlich’s diazo reagent in an aqueous solution. To allow the reaction to occur, the bilirubin must be solubilized in an organic solvent. Acetone serves this role well, dissolving bilirubin and releasing it from albumin so the diazo reagent can form the characteristic azobilirubin product. Distilled water won’t dissolve bilirubin, and caffeine isn’t a reagent in this reaction. Ether can be used in some older methods, but acetone is the standard choice for solubilizing unconjugated bilirubin before the Ehrlich diazo reaction.