Wetting agents or incorrect pH have what effect on unconjugated bilirubin?

• A. Increases
• B. Decreases
• C. No effect
• D. Converts to conjugated

Answer: (A)

Unconjugated bilirubin is hydrophobic and binds strongly in plasma, so it is not very soluble in aqueous assay reagents. In diazo-based bilirubin measurements, it must be solubilized to react with the diazo reagent. Wetting agents (surfactants) and an incorrect pH help pull bilirubin into the soluble phase, increasing its availability to react. This makes more unconjugated bilirubin participate in the color-forming reaction, so the measured amount appears higher. Conjugated bilirubin, already water-soluble, isn’t affected in the same way and isn’t converted during the assay, so the effect described is specific to solubilizing the unconjugated fraction.