Interaction of hemoglobin in elevated serum total bilirubin determinations

A spectrophotometric study was carried out in an effort to elucidate how hemoglobin in serum interferes in the diazo coupling reaction used for the determination of bilirubin. Because it is difficult to see hemoglobin in neonatal specimens in which bilirubin is elevated without obtaining spectra of the mixtures, this can be a real problem when handling these specimens while using this particular reaction. From the spectrophotometric evidence described here, one can infer that hemoglobin can act in two ways. In one way, the formation of ferriprotoporphyin generates an oxidizing agent which can convert bilirubin to an oxidized product precluding it from taking part in a diazo coupling reaction. In the other way, the same ferriprotoporphyrin seems capable of oxidizing the azobilirubin complex after it has reached equilibrium again causing some lowering of the results obtained. It may be reasonable to assume that the quicker the analytical event can occur, the more accurate the result might be. If the matrix of reaction can be changed, and the reaction of diazo coupling also speeded up as a feature of the change, then it may be possible to avoid the interference from hemoglobin almost entirely. High concentrations of hemoglobin were tested here in an attempt to clearly show how the interference might take place.