Mechanism of interference by hemoglobin in the determination of total bilirubin. I. Method of Malloy-Evelyn.

Oxyhemoglobin is the species of hemoglobin in erythrocyte hemolysates that inhibits the diazo reaction. Ferric hemoglobin derivatives and species with relatively low molecular mass do not interfere. Conversion of oxyhemoglobin to acid hematin under assay reaction conditions is associated with rapid destruction of bilirubin, which accounts for the diazo reaction error. The most probable mechanism for this destruction of bilirubin is an oxidative reaction involving H2O2, formed in the oxidation of hemoglobin, and acid hematin acting as a pseudoperoxidase. We could find no evidence for other mechanisms of interference such as spectral error or azobilirubin destruction. Addition of potassium iodide, 4.0 mmol/L final concentration in the reaction mixture, eliminates interference from hemoglobin added to give concentrations as great as 10 g/L. It also eliminated the effects of hemolysis in the method of Ertingshausen et al. (Clin. Chem. 19: 1366, 1973), in which ethylene glycol is used as the accelerator.