Mechanism of interference by hemoglobin in the determination of total bilirubin. II. Method of Jendrassik-Grof.

Oxyhemoglobin in erythrocyte hemolysates interferes with the Jendrassik-Grof assay. Destruction of azobilirubin occurs when oxyhemoglobin is oxidized to methemoglobin during diazotization or to alkaline hematin with addition of alkaline tartrate. The most probable mechanism is by oxidation with an agent such as hydrogen peroxide or a related species resulting from hemoglobin oxidation. Methemoglobin also appears to cause some destruction of azobilirubin during diazotization. Methemoglobin forms during diazotization because of reactions of oxyhemoglobin with both diazo reagent and nitrite ion. Formation of methemoglobin is, therefore, more rapid in the test than in the blank mixture and, under reaction conditions, its absorbance is less than that of oxyhemoglobin. This results in spectral interference when neutral azobilirubin is assayed. Alkaline tartrate abolishes this spectral error by causing rapid formation of alkaline hematin in both test and blank.