Quantification of plasma hemoglobin in the presence of bilirubin with bilirubin oxidase

Owing to the overlapping absorption bands of hemoglobin and bilirubin, spectrophotometric determination of plasma free hemoglobin has been plagued by the interference of bilirubin when the latter is present in significant concentrations. To resolve this problem, bilirubin oxidase from Myrothecium verrucaria has been used to remove bilirubin in icteric samples. The enzyme converts bilirubin to biliverdin and eliminates its absorption in the 400 nm region. The absorbance of hemoglobin at 415 nm after Allen baseline correction is used to quantify its concentration. Intra-assay precision of this method on a sample containing 200 mg/L of free hemoglobin and 200 mg/L of bilirubin is 3.1 percent (n = 10), while the between-run precision is 3.8 percent (n = 10). Accuracy studies with samples containing 200 mg/L of bilirubin yielded a straight line: y = 0.90x + 2.31, Sy.x = 3.3, r = 0.99. These results demonstrate that this method can be used to determine free hemoglobin in icteric specimens.