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Investigation of the Use of Bilirubin Oxidase to Measure the Apparent Unbound Bilirubin Concentration in Human Plasma

An investigation into the use of bilirubin oxidase to measure the apparent concentration of unbound bilirubin in solutions of bilirubin or bilirubin/human serum albumin demonstrated (1) near linearity of oxidation rate with bilirubin concentration up to 16 μmol/L, (2) linearity with respect to enzyme concentration up to 75 mg/L, (3) specificity of the enzyme for unbound bilirubin, as opposed to bound bilirubin, (4) poor precision. Very small absorbance changes leading to poor precision and the potential for interference by conjugated bilirubin meant that the method was unsuitable for patient samples.