Quantitative effects of bilirubin photoisomers on the measurement of direct bilirubin by the enzymatic bilirubin oxidase method

Abstract

Background Bilirubin photoisomers, generated during phototherapy or incidental light exposure, may interfere with direct bilirubin (DB) measurement using the bilirubin oxidase method. This interference is particularly relevant in neonates, who physiologically exhibit elevated levels of unconjugated bilirubin. Methods Residual serum samples from 30 neonates were irradiated under controlled conditions to selectively produce bilirubin configurational isomers (BCIs) and structural isomers (BSIs). DB and total bilirubin (TB) were measured pre- and post- irradiation using the bilirubin oxidase method. BCI and BSI concentrations were quantified using high-performance liquid chromatography (HPLC), and their contributions to DB values were evaluated using linear and multiple regression analyses. Results Post-irradiation, DB levels increased significantly in correlation with BCI and BSI concentrations. Approximately 11% of BCI and 32% of BSI were quantified as DB using the bilirubin oxidase method. These findings were consistent across both individual and multiple regression models. Conclusions Bilirubin photoisomers significantly influence DB values measured by the bilirubin oxidase method, potentially leading to overestimation of conjugated bilirubin. In neonatal care, accurate interpretation of DB values requires attention to sample handling and awareness of photoisomer interference, particularly under light-expose conditions.