A Novel Method for Measuring Serum Unbound Bilirubin Levels Using Glucose Oxidase–Peroxidase and Bilirubin-Inducible Fluorescent Protein (UnaG): No Influence of Direct Bilirubin
Sota Iwatani, Keiji Yamana, Hajime Nakamura, Kosuke Nishida, Takeshi Morisawa, Masami Mizobuchi, Kayo Osawa, Kazumoto Iijima, Ichiro Morioka
Abstract
The glucose oxidase–peroxidase (GOD–POD) method used to measure serum unbound bilirubin (UB) suffers from direct bilirubin (DB) interference. Using a bilirubin-inducible fluorescent protein from eel muscle (UnaG), a novel GOD–POD–UnaG method for measuring UB was developed. Newborn sera with an indirect bilirubin/albumin (iDB/A) molar ratio of <0.5 were classified into four groups of DB/total serum bilirubin (TB) ratios (<5%, 5–10%, 10–20%, and ≥20%), and the correlation between the UB levels and iDB/A ratio was examined. Linear regression analysis was performed to compare UB values from both methods with the iDB/A ratio from 38 sera samples with DB/TB ratio <5% and 11 samples with DB/TB ratio ≥5%. The correlation coefficient (r) between UB values and the iDB/A ratio for the GOD–POD method was 0.8096 (DB/TB ratio <5%, n = 239), 0.7265 (5–10%, n = 29), 0.7165 (10–20%, n = 17), and 0.4816 (≥20%, n = 16). UB values using the GOD–POD–UnaG method highly correlated with the iDB/A ratio in both <5% and ≥5% DB/TB ratio sera (r = 0.887 and 0.806, respectively), whereas a low correlation (r = 0.428) occurred for ≥5% DB/TB ratio sera using the GOD–POD method. Our GOD–POD–UnaG method can measure UB levels regardless of the presence of DB.