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Comparative Study
. 2020 Sep 16;21(18):6778.
doi: 10.3390/ijms21186778.

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  1 , Keiji Yamana  2 , Hajime Nakamura  3 , Kosuke Nishida  3 , Takeshi Morisawa  2 , Masami Mizobuchi  4 , Kayo Osawa  5 , Kazumoto Iijima  3 , Ichiro Morioka  6
Affiliations
Comparative Study

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 et al. Int J Mol Sci. .

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.

Keywords: albumin; analyzer; bilirubin encephalopathy; glucose oxidase-peroxidase method; indirect bilirubin; neonatal intensive care unit.

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Conflict of interest statement

The authors declare no conflict of interest. I.M. has received study grants and lecture and manuscript fees from Atom Medical Corp. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Correlations between the iDB/A ratio and UB classified by DB/TB ratio in clinical serum samples. The 301 serum samples were classified into four groups according to the DB/TB ratio for <5%, n = 239; 5–10%, n = 29; 10–20%, n = 17; and ≥20%, n = 16. A, albumin; DB, direct bilirubin; iDB, indirect bilirubin; TB, total serum bilirubin; UB, unbound bilirubin.
Figure 2
Figure 2
Comparison of the number of samples higher than the upper limit line of the 95% confidence interval of the DB/TB ratio <5% (outliers). CI, confidence interval; DB, direct bilirubin; TB, total serum bilirubin.
Figure 3
Figure 3
Correlations between the iDB/A ratio and serum UB values measured with the GOD–POD and GOD–POD–UnaG methods. A, albumin; DB, direct bilirubin; GOD, glucose oxidase; iDB, indirect bilirubin; POD, peroxidase; TB, total serum bilirubin; UB, unbound bilirubin; UnaG, bilirubin-inducible fluorescent protein from eel muscle.
Figure 4
Figure 4
Correlation analysis of UB values determined from the GOD–POD and GOD–POD–UnaG methods. DB, direct bilirubin; GOD, glucose oxidase; POD, peroxidase; TB, total serum bilirubin; UB, unbound bilirubin; UnaG, bilirubin-inducible fluorescent protein from eel muscle.
Figure 5
Figure 5
Calculation method of UB using estimated initial velocity of the GOD–POD and GOD–POD–UnaG methods. (a) In the GOD–POD method, the UB value is calculated by estimating the initial velocity based on the time taken for TB to reduce by 20% (Δt). (b) In the GOD–POD–UnaG method, the UB value is calculated based on the rate of reduction of iDB (ΔiDB) over 20 s. GOD, glucose oxidase; iDB, indirect bilirubin; POD, peroxidase; UB, unbound bilirubin; UnaG, bilirubin-inducible fluorescent protein from eel muscle.
Figure 6
Figure 6
GOD–POD–UnaG method protocol. GOD, glucose oxidase; POD, peroxidase; UnaG, bilirubin-inducible fluorescent protein from eel muscle.
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