An increase in serum unconjugated bilirubin (indirect bilirubin, iDB) levels, most importantly free bilirubin not bound to albumin (unbound bilirubin, UB), is associated with the development of serious brain injury in newborns, called bilirubin encephalopathy [1
]. In Japan, bilirubin encephalopathy is an etiology of dyskinetic cerebral palsy and abnormal auditory brainstem response. This is of particular concern for extremely preterm infants [3
], of which the prevalence is at least two per 1000 live births less than 30 weeks of gestational age [5
]. Furthermore, children with bilirubin encephalopathy suffer from serious challenges in their daily lives [3
An automated analyzer designed to measure UB serum/plasma levels was developed in 1982 (UB-Analyzer; UA-2, Arrows Co., Ltd., Osaka, Japan) using the glucose oxidase and peroxidase (GOD–POD) method [6
]. To date, this is the only method approved for clinical use by the US Food and Drug Administration and the Ministry of Health, Labor, and Welfare in Japan. In this method using absorbance spectroscopy, albumin-unbound bilirubin is oxidatively degraded and readily converted into a colorless substance, whereas albumin-bound bilirubin tends not to be oxidatively degraded. Therefore, UB levels are calculated from the initial rate of oxidative degradation [6
]. More specifically, UB levels are determined by colorimetrically monitoring the rate of decreasing absorbance associated with bilirubin pigments [1
]. However, several factors may interfere with UB measurements using the GOD–POD method, resulting in the underestimation (from either sample dilution, ZE-bilirubin, or vitamin C) or overestimation (from conjugated bilirubin (direct bilirubin, DB), EZ-cyclobilirubin, or hemoglobin) [1
]. Most importantly, DB affects the accuracy of UB measurement because DB is also easily changed into a colorless compound by POD. Consequently, this can lead to misjudgments and wrong decisions in clinical practice when planning treatments. The UB values displayed by the UB-Analyzer using the GOD–POD method can register higher than is actually present when the DB/TB ratio is high; a DB/TB ratio >10% overestimates UB levels compared to a ratio <10%, although the specific effect of DB on UB measurements by the GOD–POD method remains unclear [1
]. This problem is more significant for newborn care in the neonatal intensive care unit (NICU) because >20% of hospitalized infants in Japanese NICUs have conjugated hyperbilirubinemia, which is especially prevalent among extremely preterm infants, small-for-gestational infants, and infants with gastrointestinal anomalies or chromosomal disorders [8
In 2013, a bilirubin-inducible fluorescent protein from eel muscle (UnaG) was cloned, which is characterized as having high affinity and specific binding to iDB rather than DB [9
]. In newborn sera, we confirmed the measurement of iDB directly using UnaG regardless of the presence of DB, bilirubin photoisomers, hemoglobin, or lipid emulsions [10
]. To overcome the challenges of the GOD–POD method, we recently developed a novel method for measuring UB levels using GOD–POD in conjunction with UnaG, referred to as the GOD–POD–UnaG method, which was registered with the patent office on 12 June 2020 (registration number: 6716108).
The aims of our study involve further delineating the strengths of the GOD–POD–UnaG method with respect to the established GOD–POD method. First, we studied clinical serum samples to determine how much the DB/TB ratio would affect UB values when using the GOD–POD method (Study A). Next, we tested whether the GOD–POD–UnaG method could be used for UB measurements in newborn sera, regardless of those with a high DB/TB ratio (Study B).
In Study A, the GOD–POD method had a lower correlation with the UB value in the presence of DB with a DB/TB ratio ≥5% in newborn serum. This impact became more profound as the DB/TB ratio became larger and the number of outliers increased. It is reported that it is difficult to obtain accurate UB levels from the GOD–POD method when the DB/TB ratio is ≥10% [1
]. Our findings reveal that the influence of the DB/TB ratio indeed occurs at ratios lower than 10% (and at least 5%). In Study B, we validated the GOD–POD–UnaG method that we developed for comparison with the established GOD–POD method using clinically-obtained neonatal sera. With low DB/TB ratio (< 5%) sera, the UB values from the GOD–POD–UnaG method were similar to those of the GOD–POD method. Notably, when using the GOD–POD–UnaG method, we demonstrated for the first time that even a high DB/TB ratio (≥5%) of serum does not affect the UB value.
Due to the development of perinatal medical care in recent years, severely ill infants (e.g., extremely preterm infants and infants with gastrointestinal anomalies or chromosomal disorders) can now survive. Neonatologists are encountering an increase in opportunities to treat and care for newborn patients exhibiting high DB/TB ratios in NICUs [8
]. These newborn patients are more likely to develop hypoalbuminemia, with many possibilities to use drugs that alter albumin binding (e.g., antimicrobial drugs, fat formulations, and indomethacin), which consequently affect bilirubin binding to albumin to result in hyper-unbound bilirubinemia [1
]. Therefore, accurate UB measurements have become paramount for such patients for a favorable prognosis. In infants with conjugated hyperbilirubinemia, there is an urgent need to develop a methodology that can measure UB levels regardless of the presence of DB.
Analysis of the correlation between UB and the iDB/A ratio was limited to serum with an iDB/A ratio <0.5; the relationship between the two certainly remains linear when the iDB/A ratio <0.5 [11
], i.e., the equilibrium state of the first binding site of albumin is established. In fact, when the ratio was ≥0.6, the linear relationship between UB and the iDB/A ratio may not hold due to an equilibrium condition associated with the second binding site of albumin. As this study concerned the influence of DB in the GOD–POD method, we limited the analysis to serum samples with an iDB/A ratio <0.5 so that the analysis was simpler and easier to understand.
The UB-Analyzer (UA-2) that uses the GOD–POD method can measure serum TB and UB levels [7
]. UB is rapidly oxidized to colorless compounds by POD in the presence of hydrogen peroxide derived from glucose by mediation of GOD. First, TB levels are determined by direct absorbance measurement at 460 nm. Next, under experimental conditions where bilirubin oxidation follows first-order kinetics, the rate constant is determined by measuring the oxidation velocity of bilirubin in the absence of albumin. The initial velocity is estimated from the time required for a 20% decrease in concentration from the initial TB concentration. UB is calculated from the initial velocity of bilirubin degradation and the ratio of the POD concentration to that in the standard assay of the albumin-free bilirubin solution [1
] (Figure 5
a). As UnaG is a protein characterized by iDB concentration-dependent fluorescence [9
], the GOD–POD–UnaG method involves the division of the serum sample into two samples to calculate the difference in TB level initially and after 20 s of POD reaction (TB reduction rate). Therefore, it is necessary to stop the POD reaction after 20 s, which can be accomplished by adding ascorbic acid (patent registration number: 6716108) (Figure 5
b). As a result, we were able to establish a GOD–POD–UnaG method with a fixed reaction time (20 s), which is beneficial for the development of an automated measuring device that is currently under development in our laboratories.
Because UnaG emits very intense fluorescence and accurate measurements are hindered by an internal shielding effect when a high concentration of iDB is present, we diluted the solutions 800-fold. However, if we consider the equilibrium relationship between bilirubin and albumin, it is important to use a more concentrated solution for the UB measurement requiring the reaction with POD [12
]; an 800-fold dilution for the reaction with POD would result in inaccurate UB levels. Therefore, a key feature of this GOD–POD–UnaG method is the use of a two-step dilution process, whereby the POD reaction proceeds first with a 51-fold dilution in the same manner as the GOD–POD method, and then a subsequent 800-fold dilution is used for the UnaG fluorescence measurement.
In clinical practice, a newborn patient with a high serum DB/TB ratio may require phototherapy and exchange transfusion for treatment, which is administered based on the UB values measured by the GOD–POD method; however, if judged by the results from the GOD–POD–UnaG method, this patient does not need such treatments. Out of the 11 high DB/TB ratio serum samples in Table 1
, three serum samples (Samples 1, 4, and 11) had UB values ≥1.0 from the GOD–POD method, which indicates the need for exchange transfusion according to the 1992 Kobe University Treatment Criteria [13
]. However, the GOD–POD–UnaG results for these samples indicated that not only exchange transfusion but also phototherapy would be unnecessary for the patients of serum samples 4 and 11. The results from the GOD–POD–UnaG method for serum sample 1 indicate criteria for exchange transfusion [13
], even when taking the influence of DB into consideration. Therefore, the patient with serum sample 1 could be clearly diagnosed with serious unconjugated hyperbilirubinemia. Furthermore, while the results from the GOD–POD method for three other samples (samples 6, 7, and 8) indicated the need for phototherapy (UB ≥0.6) for these patients based on the 1992 Kobe University Treatment Criteria [13
], the results from the GOD–POD–UnaG method indicate that phototherapy is unnecessary. Indeed, a clinical issue in Japan is the overestimation of UB values from the GOD–POD method that can lead to the overtreatment of hyperbilirubinemia. As there were patients with serious UB levels even after removing the impact of DB, it is desirable to confirm UB values of high DB/TB ratio serum samples with the GOD–POD–UnaG method.
A limitation of this study was the number of cases examined. However, we were able to show clearly from both clinical and fundamental science perspectives that the GOD–POD method affects the UB levels when the DB/TB ratio in patient serum is ≥5%. As the GOD–POD–UnaG method in this study involved manual labor and the use of expensive microplate readers, we were unable to examine numerous serum samples. Going forward, an automated device needs to be developed to perform GOD–POD–UnaG measurements with a larger number of newborn sera to verify these results and provide a clinically feasible alternative to the GOD–POD method. Finally, because other novel UB measurement methods using a fluorescence sensor or potentiometric sensor are developed [15
], further studies are needed to compare the UB levels between their methods and our GOD–POD–UnaG method.