Noninvasive monitoring of bilirubin photoisomer excretion during phototherapy

Abstract

Lumirubin is the most prevalently excreted hydrophilic bilirubin photoisomer in phototherapy for neonatal jaundice caused by excess hydrophobic unconjugated bilirubin (ZZ-bilirubin). We developed a simple method to estimate the amount of lumirubin by monitoring the reverse photoisomerization of lumirubin to ZZ-bilirubin. Although lumirubin formation was long considered irreversible, exposure to blue light in the presence of the fluorescent protein UnaG, which binds specifically and tightly to ZZ-bilirubin, enables the reverse photoisomerization of lumirubin. This reaction was first detected using a fluorescence assay of neonatal urine sampled during phototherapy and purified lumirubin. The phenomenon of reverse photoisomerization of lumirubin was validated using liquid chromatography-mass spectrometry, which confirmed that lumirubin is reconverted to ZZ-bilirubin in the presence of UnaG. Analyses of 20 urine samples from 17 neonates revealed a significant correlation (correlation coefficient [r] = 0.978; 95% confidence interval 0.867-0.979; P < .001) between lumirubin and ZZ-bilirubin concentration before and after reverse photoisomerization. In general, the rate of photo-reconversion of lumirubin to ZZ-bilirubin is approximately 40%. In conclusion, we demonstrate here that lumirubin can be photo-reconverted to ZZ-bilirubin via exposure to blue light in the presence of UnaG. Utilizing this approach, urinary lumirubin levels can be estimated using an easy-to-perform fluorescence assay.

Figure 1

Fluorescence assay. Fluorescence intensity over time of 2 μmol/L purified LR and urine collected during phototherapy, each mixed with UnaG mixture (see Methods in the text) and then exposed to blue light. In both samples, the fluorescence intensity representing the UnaG–ZZ-BR complex increased with duration of exposure and plateaued at 60–75 min. LR, lumirubin; ZZ-BR, ZZ-bilirubin.

Figure 2

Bilirubin photoisomer calibration curves. (a) Fluorescence assay. Calibration curve was prepared from the fluorescence intensity of reference ZZ-BR adjusted to concentrations in the range of 0–5 μmol/L along with UnaG mixture (see Methods in the text). (b) Liquid chromatography–mass spectrometry. ZZ-BR and LR are expressed as the ratio of ion intensity to that of the internal standard, MBR. ZZ-BR, ZZ-bilirubin; LR, lumirubin; MBR, mesobilirubin.

Figure 3

Reverse photoisomerization in LC–MS/MS. MRM spectra of purified LR and urine. The retention times of MBR, ZZ-BR, and LR were 17.22, 16.53, and 2.00 min, respectively. As MBR was used as an internal standard, a signal at 17.22 min was observed in all analyses. (a) Before blue light exposure. All MRM spectra showed the concentration of LR but not that of ZZ-BR. (b) After blue light exposure. ZZ-BR (16.53 min) appeared with apoUnaG in both purified LR and urine. Without apoUnaG, however, only LR decreased, and ZZ-BR was not detected, even after exposure to blue light. LC–MS/MS, liquid chromatography–mass spectrometry; MRM, multiple reaction monitoring; MBR, mesobilirubin; ZZ-BR, ZZ-bilirubin; LR, lumirubin.

Figure 4

Correlation and efficiency of conversion of urinary LR to ZZ-B in the presence of apoUnaG following a 90-min blue light exposure. Single linear regression analysis of the increase in ZZ-BR concentration and decrease in LR concentration in urine samples during blue light exposure in the presence of apoUnaG. ΔZZ-BR is defined as the difference in ZZ-BR concentration before and after blue light exposure. ΔLR is defined as the absolute difference in LR concentration before and after blue light exposure. A strong positive correlation between ΔZZ-BR and ΔLR was observed. ZZ-BR, ZZ-bilirubin; LR, lumirubin.