The Biological Effects of Bilirubin Photoisomers

Abstract

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.

Characterisation and the effects of bilirubin binding to human fibrinogen

“…Therefore, the results presented in this work suggest that bilirubin bound to fibrinogen probably exists in a more cyclic conformation. A similar shift was observed upon bilirubin binding to HSA [24][25][26]. In contrast to the HSA/bilirubin complex, which was characterised by a large increase in the fluorescence of bilirubin [27], this phenomenon was not observed in the case of the fibrinogen/bilirubin complex (data not shown).…”

Update on phototherapy in jaundiced neonates

“…Isolated photobilirubins formed in vitro may be biologically inert and non-toxic to neuroblastoma cells [20]. This finding is consistent with chemical and biological arguments, which posit that photoisomers of bilirubin must be less toxic than the predominant native Z,Z-bilirubin.…”

A novel accurate LC-MS/MS method for quantitative determination of Z-lumirubin

“…In fact, it has recently been reported that neonates with extremely low birth weights treated with aggressive PT have higher morbidity and mortality [7][8][9] . In addition, as we demonstrated in our previous in vitro/ex vivo studies, LR, although not affecting the viability of neuronal cells 24 , can produce pro-inflammatory cytokines 19 , which may certainly account for certain of the clinical observations. These facts emphasize the need for detailed studies of bilirubin photochemistry using proper analytical tools.…”

“…In our current study, we describe a much improved, analytical LC-MS/MS method for simultaneous determination of LR, the major bilirubin photoisomer; and UCB, with a sufficient sensitivity and accuracy to be used in clinical research. For this method, we used a standard of LR, whose isolation was originally described by McDonagh 15 and modified in our previous in vitro studies 24 . Despite this achievement and all the effort made, it must be noted that isolated LR is still not sufficiently pure, and this is one of the reasons for suboptimal variability in the LR concentrations measured in clinical serum samples (the other reason is related to the chemical instabilities of the analytes).…”