Background
Oxidative stress plays an important role in the pathogenesis of cardiovascular degenerative diseases. Bilirubin is
known to be a potent endogenous antioxidant, both in in
vitro and in vivo. The latter can be ascribed to an efficient
amplification cycle whereby bilirubin, acting as antioxidant, is itself oxidized to biliverdin and then recycled back
to bilirubin by NADPH-dependent biliverdin reductase.
Bilitranslocase, a bilirubin-specific membrane carrier that
mediates cellular uptake of bilirubin, has recently been
found in the vascular endothelium. However, the levels of
albumin-free bilirubin in plasma and tissues are only 10
to 50 nM. Therefore, the objective of this study was to elucidate the antioxidant activity of low concentrations of
bilirubin and the involvement of bilitranslocase-mediated plasma membrane transport in human endothelial
cells.
Methods
In this study we used the cellular antioxidant activity
(CAA) assay developed by Wolfe and Liu [1]. Briefly, the
assay is designed to trigger an acute oxidative stress into
cells (by adding the radical initiator ABAP to the cell
medium) and to fluorimetrically follow the subsequent
increase of an intracellular radical-sensitive fluorescent
dye. Substances with antioxidative properties that have
free oxygen radical scavenging properties have been found
to decrease the formation of fluorescence. By using the
CAA assay, we have quantitatively evaluated the antioxidant activity of bilirubin in the endothelial cell line EA.hy
926. In our experiments, the cells were pre-incubated with
anti-bilitranslocase antibodies (studied group) or bovine
IgG (control group) before starting the CAA assay with
bilirubin (0.5-100 nM).
Results
The intra-cellular antioxidant activity of bilirubin was
concentration-dependent with an apparent saturation
obtained at higher concentrations. The half-maximal
effect was obtained at concentrations as low as 5 nM. The
pre-incubation of the cells with anti-bilitranslocase antibodies reduced the antioxidant activity by about 50%.
Conclusion
Bilirubin has a potent intracellular antioxidant activity if
applied to cells at concentrations close to its albumin-free
plasma levels. The observed cellular antioxidant activity
depends on bilitranslocase-mediated plasma membrane
transport and therefore confirms the functional role of
bilitranslocase as a membrane transporter in the endothelium.
