title: Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells.
abstract: Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9-1.5 pmol mg(-1) protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3-5 pmol mg(-1) protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5-13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and may thus be a dynamic factor of the endothelial function.
authors.affiliation: University of Trieste, Department of Life Sciences, via L. Giorgeri 1, 34127 Trieste, Italy.|Univesity of Ljubljana, Faculty of Medicine, Institute of Pharmacology and Experimental Toxicology, Korytkova 2, 1000 Ljubjana, Slovenia.
authors.forename: Lovro
authors.initials: L
authors.lastname: Ziberna
authors.affiliation: University of Nova Gorica, The Laboratory for Environmental Research, Vipavska 13, POB 301, 5001 Nova Gorica, Slovenia.
authors.forename: Mitja
authors.initials: M
authors.lastname: Martelanc
authors.affiliation: University of Nova Gorica, The Laboratory for Environmental Research, Vipavska 13, POB 301, 5001 Nova Gorica, Slovenia.
authors.forename: Mladen
authors.initials: M
authors.lastname: Franko
authors.affiliation: University of Trieste, Department of Life Sciences, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Sabina
authors.initials: S
authors.lastname: Passamonti
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000032:analysis* / Q000494:pharmacology; D001663:Bilirubin / Q000032:analysis* / Q000494:pharmacology; D002460:Cell Line; D002853:Chromatography, Liquid; D042783:Endothelial Cells / Q000737:chemistry* / Q000502:physiology*; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D000975:Antioxidants; D051547:Heme Oxygenase-1; D001663:Bilirubin