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.
Sci Rep. 2016 Jul 6;6:29240. doi: 10.1038/srep29240.
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.
胆红素是肝功能的标准血清生物标志物。但令人费解的是，它与心血管疾病风险呈负相关。鉴于内皮功能障碍在引发心血管疾病中的作用，如果能直接分析血管内皮中的胆红素，可能有助于阐明这些关系。因此，我们使用高效液相色谱法与热透镜光谱检测法和二极管阵列检测法来测定内源性细胞 IXα-胆红素。为了确认异构体 IXα-胆红素，我们使用超高效液相色谱法与采用电喷雾电离源的高分辨率质谱仪以及串联质谱检测法。我们测量了动脉和静脉大鼠内皮中的胆红素（0.9-1.5 pmol mg(-1) 蛋白）。在人内皮 Ea.hy926 细胞系中，我们证明细胞内胆红素（3-5 pmol mg(-1) 蛋白）可以通过细胞外胆红素摄取或细胞血红素加氧酶-1（HO-1）的上调来调节，HO-1 是一种与内源性胆红素合成相关的细胞酶。此外，我们通过胆红素测定了细胞内抗氧化活性，其 EC50=11.4±0.2 nM，处于报道的游离血清胆红素（8.5-13.1 nM）范围内。胆绿素显示出与胆红素相似的抗氧化特性。从这些观察结果推断，内皮内胆红素呈波动变化，因此可能是内皮功能的一个动态因素。
