Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes
Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes
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DOI：
10.1042/bj20051496
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发表时间：
2006-04-01
影响因子：
4.1
通讯作者：
Petrat, F
中科院分区：
文献类型：
--
作者：
Ma, YM;De Groot, H;Petrat, F
已经合成了一系列荧光铁螯合剂，使得荧光官能团与3-羟基吡啶-4-酮共价连接。在本研究中，将荧光铁螯合剂加载到分离的大鼠肝细胞中。细胞内的荧光不仅淬灭通过添加一个高度亲脂性的8-羟基喹啉-铁（III）复合物，但也通过添加过量的膜渗透性铁螯合剂CP 94（1，2-二乙基-3-羟基吡啶-4-酮）dequenched。通过使用数字荧光显微镜在线记录单个完整细胞中铁和铁螯合的摄取的时间过程。各种荧光铁螯合剂的细胞内浓度测定通过使用荧光分光光度计裂解后的探针加载的细胞，并被发现取决于它们的分配系数;更疏水的化合物，更高的细胞内浓度。采用离体校正法测定培养大鼠肝细胞的可螯合铁库。CP 655（7-二乙基氨基-N-[（5-羟基-6-甲基-4-氧代-1，4-二氢吡啶-3-基）甲基]-N-甲基-2-氧代-2H-苯并吡喃-3-甲酰胺）是一种中等亲脂性的荧光螯合剂，被发现是监测可螯合铁的最灵敏的探针，如通过加入CP 94诱导的细胞内荧光增加所确定的。通过该探针测定肝细胞中细胞内可螯合铁池的浓度为5.4 +/-1.3 μ M。
A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline-iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)niethyll-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4 +/- 1.3 mu M.