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)methyl]-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 μM.
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Research Article|
March 15 2006
Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes
Yongmin Ma;
Yongmin Ma
*Department of Pharmacy, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, U.K.
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Herbert de Groot;
Herbert de Groot
†Institut für Physiologische Chemie, Universitätsklinikum, Hufelandstr. 55, D-45122 Essen, Germany
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Zudong Liu;
Zudong Liu
*Department of Pharmacy, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, U.K.
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Robert C. Hider;
Robert C. Hider
1
*Department of Pharmacy, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, U.K.
1To whom correspondence should be addressed (email robert.hider@kcl.ac.uk).
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Frank Petrat
Frank Petrat
†Institut für Physiologische Chemie, Universitätsklinikum, Hufelandstr. 55, D-45122 Essen, Germany
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Publisher: Portland Press Ltd
Received:
September 09 2005
Revision Received:
November 21 2005
Accepted:
December 09 2005
Accepted Manuscript online:
December 09 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 395 (1): 49–55.
Article history
Received:
September 09 2005
Revision Received:
November 21 2005
Accepted:
December 09 2005
Accepted Manuscript online:
December 09 2005
Citation
Yongmin Ma, Herbert de Groot, Zudong Liu, Robert C. Hider, Frank Petrat; Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes. Biochem J 1 April 2006; 395 (1): 49–55. doi: https://doi.org/10.1042/BJ20051496
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