Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK

The intracellular pool of chelatable iron is considered to be a decisive pa thogenetic factor for various kinds of cell injury. We therefore set about establishing a method of detecting chelatable iron in isolated hepatocytes based on digital fluorescence microscopy. The fluorescence of hepatocytes l oaded with the fluorescent metal indicators, phen green SK (PG SK), phen gr een FL (PG FL), calcein, or fluorescein desferrioxamine (FL-DFO), was quenc hed when iron was added to the cells in a membrane-permeable form. It incre ased when cellular chelatable iron available to the probe was experimentall y decreased by an excess of various membrane-permeable transition metal che lators. The quenching by means of the ferrous ammonium sulfate + citrate co mplex and also the "dequenching" using 2,2'-dipyridyl (2,2'-DPD) were large st for PG, We therefore optimized the conditions for its use in hepatocytes and tested the influence of possible confounding factors. An ex situ calib ration method was set up to determine the chelatable iron pool of cultured hepatocytes from the increase of PG SK fluorescence after the addition of e xcess 2,2'-DPD. Using this method, we found 9.8 +/- 2.9 mu mol/L (mean +/- SEM; n = 18) chelatable iron in rat hepatocytes, which constituted 1.0% +/- 0.3% of the total iron content of the cells as determined by atomic absorp tion spectroscopy. The concentration of chelatable iron in hepatocytes was higher than the one in K562 cells (4.0 +/- 1.3 mu mol/L; mean +/- SEM; n = 8), which were used for comparison. This method allowed us to record time c ourses of iron uptake and of iron chelation by different chelators (e.g., d eferoxamine, 1,10-phenanthroline) in single, intact cells.