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

Abstract

The intracellular pool of chelatable iron is considered to be a decisive pathogenetic 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 loaded with the fluorescent metal indicators, phen green SK (PG SK), phen green FL (PG FL), calcein, or fluorescein desferrioxamine (FL-DFO), was quenched when iron was added to the cells in a membrane-permeable form. It increased when cellular chelatable iron available to the probe was experimentally decreased by an excess of various membrane-permeable transition metal chelators. The quenching by means of the ferrous ammonium sulfate ؉ citrate complex and also the ''dequenching'' using 2,2Ј-dipyridyl (2,2Ј-DPD) were largest for PG. We therefore optimized the conditions for its use in hepatocytes and tested the influence of possible confounding factors. An ex situ calibration method was set up to determine the chelatable iron pool of cultured hepatocytes from the increase of PG SK fluorescence after the addition of excess 2,2Ј-DPD. Using this method, we found 9.8 ؎ 2.9 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 absorption spectroscopy. The concentration of chelatable iron in hepatocytes was higher than the one in K562 cells (4.0 ؎ 1.3 mol/L; mean ؎ SEM; n ‫؍‬ 8), which were used for comparison. This method allowed us to record time courses of iron uptake and of iron chelation by different chelators (e.g., deferoxamine, 1,10-phenanthroline) in single, intact cells. (HEPATOLOGY 1999;29: 1171-1179.)

Copper Transport Across Pea Thylakoid Membranes

“…This is within the same magnitude of the reported K m for Cu 21 uptake in Chlamydomonas reinhardtii (Hill et al, 1996). The reported ratio of PGSK to Fe 21 interaction is 3:1 (Petrat et al, 1999). If this is also the case for Cu 21 , with 50 mM PGSK loaded inside the membranes, Cu 21 transport reaches completion before the probe is completely complexed.…”

“…Phen Green SK was first used as a fluorescent probe to measure free iron levels in hepatocyte and human erythroleukemia K562 cells, which had been preloaded with the fluorophore (Petrat et al, 1999(Petrat et al, , 2000. We showed that PGSK entrapped in isolated vesicles could be used to measure the transport of ferrous iron across chloroplast inner envelope membranes (Shingles et al, 2001).…”

Enhancement of iron toxicity in L929 cells by d-glucose: accelerated(re-)reduction

“…The chelation to the fairly small and lipophilic 8HQ made the iron membrane permeable and thus allowed us to bypass the highly regulated cellular uptake of iron, whereas iron salts such as FeCl $ , FeSO % and Fe(NH % ) # (SO % ) # or iron bound to nitrilotriacetic acid did not enter the cells with comparable ease (experiments in isolated hepatocytes ; [16] and F. Petrat and U. Rauen, unpublished work) and did, in contrast to the membranepermeable iron complex, not exert considerable toxicity in L929 fibroblasts (I. Lehnen-Beyel and U. Rauen, unpublished work).…”

“…The response of PhenGreen SK fluorescence to ferrous and ferric iron was tested in a chelex-treated cytosolic medium described in detail before [15,16] using a laser-scanning microscope (LSM 510 ; Zeiss). PhenGreen SK (30 µM ; dipotassium salt) was added to 2 ml of cytosolic medium at 37 mC.…”

“…Unfortunately, the determination of the redox state of intracellular chelatable iron is highly problematic. To estimate a possible influence of -glucose (or other reducing agents) on redox state and\or reduction rate of intracellular iron we therefore established a new application of the fluorescent probe PhenGreen SK, which has been used as an intracellular iron indicator [15,16]. Its response to ferrous and ferric iron is very different : in the presence of only 2 µM ferrous iron, the fluorescence of 30 µM PhenGreen SK was already clearly decreased in a cell-free system, whereas even 10 µM ferric iron did not significantly influence PhenGreen SK fluorescence (Table 1).…”