Intracellular determination of elements in mammalian cultured cells by total reflection X-ray fluorescence spectrometry

Abstract

TXRF (total reflection X-ray fluorescence spectrometry), a rapid and reliable multi-element method, was applied to quantify intracellular concentrations of trace and non-trace elements in different mammalian cell lines. The coordinated variations in their intracellular contents were determined when the extracellular concentration of one of them was modified. The results indicate that TXRF permits the detection of total trace metal contents using a minimum amount of cells [(1-2)×10 6 ), while (4-6)×10 6 cells were sufficient to determine their cytosol/pellet distribution. In the six cell lines analyzed, the order of relative abundance for trace and non-trace elements was Cu<Fe<Zn and Ca<S, respectively. One of the cell lines was exposed to increased amounts of extracellular copper (0.44 to 100 µmol l –1 ), and then transferred to a culture medium containing a minimum concentration of the metal (0.44 µmol l –1 ). Under this condition, it was possible to measure both the rise (28-fold) in the intracellular copper content and its recovery to a base concentration. In addition, intracellular levels of other elements were measured, indicating that Fe and Ca showed the highest increase (1.7- and 2.2-fold respectively). On the other hand, the Zn/Fe ratio was hardly affected, suggesting that this ratio can be used as an internal marker of metabolic integrity in cells exposed to different extracellular copper concentrations. These results indicated that TXRF is an adequate method to measure simultaneously the intracellular concentration of different elements and permits the evaluation of their variations in response to modified concentrations in the extracellular medium of one element.