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Method diversity can explain discrepancies between iron uptake in CaCo‐2 cells incubated with Na2EDTA. Are there two iron pools?
Author(s) -
Viteri Fernando Eduardo,
Casanueva Esther,
de Regil Luz Maria,
Killilea David W
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a624-a
Subject(s) - incubation , ferritin , extracellular , ascorbic acid , absorption (acoustics) , chemistry , biochemistry , cell , nuclear chemistry , biophysics , food science , biology , materials science , composite material
Background Reports are conflicting on the effect of adding Na2EDTA to FeSO4 while incubating CaCo2 cells. Studies indicate that EDTA impairs Fe uptake by these cells, opposite of its effect in humans. No standardized method has been used, making comparisons difficult. We developed a set of CaCo2 incubation conditions where EDTA and ascorbic acid addition to different Fe compounds resemble their behavior in humans. Methods We examined the following conditions in Fe incubations: effect of passage/differentiation (27–29 vs 48–59); media (PBS or MEM); incubation time (1, 2, 8, 24 h); pH (6 vs 7.2); preincubation of compounds prior to addition to the media; removal of extracellular Fe during harvesting. 18 μM of Fe was used. Response variables: protein, ferritin, cell Fe and their ratios. Results Older cells behave similarly to observed human Fe absorption; not so younger cells; PBS is OK for 2h incubations but MEM is needed for ferritin synthesis after; 8h incubation with Fe is optimal; Fe leaches out of the cells when incubated with EDTA for 24h; Cells withstand pH6 for 2h, but pH 7.2 is essential for cell survival for 24h; Ferritin synthesis increases 12% but 59Fe uptake decreases by 85% when premixed EDTA+Fe+59Fe is added to the media: are there 2 Fe pools?; removal of extracellular Fe is essential. Conclusions A standardized incubation method results in similar cell uptakes to Fe absorption in humans. Partially supported by International Agency of Atomic Energy (12406 IAEA), CONACyT‐salud 2002‐C01‐8109, Mexico, NCMHD/NIH 1 P60 MD00222‐02‐03, CHORI SPO BB.