title: Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism.
abstract: Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased (59)Fe uptake from transferrin by 1.5-2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased (59)Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated (59)Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of (59)Fe uptake from (59)Fe-citrate, only intracellular ascorbate is needed for transferrin (59)Fe uptake. Additionally, experiments with l-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent (59)Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or (125)I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism.
authors.affiliation: Department of Pathology, University of Sydney, Sydney, New South Wales, Australia. darius.lane@sydney.edu.au
authors.forename: Darius J R
authors.initials: DJ
authors.lastname: Lane
authors.forename: Sherin
authors.initials: S
authors.lastname: Chikhani
authors.forename: Vera
authors.initials: V
authors.lastname: Richardson
authors.forename: Des R
authors.initials: DR
authors.lastname: Richardson
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology*; D001692:Biological Transport; D015153:Blotting, Western; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology*; D005290:Ferric Compounds / Q000378:metabolism*; D005293:Ferritins / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism*
chemical_list: D005290:Ferric Compounds; D011990:Receptors, Transferrin; D014168:Transferrin; D005293:Ferritins; D007501:Iron; D001205:Ascorbic Acid