Abstract
Ascorbic acid retards ferritin degradation in K562 erythroleukemia cells leading to an increase in the availability of cellular iron (Bridges, K. R., and Hoffman, K. E. (1986) J. Biol. Chem. 261, 14273-14277). To explore the mechanism of this effect, the influence of ascorbate on subcellular ferritin distribution was examined. Cellular ferritin was pulse-labeled with 59Fe for 2 h, after which the cells were hypotonically lysed and fractionated on an 8% Percoll density gradient. Immediately after the labeling, all of the ferritin was in the cytoplasmic fractions at the top of the gradient. When the labeling was followed by a 24-h period of growth, a portion of the ferritin shifted to the lysosome-associated fractions at the bottom of the gradient, consistent with lysosomal autophagy of cytoplasmic ferritin. When ascorbate was added to the culture medium during the 24-h incubation, the magnitude of the shift was reduced. This process was also examined by size-fractionation of the contents of labeled cells using a Sepharose CL-6B column. Immediately after labeling, ferritin emerged from the column in two peaks, indicating the existence of both ferritin monomer and aggregates within the cytoplasm. After a 24-h period of growth, the monomer peak disappeared, while a new ferritin peak coincident with lysosomes emerged again, indicative of lysosomal autophagy of ferritin. In cells cultured with ascorbate for 24-h, there was a marked attenuation of the shift of ferritin to the lysosomal fractions. The monomer peak disappeared, as in the controls, but there was instead, an accumulation of ferritin as cytoplasmic aggregates. The total ferritin content of the ascorbate-treated cells was increased by 4-fold over that of the control. These experiments indicate that ascorbate blocks the degradation of cytoplasmic ferritin by reducing lysosomal autophagy of the protein. The access to the cell of the potentially toxic iron stored within the ferritin molecule is thereby increased.
Highlights
Ascorbic acid retards ferritin degradation in K562 shell and thereby is rendered innocuous
A number of observations indicate an interplay between the metabolism of iron andascorbic acid [6]
Ascorbate directly mobilizes iron from the crystal core of ferritin in vitro by reducing Fe3+to Fez+ [7]. This inforconsistent with lysosomal autophagy of cytoplasmic mation provided the rationalefor the clinical useof ascorbate ferritin
Summary
From the Departmentof Medicine, Laboratory ofthe Howard Hughes Medical institute, Division of Hematobgy, Brigham and Women’s Hospital, Harvard Medical School, BostonM, assachusetts 02115. Ascorbate directly mobilizes iron from the crystal core of ferritin in vitro by reducing Fe3+to Fez+ [7]. This inforconsistent with lysosomal autophagy of cytoplasmic mation provided the rationalefor the clinical useof ascorbate ferritin. Individuals with dium during the 24-ihncubation, the magnitude of the uncorrectableanemiarequirerepeated blood transfusions, shift wasreduced This process was examined by leading eventually to massive iron deposition and injury to size-fractionation of the contentsof labeled cells using manyorgans,such as theheart,pancreas,and liver [8]. Dicate that ascorbate blocks the degradation of cyto- We have examined the role of ascorbic acid in the cellular plasmic ferritin by reducing lysosomal autophagy of metabolism of ferritin and iron, using the K562 erythroleuthe protein.
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