Can Ferritin Provide Iron for Hemoglobin Synthesis?

To the Editor:

We have read with great interest a recent report by Gelvan et al1 attempting to resurrect an old idea that immature erythroid cells obtain iron (Fe) from ferritin. We wish to comment on one single and extremely important issue in this report, viz, that Fe from internalized ferritin can be used for hemoglobin synthesis. We believe that neither the results of previous studies nor experiments presented by Gelvan et al1 provide adequate evidence supporting this claim.

In 1957 Bessis and Breton-Gorius2 presented electron micrographs depicting erythroblastic islands in the bone marrow, in which a central reticulum cell (“nurse cell”) was surrounded by a ring of erythroblasts. In the region of contact between these cells the authors observed ferritin and proposed that it was transferred from the reticulum cell to the erythroblasts by a form of micropinocytosis termed “rhopheocytosis” (for review see ref 3). Somewhat later, Jandl, Katz, and coworkers4,5 showed that immature erythroid cells take up Fe from plasma Fe-binding protein, transferrin (Tf ), and suggested the existence of a membrane-bound Tf receptor which may be involved in Fe uptake. The many studies and discussions that followed have led to a general consensus that Tf, not ferritin, is the source of Fe for hemoglobin synthesis, and this view has been supported by the following evidence. First, under normal conditions all plasma Fe is associated with Tf, and ferrokinetic studies have provided clear evidence that all Fe used for hemoglobin synthesis passes through the plasma.6 Hence, if the reticulum cell ferritin was the source of hemoglobin Fe, these cells would have to acquire it from Tf. However, virtually no Fe enters the reticuloendothelial system from plasma Tf.7 Second, it should also be pointed out that plasma ferritin has a very low Fe content, and no ferrokinetic evidence supports its role in Fe transport.7 Third, a specific relationship between Tf and erythroid cells is documented by in vitro studies showing that this molecule is the only physiologically relevant Fe complex capable of providing Fe for hemoglobin synthesis.8 Fourth, normally developing erythroid cells take up 26 mg Fe/d from plasma Tf,7 a rate that matches almost perfectly with the daily production of hemoglobin (∼6.2 g). The final and most convincing evidence of an absolute requirement for Tf by erythroid cells comes from observations that both humans and mice with hereditary atransferrinemia have severe hypochromic microcytic anemias.9-11 Furthermore, when compared with wild-type animals, hypotransferrinemic mice show an extremely restricted uptake of ⁵⁹Fe by the erythron.12,13 Collectively, the above studies document the stringent dependency of erythroid cells on Tf-bound Fe. Interestingly, in 1962 Bessis and Breton-Gorius themselves reviewed critically their original hypothesis, and did not exclude the possibility that the phenomenon of “rhopheocytosis” takes place in the opposite direction, ie, “the erythroblast imparts the ferritin to the reticular cell.”¹⁴

Ferritin is not only an improbable source of Fe for the erythroid cells, but it is also an unlikely mediator involved in intracellular translocation of Fe for heme synthesis. Although ferritin has been postulated to act as an intermediate for heme synthesis in erythroid cells,15,16 several studies failed to show that ⁵⁹Fe from ⁵⁹Fe-ferritin could be incorporated into hemoglobin.17,18 When heme synthesis was inhibited in erythroid cells incubated with ⁵⁹Fe-transferrin, very little19,20 or no21,⁵⁹Fe accumulates in ferritin. More importantly, when heme synthesis is restored in these cells, no ⁵⁹Fe in ferritin was used for heme synthesis.20 These findings concur with observations that the intracellular Fe release from ferritin may require its catabolism,22 and suggests limited availability of ferritin Fe for metabolic purposes.

The evidence from previous studies showing that Tf and not ferritin is the Fe donor for hemoglobin synthesis must now be considered with the recent investigation by Gelvan et al.1 In this latter study the authors have incubated erythroid precursors with ⁵⁹Fe-labeled ferritin for 21 hours, at which time they detected some ⁵⁹Fe in the cells and in heme. Unfortunately, a number of technical flaws prevent the conclusion that ⁵⁹Fe found in heme comes from ⁵⁹Fe in the ferritin core. To prepare ⁵⁹Fe-labeled ferritin, Gelvan and associates have exposed apoferritin to a mixture of 5% ⁵⁹Fe(III) (as ⁵⁹FeCl₃ ) and 95% ⁵⁶Fe(II) in an oxygenated buffer at pH 7.0. It is essential to point out that apoferritin shells can be loaded with Fe(II), whereas attempts to load Fe(III) into the core have failed.23 In their study, Gelvan et al probably presumed that ⁵⁹Fe(III), in the presence of a large excess of unlabeled Fe(II), would be converted to ⁵⁹Fe(II). However, in the absence of Fe-binding ligands such a conversion has been shown to occur only at an extremely low pH (<1.0, ref 24). From method description1 it is not apparent what ligand was present in the MOPS buffer that would stabilize both Fe(II) and Fe(III) in aqueous solution at pH 7.0 to allow electron self-exchange between the two redox states of Fe to occur. MOPS buffers are sometimes supplemented with EDTA (although this is not specified in ref 1) which, however, has almost 11 orders of magnitude higher formation constant for Fe(III) than for Fe(II).25 Hence, under aerobic conditions the Fe(II)-EDTA complex should be rapidly oxidized to Fe(III)-EDTA complex, but no reduction of ⁵⁹Fe(III)-EDTA would occur. On the other hand, in the absence of any ligand, at pH 7.0, ⁵⁹Fe(III) would be rapidly hydrolyzed into virtually insoluble ferric hydroxide polymers that could become nonspecifically associated with the external surface of ferritin shells.

The most troubling aspect of this study is that no attempt was made to determine that the ⁵⁹Fe was actually present within the core of the ferritin molecule in contrast to nonspecifically bound to the surface. Hence, the physiological relevance of this ferritin preparation must be questioned. This poorly defined ⁵⁹Fe-ferritin was then used for experiments at an Fe concentration of 6.5 mmol/L, ie, about 1,000-fold higher than the concentration of Fe found in the plasma under physiologic conditions. In addition, because the erythroblasts used in this study were not depleted of internalized Tf, it is possible that endocytosed Tf present within the cells could remove ⁵⁹Fe from the surface of ferritin and donate it to the cells. It is also conceivable that some of the ⁵⁹Fe(OH)₃-polymers could be internalized by cells via nonspecific pinocytosis. Moreover, because of the aforementioned problems it is impossible to determine the specific radioactivity of ⁵⁹Fe available for cellular uptake. Hence, it is impossible to estimate net Fe uptakes in terms of picomoles/10⁶ cells, and no reasonable comparisons with Fe uptakes from Tf can be made. Apart from these problems, a number of important control experiments need to be completed before the physiological relevance of Fe uptake from ferritin can be postulated. These include time kinetics of ⁵⁹Fe-ferritin uptake and saturability of ⁵⁹Fe-ferritin uptake as a function of ferritin concentration. These latter studies need to be done with ferritin containing ⁵⁹Fe in its core.

In conclusion, the experiments by Gelvan et al1 have not been adequately performed to conclude that ferritin plays a role as an Fe donor for hemoglobin synthesis. We strongly believe the investigators should repeat their experiments with ferritin, which has been definitely shown to contain ⁵⁹Fe in its core. In combination with the additional controls described above, this strategy will then be appropriate to determine the role of ferritin in heme synthesis by erythroid cells.