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Center for BioIron
Children's Hospital Oakland Research Institute
5700 Martin Luther King Jr Way
Oakland, CA 94609
Department of Nutrition
University of California
One Shields Avenue
Davis, CA 95616
E-mail: bllonnerdal{at}ucdavis.edu
Dear Sir:
The comments of Hunt about our recently published article on the absorption of iron from ferritin (1) reflect the confusion that often exists over the consequences of the unique features of ferritin biology and chemistry: a solid mineral inside an organic protein shell. With the exception of calcium phosphate minerals, there is no other solid mineral in humans. The phase transition of calcium, phosphate, iron, and hydroxide from liquid to solid in biology remains poorly understood at this time.
Under physiologic conditions, the iron in solutions of ferritin—the protein with the solid iron mineral inside—appears to be physically in the same phase as iron in solutions of other nutritional, nonheme-iron complexes such as ferrous sulfate and ferric EDTA. However, the reality is much more complicated. Adding iron to ferritin involves a phase transition that makes full equilibration of exogenous iron very slow (2). In addition, ferritin protein varies in different tissues or under different physiologic conditions and controls the entry and exit of iron to and from the solid phase (3). Finally, the structure of the iron mineral is sensitive to physiologic conditions (4).
The discussion of the causes of the apparent inconsistencies in the results of using isotopically labeled ferritin in iron-absorption experiments appears in our first article on iron absorption from ferritin (5) and again in a recent review (6). Briefly, when labeled iron atoms are added to the unlabeled, solid mineral in ferritin, equilibration is very slow (days to months). In the 1997 study annotated in Hunt's letter (7), the label was added to ferritin (bovine), which had endogenous iron both in vitro and in vivo. In addition, in that study the estimate of the iron:protein ratio in the isolated ferritin was very high, based on the data in the article, which suggested the presence of denatured ferritin protein/hemosiderin to which some labeled iron added in vitro may have been adsorbed, giving rise to the inappropriately high absorption observed (7). None of these possibilities would have been detected in the protein analyses described in the article by Hunt, because the effect is on the location of the labeled iron in the iron mineral.
The assertion in Hunt's letter about the plant ferritin mineral in reconstituted horse spleen ferritin is incorrect. We previously compared the mineral in pea ferritin with that in horse spleen ferritin reconstituted to have a plant ferritin mineral composition and found, using EXAFS analysis, that the high phosphate mineral content in horse spleen ferritin was similar to that in natural pea ferritin (8) and distinct from that in animal ferritins; the work was annotated in our recent paper. For Hunt's assertion to be correct, the nonconserved structural features would have had to dominate the many conserved structural features of ferritin protein in iron absorption. In our recent article, we were merely exploring whether the structure of the plant ferritin mineral influenced iron absorption in humans. Studies currently in progress will explore the influence, if any, of the plant ferritin protein on iron absorption. Hunt misunderstood our statement about soybean cultivars, which simply indicated that soybeans with more ferritin would likely have a greater proportion of the bean iron in ferritin. Under the hydroponic, nonnodulating conditions used in the study described (9), the percentage of bean iron in ferritin was lower than that in field-grown beans. All soybean cultivars have a large fraction of the iron in ferritin when grown under field conditions.
In contrast with the statement in Hunt's letter, we did not "conclude" that iron from ferritin or ferrous sulfate follow different metabolic pathways after absorption, we merely suggested this as a possible explanation for the differences observed. Further molecular studies at a cellular level are needed to verify this, but our own preliminary data from Caco-2 cells (presented at Experimental Biology 2004) support such a scenario. We did not intend to state that the retention in erythrocytes was greater than that in the whole body. We used the conventional approach for estimating whole-body retention from red blood cell (RBC) incorporation, ie, assuming 80% incorporation and calculating whole-body retention from blood volume estimated from body weight. This was stated in the text as the well-known "RBC incorporation method for estimating iron absorption" rather than in explicated detail in the Methods, which, unfortunately, may have caused confusion. We obtained a higher absorption using this method than when using whole-body counting. In Hunt's previous work (10), a lower value was obtained with the RBC method (calculated from Figure 1 in reference 10) than with whole-body counting. These differences between the 2 methods for determining iron absorption may be related to differences in the geometry of the counter when determining radioactivity from a point source (blood) rather than from a whole body. However, in our study, the same method was used in the 2 groups of subjects. We observed a difference for ferritin iron and ferrous sulfate, which suggested a difference in metabolic handling of the 2 types of iron. Further studies are needed to explore this in more detail.
To summarize, we showed an efficient absorption (20–25%) of ferritin iron in several studies using different analytic methods to measure iron incorporation. We avoided the problem of equilibration of unlabeled, solid, mineral iron in ferritin with added labeled iron in 3 ways: 1) using no label (5), 2) using soybean plants to which the label was added before seed ferritin formation (9, 11), and 3) using ferritin from which iron was removed before reconstituting the mineral with labeled iron (1). Earlier, we suggested that both the iron status of the subjects and the labeling method may have contributed to the apparent inconsistencies among the various studies (5, 9). Now it appears more likely that the apparent inconsistencies among previous studies depend most heavily on poor equilibration of labeled iron with endogenous, solid, mineral ferritin in vitro or in vivo if the label is added late in bean development, because the women in our recent study had normal iron status (1).
ACKNOWLEDGMENTS
There was no conflict of interest.
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