Absorption of iron from ferritin.

I would like to comment on the article “Iron in ferritin or in salts (ferrous sulfate) is equally bioavailable in nonanemic women,” by Davila-Hicks et al (1). The conclusion indicated in the title is based on measurements of iron absorption from horse spleen ferritin that was radiolabeled in vitro and appears to contrast with the results of others whose studies using ferritin radiolabeled in vivo were not cited (2–4). For example, Skikne et al (4) also found that iron from ferritin radiolabeled in vitro was absorbed similarly to iron from ferrous sulfate. However, the same group further reported that radioiron incorporated into bovine spleen ferritin in vivo was significantly less absorbed than was iron from ferrous sulfate: 3.2% compared with 8.2% from a 3-mg dose with food, 3.8% compared with 24.1% from a 3-mg dose without food, 0.6% compared with 2.6% from a 50-mg dose with food, and 0.7% compared with 7.9% from a 50-mg dose without food (4). Those who have studied ferritin radiolabeled in vivo have concluded that ferritin iron is poorly absorbed and that it is not part of the nonheme pool of dietary iron that is readily exchangeable in and is similarly absorbed from the intestinal lumen (2–4). For instance, in vivo–labeled ferritin Fe was only 36% as well absorbed as was Fe from intrinsically labeled soybeans consumed in the same meal (2). It is possible that a lower absorption of ferritin iron may explain the slightly greater (10%) absorption of nonheme iron from extrinsically than from intrinsically labeled foods (5), which suggests that the ferritin iron content of food is only a minor portion of total food iron. It is worth noting that the ferritin iron content of foods has not been widely determined because of the lack of species-specific antibodies as well as the insolubility and possible time-dependent molecular changes that may make ferritin iron less exchangeable (6). Each labeling method has potential problems. On the one hand, the in vivo labeling of animal ferritin has in some (2, 4), but not in all (3), reports involved procedures to limit the radiolabel incorporation into blood by reducing erythrocyte synthesis or increasing erythrocyte breakdown, and it is not known whether these techniques alter ferritin isomerization. It is clear that the in vivo procedure does not uniformly label all of the iron in ferritin, but this would not necessarily explain the reduced iron bioavailability because the portion that is unlabeled may be less, not more, exchangeable or absorbable. On the other hand, in vitro labeling results in higher bioavailability regardless of whether the ferritin has first been depleted of iron (1) or not (4), and in vitro iron exchange can induce ferritin degradation through Fenton chemistry (6). Skikne et al (4) observed a minor small molecular peak in the Sepharose 6B elution pattern of in vitro, but not in vivo, labeled ferritin, that they proposed to be denatured ferritin. Those investigators (4) determined that in vitro procedures labeled a full range of isoferritins but that isotope incorporation into the more acidic forms was slightly higher (4). It is unlikely that horse spleen ferritin labeled with extra phosphorus in vitro (1) is comparable with plant ferritin. Using Mössbauer spectroscopy, Ambe et al (7) found that the form of ferric iron, representing 95% of the iron in soybeans, was clearly distinguishable from, but more similar to, horse spleen ferritin than to ferric phytate. Although physicochemical methods detected only minor alterations in ferritin labeled in vitro (1, 4), the human absorption results provide a distinguishing bioassay for ferritin labeled in vitro compared with in vivo. Davila-Hicks et al (1) proposed that a high absorption of iron from the Tokyo soybean cultivar is partially explained by a high ferritin content of this cultivar, in addition to the low iron status of the subjects (8). After logarithmic transformations of both variables, absorption of iron is inversely related to body iron stores, varying 10–15-fold between subjects (see Figure 1 of reference 9). This relation alone is sufficient to account for the differences in iron absorption from soybeans cited by Davila-Hicks et al (1): 26% in women with borderline iron deficiency (assuming 80% red blood cell incorporation of absorbed isotope) (8), 20% in women with iron deficiency (assuming 100% red blood cell incorporation; the absorption calculation is increased to 25% if red blood cell incorporation is assumed to be 80%) (10), and 2.8% in iron-replete men (11). Lacking a direct comparison of cultivars with the same subjects, the similar results obtained by Murry-Kolb et al (8) and Sayers et al (10) do not support the hypothesis that the iron from the high-ferritin Tokyo soybean cultivar was more bioavailable than was that from commonly used soybean cultivars. Davila-Hicks et al (1) concluded that iron from ferritin or ferrous sulfate follow different metabolic pathways after absorption. This was based on similarities in iron absorption when measured by whole-body scintillation counting (22% and 22% from ferritin and ferrous sulfate, respectively; see Table 1) but differences in absorption when measured from erythrocyte iron incorporation (27% and 48%). The greater retention of isotope in the erythrocytes than in the whole body suggests methodologic difficulties. The specific method and assumptions used were not delineated. With the use of commonly used methods (see citations in reference 9) and an assumption of 80% incorporation of the absorbed isotope into blood, we repeatedly obtained similar absorption results between the 2 methods, including results with added ferrous sulfate (9). For instance, nonheme-iron absorption from a hamburger meal supplemented with 20 mg Fe as ferrous sulfate was 8.4% (geometric x 1 SE: 6.8, 10.3) by whole-body counting (see data in Figure 1 of reference 9) and 8.5% (6.7, 10.7) by the erythrocyte incorporation method, and the assumption of 80% incorporation of the absorbed isotope into blood was confirmed (9). Note that the blood incorporation data in reference 9 was incorrectly labeled as the incorporation of the ingested rather than of the absorbed isotope dose; an erratum was submitted. Davila-Hicks et al (1) appear to have expressed the blood incorporation as a percentage of the ingested dose; expressed as such, their values seem excessive, but when expressed as the percentage absorbed seem insufficient. With the use of data from individual subjects (n 23), the blood incorporation method was highly correlated with the whole-body counting method (R 0.98) (9). These data do not confirm the finding that iron from ferrous sulfate is more extensively incorporated into blood than is apparent from whole-body counting measurements. In conclusion, research on iron bioavailability from ferritin labeled in vitro must be interpreted with caution. The evidence does not support the conclusion that iron absorbed from ferrous sulfate 1178 LETTERS TO THE EDITOR