Kinetics of Iron Import into Developing Mouse Organs Determined by a Pup-swapping Method

Background: Rates of iron import from blood to developing organs were measured.

Results: Two distinct iron species in the blood are imported.

Conclusion: Nontransferrin-bound iron dominates the iron import process in young mice.

Significance: The role of transferrin-bound iron in import should be reevaluated.

Abstract

The kinetics of dietary iron import into various organs of mice were evaluated using a novel pup-swapping approach. Newborn pups whose bodies primarily contained ⁵⁶Fe or ⁵⁷Fe were swapped at birth such that each nursed on milk containing the opposite isotope. A pup from each litter was euthanized weekly over a 7-week period. Blood plasma was obtained, and organs were isolated typically after flushing with Ringer's buffer. ⁵⁶Fe and ⁵⁷Fe concentrations were determined for organs and plasma; organ volumes were also determined. Mössbauer spectra of equivalent ⁵⁷Fe-enriched samples were used to quantify residual blood in organs; this fraction was excluded from later analysis. Rates of import into brain, spleen, heart, and kidneys were highest during the first 2 weeks of life. In contrast, half of iron in the newborn liver exited during that time, and influx peaked later. Two mathematical models were developed to analyze the import kinetics. The only model that simulated the data adequately assumed that an iron-containing species enters the plasma and converts into a second species and that both are independently imported into organs. Consistent with this, liquid chromatography with an on-line ICP-MS detector revealed numerous iron species in plasma besides transferrin. Model fitting required that the first species, assigned to non-transferrin-bound iron, imports faster into organs than the second, assigned to transferrin-bound-iron. Non-transferrin-bound iron rather than transferrin-bound-iron appears to play the dominant role in importing iron into organs during early development of healthy mice.

Introduction

Iron is a redox-active metal that plays essential roles in mammalian physiology and disease (1). Iron-containing hemoglobin binds and transports O₂ to tissues where mitochondrial respiratory complexes, packed with iron-rich cytochromes and Fe/S clusters, catalyze O₂ reduction. Iron in the brain helps synthesize neurotransmitters and myelin that insulates neurons (2). Iron is critical for the developing hippocampus (3). Young rodent brains experience a growth-associated iron deficiency due to the inability of iron to be imported fast enough to counterbalance the diluting effect of brain growth (4, 5).

The liver plays a central role in iron metabolism (6). Hepatocytes synthesize hepcidin, the master regulator of iron in the body. This peptide hormone controls the stability of ferroportin, a membrane-bound protein through which duodenal enterocytes, hepatocytes and macrophages export Fe^II into the blood. Hepatocytes also synthesize transferrin, a blood protein that binds Fe^III in the plasma. Transferrin is widely considered to be the dominant (or even exclusive) iron component of the plasma, and the major (or exclusive) protein that transports plasma iron into healthy tissues. TBI³ enters cells through the receptor TfR1 (1). Ferritin is a spherically-shaped iron storage protein complex. Liver macrophages contain high levels of ferritin that can be exported into the blood (6, 7). Hepatocytes also synthesize hephaestin, which oxidizes the ferroportin-associated Fe^II that enters the blood. This oxidation to Fe^III is required for transferrin binding.

A significant portion of splenic iron is found in red-pulp macrophages that degrade senescent erythrocytes (8). The resulting non-heme iron is released into the blood, coordinated to transferrin, and sent to the bone marrow for installation into nascent heme groups. As a result, plasma iron exchanges rapidly. The concentration of iron in the plasma (20–50 μm) is largely controlled by ferroportin and hepcidin (1, 7–10).

Transferrin has a high affinity for aqueous Fe^III, yet under normal dietary conditions, only ∼30% of plasma transferrin is iron-bound (9) which implies that the concentration of aqueous Fe^III in the blood must be very low. Indeed, apotransferrin scavenges free iron in the plasma. As long as sufficient apotransferrin is available, virtually all ⁵⁹Fe^IIICl₃ or ⁵⁹Fe^III citrate added to plasma will coordinate in minutes (11–15). These results have been interpreted to mean that virtually all iron in normal plasma is TBI. NTBI is thought to play an important and damaging metabolic role, but only when transferrin is fully saturated, e.g. in iron overload diseases (9, 16, 17) for which NTBI concentrations are high (18). However, healthy plasma reportedly contains other forms of iron besides transferrin (19). This contradiction stems from uncertainty or disagreement as to the composition, concentration, and stability of NTBI. NTBI might be a population of iron complexes, including heme groups, heme:hemopexin complexes, hemoglobin:haptoglobin complexes, ferritin (9, 10, 14) and/or iron coordinated to citrate, ascorbate, acetate, or albumin (9, 20–22).

NTBI rapidly enters tissues, especially the liver (12, 13). In heart cell cultures, NTBI is imported 300 times faster than TBI (23). The receptors that import NTBI include DMT1, ZIP14, and voltage-dependent calcium channels (9, 24). The liver and kidney rapidly absorb ferritin via TIM2 (25) and Scara5 receptors, respectively (26). The NTBI importer in hepatocytes exhibits an apparent Michaelis-Menten K_m of 1.25 μm (27).

The kinetics of iron import into organs have been evaluated by injecting radioactive ⁵⁹Fe into rodents, sacrificing the animals at various times, dissecting organs, and measuring how much radioactivity was incorporated. Dallman and Spirito (28) reported that the amount of radioactive ⁵⁹FeCl₃ taken up per brain maximized at postnatal day 18 (P18). Taylor and Morgan (10) obtained similar results using ⁵⁹TBI. Both groups concluded that the concentration of receptors for iron import in the brain increased during the first 2 weeks of life and then declined. Craven et al. (13) saturated plasma transferrin in rodents and then injected ⁵⁹Fe citrate. ⁵⁹Fe citrate was absorbed into organs with a t_1/2 < 30 s, compared with TBI (in controls) which had t_1/2 ∼ 50 min. In a related study, Ueda et al. (11) blocked transferrin receptors in mice and then injected ⁵⁹FeCl₃ into the blood. Within minutes, essentially all of this iron converted into TBI as monitored by gel filtration LC. Malecki et al. (29) injected ⁵⁹FeCl₃ into hypotransferrinemic mice. The rate of ⁵⁹Fe uptake into the brain was nearly 100 times faster than with WT mice, confirming that NTBI imports faster than TBI (30, 31).

The most critical period of mammalian development is from birth to young adulthood, yet this is also the most difficult to study using tracers. We developed a pup-swapping method to determine the kinetics of iron uptake during this period. ⁵⁶Fe- and ⁵⁷Fe-enriched pups were swapped at birth, such that each nursed on milk containing the opposite isotope. Mathematical models were developed to quantify the rates of iron import from the plasma into major organs. Surprisingly, NTBI rather than TBI was the dominant iron-containing species that incorporated into the organs of healthy non-iron-overloaded mice at early stages of development.

EXPERIMENTAL PROCEDURES

C57BL/6 mice were raised in an iron-deficient environment and on iron-deficient chow (Harlan Teklad no. 80396) supplemented with 50 mg of ⁵⁷Fe^III citrate (96.3%, Isoflex USA) per Kg chow, as described (5). Another colony was raised equivalently but on chow supplemented with natural abundance Fe^III citrate containing ∼96% ⁵⁶Fe. After more than three generations on these diets, the mice were >90% enriched in either isotope. For simplicity, only these two isotopes were measured and included in the analysis (5). On the same day, two ⁵⁷Fe-enriched males were placed in the cages of two ⁵⁷Fe-enriched females, one in each cage. Similarly, two ⁵⁶Fe-enriched males were placed in the cages of two ⁵⁶Fe-enriched females. Approximately 19 days later, the females gave birth to litters 1–4, consisting of 6, 5, 7, and 7 pups, respectively. Females from litters 1 and 2 (called ⁵⁶F1 and ⁵⁷F2) gave birth within 1 day of each other, as did ⁵⁶F3 and ⁵⁷F4. The ⁵⁶Fe-enriched pups from litter 1 (called ⁵⁶M1 pups) were swapped with the ⁵⁷M2 pups, such that ⁵⁶M1 pups were nursed on ⁵⁷Fe-enriched milk and ⁵⁷M2 pups were nursed on ⁵⁶Fe-enriched milk. ⁵⁶M3 and ⁵⁷M4 pups were similarly swapped. Swapped pups were designated as 56→57 and 57→56. The first number refers to the starting isotope and the last to the enriching isotope. Seven pups from another ⁵⁷Fe litter were euthanized within 24 h of birth, called “day 0”; they served as newborn controls. Eleven pups from two other ⁵⁶Fe litters were also used as controls, but only volumes and iron concentrations in brains were determined. After weaning, pups were fed the diets described above.

Starting 1 week after birth, and continuing on the same day for 6 additional weeks, one pup from each of the four swapped litters was euthanized. Brain, liver, spleen, heart, and kidneys were dissected and weighed. The concentration of iron in each organ was determined by ICP-MS, as described (5). Starting at 2 weeks and continuing each week thereafter, ∼50 μl of blood was removed from each euthanized animal. The supernatant obtained by blood centrifugation was defined as plasma. Blood was extensively flushed from animals using Ringer's buffer (5). Sufficient blood could not be collected from younger animals, and flushing was not possible. ⁵⁶Fe and ⁵⁷Fe concentrations in the plasma were determined. For LC traces, plasma was injected onto a Superdex 200 column equilibrated in 50 mm Tris-HCl and 150 mm NaCl, pH 7.4. Analysis of the time-dependent changes in ⁵⁶Fe and ⁵⁷Fe concentrations in plasma and organs did not depend on whether the starting or enriching isotope was ⁵⁶Fe or ⁵⁷Fe. This symmetry allowed us to combine 56→57 and 57→56 data and relabel isotopes as starting and enriching. Doing so simplified the analysis and improved the statistics.

RESULTS

Characterizing the kinetics of iron import in mammals during early development is challenging because the volumes of organs and plasma increase significantly. We accounted for this by measuring organ volumes and by developing mathematical models that took volume-associated dilution into account. The volume of the newborn brain was 20% of the adult-brain volume, whereas those of the other organs were percentage-wise less (Fig. 1, top panel, and supplemental Tables S1 and S3). The newborn spleen grew the fastest of the considered organs. Initially, the liver grew slowest, but it experienced a burst of growth at around P20. Brain, heart, and kidney growth rates were intermediate. By P49, organ growth rates had slowed, with brain and spleen volumes approaching those of the adult; other organs were not fully developed.

FIGURE 1.

Organ volumes (top) and growth rates (bottom) during development. Black, brain; red, liver; blue, heart; cyan, kidneys; green, spleen; and pink, plasma. Plots of α functions were offset by 0.10 day⁻¹ for display.

The concentration of iron in blood is much higher than the endogenous iron concentration in the organs. Thus, removing that contribution was critical to determining endogenous iron concentrations. We flushed the organs extensively with buffer and estimated the residual blood contributions using Mössbauer spectroscopy (Fig. 2). Newborn (P0) and P7 mice could not be flushed, and so the measured concentrations of ⁵⁶Fe and ⁵⁷Fe in those organs (supplemental Table S2) included significant blood contributions.

FIGURE 2.

Mössbauer spectra of organs before and after perfusion. A, flushed brain; B, blood; C, same as A but after subtracting blood contribution (15% spectral intensity); D, flushed liver. Spectra were obtained at 5 K and 0.05 Tesla applied parallel to the gamma radiation. The dashed line shows the high-energy line of the heme doublet. In spectrum B, the heme doublet fit to δ = 0.94 mm/s and ΔE_Q = 2.35 mm/s. The heme doublet in spleen required δ = 0.94 mm/s and ΔE_Q ≈ 2.32 mm/s (not shown).

Blood exhibited a quadrupole doublet (Fig. 2B) with parameters typical of deoxyhemoglobin. A similar doublet was evident in organ spectra. Percentages of the heme doublet for the brain (Fig. 2A) and heart spectra were lower than for the spleen, liver (Fig. 2D), or kidneys (supplemental Table S4). Parameters associated with the heme doublet in the spleen spectrum differed slightly from that of blood, raising the possibility that some of the intensity was due to modified heme centers, perhaps in macrophages associated with the organ. Because the macrophage contribution in the brain is small, we assumed that all of the heme contribution in the flushed brain was due to blood, generated the corrected spectrum in Fig. 2C. For the other organs, the same percentage of the heme doublet was assumed to be due to blood. Anything beyond that was assumed to be associated with macrophages. This excess heme-associated fraction of iron was not removed in determining the endogenous iron concentrations in the organs (supplemental Table S5). Our estimates of residual heme iron in flushed organs were similar to previous results (32).

As expected, the starting isotope concentration in the plasma, [^sFe_P], decreased with age while that of the enriching isotope, [^eFe_P], increased. The sum of these concentrations, [^tFe_P], increased slightly with time. Extrapolation to P0 and P7 afforded estimates of [^tFe_P] for P0 and P7 plasma. Details of how this was done are given in supplemental Appendix A.

We next examined the content of mouse plasma using liquid chromatography with an on-line ICP-MS detector. The liquid chromatogram exhibited about six iron-associated peaks (Fig. 3A). Peaks at >300, 90, and 55 kDa were assigned to ferritin, transferrin, and hemopexin, respectively; the other three peaks could not be assigned. The LC-ICP-MS experiment demonstrates that numerous iron-containing species coexist with transferrin in healthy WT mouse plasma.

FIGURE 3.

Iron-detected liquid chromatograms of mouse blood plasma (A), transferrin (B), ferritin (C), and hemopexin (D). Black lines are data, and red lines are simulations. The blue line is a composite simulation, including, from left to right, ferritin (6% of the iron), transferrin (17%), unidentified protein 1 (UP1, 6%), hemopexin (69%), and unidentified proteins UP2 and UP3 (1% each). UP1 has a molecular mass of 60–80 kDa, whereas UP2 and UP3 have masses < 7 kDa. Trace E is from the same injection of plasma as in A, but detected for copper rather than iron. The only species observed migrated with a mass of 150 kDa; it was assigned to ceruloplasmin.

Brain

The iron concentration in the brain, averaged over the study, was three to four times lower than in liver, heart, and kidneys, and nearly 10 times lower than in spleen. The red, black, and blue circles in Fig. 4, A and B, represent [^tFe_B], [^sFe_B], and [^eFe_B], respectively. From P0 to P14, [^tFe_B] declined 2-fold and then plateaued. There was no loss of moles of starting isotope during the decline (see supplemental data), indicating that the decline in concentration arose because the import rate of ^eFe_B was insufficient to counterbalance the effect of increasing brain volume. We evaluated whether iron exited the brain by calculating whether the amount of ^sFe_B in the organ declined after the plasma was essentially devoid of ^sFe_B, namely at P35 and beyond. Prior to that, the plasma was dominated by ^sFe_P (Fig. 4D) so evaluating whether ^sFe_B exited the brain was complicated by the simultaneous import of ^sFe_P. There was a loss of ∼12 nmol of ^sFe_B from the brain between P7 and P49. Half of that exodus occurred after P35. During the entire 7-week period, only ∼30 nmol of ^eFe_B were imported. Thus, iron exited the brain at rates that were comparable with that at which “new” iron was imported. This demonstrates a dynamic iron import/export process in the developing brain.

FIGURE 4.

Concentrations of starting and enriching isotopes of iron in the brain (A and B), liver (C), and plasma (D). Red, blue, and black circles are total, enriching, and starting isotope concentrations, respectively. Solid lines are simulations. A, brain, OCM simulations; B, brain, TCM simulations; C, liver, TCM simulations; D, plasma, TCM simulations.

Liver

The newborn liver had the highest endogenous iron concentration of the examined organs (Fig. 4C). [^tFe_L] declined 5-fold during the first 3 weeks of life. This was due primarily to the net exodus of ^sFe_L; in fact, over half of the ^sFe_L in the newborn liver exited during the first week. During this same period, there was a net import of ^eFe_P; the amount imported nearly matched the net amount of ^sFe_L lost. These processes, along with a doubling of liver volume, caused the overall concentration of iron to decline ∼2-fold during the first week. [^tFe_L] declined to ∼300 μm in the next few weeks and then recovered slightly. Averaged over the study, the endogenous iron concentration in the liver was only 630 ± 440 μm, comparable with heart and kidneys. Much of the iron in “iron-rich” liver originates from blood.

For the first few weeks, the liver enriched faster than the brain or plasma (Fig. 5 and supplemental Table S2). The enrichment rate eventually slowed, but the liver was fully enriched by the end of the study. How could the liver (or any organ) enrich faster than the plasma, the region which supplies iron to the organs? This result implies that the liver (and actually all organs examined) preferentially import ^eFe_P instead of ^sFe_P. This selectivity was most extreme for the spleen, followed by kidney, liver, and heart. The brain showed the least isotope selectivity.

FIGURE 5.

Enrichment percentages into organs and plasma. Black, brain; red, liver; blue, heart; cyan, kidneys; green, spleen; pink, plasma.

Other Organs

The average iron concentration in the buffer-flushed spleen was the highest of the examined organs, and it was fairly constant throughout the study (Fig. 6, left panel). There was no growth-related decline in total endogenous iron concentration. The percent enrichment increased fastest of any organ, from 2 to 61%, during the first week of life (Fig. 5, green line). This enrichment rate was 3 times faster than that of the plasma. MB spectra revealed that ∼85% of the iron in the flushed spleen was heme-associated. Virtually all of the iron imported by the newborn spleen was the enriching isotope, and there was a slow exodus of iron. The iron concentrations in the heart and kidney increased gradually (Fig. 6, middle and right panels). The amount of starting isotope in these organs decreased from P0 → P35 suggesting some iron export. Enrichment rates of the heart and kidneys were also similar: faster than for the liver and brain, but slower than for the spleen.

FIGURE 6.

Plots of endogenous iron concentrations and simulations for spleen (A), heart (B), and kidneys (C). For each organ, red, blue, and black circles are total, enriching, and starting isotope concentrations ([^tFe_O], [^eFe_O], and [^sFe_O]), respectively. Solid lines are simulations. The top panel shows simulations assuming the OCM, whereas the bottom panel simulations assume the TCM. Data in both top and bottom panels are the same.

Models

We developed two closely related mathematical models based on the mechanisms illustrated in Fig. 7, which described how the concentrations of starting and enriching iron isotopes change in each organ and in the plasma during development and as organ and plasma volumes increase. Concentrations changed according to the ordinary differential equation (ODE) 1, (dC_i/dt) = R_chem − α_OC_i, where R_chem represents terms associated with chemical changes of C_i in organ (O) = brain (B), liver (L), spleen (S), heart (H), kidney (K), and plasma (P). The last term reflects the dilution of C_i caused by the growth of that organ/plasma. Time-dependent functions of the form, α_O(t) = (1/V_O)(dV_O/dt), describe the normalized volume growth rates for each organ or plasma.

FIGURE 7.

OCM (top) and TCM (bottom) describing iron import into mouse organs. For the OCM, enriching and starting isotopes are imported from the gut to the plasma to form species Fe_T, which is imported into organs through a receptor. The resulting species Fe_O can be exported. The TCM is similar except that the enriching isotope enters the plasma as a second species Fe_NT which converts to Fe_T. Both Fe_NT and Fe_T can be imported into organs at independent rates.

α_O(t) functions were determined by fitting to weekly experimental values (see supplemental Appendix A). Resulting α_O(t) functions, generated using the coefficients in supplemental Table S6, are shown in Fig. 1, lower panel. They quantify the growth rates of the organs, including the growth-spurt in the liver at P20 and the rapid initial growth rate of the spleen. Uncertainties in α_P(t) were greater than for the organs because plasma volume V_P was not measured. A range of blood volumes in mice has been reported (32, 33). The value used, ∼9% of body weight (supplemental Table S7), is within the normal range.

The one-component model (OCM) assumed that a single iron-containing species called Fe_T was imported into the plasma and that Fe_T alone was deposited into the organs (Fig. 7, top). At birth, all Fe_T in the plasma and tissues were assumed to be of the starting isotope. The OCM included 12 chemical components, namely ^sFe_T and ^eFe_T (plasma) and ^sFe_O and ^eFe_O. Concentration changes of each component were determined by ODEs 1 and 2.

Terms involving k_TO reflect import of Fe_T into each organ. Both ^eFe_T and ^sFe_T were assumed to compete for import into organs via a common receptor at the interface with the blood. Receptors were treated as Michaelis-Menten enzymes acting on substrates ^eFe_T and ^sFe_T. Steady-state import rate expressions of each substrate are derived in supplemental Appendix B. Facilitated transport was assumed. Rates of interfacial reactions were augmented by the ratio V_P/V_O (see supplemental Appendix C). Functions k_XO reflect rate-constants associated with the export of iron from the organs. S_feed reflects the rate of ^sFe_T entering the plasma from the gut or other sources. ^eFe_T enters the blood at rate E_feed.

ODEs (Equations 1 and 2) were solved piecewise for each weekly period, with time-dependent parameters constant for a given period. The initial data for numerical integrations were the beginning concentrations for each period. Parameters were adjusted to minimize the relative error between ending concentration data and corresponding simulation points. We tried to minimize S_feed and k_XO. Final simulated concentrations, obtained using parameters in supplemental Tables S6 and S8, are shown in Figs. 4A and 6, top panels. Simulations could not match observed enrichment rates for which organs enriched faster than plasma. Achieving this was impossible for the OCM, because the model requires that iron in the gut exclusively feeds the blood and that iron in the blood exclusively feeds the organs.

The two-component model (TCM) (Fig. 7, bottom panel) resolved this conundrum by assuming that: (a) iron enters the plasma exclusively as second species called Fe_NT; (b) Fe_NT in the plasma converts to Fe_T; and (c) Fe_NT and Fe_T are imported independently into the organs. Fe_NT might represent one species or many. These modifications do not affect starting ODEs (Equation 1), but the enriching ODEs change to Equation 3.

Compared with the OCM, the TCM included an additional component in the plasma (^eFe_NT) and an additional ODE associated with it. The TCM included seven additional reactions and time-dependent rate functions, associated with the import of Fe_NT and the conversion of ^eFe_NT → ^eFe_T. Reactions and parameters are given in supplemental Table S9. Equations 1 and 3 were integrated piecewise as above (see Appendix A). Optimized simulations, generated using parameters in supplemental Tables S9 and S6, are shown in Fig. 4, B–D, and Fig. 6, bottom panels. Residual errors associated with best-fit simulations were about six times lower than those of the OCM.

The rate of Fe_NT import into the brain was seven times greater on average than that for Fe_T. This is evident from a plot showing estimated k_NTB versus k_TB functions (Fig. 8, solid line versus dashed line). Both rates were highest during the first 2 weeks (collectively ∼18 μm/day) and lowest during the last 3 weeks. Time-dependent differences between k_NTB and k_TB may reflect changes in the expression level of the corresponding receptors in the brain. The average rate of iron export from the brain was 2–3 μm/day, which was less than but comparable with the import rate (∼8 μm/day). Import rates declined gradually to ∼2 μm/day, similar to export. Import and export rates should become similar as development slows because the overall iron concentration in the adult brain is roughly invariant.

FIGURE 8.

k_NTO and k_TO functions associated with the TCM. The ODE system was piecewise solved using different optimized k_NTO and k_TO values for each period (supplemental Table S8). These values were fitted with smoothing functions (MATLAB, cftool), yielding the plots shown. The artifactually large value of k_TL at 35 days was not included in smoothing.

Rates of iron import to and export from the liver were higher than those of the brain, and the developmental changes differed. The rate associated with Fe_NT import into the liver was highest (Fig. 8, solid line), on average, than for any other organ (>16 times higher than for the brain). This rate was approximately constant for the first 3 weeks, increased 2–3-fold during weeks 4–5, and then declined. This burst of Fe_NT import approximately correlates to the period of rapid growth of the liver at 21–28 days. Virtually all of the iron imported into the young liver originated from Fe_NT, not Fe_T. The rate associated with Fe_T import into the liver was near zero for the first 4 weeks. Our data and simulations indicate that Fe_T plays little role in iron import into the young (newborn to 1 month) liver.

The newborn liver not only experienced an influx of iron during the first few weeks of life but a massive efflux. The efflux rate was greatest during the first 2 weeks, ∼100 μm/day, and then it declined. In later developmental stages, import and export rates slowed. The developmental changes associated with iron import and export into the liver appear to be more complex than for the other organs. Iron import/export dynamics in the young liver were about five times faster than that for the brain. The rate for Fe_NT import into the spleen was high during the first few weeks of life, followed by a gradual decline (Fig. 8, solid line). These changes were mirrored for the import of Fe_T, but at one-fifth the average rate. The newborn spleen imported Fe_NT at a rate that was slower than for the liver but faster than for the brain. The rate of Fe_T import was comparable with that of the newborn brain in that both declined after the first few weeks. The efflux rate of iron from the spleen was variable but generally comparable with that of other organs. The situation for the heart and kidneys were similar; however, initial import rates and subsequent declines were less dramatic. The average Fe_NT:Fe_T import rate ratios were 6:1 for both heart and kidneys, similar to the other organs. Iron efflux rates from the heart and kidneys were evident only near the end of the study.

The average Fe_NT:Fe_T import rate ratios for all organs, during the entire study, were remarkably similar (6 ± 0.5). We interpret this to mean that the ratios of the expression levels of the NT and T receptors are similar in each organ. Both rates (and perhaps both receptor expression levels) declined proportionately with development. The exception was the newborn liver, in that the rate of Fe_NT import was high for much of the study (70–250 μm/day), whereas that for Fe_T import was near zero. Another common feature, excepting the liver, was that the import rates were the fastest during the first week of life, followed by sharp declines. Iron import into the brain shut down most severely relative to the other organs, probably because the brain reached full size earliest, before the end of the study. Iron import rates into the liver did not follow this trend; import rates increased significantly during the first 5 weeks of life.

DISCUSSION

In this study, we evaluated the kinetics of iron import from the mouse gut into the plasma and then into various organs. We focused on the first 7 weeks of life when the volumes of organs and plasma increased dramatically. This is the most important period developmentally but also the most difficult to study. We invented a pup-swapping method in which the nutrients received by nursing and weaned pups exclusively contained an isotope of iron that differed from the one contained in the bodies of animals at the beginning of the study. ODE-based models were developed to quantify the rates at which the enriching isotope was imported, and the rates at which the starting isotope was exported, all while the organ and plasma volumes increased. Only the model that assumed two forms of iron in the plasma could simulate the results. Accordingly, the form of iron that enters the plasma from the gut (Fe_NT) converts slowly into a second form of plasma iron (Fe_T); then Fe_NT imports faster into organs than Fe_T.

Advantages of Pup Swapping

Pup swapping has advantages relative to tracer radioactive isotope injection studies. First, it allows the most critical period of animal development to be studied, namely from birth to young adulthood. Injecting newborns and very young rodents with tiny volumes of tracer solutions is difficult and error-prone. Second, pup-swapping measures absolute (μm) concentrations of each iron isotope, allowing quantitative analysis. Tracer studies measure relative concentrations of the label, with the vast majority of tissue iron left undetected. This is problematic because chemical kinetics generally reflects bulk concentrations. Third, there is no question that the form of iron ingested by the pups is physiologically relevant. In contrast, injecting mice with iron salts that are not normally present in plasma (e.g. ⁵⁹FeCl₃) represents a significant perturbation. Injecting ⁵⁹Fe-TBI is more “natural,” but the possibility of NTBI import is ignored. Finally, pup swapping allows the rate of iron export from organs to be evaluated. This is not possible in tracer studies because organs initially lack the radiolabel. One limitation of pup swapping is that the measured rates of iron import reflect more than just the biochemical events involved. With each swallow of food, ingested iron travels through membrane-bound importers and diffuses across various regions on its journey to an organ. The rates determined in this study include these processes, but they also include periods when the animal is not eating. This causes the rates reported here to be far slower than those associated exclusively with iron import.

Import of Iron from the Gut to the Plasma

The form(s) of iron that enter the plasma from the gut of nursing and just weaned pups is uncertain. The iron in breast milk is highly bioavailable and is associated with immunoglobulins, including lactoferrin (34, 35). During nursing, some lactoferrin is not digested as it travels into the blood and binds to macrophages (35). This might have contributed to the high rates of enrichment in the organs.

Iron Export

Our results indicate that there is a massive exodus of iron from the liver during the first week of life. The released iron may be absorbed by other organs (and/or used in erythropoiesis), ensuring sufficient iron for animal development regardless of the supply of nutrient iron after birth. This highlights the fact that the liver does not merely store iron, but also releases it. We also presented some evidence that iron is exported from other organs. The rate of iron export required by our model varied for each organ and for each time period. Our fitting strategy was to simulate the data for a particular organ and period assuming no export and add an export rate only if that assumption proved untenable. A nonzero export rate was needed to fit about half of the 35 data points used for fitting. During a 4-month period, adult rats imported from the diet 27, 24, 12, and 9% of new iron into the liver, kidney, heart, and brain, respectively (36). This suggests significant import and export activity in fully developed organs whose volumes and concentrations are roughly invariant.

Importance of NTBI in Developing Mammalian Organs

We assign Fe_NT in our model to NTBI, and Fe_T to TBI, justified as follows. First, iron does not enter the plasma from the gut as TBI; nonheme iron enters through DMT1 as Fe^II which is then oxidized to Fe^III by hephaestin and then coordinated to apotransferrin. These processes collectively account for the Fe_NT → Fe_T reaction. Second, NTBI is imported into organs faster than TBI (12, 13, 23), consistent with the more rapid import of Fe_NT versus Fe_T as required by our simulations. Third, our analysis requires that plasma contain at least two forms of iron; by LC, we found that plasma contains approximately six iron-containing species only one of which was TBI. Whether any of the other species detected reflects NTBI is uncertain; the concentration of the NTBI in the plasma, which is imported rapidly into organs, may be below the detection limit.

Our results for young developing mice do not speak to the importance of NTBI versus TBI in the iron import of adults. Here, the TBI system may play a greater role. The importance of each form of iron in importing into organs is probably dictated by the expression level of T and NT receptors on the organ:plasma interfaces during various stages of life. NTBI may play the major role in importing iron into young developing healthy organs and into organs of iron-overloaded patients. The transferrin system may function as a slower but perhaps better regulated iron import system. The TBI system also likely functions as a buffer that soaks up additional NTBI in the plasma so as to avoid iron overload of healthy adult organs.

Comparison with Previous Studies

If the increasing volumes of developing organs were not considered, calculated import rates would be slower than they actually are. This is especially important in the first few weeks of life when organs are growing quickly. Previous studies did not appear to take this into account, giving rise to the conclusion that the import rate of iron into the brain maximizes at around P15 (4, 10). These conclusions were based on plotting data (amount of radioactivity per brain per injection versus age) that do not appear to have been normalized to increasing brain volume. Their results, once normalized, would be consistent with our data, with the fastest iron import rates into the newborn brain (and other examined newborn organs except for the liver).

More generally, our conclusions conflict with the established view that (a) TBI is the major (or even the exclusive) iron species in the plasma; (b) TBI is the only iron species in the plasma that is imported into healthy mammalian organs; and (c) NTBI is only physiologically important in iron overload-diseased states when the transferrin system is saturated. In hindsight, we can perceive some philosophical cracks in these foundational pillars. The rate by which added ⁵⁹Fe salts convert into TBI is characterized as being “fast,” but this is relative to a reference rate that is slower. When compared with the rate at which NTBI imports into organs (seconds), the rate by which NTBI converts to TBI (minutes) is indeed slow. According to our analysis, the partitioning between the conversion of NTBI into TBI versus the import of NTBI into organs is kinetically controlled, with the latter process dominating because it is faster. Another crack in the established foundation is the implicit assumption that the absence of NTBI in healthy plasma reflects the unimportance of this (these) species in the import of iron in healthy mammals. The steady-state concentration of NTBI in the plasma is dictated by competing rates of import from the gut and export to the organs. A near-zero steady-state concentration of NTBI can easily be achieved using an export rate that vastly exceeds the import rate. Species with undetectably low concentrations can be very important physiologically. With this new perspective, we are designing experiments to detect and identify NTBI in plasma and more rigorously characterize its role in human health.