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Iron excretion in iron excess
ly. In contrast, the primary defect in β-thalassemia mouse models - β-globin mutations - cannot. Red cell transfu- sions can reverse the anemia in this disease but also intro- duce a large burden of exogenous iron.
As mentioned above, organ iron concentrations decreased in transferrin-treated Trfhpx/hpx mice. A similar phenomenon has been described in mouse models of hereditary hemochromatosis and β-thalassemia. Hepcidin deficiency is a key characteristic of both dis- eases. Administration of pharmacological agents that induce hepcidin expression or mimic hepcidin activity decreased iron levels in mouse models of these diseases.21– 25 Whether correction of hepcidin deficiency simply pre- vented worsening of organ iron excess or led to mobiliza- tion of iron from these organs and excretion from the body remains to be determined. We propose that the decreased organ iron levels in these models reflects a combination of normalized iron absorption and increased excretion rates.
The increased excretion rates we estimated for Trf mice are similar to those previously reported. In two of the ear- liest studies on iron excretion in mice, injected 55Fe cleared from the body of Swiss mice with a half-life of 140 days, which is equivalent to a loss of 0.5% body 55Fe per day.17,18 We observed that 59Fe cleared from all Trf mice except untreated male Trfhpx/hpx mice with a half-life of 80-120 days and a loss of 0.6-0.8% body 59Fe per day (Figure 4D,E). In the older studies, iron-sufficient Swiss mice excreted 11.5 mg iron/day, while mice with increased body iron secondary to dietary or intravenous iron load- ing excreted 14-57 mg iron/day. These values were similar to those we predicted for Trf mice based on our 59Fe stud- ies (Figure 5C).
We also used rates of body 59Fe loss in urine and feces to estimate the rate at which iron was excreted via urine and feces. Measured urinary iron levels agreed with our 59Fe- based estimates of urinary iron levels except for untreated mutant mice, in which actual iron levels were much higher than predicted. The reason for this underestimation is not clear, although it may reflect the fact that perturbations in iron homeostasis in untreated Trfhpx/hpx mice are quite severe compared to those in Trf+/+ and treated Trfhpx/hpx mice. We also estimated the levels of fecally excreted iron in all mouse groups. While total fecal iron levels were not informative, the relative abundance of fecal ferritin was matched by the relative abundance of fecally excreted iron. Whether fecal ferritin solely represents a marker of iron excess in Trf mice or plays a mechanistic role in iron excre- tion remains to be determined. The source of fecal ferritin is also not known at this time. Possible sources include sloughed epithelial cells and biliary excretion.
We propose that our study can be used as an initial step in a reconsideration of the physiological basis of iron excretion and the significance of its role in iron homeosta- sis. While humans and rodents may differ in their rates and routes of iron excretion, the possibility that iron excretion affects body iron levels has implications for treatment of human disease. Notably, a seminal work by Green et al. in 1968 indicated that adult men of Bantu ori- gin, a population with increased iron stores, have increased daily iron losses.26 Development of hepcidin mimetics or agonists is an active area of research and may lead to novel treatments for hereditary hemochromatosis, β-thalassemia, and other diseases of iron excess.27 If body iron levels are regulated largely by absorption, treatment
of patients with hepcidin agonists or mimetics will pre- vent worsening of iron excess but will not reverse it - additional treatment modalities such as chelation will be required to clear excess iron from the body. If body iron levels do influence iron excretion, treatment of patients with hepcidin agonists should result in decreased body iron burden - with rates of iron absorption normalized, excess iron will clear from the body through physiologi- cal mechanisms.
The means by which iron is excreted from the body have not yet been established. Iron excretion is currently attributed to multiple processes including exfoliation of dead skin, blood loss, and turnover of intestinal epitheli- um (Figure 7). Our data indicating that iron is excreted largely via the gastrointestinal tract suggest that skin exfoliation does not play a prominent role. The possibili- ty that increased blood loss contributes prominently to excretion in Trfhpx/hpx mice is also unlikely given that 59Fe half-lives did not decrease in Trfhpx/hpx mice relative to those in Trf+/+ mice (Figure 4D). The possibility that turnover of intestinal epithelium is a major route of iron excretion is stronger. It is supported by the fact that intestinal epithe- lium in mammals turns over in less than 1 week.28–30 Trfhpx/hpx mice do load excess iron into gastrointestinal organs (Table 1) but histological iron staining indicates that a considerable fraction of this iron in younger mutant mice resides in smooth muscle, not enterocytes (Figure 2). Pountney et al. previously demonstrated that non-heme iron levels are similar in enterocytes isolated from Trf+/+ mice and treated Trfhpx/hpx mice.31 Based on this, we suggest that the increased duodenal iron levels we measured in Trfhpx/hpx mice largely reflect smooth muscle iron loading (Figure 2). The observation by Pountney et al. that trans- ferrin can be internalized by enterocytes isolated from Trfhpx/hpx mice may also explain stainable iron observed in enterocytes of treated but not untreated Trfhpx/hpx mice - it may represent uptake of diferric transferrin across the basolateral membrane of enterocytes. Overall, a careful investigation of the potential role of epithelial turnover to iron excretion would require a quantitative assessment of multiple factors: enterocyte iron levels, the biochemical form of enterocyte iron, and the rate of epithelial turnover in gastrointestinal organs in multiple models of iron excess and deficiency.
Another potential contributor to gastrointestinal iron excretion is hepatobiliary excretion. This process is large- ly ignored by the current view of mammalian iron biolo- gy. The reason for this is not apparent. One study in rats excluded bile as a route of excretion, but this was based on the observation that bile duct ligation did not impair the decrease in body iron levels in rats switched from an iron-rich to an iron-deficient diet.32 The use of bile duct ligation is a concern given that this is an established method for inducing liver cirrhosis.33 Multiple studies, most of which were performed in rats, have shown that iron is readily detectable in bile and that biliary iron levels decrease in conditions of iron deficiency and increase in conditions of iron excess.34–52 Several of these studies involved the use of chelators - our statement that iron is readily detectable in bile refers to the measurement of bil- iary iron in control animals not exposed to chelators. Overall, a full investigation of the contribution of biliary excretion to systemic iron excretion would require meas- urement of multiple parameters. While our preliminary analysis indicates that biliary iron levels are increased in
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