Iron Absorption in Disorders of Iron Metabolism

The mechanism of iron absorption, and the involvement of recently detected proteins in iron transport, is described in detail in Chapter 8. Our understanding of iron absorption at the molecular level is growing rapidly. This knowledge is essentially based on detailed quantitative information on iron absorption in normal subjects, in patients with iron deficiency, hereditary and secondary iron overload, and inflammation (Heinrich et al., 1966; Marx, 1979a; Werner and Kaltwasser, 1987; Wienk etal., 1999). By using a whole body counter, with 59Fe as a tracer, and [51Cr]CrCl3 as a non-absorbable indicator, it was possible to analyse different steps of iron absorption: the uptake of iron by the mucosal microvillus membrane, the proportional transport of iron by the basolateral membrane, and the final retention of iron in the body at about two weeks after ingestion of an oral test dose (Marx, 1979b; Marx et al., 1980; Marx and van den Bent 1981). The method was adapted for use in animals like rats (Wienk et al., 1997), mice (Santos et al., 1997a), calves (Miltenberg et al., 1993) and birds (Mete et al., 2001). The double-isotope approach allows identification of iron transport modifications at the microvillus membrane, in intracellular pathways and at the basolateral membrane, and can also be applied to knock-out mice. Site-specific effects of molecular defects of transport proteins on iron absorption can be evaluated in vivo in man and in animals. If an animal model were to be claimed to exhibit similar defects to those observed in man, it would be proof that it had an identical pattern of iron absorption to the corresponding human disorder. This was clearly demonstrated in b2-microglobulin knock-out mice, which showed identical defects of iron transport, in particular of iron absorption, to

Shadow Shield Whole Body Counter

Figure 9.3 The whole-body counter of University Medical Centre, Utrecht, The Netherlands. The counter has a mobile shadow shield with two NaI(Tl) scintillation detectors (4 x 6 in) placed at opposite sites of the subject. The lead shielding is 100 cm long with a diameter of 90 cm. The scanner moves on rails over a distance of 240 cm with an adjustable speed. Extreme variations in geometry yield practically the same value for 59 Fe activity. The equipment can be used for measurements in man and small animals.

Figure 9.3 The whole-body counter of University Medical Centre, Utrecht, The Netherlands. The counter has a mobile shadow shield with two NaI(Tl) scintillation detectors (4 x 6 in) placed at opposite sites of the subject. The lead shielding is 100 cm long with a diameter of 90 cm. The scanner moves on rails over a distance of 240 cm with an adjustable speed. Extreme variations in geometry yield practically the same value for 59 Fe activity. The equipment can be used for measurements in man and small animals.

those observed in patients with hereditary haemochromatosis (Santos etal., 1998). The changes could easily be distinguished from adaptive responses to anaemia, increased erythropoiesis, iron deficiency and artificial iron loading. The whole-body scanner of the University Medical Centre, Utrecht (The Netherlands), which has now been used for almost 30 years in clinical and experimental iron absorption studies, is shown in Figure 9.3 (Marx, 1979a,b).

Figure 9.4 (a) Schematic representation of iron absorption in normal subjects. The upper panel represents a mature intestinal mucosa cell at the tip of a villus. The lower panel represents an early cell in a crypt of Lieberkuhn. NRAMP2 = natural-resistance-associated macrophage protein2; DMT1 = divalent metal transporter 1; IREG1 = iron-regulated transporter 1; HaemTr = haem transporter; HaemOx = haem oxygenase I; IRE = iron responsive element; IRP = iron responsive protein; HFE = the 'haemochromatosis gene' product; b2m = ^-microglobulin; DcytB = duodenal cytochrome B; TfR = transferrin receptor.

The situation prevailing in the crypt cell at the beginning of its differentiation into an enterocyte and before it has begun to climb towards the villus is shown in the lower panel. The cell's iron requirements are supplied by receptor-mediated diferric transferrin uptake from the basolateral membrane. The TfR in turn is involved in an interaction with the HFE protein, which decreases the affinity of TfR for diferric transferrin. The level of transferrin saturation, or some other factor, determines the amount of iron taken up, and presets the IRP system at a level that corresponds to the iron requirements of the organism.

In the enterocyte as it enters the absorptive zone near to the villus tips, dietary iron is absorbed either directly as Fe(II) after reduction in the gastrointestinal tract by reductants like ascorbate, or after reduction of Fe(III) by the apical membrane ferrireductase Dcytb, via the divalent transporter Nramp2 (DCT1). Alternatively, haem is taken up at the apical surface, perhaps via a receptor, and is degraded by haem oxygenase to release Fe(II) into the same intracellular pool. The setting of IRPs (which are assumed to act as iron biosensors) determines the amount of iron that is retained within the enterocyte as ferritin, and that which is transferred to the circulation. This latter process is presumed to involve IREG 1 (ferroportin) and the GPI-linked hephaestin at the basolateral membrane with incorporation of iron into apotransferrin. (b) A representation of iron absorption in HFE-related haemochromatosis.

(apo) transferrin

(apo) transferrin

Hephaestin Fe-oxidase Q

IREG1

Iron Metabolism And Absorption

Transferrin

Hephaestin Fe-oxidase Q

IREG1

Transferrin

Absorption
(b)

In Figure 9.4(a) we have summarized iron transport by intestinal mucosa cells. Known iron transport proteins, described in more detail in Chapter 8, are included. It is essential to make the difference between mature mucosa cells at the tips of the villi, and developing cells in the crypts of Lieberkuhn. The total lifetime of duodenal mucosa cells is not more than two days. Considering the time needed for moving from the crypts towards the tips of the villi, each mucosa cell sees some iron passing by before the cell is released to the intestinal lumen only once or twice in its lifetime, eventually after phagocytosis by local macrophages. The events depicted in Figure 9.4 correspond to the situation where mucosal cells have been exposed to physiological amounts of iron from the diet. If the mucosa is flooded with iron, as may happen during oral treatment of iron deficiency or in iron intoxication, other events may prevail. Fe(II) may then not only use the physiological pathway involving NRAMP2 and IREG1, but may damage cell membranes by formation of toxic oxygen metabolites and by lipid peroxidation, allowing rapid diffusion of Fe(II) towards the plasma (Marx and Aisen, 1981; Fodor and Marx, 1988). However let us first briefly summarize the physiological pathway taken by dietary iron that reaches the microvillus surface in a water-soluble form. As transport of iron across the microvillus membrane by NRAMP2 (also described as DCT1 or DMT1), a divalent metal transporter (Gunshin et al., 1997), will only transport iron as Fe(II), transport across the apical membrane must be preceded by a Fe-reductase step in the close vicinity of the transporter (Riedel et al., 1995). This activity may be provided by a duodenal cytochrome B (DcytB). In the reducing environment of the cytosol, iron will remain long enough in the Fe(II) state to reach the basolateral membrane or to encounter a ferritin molecule, which incorporates it as Fe(II) (Chapter 6). If there are many ferritin molecules available (this is the normal situation), then much of the iron will be trapped in ferritin and retained in the mucosal cell; this will be reflected in vivo as a low mucosal transfer of iron (Marx, 1979a,b).

In iron deficiency, mucosal cells, like other cells in the body, hardly produce any ferritin and most of the iron crossing the apical membrane is available for transport to the plasma, which is effected by the newly discovered basolateral iron transporter IREG1 (McKie etal., 2000), also known as ferroportin (Donovan etal., 2000) or MTP1 (Abboud and Haile, 2000). It seems that there are hardly any quantitative restrictions for basolateral Fe(II) transport. Once Fe(II) has entered the mucosal cell it has only two choices: either to be trapped by ferritin or to be transported to the plasma. It is thought that IREG1 facilitates diffusion of Fe(II) across the basolateral membrane of the enterocyte. It has been known for many years that iron entering the portal circulation is in the ferrous state (Wollenberg et al., 1990), and the flux of iron across the basolateral membrane may be significantly driven by oxidation of the highly soluble Fe(II) to much less soluble Fe(III). This could be catalysed either by the recently discovered multicopper oxidase hephaestin, localized in the basolateral membrane, or by ceruloplasmin in the plasma. The Fe(III) so formed would be rapidly bound to plasma ligands, essentially apotransferrin, except in situations of saturation of the binding capacity of the plasma transporter when it would also bind to other ligands such as citrate, constituting the so-called non-transferrin bound iron (NTBI). Iron export from the enterocyte would also be influenced by the velocity of the blood flow in the portal system. It is obvious from numerous studies that iron deficiency results in a high mucosal uptake (with up-regulation of NRAMP2) and a high mucosal transfer of iron (with low mucosal ferritin and a considerable increase of free iron-binding sites on plasma transferrin). Iron absorption is, however, also increased in secondary iron overload due to increased erythropoiesis in severe haemolysis and dyserythropoiesis. In such conditions ferrokinetic studies have demonstrated an enormously increased plasma iron turnover, apparently induced by increased expression of transferrin receptors on erythropoietic cells (Barosi et al., 1978; Stefanelli et al., 1980). Although plasma iron saturation is rather high, this is more than compensated for by the very high plasma-iron turnover, offering a number of free iron sites on transferrin which may even exceed that found in iron deficiency (Marx, 1982). In normal subjects, and certainly in iron deficiency, all iron from mucosal cells entering plasma will bind to transferrin. Though Fe(II) can also be autoxidized to bind to transferrin, this takes time. After absorption iron can be detected in plasma as potentially toxic Fe(II) (Wollenberg etal., 1990). It is an important task of hephaestin (and possibly ceruloplasmin) to facilitate oxidation of Fe(II), which will allow rapid binding of iron to transferrin and its delivery to cells expressing IRP-regulated transferrin receptors, thus preventing endothelial damage and uptake of iron instead by hepatocytes.

Expression of proteins involved in mucosal iron transport is regulated in dividing cells in the crypts of Lieberkuhn, as hypothesized in Figure 9.4. There can be little doubt that IRPs play a decisive role here. In the crypts, abundant concentrations of transferrin receptor (TfR) can be identified on the basolateral membrane of enterocytes; once the cells have reached the tips of the villi, the transferrin receptors have disappeared. With normal plasma iron saturation, the labile iron pool in young mucosal cells will be high, IRP binding to IREs will be low, and ferritin can be synthesized. In the adult absorbing cell, this will restrict mucosal iron transfer to the plasma. As mRNAs of NRAMP2 and IREG1 carry IREs, similar to the transferrin receptor but not always in the same region, sufficient amounts of those proteins will be expressed to allow adequate mucosal uptake and basolateral transport of iron. In iron-deficient young mucosal cells, IRP binding to IREs must be increased. Despite more copies of TfR little iron is entering the cells, as transferrin has just delivered all its iron to the bone marrow. As a result also NRAMP2 and probably IREG1 will be up-regulated, allowing a much higher mucosal uptake (Canonne-Hergaux et al., 1999) and a complete transfer of all Fe(II) entering the cell to and across the basolateral membrane, the latter because no iron trap in the form of ferritin is available.

The peculiar thing in hereditary haemochromatosis (HH) is that the intestinal mucosal cell behaves essentially like an iron deficient cell. Iron absorption is always high if related to the body's iron needs. In HH subjects with normal plasma ferritin values, both mucosal uptake and mucosal transfer of iron often exceed values found in patients with uncomplicated iron deficiency (Marx, 1979b). In fact the situation with respect to iron absorption in mature intestinal mucosal cells, as depicted in Figure 9.4(b), is identical to that in iron deficiency, except for the difference in plasma iron saturation. It was already known that mucosal cells in HH contain no ferritin, explaining the high mucosal transfer of iron (Francanzani et al., 1989). Recent work has made it clear that in HH, NRAMP2 is greatly over-expressed, explaining the high mucosal uptake of iron observed in this condition (Zoller et al., 1999; Fleming et al., 1999). There are indications that IREG1, which carries an IRE in its 5'-UTR, is also regulated by IRP in a similar way in iron deficiency and HH (McKie etal., 2000)+. Although there is only circumstantial evidence, observations in HH point to a situation where there is a low concentration of iron in the labile iron pool in the preenterocyte as it leaves the crypts and begins to migrate towards the villi. Yet, iron in plasma is abundantly available, with transferrin saturated with iron, so we might conclude that binding of transferrin to its receptors on the basolateral membrane must be defective in HH. Since the TfR itself is normal in HH (Tsuchihashi et al., 1998), this might suggest insufficient expression of TfR on the basolateral membrane. In iron-poor cells, however, more then enough TfR is produced. It must be concluded, therefore, that the transport of the transferrin-TfR complex from the cell membrane is impaired. The Cys-282Tyr mutation of the HFE gene must be responsible for this functional defect. The biochemical basis of this mutation is disruption of a disul-phide bridge, which prohibits locking in of a b2-microglobulin molecule, required for membrane expression of classical and non-classical MHC class I molecules like HFE (Waheed et al., 1997). This also explains why b2-microglobulin knockout mice are virtually perfect models for human HH (Santos et al., 1996, 1997a,b). Apparently HFE functions as a chaperone protein for TfR, perhaps facilitating its migration to the cell membrane (Feder et al., 1998). There is much confusion about the affinity of transferrin for TfR compared to that of the TfR-HFE complex. Experiments have been performed in a variety of cell types, the majority of which are not relevant for studies on iron absorption (Gross et al., 1998; Roy et al., 1999). In experiments using gut crypt and villus cells from human duodenum coexpression in crypt enterocytes of HFE, b2-microglobulin and TfR was clearly observed (Waheed etal., 1999). In the design of further studies one should take into account that the interactions of normal and mutated HFE with TfR, and its role in the cellular procurement of transferrin-bound iron, may be completely different in enterocytes and macrophages as compared with, say, hepatocytes, pancreas, heart and, in particular, bone marrow. One of the classical features of HH is that iron overload, with altered iron absorption, coexists with a state of completely normal erythropoiesis. Although defective coexpression of HFE-TfR on the enterocyte basolateral membrane could explain the pathophysiology of iron absorption in HH, TfR-mediated endocytosis should be fully intact in erythrob-lasts. Because many patients with the clinical picture of HH are not carrying the homozygote Cys-282Tyr mutation of HFE, other molecular defects may lead to misinterpretation of the body iron status by crypt enterocytes, leading to increased intestinal iron transport. The outline of the puzzle of iron absorption becomes clearer, but there remain many missing pieces which must still be fitted into the puzzle.

+ This constitutes one of the many anomalies found in trying to explain how the IRP system regulates iron uptake in normal and disease conditions. An IRE located in the 5'-UTR would be expected to prevent formation of the initiation complex required for translation of the mRNA (see Chapter 7 for details), yet the results reported by McKie et al., show the opposite, i.e. when IRP binding activity is high, both IREG1 mRNA and protein levels are increased.

Iron Absorption in Disorders of Iron Metabolism 9.3.1 Genotype and Phenotype of Animal and Human Iron Disorders

Before the recent identification of many new genes involved in iron transport and homeostasis, a lot was already known about the phenotype of many iron disorders. Clinicians provided detailed descriptions of diseases related to iron deficiency and iron overload, and animal models allowed us to gain even more insight into the biochemistry and pathophysiology of the human disorders (Santos etal., 2000b; Ramm, 2000; Smith, 2000). An overview of genes that may affect iron metabolism in man, together with the relevant animal model, is provided in Table 9.1. If the gene products are projected into Figures 9.1 and 9.4, a clear picture of the variety of phenotypes in patients with mutations of those genes emerges. It is possible to predict phenotypes of not-yet-identified disorders of iron transport and storage. Patients with all those disorders will exist, sometimes with a diagnosis of iron deficiency or iron overload of unknown origin! Close cooperation of clinical scientists and biochemists is needed to fill in all gaps of our knowledge of iron disorders in the near future.

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