Disorders of iron metabolism. Part II: iron deficiency and iron overload

MAIN DISORDERS OF IRON METABOLISM: Increased iron requirements, limited external supply, and increased blood loss may lead to iron deficiency (ID) and iron deficiency anaemia. In chronic inflammation, the excess of hepcidin decreases iron absorption and prevents iron recycling, resulting in hypoferraemia and iron restricted erythropoiesis, despite normal iron stores (functional iron deficiency), and finally anaemia of chronic disease (ACD), which can evolve to ACD plus true ID (ACD+ID). In contrast, low hepcidin expression may lead to hereditary haemochromatosis (HH type I, mutations of the HFE gene) and type II (mutations of the hemojuvelin and hepcidin genes). Mutations of transferrin receptor 2 lead to HH type III, whereas those of the ferroportin gene lead to HH type IV. All these syndromes are characterised by iron overload. As transferrin becomes saturated in iron overload states, non-transferrin bound iron appears. Part of this iron is highly reactive (labile plasma iron), inducing free radical formation. Free radicals are responsible for the parenchymal cell injury associated with iron overload syndromes. ROLE OF LABORATORY TESTING IN DIAGNOSIS: In iron deficiency status, laboratory tests may provide evidence of iron depletion in the body or reflect iron deficient red cell production. Increased transferrin saturation and/or ferritin levels are the main cues for further investigation of iron overload. The appropriate combination of different laboratory tests with an integrated algorithm will help to establish a correct diagnosis of iron overload, iron deficiency and anaemia. REVIEW OF TREATMENT OPTIONS: Indications, advantages and side effects of the different options for treating iron overload (phlebotomy and iron chelators) and iron deficiency (oral or intravenous iron formulations) will be discussed.     

Determination of iron in solutions containing iron complexes

A method of estimating the iron content of solutions containing haemoglobin, ferritin, or ferrioxamine is described. Iron is released by treatment with acid permanganate and ascorbic acid before conventional determination by an Auto Analyzer technique.     

Urinary iron excretion test in iron deficiency anemia

A urinary iron excretion test was carried out in 22 patients with iron deficiency anemia. The iron excretion index was significantly higher in patients with intractable iron deficiency anemia compared with normal subjects and anemic patients who were responsive to iron therapy. The findings suggest that iron excretion may be a factor that modulates the response of patients to iron therapy.     

Synovial iron deposits in black subjects with iron overload

The traditional alcoholic beverages drunk by many South African blacks are prepared in iron drums, and, as a result, iron overload is common in this population. The typical pattern of iron distribution in such persons with severe iron overload is one in which the major deposits are in hepatocytes and the reticuloendothelial system. However, when cirrhosis is present, significant epithelial deposits are found in a number of organs. In the present study, the synovium of the knee joint was examined in 41 black subjects and synovial iron deposits, when present, were correlated with those in other organs. Significant amounts of iron in the synovial-lining cells were not found in any subject in whom the hepatic iron concentration was less than 1% dry weight. Heavy deposits, which were found in six of 19 subjects with concentrations above this figure, only occurred in those exhibiting cirrhosis and significant levels of histological iron in a number of epithelial tissues. Insofar as iron uptake is concerned, these findings suggest that there are cells within the synovium that have the functional characteristics of epithelial cells.     

The role of iron and iron chelators in zygomycosis

Iron is an essential element for cell growth and development, contributing to DNA synthesis and regulating the G₁-phase to S-phase transition. Moreover, iron is important for the virulence of the majority of microorganisms, and the function of the genes regulating iron uptake is coupled with the manifestations of the virulence phenotype. All fungi elaborate specific uptake mechanisms to sequester iron, and most commonly produce small molecules with high affinity for iron, the siderophores. The importance of iron appears to be particularly high for Zygomycetes, which grow abundantly in iron-rich media, and all the known predisposing factors for zygomycosis have, as a common feature, the increased availability of free iron. Among the known iron chelators, deferoxamine supports the growth of Zygomycetes because it acts as xenosiderophore, delivering iron to iron-uptaking molecules of these species. Conversely, the newer iron chelators deferiprone and deferasirox do not exhibit similar activity, apparently because they share higher affinity constants for iron and, as a result, deprive the fungi of iron, inhibiting their growth. This activity has been documented in various culture systems and in many animal models of zygomycosis, and therefore suggests that these drugs might be used as adjuvant treatment for systemic zygomycosis. There are few case reports in which the newer iron chelators have been used as antifungals, and their possible benefit must be verified in a prospective randomized trial.     

Hepatic iron in dialysed patients given intravenous iron dextran.

Five percutaneous biopsy and 17 necropsy liver specimens were analysed histologically and chemically for iron content in 22 patients receiving dialysis for chronic renal failure, 13 of whom were given intravenous iron-dextran. Brissot scores for assessing histological hepatic iron deposition and chemically measured liver iron concentrations correlated closely. Both variables depended on total cumulative dose of iron, and to a lesser extent, on time since the last dose. Fibrosis (seen in five patients) was minimal and non-specific. Electron microscopic examination showed that there was no generalised damage and confirmed the presence of iron in the hepatocytes in the form of ferritin. High liver iron concentrations, in excess of 1000 micrograms/100 mg dry weight, were seen in two patients. Four others given comparable cumulated amounts (18-23 g iron) did not have such high concentrations. Plasma ferritin concentrations were high in eight patients, some with and some without fibrosis. The risk of temporarily high iron deposition in the liver causing damage seemed to be minimal when weighed against the benefit of increased haemoglobin in most of the patients. Intravenous iron treatment merits further evaluation, particularly with the advent of erythropoietin treatment, which requires continuously available iron.     

Impact of iron loading and iron chelation on murine tuberculosis

Objective: To demonstrate in a mouse model of tuberculosis that excess iron load enhances the growth of Mycobacterium tuberculosis and to assess whether or not iron chelation may abrogate the effect of iron loading on Mycobacterium tuberculosis growth in the mouse model.
Methods: In the first experiment, female BALB/C mice were infected intravenously with 5.4 × 10⁴ CPU of M. tuberculosis H37Rv per mouse. Before infection, half of them were treated for 2 weeks with 50 mg/kg polymaltose ferric hydroxide, a source of iron. In a second experiment, female BALB/C mice were infected intravenously with 9 × 10⁴ CPU per mouse and half of them were iron loaded for 2 weeks before infection. Both iron-loaded and non-iron-loaded mice were treated with desferrioxamine (DFO), an iron chelator, or isoniazid. At each sacrifice, mice and their spleens were weighed, lung lesions were noted, and the number of M. tuberculosis CFU determined by quantitative cultures of spleen and lungs.
Results: In the first experiment, the number of CFU was significantly higher in the spleen of iron-treated mice than in non-iron-loaded mice at days 14 and 28 after infection. In the second experiment, iron loading enhanced the multiplication of M. tuberculosis in the spleen but not in the lungs, DFO displayed a modest but significant effect on the multiplication of M. tuberculosis in iron-loaded mice, and isoniazid therapy was effective in both iron-loaded and non-iron-loaded mice.
Conclusions: Iron loading of BALB/C mice enhanced the multiplication of M. tuberculosis in the spleens but not in the lungs. DFO exhibited significant activity against M. tuberculosis in iron-loaded mice, and isoniazid therapy was strongly bactericidal in both iron-loaded and non-iron-loaded mice.     

Are iron supplements appropriate for iron replete pregnant women?

Iron replete pregnant women often are routinely advised to take a daily supplement of 30–40 mg iron. An extensive review of controlled trials has failed to demonstrate that this practice improves clinical outcome of mother or newborn. However, this iron loading has long been assumed to be harmless. Recently, two hazardous complications of pregnancy, gestational diabetes and pre-eclampsia, have been recognized to be associated with iron loading. Accordingly, it may be appropriate to consider performing, at the patient’s initial prenatal medical visit, a serum ferritin test to ascertain iron status. This simple procedure would enable evidence-based medical practice to replace mass medication with iron, a potentially toxic element.     

Brain iron homeostasis

Iron is essential for virtually all types of cells and organisms. The significance of the iron for brain function is reflected by the presence of receptors for transferrin on brain capillary endothelial cells. The transport of iron into the brain from the circulation is regulated so that the extraction of iron by brain capillary endothelial cells is low in iron-replete conditions and the reverse when the iron need of the brain is high as in conditions with iron deficiency and during development of the brain. Whereas there is good agreement that iron is taken up by means of receptor-mediated uptake of iron-transferrin at the brain barriers, there are contradictory views on how iron is transported further on from the brain barriers and into the brain extracellular space. The prevailing hypothesis for transport of iron across the BBB suggests a mechanism that involves detachment of iron from transferrin within barrier cells followed by recycling of apo-transferrin to blood plasma and release of iron as non-transferrin-bound iron into the brain interstitium from where the iron is taken up by neurons and glial cells. Another hypothesis claims that iron-transferrin is transported into the brain by means of transcytosis through the BBB. This thesis deals with the topic "brain iron homeostasis" defined as the attempts to maintain constant concentrations of iron in the brain internal environment via regulation of iron transport through brain barriers, cellular iron uptake by neurons and glia, and export of iron from brain to blood. The first part deals with transport of iron-transferrin complexes from blood to brain either by transport across the brain barriers or by uptake and retrograde axonal transport in motor neurons projecting beyond the blood-brain barrier. The transport of iron and transport into the brain was examined using radiolabeled iron-transferrin. Intravenous injection of [59Fe-125]transferrin led to an almost two-fold higher accumulation of 59Fe than of [125I]transferrin in the brain. Some of the 59Fe was detected in CSF in a fraction less than 30 kDa (III). It was estimated that the iron-binding capacity of transferrin in CSF was exceeded, suggesting that iron is transported into the brain in a quantity that exceeds that of transferrin. Accordingly, it was concluded that the paramount iron transport across the BBB is the result of receptor-mediated endocytosis of iron-containing transferrin by capillary endothelial cells, followed by recycling of transferrin to the blood and transport of non-transferrin-bound iron into the brain. It was found that retrograde axonal transport in a cranial motor nerve is age-dependent, varying from almost negligible in the neonatal brain to high in the adult brain. The principle sources of extracellular transferrin in the brain are hepatocytes, oligodendrocytes, and the choroid plexus. As the passage of liver-derived transferrin into the brain is restricted due to the BBB, other candidates for binding iron in the interstitium should be considered. In vitro studies have revealed secretion of transferrin from the choroid plexus and oligodendrocytes. The second part of the thesis encompasses the circulation of iron in the extracellular fluids of the brain, i.e. the brain interstitial fluid and the CSF. As the latter receives drainage from the interstitial fluid, the CSF of the ventricles can be considered a mixture of these fluids, which may allow for analysis of CSF in matters that relate to the brain interstitial fluid. As the choroid plexus is known to synthesize transferrin, a key question is whether transferrin of the CSF might play a role for iron homeostasis by diffusing from the ventricles and subarachnoid space to the brain interstitium. Intracerebroventricular injection of [59Fe125I]transferrin led to a higher accumulation of 59Fe than of [125I]transferrin in the brain. Except for uptake and axonal transport by certain neurons with access to the ventricular CSF, both iron and transferrin were, however, restricted to areas situated in close proximity to the ventricular and pial surfaces. In particular, transferrin injected into the ventricles was never observed in regions distant from the CSF. It was concluded that choroid plexus-derived transferrin is not likely to play a significant role for binding and transporting iron in the brain interstitium. Transferrin secretion from oligodendrocytes probably plays the key role in this process. In the third part of the thesis, the uptake of iron by neurons devoid of projections beyond the blood-brain barrier and glia is addressed. Given the fact that the demonstration of plasma proteins in brain sections can be hampered by several methodological factors, a mapping of the cellular distribution of transferrin in the brain was performed employing extensive use of tissue-processing and staining protocols. In order to aid in the understanding of cellular iron uptake in the intact brain, attempts were made to identify iron, transferrin, and transferrin receptors at the light microscopic level. Consistent with the widespread distribution of transferrin receptors in neurons, the ligand transferrin was also found in neurons throughout the CNS. When examined at high resolution, transferrin was found to be distributed to the cytoplasm of neurons, exhibiting a dotted appearance, which is probably consistent with a distribution in the endosomallysosomal system. In contrast to the consistent presence of transferrin receptors on neurons, it was not possible to detect transferrin receptors on glial cells. Related to these observations, the presence of non-transferrin-bound iron in the brain suggests that glial cells may take it up by a mechanism that does not involve the transferrin receptor. The widespread distribution of ferritin in glial cells clearly indicates that the glial cells acquire iron. Dietary iron-overload did not change the distribution of transferrin receptors or ferritin in the brain. By contrast, iron deficiency altered the cellular content of these proteins so that transferrin receptors were higher and ferritin lower. The transport of iron from brain to blood was addressed in the last part of the thesis. It was found that in the case of iron and transferrin, there is no evidence showing other significant routes of transport from the brain extracellular fluid into the blood than drainage to the ventricular system followed by export to the blood via the arachnoid villi. The turnover of transferrin in the CSF was found to be very high. For reasons mentioned above, transferrin of the CSF is of little significance for transport and cellular delivery of iron to transferrin receptor-expressing neurons. Instead, transferrin of the CSF probably plays a significant role for neutralization and export to the blood of metals, including iron. Once appearing in blood, transferrin of the CSF was degraded at the same rate as intravenously injected transferrin, which indicates that the transferrin of CSF is not altered to an extent that changes its catabolism during the passage from CSF to blood plasma. The metabolism of iron in the developing brain was found to differ markedly when compared to that of the adult brain. A developing regulated transfer of iron to the brain was reflected morphologically by a higher content of transferrin receptors and non-heme iron in endothelial cells of the developing rat brain than in the adult. Neurons had a very low level of transferrin receptors. After about 20 days of age, iron transport into the brain decreased rapidly, and transferrin receptors appeared on neurons. Iron and transferrin injected into the ventricular system of the developing brain were much more widely distributed in the brain parenchyma than in the adult brain. This high accumulation of substances injected into the ventricles in young animals is probably due to the lower rate of production and turnover of CSF, which will increase the time available for diffusion of proteins into the brain parenchyma, thus giving neurons of the developing brain the opportunity to take up transferrin originating from the CSF.     

Hepatic iron concentration and total body iron stores in thalassemia major

BACKGROUND AND METHODS: We tested the usefulness of measuring the hepatic iron concentration to evaluate total body iron stores in patients who had been cured of thalassemia major by bone marrow transplantation and who were undergoing phlebotomy treatment to remove excess iron. RESULTS: We began treatment with phlebotomy a mean (+/-SD) of 4.3+/-2.7 years after transplantation in 48 patients without hepatic cirrhosis. In the group of 25 patients with liver-biopsy samples that were at least 1.0 mg in dry weight, there was a significant correlation between the decrease in the hepatic iron concentration and total body iron stores (r=0.98, P<0.001). Assuming that the hepatic iron concentration is reduced to zero with complete removal of body iron stores during phlebotomy, the amount of total body iron stores (in milligrams per kilogram of body weight) is equivalent to 10.6 times the hepatic iron concentration (in milligrams per gram of liver, dry weight). With the use of this equation, we could reliably estimate total body iron stores as high as 250 mg per kilogram of body weight, with a standard error of less than 7.9. CONCLUSIONS: The hepatic iron concentration is a reliable indicator of total body iron stores in patients with thalassemia major. In patients with transfusion-related iron overload, repeated determinations of the hepatic iron concentration can provide a quantitative means of measuring the long-term iron balance.